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TRANSCRIPT
SUPPORTING INFORMATION
An electrochemical immunosensor using gold nanoparticles-PAMAM-
nanostructured screen-printed carbon electrodes for tau protein
determination in plasma and brain tissues from Alzheimer patients
Claudia A Razzinoabζ Veroacutenica Serafiacutena ζ Maria Gamellaa ζ Mariacutea Pedreroa Ana Montero-
Callec Rodrigo Barderasc Miguel Calerod Anderson O Loboe Paloma Yaacutentildeez-Sedentildeoa
Susana Campuzanoa Joseacute M Pingarroacutena
ζThese authors contributed equally to this work
To whom correspondence should be addressed (susanacrquimucmes
pingarroquimucmes)
CONTENTS PAGES
MATERIALS AND METHODS S2-S6
Apparatus and electrodes S2-S3
Reagents and solutions S3-S4
Synthesis of 3D-Au-PAMAM S4
Immunosensor preparation S4-S5
Electrochemical measurements S5-S6
Analysis of real samples S6
RESULTS AND DISCUSSION S7-S18
Characterization of 3D-Au-PAMAM S7-S10
Fig S1 S7-S8
Fig S2 S9
Fig S3 S10
Optimization of experimental variables S10-S13
S1
Fig S4 S10
Table S1 S12
Fig S5 S13
Fig S6 S14
Table S2 S15
References S15-S16
MATERIALS AND METHODS
Apparatus and electrodes
Amperometric measurements were made with a CH Instruments potentiostat (model 812B
Austin TX) controlled by the CHI812B software Cyclic voltammetry (CV) and
electrochemical impedance spectroscopy (EIS) measurements were carried out using a FRA2
microAutolab Type III potentiostatgalvanostat (Metrohm Autolab BV The Netherlands)
controlled by the GPES amp FRA software (Eco Chemie BV The Netherlands) Screen-
printed carbon electrodes (SPCEs ref DRP-110 ϕ = 4 mm) and the connector cable (ref
DRP-CAC) were purchased from Metrohm DropSens SL (Spain) All the electrochemical
measurements were at room temperature
A vortex mixer (Velp Scientifica model Wizard IR Infrared) a pH-meter (Crison model
Basic 20+) a centrifuge (Med Instruments model MPW-65R) a thermomixer MT100
incubator shaker (Universal Labortechnik) and a magnetic stirrer (Metrohm model 728)
were also employed
The UV-Vis spectroscopy studies were carried out with UV-Vis spectrophotometers (Jasco
models V-630 and V-670) controlled by Spectra manager II software and the transmission
electron microscopy (TEM) and Energy-dispersive X-ray (EDX) characterization was
S2
performed using a transmission electron microscope (FEI TECNAI Gsup2F20 HRTEM)
operating at 120 and 200 kV
Reagents and solutions
All reagents were of the highest available analytical grade and used without further
purification Sodium nitrite (NaNO2) from Panreac 4-aminobenzoic acid (p-ABA) and
aminothiophenol (S-Phe) from Across and hydrogen tetrachloroaurate (III) trihydrate
(HAuCl43H2O) from Alfa Aesar were used PAMAM dendrimer ethylenediamine core
generation 40 solution (PAMAM) sodium borohydride (NaBH4) N-(3-dimethyl-
aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) N-hydroxysulfo-succinimide (Sulfo-NHS)
hydroquinone (HQ) Tweenreg 20 hydrogen peroxide (H2O2) (30 wv) human serum
albumin (HAS) hemoglobin (Hb) and human IgG were purchased from SigmandashAldrich
Bovine serum albumin (BSA) was purchased from Gerbu BlockerTM Casein in phosphate
buffered saline (PBS) (Ref 37528 casein blocking buffer CBB solution) and Piercetrade
Protein-Free PBS (Ref 37572 protein-free blocking buffer PFBB solution) solutions were
purchased from Thermo Scientific Recombinant human tau-441 (2N4R) (Ref 842501 tau)
anti-tau antibody (Ref 806503 used as capture antibody CAb) and HRP-labeled anti-tau
antibody (Ref 814306 used as detector antibody HRP-DAb) were purchased from
BioLegend Inc (San Diego CA)
The following buffer solutions were used phosphate buffer saline (PBS) consisting of 001
mol Lminus1 phosphate buffer solution containing 0137 mol Lminus1 NaCl and 00027 mol Lminus1 KCl pH
75 005 mol Lminus1 phosphate buffer (PB) pH 60 PBS supplemented with 005 (wv)
Tweenreg 20 (PBST) 25 mmol Lminus1 MES buffer pH 50 Other solutions used were 100 μmol
Lminus1 PAMAM 20 mmol Lminus1 HAuCl4 03 mol Lminus1 NaBH4 prepared in 03 mol Lminus1 NaOH All
S3
solutions were prepared with ultrapure water (182 MΩ cm) from a Milli-Qtrade Element A10
System
Procedures
Synthesis of 3D-Au-PAMAM
Dendrimer-encapsulated gold nanoparticles were synthesized according to Kim et al (Kim et
al 2004) through reduction of dendrimer-encapsulated Au3+ ions by H2 generated by sodium
borohydride solution Briefly 35 mL of a 20 mM HAuCl4 aqueous solution were added into
a beaker containing 5 mL of 10 microM PAMAM aqueous solution protected from light
Magnetic stirring was kept for 20 min Under magnetic stirring a 43-microL aliquot of a freshly
prepared 03 M NaBH4 basic solution was then added The resulting 3D-Au-PAMAM
suspension stable at least for 2 months was stored in a refrigerator and used after 24 h
without purification The synthetized 3D-Au-PAMAM nanocomposite was characterized by
UV-Vis spectroscopy Transmission Electron Microscopy (TEM) High Resolution
Transmission Microscopy (HRTEM) Energy Dispersive X-ray (EDX) and Cyclic
Voltammetry (CV)
Immunosensor preparation
The aryl diazonium salt was prepared by adding dropwise under constant magnetic stirring
2 mmol L-1 NaNO2 aqueous solution to 10 mg of p-ABA dissolved in 10 mL of 1 M HCl
cooled in an ice bath (38 μL of NaNO2 aqueous solution for each 200 μL of p-ABA acid
solution) The SPCEs were immersed in 1 mL of this resulting solution and ten successive
cyclic voltammetric scans were made between 00 and minus10 V (v = 200 mV sminus1) (Moreno-
Guzmaacuten et al 2012) The SPCEs functionalized by p-ABA electrografting (p-ABA-SPCEs)
were washed thoroughly with ultrapure water dried at room temperature and activated by
S4
coating the working electrode with a 10-microL drop of a EDCSulfo-NHS mixture solution (01
mol Lminus1 each and freshly prepared in 25 mmol Lminus1 MES buffer solution pH 50) and let to
stand for 30 min at room temperature in a humid chamber ndash this condition was used for all
incubation steps After rinsing with 25 mmol Lminus1 MES buffer solution pH 50 and drying at
room temperature 10 μL of the 3D-Au-PAMAM suspension were casted on the EDCSulfo-
NHS-activated p-ABA-SPCEs and let to react for 30 min Then the 3D-Au-PAMAM-p-
ABA-SPCEs were rinsed with ultrapure water dried at room temperature and incubated with
5 microL of a 05 (vv) GA solution (prepared in PBS) for 60 min After rinsing the GA-
activated-3D-Au-PAMAM-p-ABA-SPCEs with PBST a 5-microL aliquot of a 10 microg mLminus1 CAb
solution was added and let to react for 60 min The CAb-3D-Au-PAMAM-p-ABA-SPCEs
were rinsed with PBS and a blocking step with 10 microL of PFBB solution was carried out for 45
