Nanocapsules

Magnetic Graphitic Nanocapsules for Programmed DNA Fishing and Detection

Zhi-Ling Song , Xu-Hua Zhao , Wei-Na Liu , Ding Ding , Xia Bian , Hao Liang , Xiao-Bing Zhang , Zhuo Chen , * and Weihong Tan *

Graphene nanomaterials are typically used in biosensing applications, and they have been demonstrated as good fl uorescence quenchers. While many conventional amplifi cation platforms are available, developing new nanomaterials and establishing simple, enzyme-free and low-cost strategies for high sensitivity biosensing is still challenging. Therefore, in this work, a core–shell magnetic graphitic nanocapsule (MGN) material is synthesized and its capabilities for the detection of biomolecules are investigated. MGN combines the unique properties of graphene and magnetic particles into one simple and sensitive biosensing platform, which quenches around 98% of the dye fl uorescence within minutes. Based on a programmed multipurpose DNA capturing and releasing strategy, the MGN sensing platform demonstrates an outstanding capacity to fi sh, enrich, and detect DNA. Target DNA molecules as low as 50 pM could be detected, which is 3-fold lower than the limit of detection commonly achieved by carbon nanotube and graphene-based fl uorescent biosensors. Moreover, the MGN platform exhibits good sensing specifi city against DNA mismatch tests. Overall, therefore, these magnetic graphitic nanocapsules demonstrate a promising tool for molecular disease diagnosis and biomedicine. This simple fi shing and enrichment strategy may also be extended to other biological and environmental applications and systems.

1. Introduction

Graphene is a 2D nanomaterial composed of a honeycomb-

like single layer of carbon atoms. It has distinct electrical [ 1 ]

and spectroscopic properties, [ 2 ] rich chemical and robust

mechanical properties, [ 3 ] and it has emerged as one of the

most extensively studied nanomaterials. [ 4 ] Numerous applica-

tions have been developed based on graphene, such as optical

modulators, [ 5 ] integrated circuits, [ 6 ] transparent conducting

© 2013 Wiley-VCH Verlag Gm

DOI: 10.1002/smll.201201975

Z.-L. Song, X.-H. Zhao, W.-N. Liu, D. Ding, X. Bian, H. Liang, Prof. X.-B. Zhang, Prof. Z. Chen, Prof. W. H. TanMolecular Science and Biomedicine LaboratoryState Key Laboratory of Chemo/Bio-Sensing and ChemometricsCollege of Chemistry and Chemical EngineeringHunan UniversityChangsha, 410082, PR China E-mail: zhuochen@hnu.edu.cn; tan@chem.ufl .edu

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electrodes, [ 7 ] ultracapacitors, [ 8 ] detection and diagnostic biode-

vices, [ 9 ] drug delivery [ 10 ] and DNA sequencing. [ 11 ] Recently,

researchers have utilized graphene as a nanoquencher and

nanoscaffold for biomolecular sensing, and it has consistently

exhibited considerable potential. [ 12 ] Graphene nanomaterials

have been demonstrated as good fl uorescence quenchers. [ 9,12 ]

Florescence dye-labeled single-stranded DNA or aptamer mol-

ecules can nonconvalently bind to graphene surfaces and can

be used as a platform for the detection of different analytes.

Various sensing strategies and systems have been explored,

and they have all demonstrated good selectivity and sensi-

tivity. [ 9,12 ] Nonetheless, the application of a graphene sensing

platform in complicated matrices remains problematic. Thus,

investigators are seeking ways to further improve selectivity

and sensitivity, reduce detection time and lower the cost of

new graphene-based biosensors. DNA enzymes, rolling circle

and polymerase chain reaction (PCR) amplifi cation strate-

gies have thus far been developed, and they have improved

the limit of detection (LOD) of DNA biosensors. [ 13 − 15 ] How-

ever, they are all relatively expensive enzyme-based sensing

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Figure 1 . Advanced structural analysis and magnetic enrichment of MGN. (A) High resolution TEM image of MGN. (B) A Raman spectrum (excitation 632 nm) of the MGN with the G and D bands of graphitic carbon. (C) Suspensions of MGN aqueous solution before (left) and after (right) magnetic enrichment under the external magnet.

