(His)6!Ni

Stroma

Lumen

PsbO CP43

CP47

Fe Cyt c-550 PsbV

Mn-Cluster

Fe(Cyt b-559)

Gold

SAM

Ni-NTA

His6xtag

Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni

PSII

Umbrella Model

Gold

hν

PSII

C18H37SH

iii) Conducting molecular wires (PAN): Fig.3 reports typical PSII-PAN-NTA-SAM-Au and PAN-NTA-SAM-Au dark scans measured under N2 at 20mV/s scan rate in MES buffer pH=6.5. A reversible peak pair (4 electrons) is observed in the presence of immobilised PSII.

CONCLUSIONS: Electrochemical deposition of CYS-SAM on Au surfaces results a good method to lower treatment time (1-12’ with respect to 16h of the chemical treatment), and address the synthes is on on ly one Au electrode in a sensor µ-array. With screen printed Au-graphite or Pt-graphite composites, selective deposition of CYS-SAM on Au or Pt particles can be achieved. The number of Ni(II) heads available f o r h i s - t a g m e d i a t e d immobilisation can be decreased and different molecules deposed (i.e. PAN conducting films or mixed hydrophobic SAMs) on the Au surface, hopefully obtaining better performances of the biosensor in terms of substrate diffusion or direct electron transfer onto the electrode.

ACKNOWLEDGEMENTS: This work was supported partly by the ROSEPROMILK Project (QLK1-CT2001-01617, European Community, the 5th FW, Quality of Life Program), partly by COSMIC Project (ENEA Target Project on Biosensors and Bioelectronics) and by the project 522/001274 of Grant Agency of the Czech Republic

Fig.3: Typical (____)PSII-PAN-NTA-SAM-Au and (- - - -) PAN-NTA-SAM-Au dark scans

-3

-3 -0.200x10

-3 -0.100x10

0

-3 0.100x10

-3 0.200x10

-3 0.300x10

-3 0.400x10

-3 0.500x10

i / A

Electrochemical deposition of PAN conducting molecular wires on CYS-SAM-Au

Electrochemical deposition of PAN conducting molecular wires on CYS-SAM-Au/graphite

REFERENCES 1 M.Sugiura & Y.Inoue (1999) Plant Cell Physiol 40, 1219-1231 2 J. Maly, E. Illiano, M. Sabato, M. De Francesco, V. Pinto, A. Masci, D. Masci, J. Masojidek, M. Sugiura, R. Franconi, R. Pilloton, (2002) Material Science and Engineering C; 22/2 pp. 257-261

3 R.Garjonyte, A.Malinauskas; Biosensors and Bioelectronics, 15 (2000) 445-45 4 D.I. Arnon. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24 (1949) 1-15

5 M. Koblizek, J. Komenda, T. Kucera, J. Masojidek, R. Pilloton, A.K. Mattoo and M.T. Giardi; Biotech.Bioeng 664-669, VOL. 60, December 20, 1998

6 M.Koblízek, J. Malý, J.Masojídek, J.Komenda, T.Kucera, M.T.Giardi, A.K. Mattoo and R Pilloton; Biotech.Bioeng, 110-116, VOL. 78, No. 1, April 5, 2002

COSMIC: Coupling Smart Molecules Into Chips Enea Target Project on Biosensors & Bioelectronics http://www.biosensing.net/cosmic.htm

REVERSIBLE IMMOBILISATION OF ENGINEERED MOLECULES BY Ni-NTA CHELATORS J.Maly1, C.Di Meo, M.De Francesco, A.Masci, J.Masojidek2, M.Sugiura3, A.Volpe, R.Pilloton

uENEA, SP061, Via Anguillarese,301, 00060 Santa Maria di Galeria, Roma, Italy, 1uUniversity of J.E.Purkyne, Dep. of Biology, 40001 Usti nad Labem, Czech Rep., 2uAcademy of Sciences, Inst. of Microbiology,379 81 Trebon, Czech Rep..3uOsaka Prefecture University, Osaka, Japan

An original procedure suitable for chemical immobilisation of engineered (His)6-tagged proteins on different sensor materials was previously reported2 on Au or graphite obtaining uoriented and highly specific immobilisation of two engineered proteins1, urenewable specific binding of proteins to sensor surfaces, ufast and sensitive detection of analytes, uon chip protein pre-concentration from crude extracts. In this paper electrochemical synthesis of the chelator on Au or Au-graphite electrodes is reported with respect to chemical synthesis, obtaining: uhigher Ni-NTA surface density (increased 30 fold), ulower treatment time (1’-12’ with respect to 16h), uability to address the chelator on one electrode in a sensor µ-array, udeposition of CYS-SAM on Au or Pt particles dispersed in a carbon matrix. Other molecules were deposed on Au electrode for ufaster diffusion of inhibitors and mediators (OCT) or u mediatorless direct electron transfer (PAN) from PSII to Au electrode.