min Finally the modified electrodes were washed several times and kept at 4 ordmC until use
The sandwich immunoassay was carried out by incubating for 60 min the CAb-3D-Au-
PAMAM-p-ABA-SPCE with 5 microL of a mixture solution containing the tau standard (or the
sample to be analyzed) and 01 microg mLminus1 of HRP-DAb prepared in blocker casein solution
(CBB) The modified electrodes were washed with PBS solution and kept in a 10 microL-drop of
PBS pH 75 at room temperature until the electrochemical measurements were carried out
Electrochemical measurements
The stepwise preparation of the immunosensor was electrochemically characterized by CV
and EIS CV experiments were performed at 50 mV sminus1 over a potential range of either 00 to
+14 V or minus03 to +10 V in either 01 M H2SO4 or 01 mol Lminus1 KCl containing 50 mmol Lminus1
[Fe(CN)6]3minus4minus respectively The EIS measurements were carried out under open circuit
conditions in a frequency range of 100 kHz to 004 Hz with a sinusoidal signal of 10 mV
The frequency interval was divided into 50 logarithmically equidistant measure points
S5
Amperometric measurements were conducted in stirred solutions at minus020 V vs Ag pseudo-
reference electrodes after immersing the immunosensor into an electrochemical cell
containing 10 mL of 50 mmol Lminus1 PB solution pH 60 supplemented with 10 mmol Lminus1 HQ
(freshly prepared) After stabilization of the background current 50 μL of a 01 mol Lminus1 H2O2
fresh solution were added and the variation in the cathodic current was recorded until
reaching the steady-state current
All the error bars shown in the Figures were estimated as a triple of the standard deviation
(n=3)
Analysis of real samples
Plasma and brain tissue samples were obtained from the CIEN Foundationrsquos Tissue Bank
(BT-CIEN) According to the brain bankrsquos protocols neuropathological diagnosis and
classification of cases was performed on the basis of international consensus criteria (Thal et
al 2002 Mirra et al 1993) Written informed consent was obtained from all patients
Brain tissue protein extraction was performed as previously reported (Barderas et al 2012
Barderas et al 2013) Briefly tissue samples were cut in small pieces in dry ice and
mechanically disaggregated with 05 SDS in phosphate buffered saline (PBS) with a
protease inhibition cocktail (Sigma) and finally clarified by centrifugation at 10000 rpm
Brain tissue protein extracts were stored at ndash 80 ordmC until use Protein extracts quality was
assessed prior to be used in any experiment by Coomassie staining of SDS-PAGE 10 gels
RESULTS AND DISCUSSION
Characterization of 3D-Au-PAMAM
The synthesized 3D-Au-PAMAM nanocomposite was characterized by UVndashvis absorption
EDX TEM HRTEM and CV
S6
Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-
PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220
nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-
charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is
featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black
curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the
band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost
disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated
the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the
addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was
attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm
(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of
AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-
Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis
of the 3D-Au-PAMAM nanocomposite
S7
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Fig S4 S10
Table S1 S12
Fig S5 S13
Fig S6 S14
Table S2 S15
References S15-S16
MATERIALS AND METHODS
Apparatus and electrodes
Amperometric measurements were made with a CH Instruments potentiostat (model 812B
Austin TX) controlled by the CHI812B software Cyclic voltammetry (CV) and
electrochemical impedance spectroscopy (EIS) measurements were carried out using a FRA2
microAutolab Type III potentiostatgalvanostat (Metrohm Autolab BV The Netherlands)
controlled by the GPES amp FRA software (Eco Chemie BV The Netherlands) Screen-
printed carbon electrodes (SPCEs ref DRP-110 ϕ = 4 mm) and the connector cable (ref
DRP-CAC) were purchased from Metrohm DropSens SL (Spain) All the electrochemical
measurements were at room temperature
A vortex mixer (Velp Scientifica model Wizard IR Infrared) a pH-meter (Crison model
Basic 20+) a centrifuge (Med Instruments model MPW-65R) a thermomixer MT100
incubator shaker (Universal Labortechnik) and a magnetic stirrer (Metrohm model 728)
were also employed
The UV-Vis spectroscopy studies were carried out with UV-Vis spectrophotometers (Jasco
models V-630 and V-670) controlled by Spectra manager II software and the transmission
electron microscopy (TEM) and Energy-dispersive X-ray (EDX) characterization was
S2
performed using a transmission electron microscope (FEI TECNAI Gsup2F20 HRTEM)
operating at 120 and 200 kV
Reagents and solutions
All reagents were of the highest available analytical grade and used without further
purification Sodium nitrite (NaNO2) from Panreac 4-aminobenzoic acid (p-ABA) and
aminothiophenol (S-Phe) from Across and hydrogen tetrachloroaurate (III) trihydrate
(HAuCl43H2O) from Alfa Aesar were used PAMAM dendrimer ethylenediamine core
generation 40 solution (PAMAM) sodium borohydride (NaBH4) N-(3-dimethyl-
aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) N-hydroxysulfo-succinimide (Sulfo-NHS)
hydroquinone (HQ) Tweenreg 20 hydrogen peroxide (H2O2) (30 wv) human serum
albumin (HAS) hemoglobin (Hb) and human IgG were purchased from SigmandashAldrich
Bovine serum albumin (BSA) was purchased from Gerbu BlockerTM Casein in phosphate
buffered saline (PBS) (Ref 37528 casein blocking buffer CBB solution) and Piercetrade
Protein-Free PBS (Ref 37572 protein-free blocking buffer PFBB solution) solutions were
purchased from Thermo Scientific Recombinant human tau-441 (2N4R) (Ref 842501 tau)
anti-tau antibody (Ref 806503 used as capture antibody CAb) and HRP-labeled anti-tau
antibody (Ref 814306 used as detector antibody HRP-DAb) were purchased from
BioLegend Inc (San Diego CA)
The following buffer solutions were used phosphate buffer saline (PBS) consisting of 001
mol Lminus1 phosphate buffer solution containing 0137 mol Lminus1 NaCl and 00027 mol Lminus1 KCl pH
75 005 mol Lminus1 phosphate buffer (PB) pH 60 PBS supplemented with 005 (wv)
Tweenreg 20 (PBST) 25 mmol Lminus1 MES buffer pH 50 Other solutions used were 100 μmol
Lminus1 PAMAM 20 mmol Lminus1 HAuCl4 03 mol Lminus1 NaBH4 prepared in 03 mol Lminus1 NaOH All