platforms, and the enzyme is easily deacti-

vated in complicated systems. [ 16 ] Although

rolling circle and PCR amplifi cation can

rapidly synthesize multiple copies of DNA

or RNA molecules, DNA replication

involves an elaborate process and requires

specifi c instrumentation. [ 17 ] Therefore,

developing new nanomaterials and estab-

lishing simple, enzyme-free and low-cost

strategies for high-sensitivity biosensing

remain challenging.

Magnetic materials, especially mag-

netic nanoparticles, are of particular signif-

icance, and a wide variety of applications

have been envisaged for them. [ 18 ] Magnetic

nanoparticles have been used in the sepa-

ration and detection of DNA, cells and

other biological entities; [ 19 ] therapeutic

drug, gene and radionuclide delivery; [ 20 ]

contrast agents for magnetic resonance

molecular imaging; [ 21 ] and as heat media-

tors for the catabolism of tumors via

hyperthermia applications. [ 22 ] Combining

the unique properties of magnetic nano-

particles and graphene will offer some

attractive possibilities in biomedicine.

Different magnetic nanoparticle carbon

coating strategies have been developed,

such as sonochemical procedures, pyrolysis of iron stearate,

and vapour deposition approaches, and they have all demon-

strated great potentials. [ 19,21,23,24 ]

Herein, we developed a magnetic graphitic nanocap-

sule nanomaterial for nucleic acid detection. Combining the

unique properties of graphene and magnetic nanoparticles,

a core–shell magnetic graphitic nanocapsule material was

fi rst synthesized. Then, by means of π -stacking interactions

between the nucleotide bases and the magnetic graphitic

nanocapsule (MGN) outer shells, stable ssDNA/MGN com-

plexes could be formed. The heart of this biosensor is the

effi cient quenching for NA detection. However, to further

functionalize this biosensor, we designed a multipurpose

DNA capturing and releasing strategy for programmed and

targeted fi shing, enrichment and detection of DNA. With

MGN nanomaterials and this simple, nonenzymatic strategy,

the LOD of our snesing platform was observed down to

50 pM, approximately three orders of magnitude better than

common carbon nanotube (CNT)- and graphene-based fl uo-

rescent biosensors. [ 9,12,25 ]

2. Results and Discussion

2.1. Characterization of Magnetic Graphitic Nanocapsules

Magnetic graphitic nanocapsules prepared after mixed-acid

polishing display high water-solubility by the massive sus-

pended hydroxyl and carboxyl groups present at the MGN

surface. Transmission electron microscopy (TEM) was utilized

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to characterize MGN morphology and construction. As

shown in Figure 1 A, the average size of the MGN is around

29 nm, and its structure consisted of a metal nanocrystal core

with several graphite shells. DLS measurement further con-

fi rmed the average size of the MGN, and ζ -Potential meas-

urements indicated the negative charge of the MGN which

originated from the carboxyl groups present on the MGN

surface (Figure S1 and S2 in the Supporting Information).

The graphite shell was further proved through Raman spec-

troscopy (Figure 1 B), showing a graphitic carbon (G) peak

at ∼ 1590 cm − 1 and a disordered (D) peak at ∼ 1300 cm − 1 . The

high Raman D peak refl ects the high strain of the graphite

shells caused by the small size of the MGN. Figure 1 C dem-

onstrated the enrichment of MGN under an external mag-

netic fi eld. The acid-treated MGN was suspended in water as

a stable black solution (Figure 1 C, left), which could remain

stable for weeks. After magnetic manipulation, the MGNs

were enriched around the sidewall of the vial (Figure 1 C,

right). The strong magnetic force overcame the gravitational

force and attracted the MGN around the magnet, aligned

according to the magnetic fi eld gradient. This effectively dem-

onstrated the detection and enrichment capabilities of MGN.