PROTEIN Ni-NTA

N H

C H - C H 2

N H

N H

C H - C H 2

(His)4

N

N

N

O -

O -

O - C H 2

C H 2

O

C

C H

C

O

C

O

C O

C

O

N i 2 +

N

N

(His)6x-tag Carbon or Gold or

Carbon/gold

Log K = 11.5

SAM

Synthesis of the Ni-NTA chelator on gold: with (III) or without (II) Lys-GA arm

S H N H 2

S N H 2

C H O O H C

S N C H O Au Au Au

( I) S

N N H 2 N

O O

Au

N H 2 N H 2

O O

C H O O H C

S N N N

O O

C H O Au

O

O

N H 2 O

N O

O

O

S N N N

O O O

O

N

O

O

N O

O

Au

( III)

S N

O O

N O

O N O

O

Au ( II)

1st derivative 2nd derivative

6.7 s

3.3 s

30 s

d(I/A

)/d(t/

s),

d(d(

I/A)/d

(t/s)

)/d(t/

s)

1st SCAN 10th SCAN

Electrochemical Synthesis of CYS-SAM on gold surfaces

0 250 500 750 1000 -3

0.10x10

0

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t/s

CYS-SAM deposition on Au/graphite composite Screen Printed Electrodes

70°C, 1h

GOLD PLATING ON Cu PATHS

Au

PRINTING

ETCHING (FeCl3)

Cu

Cu(CN)42ˉ aq + 2Au(s) f 2Au(CN)2ˉaq + Cu(s)

EXPERIMENTAL: a) Chemical Au plating on Cu paths: Au electrodes were obtained from a commercial Cu sheet deposited on fiberglass. Real surface (Ar) of Au electrodes was chronocoulometrically obtained and a roughness factor of 1.13 was determined.

c) Synthesis of PAN molecular wires3

d) Purification and immobilisation of PSII: Thermophilic cyanobacterial Synechococcus elongatus 43H cells expressing psbC with an (His)6 extension were used1. Immobilisation of PSII was obtained by incubation of electrodes in MESB containing PSII equivalent of 300 µg Chl mL-1 at 4 °C in complete darkness for 20’. e) CV of the Au-CYS-SAM-NTA-PAN-PSII electrodes: Measurements were done in mediatorless buffer solution under N2 and complete darkness. f) amperometric measurement of PSII activity4 on the electrode were done in a home-made flow-cell (flow rate 0.25 mL/min).

b) Deposition of CYS-SAM and synthesis of Ni-NTA: CYS 20 mM in PB for 16h was used for chemical deposition2. Screen printed Au-graphite or Pt-graphite composites allowed deposition of CYS only on the metal particles dispersed in the carbon pastes at 0.85V vs RE. Ni-NTA chelator2 was prepared as reported below. The surface density of Ni+2 (513 pmol/mm2) achieved electrochemically for 20’ resulted greater (15 fold) than that one obtained with a chemical treatment of 16h) and 30 fold after 20’.

time [min]

0 100 200 1000 1100 1200

Pea

k he

ight

[r.u

.]

20

40

60

80

100

120

140

160no detergentdetergent

time [s]

0 20 40 60 80

I [nA

]

0.00

0.05

0.10

0.15

0.20

0.25

I [nA

]

0.0

0.5

1.0

1.5

2.0

2.5 Chemically deposited SAM with PSIIPSII immobilised in BSA-GA gel matrixElectrochemically deposited SAM with PSII

Fig.2: DM detergent significantly prolonged life-time

Fig.1: Current increase after illumination (5s) due reoxidation of DQ.

ii) Surface density of CYS, Ni(II) and (His)6-PSII: In Tab.1 an exstimation of surface density is reported by experimental data and theoretical calculation. With an electrochemical treatment of tmax=12’ (or higher) a ratio Ni:(His)6‑PSII~39500 was experimentally determined. This means that a single (His)6‑PSII

RESULTS AND DISCUSSION: i) Comparison of PSII monolayer vs crosslinked PSII in BSA-GA matrix,5,6. Fig.1 reports the velocity of reoxidation of reduced DQ in the case of BSA-GA-PSII electrode5,6 and CYS-SAM-NTA-PSII. In the case of PSII monolayer, again a rapid inhibition of PSII electrode is observable directly after the addition of herbicide. On the contrary, for BSA-GA-PSII gel matrix a stable signal of inhibited electrode is obtained after 15’ of herbicide exposition. I50 values were then compared for atrazine. Au-CYS-SAM-NTA-PSII electrode has shown a lower I50 (I50=2x10-8 mol/L) compared to the electrode with BSA-GA-PSII gel matrix (I50=9x10-8 mol/L). A striking difference (I50=5x10-10 mol/L), has been observed in the electrode consisting of a mixed SAM layer (CYS+OCT) with increased hydrophobic properties. DM significantly prolonged the life-time of the Au-CYS-SAM-NTA-PSII electrode (Fig.2). On the contrary, BSA-GA-PSII was stable also without detergent in the measuring buffer, probably because of the stabilisation of PSII provided by croslinking.

covers ~39500 Ni(II) heads but binds only one of them through the his6-tag. A “beach umbrella model” should be more convenient because other functional groups or molecules (mixed SAM layers, i.e. CYS+OCT, or conductive molecular wires could be used, still obtaining the same surface density of immobilized (His)6-PSII.

ABBREVIATIONS: AdCSV:Adsorptive Cathodic Stripping Voltammetry; CV:Cyclic Voltammetry; CYS:cysteamine; atrazine:2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine; LOD:limit of detection; His:histidine; BSA:bovine serum albumin; DM:Dodecylmaltoside; DQ:tetramethyl-p-benzoquinone; LYS:Lysine; GA:Glutaraldehyde; LED:light-emitting diode; MES:2-(N-morpholino)ethanesulfonic acid; MESB:40mM MES, 0.1M NaCl, 15mM CaCl2, 15mM MgCl2, 50µM chloramphenicol, 0.03% DM, pH=6.5; Ni-NTA:nickel-nitrilotriacetic acid; PAN:polyaniline; PB:phosphate buffer 0.1M, pH=7.0; PSII:photosystem II; OCT:octadecanethiol; RE, WE:reference (Ag/AgCl), working electrodes; SAM: self assembled monolayer