S3
solutions were prepared with ultrapure water (182 MΩ cm) from a Milli-Qtrade Element A10
System
Procedures
Synthesis of 3D-Au-PAMAM
Dendrimer-encapsulated gold nanoparticles were synthesized according to Kim et al (Kim et
al 2004) through reduction of dendrimer-encapsulated Au3+ ions by H2 generated by sodium
borohydride solution Briefly 35 mL of a 20 mM HAuCl4 aqueous solution were added into
a beaker containing 5 mL of 10 microM PAMAM aqueous solution protected from light
Magnetic stirring was kept for 20 min Under magnetic stirring a 43-microL aliquot of a freshly
prepared 03 M NaBH4 basic solution was then added The resulting 3D-Au-PAMAM
suspension stable at least for 2 months was stored in a refrigerator and used after 24 h
without purification The synthetized 3D-Au-PAMAM nanocomposite was characterized by
UV-Vis spectroscopy Transmission Electron Microscopy (TEM) High Resolution
Transmission Microscopy (HRTEM) Energy Dispersive X-ray (EDX) and Cyclic
Voltammetry (CV)
Immunosensor preparation
The aryl diazonium salt was prepared by adding dropwise under constant magnetic stirring
2 mmol L-1 NaNO2 aqueous solution to 10 mg of p-ABA dissolved in 10 mL of 1 M HCl
cooled in an ice bath (38 μL of NaNO2 aqueous solution for each 200 μL of p-ABA acid
solution) The SPCEs were immersed in 1 mL of this resulting solution and ten successive
cyclic voltammetric scans were made between 00 and minus10 V (v = 200 mV sminus1) (Moreno-
Guzmaacuten et al 2012) The SPCEs functionalized by p-ABA electrografting (p-ABA-SPCEs)
were washed thoroughly with ultrapure water dried at room temperature and activated by
S4
coating the working electrode with a 10-microL drop of a EDCSulfo-NHS mixture solution (01
mol Lminus1 each and freshly prepared in 25 mmol Lminus1 MES buffer solution pH 50) and let to
stand for 30 min at room temperature in a humid chamber ndash this condition was used for all
incubation steps After rinsing with 25 mmol Lminus1 MES buffer solution pH 50 and drying at
room temperature 10 μL of the 3D-Au-PAMAM suspension were casted on the EDCSulfo-
NHS-activated p-ABA-SPCEs and let to react for 30 min Then the 3D-Au-PAMAM-p-
ABA-SPCEs were rinsed with ultrapure water dried at room temperature and incubated with
5 microL of a 05 (vv) GA solution (prepared in PBS) for 60 min After rinsing the GA-
activated-3D-Au-PAMAM-p-ABA-SPCEs with PBST a 5-microL aliquot of a 10 microg mLminus1 CAb
solution was added and let to react for 60 min The CAb-3D-Au-PAMAM-p-ABA-SPCEs
were rinsed with PBS and a blocking step with 10 microL of PFBB solution was carried out for 45
min Finally the modified electrodes were washed several times and kept at 4 ordmC until use
The sandwich immunoassay was carried out by incubating for 60 min the CAb-3D-Au-
PAMAM-p-ABA-SPCE with 5 microL of a mixture solution containing the tau standard (or the
sample to be analyzed) and 01 microg mLminus1 of HRP-DAb prepared in blocker casein solution
(CBB) The modified electrodes were washed with PBS solution and kept in a 10 microL-drop of
PBS pH 75 at room temperature until the electrochemical measurements were carried out
Electrochemical measurements
The stepwise preparation of the immunosensor was electrochemically characterized by CV
and EIS CV experiments were performed at 50 mV sminus1 over a potential range of either 00 to
+14 V or minus03 to +10 V in either 01 M H2SO4 or 01 mol Lminus1 KCl containing 50 mmol Lminus1
[Fe(CN)6]3minus4minus respectively The EIS measurements were carried out under open circuit
conditions in a frequency range of 100 kHz to 004 Hz with a sinusoidal signal of 10 mV
The frequency interval was divided into 50 logarithmically equidistant measure points
S5
Amperometric measurements were conducted in stirred solutions at minus020 V vs Ag pseudo-
reference electrodes after immersing the immunosensor into an electrochemical cell
containing 10 mL of 50 mmol Lminus1 PB solution pH 60 supplemented with 10 mmol Lminus1 HQ
(freshly prepared) After stabilization of the background current 50 μL of a 01 mol Lminus1 H2O2
fresh solution were added and the variation in the cathodic current was recorded until
reaching the steady-state current
All the error bars shown in the Figures were estimated as a triple of the standard deviation
(n=3)
Analysis of real samples
Plasma and brain tissue samples were obtained from the CIEN Foundationrsquos Tissue Bank
(BT-CIEN) According to the brain bankrsquos protocols neuropathological diagnosis and
classification of cases was performed on the basis of international consensus criteria (Thal et
al 2002 Mirra et al 1993) Written informed consent was obtained from all patients
Brain tissue protein extraction was performed as previously reported (Barderas et al 2012
Barderas et al 2013) Briefly tissue samples were cut in small pieces in dry ice and
mechanically disaggregated with 05 SDS in phosphate buffered saline (PBS) with a
protease inhibition cocktail (Sigma) and finally clarified by centrifugation at 10000 rpm
Brain tissue protein extracts were stored at ndash 80 ordmC until use Protein extracts quality was
assessed prior to be used in any experiment by Coomassie staining of SDS-PAGE 10 gels
RESULTS AND DISCUSSION
Characterization of 3D-Au-PAMAM
The synthesized 3D-Au-PAMAM nanocomposite was characterized by UVndashvis absorption
EDX TEM HRTEM and CV
S6
Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-
PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220
nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-
charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is
featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black
curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the
band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost
disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated
the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the
addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was
attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm
(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of
AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-
Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis
of the 3D-Au-PAMAM nanocomposite
S7
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
performed using a transmission electron microscope (FEI TECNAI Gsup2F20 HRTEM)
operating at 120 and 200 kV
Reagents and solutions
All reagents were of the highest available analytical grade and used without further
purification Sodium nitrite (NaNO2) from Panreac 4-aminobenzoic acid (p-ABA) and
aminothiophenol (S-Phe) from Across and hydrogen tetrachloroaurate (III) trihydrate