2.2. Strategies of utilizing MGN for DNA Fishing and Detection

DNA fi shing and detection have obvious implications in clin-

ical diagnostics, and our strategy, which combines the unique

properties of magnetic NPs and graphene, was illustrated in

Scheme 1 . FAM-conjugated DNA1 chains were fi rst absorbed

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Magnetic Graphitic Nanocapsules

Scheme 1 . Strategy of the MGN for DNA fi shing and detection. (A) Fishing and enriching T1 molecules from the initial sample solution under an external magnet. (B) Detection of Target T1 molecules through fl uorescence quenching and recovery from the MGN surface.

on the MGN surfaces through π -stacking interactions between

the nucleotide bases and the MGN outer graphitic shells.

The fl uorescence of FAM is quenched by the graphitic shell

through fl uorescent resonance energy transfer (FRET). [ 9,12 ]

DNA1 was designed to have a dual function. One part of the

DNA1 molecule is able to specifi cally recognize the target

DNA (T1) molecules. The other part is used as an anchor to

keep the whole molecule (with T1 molecules bound) on the

MGN surface during the T1 fi shing process. After the T1 mol-

ecules were fi shed by the DNA1-MGN complexes, a magnet

was utilized to enrich and concentrate them, as illustrated in

Scheme 1 A. With respect to applications in clinical medicine,

this strategy also permits the fi shing of target molecules from

© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 2 . Quenching effi ciency of the magnetic graphitic nanocapsule materials. (A) Fluorescencconcentrations of MGN in 20 mM Tris-HCl buffer (pH 7.0) for 30 min. (B) Quenching effi ciency vs. cof FAM-DNA1 (50 nM) in Tris-HCl buffer by MGN as a function of time. (D) Fluorescence restorationand without (dark dots) T1 present in Tris-HCl buffer as a function of time. T1 enrichment factoconcentration: 10 nm. P1 concentration: 100 nM. Excitation wavelength: 494 nm, and emission

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a complicated sample solution like serum

and urine, even at a very low concentration.

After the fi shing and enriching process, we

next used a releasing DNA (P1) for pro-

grammed detection of the T1 DNA mol-

ecules. P1 molecule was designed to bind

to the tail of the DNA1. When T1 bound to

DNA1 and concentrated, the P1 was added

to hybridize with the FAM-DNA1/T1

complex to form a complete duplex helix.

The helix has weaker interaction with the

MGN which results in creating distance

between the FAM molecules and MGN

and, thus, recovering fl uorescence. Both T1

and P1 DNA must be present to activate

the release of FAM-DNA1 from the MGN

surface and recover fl uorescence (Scheme 1 B).

2.3. Fluorescence Quenching Effi ciency of the Magnetic Graphitic Nanocapsules

Different tests were conducted to verify the quenching effi -

ciency of the MGN nanomaterials. Increase in MGN concen-

tration correlates with increased quenching of FAM-DNA1

fl uorescence intensity ( Figure 2 A), until MGN concentration

approached 0.09 mg/mL, when the fl uorescence intensity of

50 nM FAM-DNA1 decreased sharply. As shown in Figure 2 B,

more than 98% of the fl uorescence can be quenched when

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e spectra of 50 nM FAM-DNA1 mixed with various oncentration of MGN. (C) Fluorescence quenching

of FAM-DNA1/MGN complexes with (red triangles) r is 5. FAM-DNA1 concentration: 50 nM. Initial T1 wavelength: 518 nm.

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Figure 3 . MGN for fi shing and enrichment of DNA molecules. (A) Fluorescence-emission spectra of DNA1 (fi nal concentration of 50 nM) under different conditions: a) MGN + DNA1, without enrichment; b) MGN + DNA1 + T1 (10 nM), without enrichment; c) MGN + DNA1, with 5 times enrichment; d) MGN + DNA1 + T1 (10 nM), with 5 times enrichment. (B) F/F0 (relative fl uorescence intensity, where F0 and F are the fl uorescence intensity without and with the presence of 10 nM T1, respectively) with different enrichment factors.

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the concentration of MGN is higher than 0.09 mg/mL. FAM-

DNA1 interacts with MGN through π -stacking interactions

between the nucleotide bases and the MGN outer shells to

guarantee the close proximity of FAM with MGN. The MGN

collectively quenched FAM fl uorescence through energy-

transfer or electron-transfer processes. A typical MGN

possesses an absorption spectrum that overlaps the pho-

toluminescence spectra of FAM, allowing resonant energy

transfer (RET) to occur. MGN, which contains highly delo-

calized π electrons on the outer graphitic shell, acts as the

acceptor in the energy-transfer process, and photo excited

fl uorophores act as the donors to transfer energy to the

ground-state MGN acceptor. [ 12 ]

We further investigated the quenching effi ciency of the

MGN as a function of time. Figure 2 C shows the changes of

fl uorescence intensity of 50 nM FAM-DNA1 relative to time

after addition of 0.09 mg/mL MGN. Within 2 min, the fl uores-

cence intensity dropped to around 80%. After 15 min, more

than 98% fl uorescence had been quenched by MGN, and no

signifi cant fl uorescence recovery was observed, indicating

good quenching effi ciency. Figure 2 D shows the fl uorescence

recovery curve with and without 10 nM target DNA T1.

Without target, most of the fl uorescence was still quenched,

and only a slight increase of intensity occurred as a result

of the background from the addition of P1 releasing DNAs.

However, with the addition of T1 DNA, an increase in fl uo-

rescence recovery was observed. After 1.5 h of incubation,

the recovered fl uorescence intensity reached a plateau, and

no obvious further increases were observed, indicating equi-

librium of the hybridization between the FAM-DNA1 and

T1, P1 molecules. The hybridized DNA formed a duplex helix

which had weaker interaction with the MGN. This resulted in

separating the FAM molecules from the MGN, and a strong

fl uorescence was recovered.

2.4. MGN for Fishing and Enrichment of DNA Molecules

We next investigated the concentration capacity of MGN

nanomaterials. Figure 3 shows the detection of 10 nM T1

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DNA molecules through the programmed MGN strategy.

Different enrichment factors were investigated. As shown

in Figure 3 A, curves a and b were the fl uorescence inten-

sity with and without 10 nM T1 present, respectively, when

no magnet was utilized. On the other hand, curves c and d

were the fl uorescence change after 5 times enrichment and

fi shing with the magnet. The intensity of curve d signifi cantly

increased when compared to that of curve b. Interestingly, the

fl uorescence intensities of curve c were slightly lower than

those of curve a. This decrease in background intensity after

magnetic fi shing and enrichment contributed to the improve-

ment in sensing sensitivity. The free FAM-DNA1 molecules

in the sample solution were excluded during the enrichment

step. By reducing the background signal, such exclusion of

free FAM-DNA1 molecules was speculated to be the mecha-

nism underlying the lower fl uorescence intensity. System-

atic investigation of enrichment times and DNA detection

was conducted, and the fi ndings are reported in Figure 3 B.

F/F0 was the relative fl uorescence where F0 and F corre-

spond to the fl uorescence intensity without and with target

T1 molecules, respectively. Without enrichment (1 time) and

only magnetic fi shing, the relative fl uorescence F/F0 was

similar to that without enrichment. Fishing and enriching of

T1 molecules by 2 times and 5 times resulted in signifi cantly

increasing the F/F0 value. More than 3 times relative fl uores-

cence improved after 5 times enrichment. This MGN sensing

strategy was simple, and no enzyme was needed. This also

indicated the potential of using the programmed MGN for

sensitive detection of molecules from a complicated sample

solution. Higher relative fl uorescence improvement could

certainly be achieved through optimization of the magnetic

enriching operation process, and this will be the direction of

our future studies.