(HAuCl43H2O) from Alfa Aesar were used PAMAM dendrimer ethylenediamine core
generation 40 solution (PAMAM) sodium borohydride (NaBH4) N-(3-dimethyl-
aminopropyl)-Nrsquo-ethylcarbodiimide (EDC) N-hydroxysulfo-succinimide (Sulfo-NHS)
hydroquinone (HQ) Tweenreg 20 hydrogen peroxide (H2O2) (30 wv) human serum
albumin (HAS) hemoglobin (Hb) and human IgG were purchased from SigmandashAldrich
Bovine serum albumin (BSA) was purchased from Gerbu BlockerTM Casein in phosphate
buffered saline (PBS) (Ref 37528 casein blocking buffer CBB solution) and Piercetrade
Protein-Free PBS (Ref 37572 protein-free blocking buffer PFBB solution) solutions were
purchased from Thermo Scientific Recombinant human tau-441 (2N4R) (Ref 842501 tau)
anti-tau antibody (Ref 806503 used as capture antibody CAb) and HRP-labeled anti-tau
antibody (Ref 814306 used as detector antibody HRP-DAb) were purchased from
BioLegend Inc (San Diego CA)
The following buffer solutions were used phosphate buffer saline (PBS) consisting of 001
mol Lminus1 phosphate buffer solution containing 0137 mol Lminus1 NaCl and 00027 mol Lminus1 KCl pH
75 005 mol Lminus1 phosphate buffer (PB) pH 60 PBS supplemented with 005 (wv)
Tweenreg 20 (PBST) 25 mmol Lminus1 MES buffer pH 50 Other solutions used were 100 μmol
Lminus1 PAMAM 20 mmol Lminus1 HAuCl4 03 mol Lminus1 NaBH4 prepared in 03 mol Lminus1 NaOH All
S3
solutions were prepared with ultrapure water (182 MΩ cm) from a Milli-Qtrade Element A10
System
Procedures
Synthesis of 3D-Au-PAMAM
Dendrimer-encapsulated gold nanoparticles were synthesized according to Kim et al (Kim et
al 2004) through reduction of dendrimer-encapsulated Au3+ ions by H2 generated by sodium
borohydride solution Briefly 35 mL of a 20 mM HAuCl4 aqueous solution were added into
a beaker containing 5 mL of 10 microM PAMAM aqueous solution protected from light
Magnetic stirring was kept for 20 min Under magnetic stirring a 43-microL aliquot of a freshly
prepared 03 M NaBH4 basic solution was then added The resulting 3D-Au-PAMAM
suspension stable at least for 2 months was stored in a refrigerator and used after 24 h
without purification The synthetized 3D-Au-PAMAM nanocomposite was characterized by
UV-Vis spectroscopy Transmission Electron Microscopy (TEM) High Resolution
Transmission Microscopy (HRTEM) Energy Dispersive X-ray (EDX) and Cyclic
Voltammetry (CV)
Immunosensor preparation
The aryl diazonium salt was prepared by adding dropwise under constant magnetic stirring
2 mmol L-1 NaNO2 aqueous solution to 10 mg of p-ABA dissolved in 10 mL of 1 M HCl
cooled in an ice bath (38 μL of NaNO2 aqueous solution for each 200 μL of p-ABA acid
solution) The SPCEs were immersed in 1 mL of this resulting solution and ten successive
cyclic voltammetric scans were made between 00 and minus10 V (v = 200 mV sminus1) (Moreno-
Guzmaacuten et al 2012) The SPCEs functionalized by p-ABA electrografting (p-ABA-SPCEs)
were washed thoroughly with ultrapure water dried at room temperature and activated by
S4
coating the working electrode with a 10-microL drop of a EDCSulfo-NHS mixture solution (01
mol Lminus1 each and freshly prepared in 25 mmol Lminus1 MES buffer solution pH 50) and let to
stand for 30 min at room temperature in a humid chamber ndash this condition was used for all
incubation steps After rinsing with 25 mmol Lminus1 MES buffer solution pH 50 and drying at
room temperature 10 μL of the 3D-Au-PAMAM suspension were casted on the EDCSulfo-
NHS-activated p-ABA-SPCEs and let to react for 30 min Then the 3D-Au-PAMAM-p-
ABA-SPCEs were rinsed with ultrapure water dried at room temperature and incubated with
5 microL of a 05 (vv) GA solution (prepared in PBS) for 60 min After rinsing the GA-
activated-3D-Au-PAMAM-p-ABA-SPCEs with PBST a 5-microL aliquot of a 10 microg mLminus1 CAb
solution was added and let to react for 60 min The CAb-3D-Au-PAMAM-p-ABA-SPCEs
were rinsed with PBS and a blocking step with 10 microL of PFBB solution was carried out for 45
min Finally the modified electrodes were washed several times and kept at 4 ordmC until use
The sandwich immunoassay was carried out by incubating for 60 min the CAb-3D-Au-
PAMAM-p-ABA-SPCE with 5 microL of a mixture solution containing the tau standard (or the
sample to be analyzed) and 01 microg mLminus1 of HRP-DAb prepared in blocker casein solution
(CBB) The modified electrodes were washed with PBS solution and kept in a 10 microL-drop of
PBS pH 75 at room temperature until the electrochemical measurements were carried out
Electrochemical measurements
The stepwise preparation of the immunosensor was electrochemically characterized by CV
and EIS CV experiments were performed at 50 mV sminus1 over a potential range of either 00 to
+14 V or minus03 to +10 V in either 01 M H2SO4 or 01 mol Lminus1 KCl containing 50 mmol Lminus1
[Fe(CN)6]3minus4minus respectively The EIS measurements were carried out under open circuit
conditions in a frequency range of 100 kHz to 004 Hz with a sinusoidal signal of 10 mV
The frequency interval was divided into 50 logarithmically equidistant measure points
S5
Amperometric measurements were conducted in stirred solutions at minus020 V vs Ag pseudo-
reference electrodes after immersing the immunosensor into an electrochemical cell
containing 10 mL of 50 mmol Lminus1 PB solution pH 60 supplemented with 10 mmol Lminus1 HQ
(freshly prepared) After stabilization of the background current 50 μL of a 01 mol Lminus1 H2O2
fresh solution were added and the variation in the cathodic current was recorded until
reaching the steady-state current
All the error bars shown in the Figures were estimated as a triple of the standard deviation
(n=3)
Analysis of real samples
Plasma and brain tissue samples were obtained from the CIEN Foundationrsquos Tissue Bank
(BT-CIEN) According to the brain bankrsquos protocols neuropathological diagnosis and
classification of cases was performed on the basis of international consensus criteria (Thal et
al 2002 Mirra et al 1993) Written informed consent was obtained from all patients
Brain tissue protein extraction was performed as previously reported (Barderas et al 2012
Barderas et al 2013) Briefly tissue samples were cut in small pieces in dry ice and
mechanically disaggregated with 05 SDS in phosphate buffered saline (PBS) with a
protease inhibition cocktail (Sigma) and finally clarified by centrifugation at 10000 rpm
Brain tissue protein extracts were stored at ndash 80 ordmC until use Protein extracts quality was
assessed prior to be used in any experiment by Coomassie staining of SDS-PAGE 10 gels
RESULTS AND DISCUSSION
Characterization of 3D-Au-PAMAM