2.5. DNA Detection with Programmed MGN Platform

Nucleic acid detection was utilized as the model system to

demonstrate the sensing capabilities of the MGN nanomate-

rials. The effect of the concentration ratio of P1 over DNA1

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Magnetic Graphitic Nanocapsules

Figure 4 . MGN fl uorescence sensing platform for fi shing and detection of target T1 molecules. (A) Fluorescence-emission spectra after incubation with different concentrations of T1, ranging from 0 to 50 nM. (B) The relationship between F/F0 (relative fl uorescence intensity, where F0 and F are the fl uorescence intensity without and with the presence of T1, respectively) and T1 concentration. Error bars were obtained from three parallel experiments. Inset shows the linear responses at low T1 concentrations. The buffer solution contains 20 mM Tris (pH 7.4), 100 mM NaCl, and 2 mM MgCl 2 .

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Figure 5 . Florescence intensity changes for complementary matched T1 over one base mismatched, either M1 or M2, and two bases mismatched, M3 (all at a concentration of 10 nM).

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on the sensing response was investigated. When the concen-

tration of the releasing DNA P1 was 2 times higher than that

of the capturing DNA1, the programmed MGN sensing plat-

form demonstrated its highest sensitivity (Figure 3S in Sup-

porting Information). The programmed MGN DNA fi shing

and detection assay is sensitive and specifi c. Figure 4 A shows

the fl uorescence emission spectra of the FAM-DNA1/MGN

complex upon adding target T1 DNA molecules at different

concentrations and releasing with P1 molecules. A dramatic

increase in FAM fl uorescence with the increasing concen-

tration of T1 molecules was observed. All the tests were

done under an enrichment of 5 times target molecules. The

plot of fl uorescence intensity with T1 concentration from

0 to 50 nM is shown in Figure 4 B. The linear range is from

50–1000 pM (Figure 4 B inset). The linear equation is y = 1.11

+ 0.0018 x, where y is the relative fl uorescence F/F0, and x

is the concentration of T1 (regression coeffi cient R = 0.994).

The detection limit was 50 pM, which is 3-fold lower than

LODs commonly reported for CNT- and graphene-based

fl uorescent biosensors. [ 9,12,25 ] Several possible reasons may

explain this excellent performance. First, MGN is a super-

quencher and possesses long-term nanoscale RET properties

between MGN and FAM molecules. The high fl uorescence

quenching effi ciency leads to low background and results

in high sensitivity. Second, through our programmed MGN

fi shing and enriching strategies, we reduced the background

and increased the positive signals, which further contributed

to the high sensitivity of the MGN sensing assay. Third, the

folding of DNA will be more favorable on the MGN surface,

as opposed to a 1D nanotube surface. Therefore, the DNA on

the MGN surface is more sensitive to the target molecules

vis-à-vis fl uorescence responses.

2.6. Specifi city of MGN DNA Sensor

The programmed MGN sensing assay is also specifi c. To eval-

uate this property, we challenged the system with nucleotide

polymorphisms, which represent promising disease markers

because they are stably inherited sequence variations in

© 2013 Wiley-VCH Verlag Gmbsmall 2013, 9, No. 6, 951–957

the human genome. Both single-base and double-base mis-

matches have been investigated. In a typical experiment, the

FAM-DNA1/MGN complex was incubated with 10 nM target

T1 DNA and single-base mismatch M1, M2 or double-base

mismatch M3 DNA molecules, respectively, then released

with P1 DNA molecules. The mismatched base of M1 and M2

were G and A, respectively. As shown in Figure 5 , remarkably

higher fl uorescence was observed with the complementary

target T1 than with the single- or double-mismatch nucle-

otides. These results clearly demonstrate the high specifi city

of our programmed MGN sensing assay. Double-base mis-

matches indicated better differentiation than the single-base

mismatches. For maximum effi ciency and selectivity, more

effort should be directed toward optimizing hybridization

time, designing a hairpin capturing DNA structure, or further

amplifying the sensing with other strategies.