The synthesized 3D-Au-PAMAM nanocomposite was characterized by UVndashvis absorption
EDX TEM HRTEM and CV
S6
Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-
PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220
nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-
charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is
featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black
curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the
band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost
disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated
the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the
addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was
attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm
(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of
AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-
Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis
of the 3D-Au-PAMAM nanocomposite
S7
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
solutions were prepared with ultrapure water (182 MΩ cm) from a Milli-Qtrade Element A10
System
Procedures
Synthesis of 3D-Au-PAMAM
Dendrimer-encapsulated gold nanoparticles were synthesized according to Kim et al (Kim et
al 2004) through reduction of dendrimer-encapsulated Au3+ ions by H2 generated by sodium
borohydride solution Briefly 35 mL of a 20 mM HAuCl4 aqueous solution were added into
a beaker containing 5 mL of 10 microM PAMAM aqueous solution protected from light
Magnetic stirring was kept for 20 min Under magnetic stirring a 43-microL aliquot of a freshly
prepared 03 M NaBH4 basic solution was then added The resulting 3D-Au-PAMAM
suspension stable at least for 2 months was stored in a refrigerator and used after 24 h
without purification The synthetized 3D-Au-PAMAM nanocomposite was characterized by
UV-Vis spectroscopy Transmission Electron Microscopy (TEM) High Resolution
Transmission Microscopy (HRTEM) Energy Dispersive X-ray (EDX) and Cyclic
Voltammetry (CV)
Immunosensor preparation
The aryl diazonium salt was prepared by adding dropwise under constant magnetic stirring
2 mmol L-1 NaNO2 aqueous solution to 10 mg of p-ABA dissolved in 10 mL of 1 M HCl
cooled in an ice bath (38 μL of NaNO2 aqueous solution for each 200 μL of p-ABA acid
solution) The SPCEs were immersed in 1 mL of this resulting solution and ten successive
cyclic voltammetric scans were made between 00 and minus10 V (v = 200 mV sminus1) (Moreno-
Guzmaacuten et al 2012) The SPCEs functionalized by p-ABA electrografting (p-ABA-SPCEs)
were washed thoroughly with ultrapure water dried at room temperature and activated by
S4
coating the working electrode with a 10-microL drop of a EDCSulfo-NHS mixture solution (01
mol Lminus1 each and freshly prepared in 25 mmol Lminus1 MES buffer solution pH 50) and let to
stand for 30 min at room temperature in a humid chamber ndash this condition was used for all
incubation steps After rinsing with 25 mmol Lminus1 MES buffer solution pH 50 and drying at
room temperature 10 μL of the 3D-Au-PAMAM suspension were casted on the EDCSulfo-
NHS-activated p-ABA-SPCEs and let to react for 30 min Then the 3D-Au-PAMAM-p-
ABA-SPCEs were rinsed with ultrapure water dried at room temperature and incubated with
5 microL of a 05 (vv) GA solution (prepared in PBS) for 60 min After rinsing the GA-
activated-3D-Au-PAMAM-p-ABA-SPCEs with PBST a 5-microL aliquot of a 10 microg mLminus1 CAb
solution was added and let to react for 60 min The CAb-3D-Au-PAMAM-p-ABA-SPCEs
were rinsed with PBS and a blocking step with 10 microL of PFBB solution was carried out for 45
min Finally the modified electrodes were washed several times and kept at 4 ordmC until use
The sandwich immunoassay was carried out by incubating for 60 min the CAb-3D-Au-
PAMAM-p-ABA-SPCE with 5 microL of a mixture solution containing the tau standard (or the
sample to be analyzed) and 01 microg mLminus1 of HRP-DAb prepared in blocker casein solution
(CBB) The modified electrodes were washed with PBS solution and kept in a 10 microL-drop of
PBS pH 75 at room temperature until the electrochemical measurements were carried out
Electrochemical measurements
The stepwise preparation of the immunosensor was electrochemically characterized by CV
and EIS CV experiments were performed at 50 mV sminus1 over a potential range of either 00 to
+14 V or minus03 to +10 V in either 01 M H2SO4 or 01 mol Lminus1 KCl containing 50 mmol Lminus1
[Fe(CN)6]3minus4minus respectively The EIS measurements were carried out under open circuit
conditions in a frequency range of 100 kHz to 004 Hz with a sinusoidal signal of 10 mV
The frequency interval was divided into 50 logarithmically equidistant measure points
S5
Amperometric measurements were conducted in stirred solutions at minus020 V vs Ag pseudo-
reference electrodes after immersing the immunosensor into an electrochemical cell
containing 10 mL of 50 mmol Lminus1 PB solution pH 60 supplemented with 10 mmol Lminus1 HQ
(freshly prepared) After stabilization of the background current 50 μL of a 01 mol Lminus1 H2O2
fresh solution were added and the variation in the cathodic current was recorded until
reaching the steady-state current
All the error bars shown in the Figures were estimated as a triple of the standard deviation
(n=3)
Analysis of real samples
Plasma and brain tissue samples were obtained from the CIEN Foundationrsquos Tissue Bank
(BT-CIEN) According to the brain bankrsquos protocols neuropathological diagnosis and
classification of cases was performed on the basis of international consensus criteria (Thal et
al 2002 Mirra et al 1993) Written informed consent was obtained from all patients
Brain tissue protein extraction was performed as previously reported (Barderas et al 2012
Barderas et al 2013) Briefly tissue samples were cut in small pieces in dry ice and
mechanically disaggregated with 05 SDS in phosphate buffered saline (PBS) with a
protease inhibition cocktail (Sigma) and finally clarified by centrifugation at 10000 rpm
Brain tissue protein extracts were stored at ndash 80 ordmC until use Protein extracts quality was
assessed prior to be used in any experiment by Coomassie staining of SDS-PAGE 10 gels
RESULTS AND DISCUSSION
Characterization of 3D-Au-PAMAM
The synthesized 3D-Au-PAMAM nanocomposite was characterized by UVndashvis absorption
EDX TEM HRTEM and CV
S6
Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-
PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220
nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-
charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is
featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black
curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the
band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost
disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated
the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the
addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was
attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm
(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of
AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-
Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis
of the 3D-Au-PAMAM nanocomposite
S7
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
coating the working electrode with a 10-microL drop of a EDCSulfo-NHS mixture solution (01
mol Lminus1 each and freshly prepared in 25 mmol Lminus1 MES buffer solution pH 50) and let to
stand for 30 min at room temperature in a humid chamber ndash this condition was used for all
incubation steps After rinsing with 25 mmol Lminus1 MES buffer solution pH 50 and drying at
room temperature 10 μL of the 3D-Au-PAMAM suspension were casted on the EDCSulfo-
NHS-activated p-ABA-SPCEs and let to react for 30 min Then the 3D-Au-PAMAM-p-
ABA-SPCEs were rinsed with ultrapure water dried at room temperature and incubated with
5 microL of a 05 (vv) GA solution (prepared in PBS) for 60 min After rinsing the GA-
activated-3D-Au-PAMAM-p-ABA-SPCEs with PBST a 5-microL aliquot of a 10 microg mLminus1 CAb
solution was added and let to react for 60 min The CAb-3D-Au-PAMAM-p-ABA-SPCEs
were rinsed with PBS and a blocking step with 10 microL of PFBB solution was carried out for 45
min Finally the modified electrodes were washed several times and kept at 4 ordmC until use
The sandwich immunoassay was carried out by incubating for 60 min the CAb-3D-Au-
PAMAM-p-ABA-SPCE with 5 microL of a mixture solution containing the tau standard (or the
sample to be analyzed) and 01 microg mLminus1 of HRP-DAb prepared in blocker casein solution
(CBB) The modified electrodes were washed with PBS solution and kept in a 10 microL-drop of
PBS pH 75 at room temperature until the electrochemical measurements were carried out
Electrochemical measurements
The stepwise preparation of the immunosensor was electrochemically characterized by CV
and EIS CV experiments were performed at 50 mV sminus1 over a potential range of either 00 to
+14 V or minus03 to +10 V in either 01 M H2SO4 or 01 mol Lminus1 KCl containing 50 mmol Lminus1
[Fe(CN)6]3minus4minus respectively The EIS measurements were carried out under open circuit
conditions in a frequency range of 100 kHz to 004 Hz with a sinusoidal signal of 10 mV
The frequency interval was divided into 50 logarithmically equidistant measure points
S5
Amperometric measurements were conducted in stirred solutions at minus020 V vs Ag pseudo-
reference electrodes after immersing the immunosensor into an electrochemical cell
containing 10 mL of 50 mmol Lminus1 PB solution pH 60 supplemented with 10 mmol Lminus1 HQ
(freshly prepared) After stabilization of the background current 50 μL of a 01 mol Lminus1 H2O2
fresh solution were added and the variation in the cathodic current was recorded until
reaching the steady-state current
All the error bars shown in the Figures were estimated as a triple of the standard deviation
(n=3)
Analysis of real samples
Plasma and brain tissue samples were obtained from the CIEN Foundationrsquos Tissue Bank
(BT-CIEN) According to the brain bankrsquos protocols neuropathological diagnosis and
classification of cases was performed on the basis of international consensus criteria (Thal et
al 2002 Mirra et al 1993) Written informed consent was obtained from all patients
Brain tissue protein extraction was performed as previously reported (Barderas et al 2012
Barderas et al 2013) Briefly tissue samples were cut in small pieces in dry ice and
mechanically disaggregated with 05 SDS in phosphate buffered saline (PBS) with a
protease inhibition cocktail (Sigma) and finally clarified by centrifugation at 10000 rpm
Brain tissue protein extracts were stored at ndash 80 ordmC until use Protein extracts quality was
assessed prior to be used in any experiment by Coomassie staining of SDS-PAGE 10 gels
RESULTS AND DISCUSSION
Characterization of 3D-Au-PAMAM
The synthesized 3D-Au-PAMAM nanocomposite was characterized by UVndashvis absorption
EDX TEM HRTEM and CV
S6
Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-
PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220
nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-
charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is
featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black
curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the
band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost
disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated
the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the
addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was
attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm
(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of
AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-
Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis
of the 3D-Au-PAMAM nanocomposite
S7
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Amperometric measurements were conducted in stirred solutions at minus020 V vs Ag pseudo-
reference electrodes after immersing the immunosensor into an electrochemical cell
containing 10 mL of 50 mmol Lminus1 PB solution pH 60 supplemented with 10 mmol Lminus1 HQ
(freshly prepared) After stabilization of the background current 50 μL of a 01 mol Lminus1 H2O2
fresh solution were added and the variation in the cathodic current was recorded until
reaching the steady-state current
All the error bars shown in the Figures were estimated as a triple of the standard deviation
(n=3)
Analysis of real samples
Plasma and brain tissue samples were obtained from the CIEN Foundationrsquos Tissue Bank
(BT-CIEN) According to the brain bankrsquos protocols neuropathological diagnosis and
classification of cases was performed on the basis of international consensus criteria (Thal et
al 2002 Mirra et al 1993) Written informed consent was obtained from all patients
Brain tissue protein extraction was performed as previously reported (Barderas et al 2012
Barderas et al 2013) Briefly tissue samples were cut in small pieces in dry ice and
mechanically disaggregated with 05 SDS in phosphate buffered saline (PBS) with a
protease inhibition cocktail (Sigma) and finally clarified by centrifugation at 10000 rpm
Brain tissue protein extracts were stored at ndash 80 ordmC until use Protein extracts quality was
assessed prior to be used in any experiment by Coomassie staining of SDS-PAGE 10 gels
RESULTS AND DISCUSSION
Characterization of 3D-Au-PAMAM
The synthesized 3D-Au-PAMAM nanocomposite was characterized by UVndashvis absorption
EDX TEM HRTEM and CV
S6
Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-
PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220
nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-
charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is
featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black
curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the
band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost
disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated
the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the
addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was
attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm
(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of
AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-
Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis
of the 3D-Au-PAMAM nanocomposite
S7
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Fig S1a compares the UV-vis spectra of PAMAM HAuCl4 PAMAM plus HAuCl4 and Au0-
PAMAM (3D-Au-PAMAM) The HAuCl4 spectrum shows a strong absorption band at 220
nm and a shoulder at 290 nm (Fig S1a red curve) which can be assigned to ligand-to-metal-
charge-transfer transitions (Esumi et al 2000) However the spectrum of PAMAM is
featureless except for a rapidly increasing absorbance below about 230 nm (Fig S1a black
curve) After mixing PAMAM and HAuCl4 the spectrum (blue curve) shows a decrease in the
band at 220 nm with a slight shifting to lower energy while the shoulder at 290 nm almost
disappears and exhibits a similar displacement (Wang et al 2003) These variations indicated
the formation of a new complex (Li and Li 2003 Luo et al 2011) Moreover after the
addition of NaBH4 (pink curve) a new absorption band appeared at 520 nm which was
attributed to the surface plasmon resonance absorption in gold nanoparticles larger than 2 nm
(Alvarez et al 1997 Rao et al 2002 Kim et al 2004) thus confirming the formation of
AuNPs encapsulated by the G4-PAMAM dendrimer (Luo et al 2011) EDX analysis of 3D-
Au-PAMAM nanocomposite (Fig S1b) is in good agreement with the successful synthesis
of the 3D-Au-PAMAM nanocomposite
S7
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Fig S1 a) UV-vis spectra of 1 micromol Lminus1 PAMAM 140 micromol Lminus1 HAuCl4 PAMAM plus
HAuCl4 and Au0-PAMAM (3D-Au-PAMAM) aqueous solutionssuspensions Inset UV-vis
spectrum of the 3D-Au-PAMAM suspension b) EDX analysis of 3D-Au-PAMAM
nanocomposite
Fig S2 shows TEM and HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The images reveal well separated Au nanoparticles due to the high charge density on the
PAMAM surface Fig S2b allows observing the 3D-Au-PAMAM crystallographic plane The
AuNPs exhibited variable diameters larger than 2 nm According to Kim et al (Kim et al
2004) the main role of the quaternary amine groups in PAMAM-G4-NH2 is to prevent
nanoparticles agglomeration and the charged surface exerts only a slight influence over the
size of the encapsulated nanoparticles The slight agglomeration intuited in the images may be
due to small particles that overlap in two-dimensional projection of images and appear as a
larger particle or even to real small agglomerations
A significant dependence of the nanocomposite structure on the dendrimer generation was
reported (Kim et al 2004 Hofman et al 2011 Camarada 2017) While low generation
dendrimers (G0ndashG4) generate inter-dendrimeric complexes in the synthesis of AuNPs these
complexes are formed inside the dendrimer for G6 or higher generation PAMAM dendrimers
Therefore AuNPs are expected to be formed on the dendrimers surface in the 3D-Au-
PAMAM and subsequently capped by other polymer units (Hofman et al 2011)
S8
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
10 nm 2 nm
a) b)
Fig S2 a) TEM and b) HRTEM images of the 3D-Au-PAMAM nanocomposite suspension
The successful modification of HOOC-p-ABA-SPCEs with 3D-Au-PAMAM through
covalent immobilization using EDCSulfo-NHS chemistry was confirmed by CV in H2SO4
As shown in Fig S3 the resulting 3D-Au-PAMAM-p-ABA-SPCE exhibits the characteristic
feature of the Au redox reaction (Hamelin 1996) with an oxidation peak at +096 V (vs the
Ag pseudo-reference electrode) corresponding to the formation of Au oxides whose reduction
is observed within this material at +045 V
For comparison purposes the CV recorded upon immobilization of the 3D-Au-PAMAM onto
a SPCE modified by grafting of p-aminothiophenol (S-Phe) is shown in Fig S3 As it can be
observed both the anodic and cathodic currents were larger at the 3D-Au-PAMAM-p-ABA-
SPCE than those obtained at the 3D-Au-PAMAM-S-Phe-SPCE most likely due to the larger
3D-Au-PAMAM loading onto the p-ABA-SPCE because of the large number of amino
groups available for immobilization in the nanocomposite
S9
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
-02 00 02 04 06 08 10 12 14 16
-40-20
020406080
100120 3D-Au-PAMAM-S-Phe-SPCE
3D-Au-PAMAM-p-ABA-SPCE p-ABA-SPCE
i
A
E V vs Ag pseudo-reference electrode
Fig S3 Cyclic voltammograms recorded in 01 M H2SO4 at p-ABA-SPCE (blue) 3D-Au-
PAMAM-S-Phe-SPCE (black) and 3D-Au-PAMAM-p-ABA-SPCE (red) (v = 50 mV sminus1)
Optimization of experimental variables
00
10
20
30
40
50
i
A
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
(-) HQ (+) HQWithout 3D-Au-PAMAM
With 3D-Au-PAMAM
Fig S4 Comparison of the amperometric responses obtained for H2O2 with and without HQ
in the presence of 00 and 50 ng mLminus1 tau standards with immunosensors prepared at p-ABA-
SPCEs or 3D-Au-PAMAM-p-ABA-SPCEs Red triangles are the resulting SB ratios
S10
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Different experimental parameters were optimized The loading of 3D-Au-PAMAM and its
incubation time the GA and CAb concentrations and the corresponding incubation times the
type and incubation time of blocking agent the number of steps involved in the immunoassay
the HRP-DAb concentration and the incubation time in the HRP-DAb-tau mixture were
checked The better ratio between the currents measured with the as prepared immunosensor
in the presence (S) of 5 ng mLminus1 tau standard and in the absence (B) (SB ratio) was taken as
the selection criterion for each tested variable The tested ranges and the selected values are
summarized in Table S1 The obtained results are shown in Figs S5 a-k in the Supporting
Information It is important to note the lack of discrimination between the absence and the
presence of tau protein observed without GA and immobilized CAb (Figs S5c and e) In
addition a similar discrimination in the absence and in the presence of tau (SB ratio) was
found by capturing on CAb-3D-Au-PAMAM-p-ABA-SPCEs and labeling the target protein
with HRP-DAb in a single (SB = 20) or in two (SB = 18) steps Other variables involved in
the p-ABA electrografting and 3D-Au-PAMAM covalent immobilization protocols (Moreno-
Guzmaacuten et al 2012) as well as the concentration of the H2O2HQ system and the applied
potential to perform the amperometric transduction (Eguiacutelaz et al 2010) were those
optimized in previous works Moreover pH and temperature two key variables in the
functioning of the HRP enzyme and therefore of the developed immunosensor were selected
according to the available literature A pH value of 60 allows maximum enzyme activity
(Conzuelo et al 2012 Sarika et al 2015 Wang et al 2018 Al-Bagmi et al 2019)
Regarding temperature the HRP activity gradually increases up to 40 ordmC and decreases for
higher temperatures (Sarika et al 2015) Since the achieved sensitivity was sufficient with
the aim to facilitate the implementation in POC devices we decided to develop the
methodology at room temperature
S11
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Table S1
Optimization of the experimental variables affecting the analytical behavior of the HRP-DAb-
tau-CAb-3D-Au-PAMAM-p-ABA-SPCE immunosensor prepared for the determination of
tau protein
Working variable Tested range Selected value
Volume of 3D-Au-PAMAM suspension microL 2540 10
Incubation time in 3D-Au-PAMAM suspension
min
15ndashon 30
GA concentration (vv) 0ndash25 05
Incubation time in GA solution min 30ndash120 60
CAb concentration microg mLminus1 0ndash25 10
Incubation time in CAb solution min 15ndashon 60
Incubation time in PFBB solution min 30ndash90 45
Number of steps 1ndash2 1
HRP-DAb concentration microg mLminus1 005ndash10 01
Incubation time in tauHRP-DAb mixture
solution min
15ndash120 60
on overnight
S12
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
0 5 10 250
20
30i A
CAb g mL-1
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
i
A
CAb incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
005 01 025 05 10
20
30
40
i
A
Ab-HRP g mL-1
0 ng mL-1 TAU 5 ng mL-1 TAU
0
1
2
3
4
SB
0
2
3
4
5
6i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20 SB0
20
30
40
50
60
i
A
0 ng mL-1 tau 5 ng mL-1 tau
00
05
10
15
20
SB
1 STEP 2 STEPS0
20
30
40
-i A
0 ng mL-1 TAU 5 ng mL-1 TAU
00
05
10
15
20
SB
30 45 60 90 1200
20
30i
A
Glutaraldehyde incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
25 5 10 40 10 (dry)0
20
30
40
i
A
Volumme3D-Au-PAMAM microL
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2 SB
15 30 45 60 90 1200
10
20
30
40
0 ng mL-1 TAU 5 ng mL-1 TAU
i
A
HRP-DAb incubation time min
0
1
2
3
SB
a)
0 025 05 10 250
20
30i A
Glutaraldehyde
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
0
20
30
40
50
i
A
3D-Au-PAMAM incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
c) d)
f) g) h)
30 45 60 900
20
30i
A
Blocking agent incubation time min
0 ng mL-1 tau 5 ng mL-1 tau
0
1
2
3
SB
i) j) k)
b)
e)
Fig S5 Effect on the amperometric responses measured in the absence (white bars) or in the presence (grey bars) of 50 ng mLminus1 tau standards and the resulting signal-to-blank ratios (SB red lines) of the volume of 3D-Au-PAMAM suspension a) and incubation time b) GA concentration c) and incubation time d) CAb concentration e) and incubation time f) type g) and incubation time of blocking agent h) number of steps involved in the immunoassay procedure i) HRP-DAb concentration j) and incubation time k) Error bars estimated at triple of the standard deviation of three replicates
S13
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
0 3 7 9 270
20
30
40
50
0 ng mL-1 tau 25 ng mL-1 tau
i
A
Days
0
1
2
3
SB
Fig S6 Storage stability of CAb-3D-Au-PAMAM-p-ABA-SPCEs (after the blocking step)
Amperometric measurements were made in the absence and in the presence of 25 ng mLminus1
tau standards (red triangles are the corresponding SB ratios) Error bars estimated as triple of
the standard deviation of three replicates
S14
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Table S2
Determination of tau in spiked plasma and brain tissue extract samples
Sample[tau]spiked
pg mLminus1
[tau]found
pg mLminus1 Recovery
Brain tissue
protein extracts
Healthy
individual
200 261 plusmn 3 125 plusmn 2
600 613 plusmn 45 101 plusmn 8
AD
patient
Broak IV
200 229 plusmn 10 114 plusmn 6
600 562 plusmn 4 94 plusmn 5
PlasmaHealthy
individual20 204 plusmn 04 102 plusmn 4
Mean values plusmn tsradicn n=3 α=005
Value obtained by subtracting the determined endogenous content shown in Table 1 from
the total found content
References
Al-Bagmi MS Khan MS Ismael MA Al-Senaidy AM Bacha AB Husain FM
Alamery SF 2019 Saudi J Biol Sci 26 301ndash307
Alvarez MM Khoury JT Schaaff TG Shafigullin MN Vezmar I
Whetten RL 1997 J Phys Chem B 101 3706ndash3712
Barderas R Babel I Diacuteaz-Uriarte R Moreno V Suaacuterez A Bonilla F Villar-Vaacutezquez
R Capellaacute G Casal JI 2012 J Proteomics 75 4647ndash55
Barderas R Villar-Vaacutezquez R Fernaacutendez-Acentildeero MJ Babel I Pelaacuteez-Garciacutea A
Torres S Casal JI 2013 Sci Rep 3 2938 doi 101038srep02938
Camarada MB 2017 J Phys Chem A 121 8124minus8135
S15
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16
Conzuelo F Gamella M Campuzano S Pinacho DG Reviejo AJ Marco MP
Pingarroacuten JM 2012 Biosens Bioelectron 36 81ndash88
Eguiacutelaz M Moreno-Guzmaacuten M Campuzano S Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2010 Biosens Bioelectron 26 517522
Esumi K Suzuki A Yamahira A Torigoe K 2000 Langmuir 16 2604ndash2608
Hamelin A 1996 J Electroanal Chem 407 1ndash11
Hoffman LW Andersson GG Sharma A Clarke SR Voelcker NH 2011 Langmuir
27 759ndash6767
Kim YG Oh SK Crooks RM 2004 Chem Mater 16 167ndash172
Li D Li J 2003 Chem Phys Lett 372 668ndash673
Luo J Dong M Lin F Liu M Tang H Li H Zhang Y Yao S 2011 Analyst 136
4500ndash4506
Mirra SS Hart MN Terry RD 1993 Arch Pathol Lab Med 117 132ndash144
Moreno-Guzmaacuten M Ojeda I Villalonga R Gonzaacutelez-Corteacutes A Yaacutentildeez-Sedentildeo P
Pingarroacuten JM 2012 Biosens Bioelectron 35 82ndash86
Rao CNR Kulkarni GU Thomas PJ 2002 Chem - Eur J 8 28ndash35
Sarika D Ashwin Kumar PSS Arshad S Sukumaran MK 2015 Int J Curr
Microbiol App Sci 4 367ndash375
Thal DR Rub U Orantes M Braak H 2002 Neurology 58 1791ndash1800
Wang J Liu G Jan MR Zhu Q 2003 Electrochem Commun 5 1000ndash1004
Wang Y Zhao K Zhang Z Jia H Chen J Fu C 2018 Int J Electrochem Sci 13
29212933
S16