3. Conclusion

In summary, we demonstrated the utilization of magnetic

graphitic nanocapsule nanomaterials for programmed DNA

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fi shing and detection. The MGN combined the unique prop-

erties of graphene and magnetic particles. Synthesized MGN

can be enriched by an external magnet and aligned according

to the magnetic fi eld gradient. The MGN exhibited good

quenching effi ciency and could quench around 98% of the

fl uorescence of the noncovalently bound FAM-DNA1 within

minutes through energy-transfer or electron-transfer proc-

esses. After the designed FAM-DNA1 functionalization, tar-

geted DNA could be fi shed and enriched from the original

sample solution with the MGN platform. Different enriching

factors were investigated, and all showed high potential for

DNA fi shing and detection. It is only in the presence of

releasing DNA P1 that the sensing platform operates, with

FAM-DNA1, T1 and P1 hybridizing to form a duplex helix.

The helix has weaker interaction with the MGN which

results in separating the FAM molecules from the MGN

and, thus, recovering fl uorescence. The programmed MGN

DNA fi shing and detection assay was demonstrated to be

sensitive and specifi c. With this simple, nonenzymatic MGN

sensing strategy, target DNA molecules down to 50 pM can

be detected, which is 3-fold lower than the limits of detection

reported for common CNT and graphene-based fl uorescent

biosensors. Moreover, DNA mismatch tests were investigated,

and the MGN platform exhibited good sensing specifi city for

both single and double mismatches. This simple fi shing and

enrichment strategy may also be extended to other compli-

cated clinical and environmental applications and systems.

The reliable, highly sensitive, and selective MGN platform

was demonstrated to be a promising tool for molecular dis-

ease diagnosis and biomedicine.

4. Experimental Section

Materials and Chemicals : All DNA sequences were synthesized by Shanghai Biotech (China). Sequences of oligonucleotide probes used in this work are as follows: Capturing DNA1: 5 ′ -FAM-TTT GTC CAG GGT CTC TAT ATT GTG CAA GG-3 ′ ; target DNA T1: 5 ′ -TAG AGA CCC TGG ACA AA-3 ′ ; releasing DNA P1: 5 ′ -CCT TGC ACA ATA-3 ′ ; single-base mismatch DNA M1: 5 ′ -TAG AGA CCC TAG ACA AA-3 ′ ; single-base mismatch DNA M2: 5 ′ -TAG AGA CCC TGG TCA AA-3 ′ ; double-base mismatch DNA M3: 5 ′ -TAG ACA CCC TGG CCA AA-3 ′ . Tris-HCl buffer solutions (20 mM Tris, 100 mM NaCl, and 2 mM MgCl 2 , pH 7.4) were used as working solutions. Magnet was pur-chased from Changsha Chemical Reagents Company (Changsha, China). All other chemicals of analytical reagent grade were obtained from Changsha Chemical Reagents Company (Changsha, China) and used as received without further purifi cation. Doubly distilled water (resistance > 18 M Ω cm − 1 ) was used throughout all experiments.

Preparation of Magnetic Graphitic Nanocapsules : Magnetic graphitic nanocapsule was produced in a chemical vapor deposi-tion (CVD) system. First, fumed silica (1.00 g, Aladdin) was impreg-nated with Co(NO 3 ) 2 · 6H 2 O (0.210 g) in methanol and sonicated for 1 h. Then the methanol was removed, the mixture was dried at 80 ° C, and the powder was ground. Typically, 0.50 g of the powder was used for methane CVD in a tube furnace. The sample grew with a methane fl ow of 200 cm 3 min − 1 for 6 min. After growth, the sample was etched with 10% HF in H 2 O (80%) and ethanol (10%)

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to dissolve the silica. The graphite-coated magnetic nanoparticle solid product was then washed thoroughly and collected through centrifugation. Before DNA detection, a solution of sulfuric and nitric acid was utilized to polish the as-grown MGN and solubilize it in water.

DNA Fishing and Sensing : DNA1 was prepared as 10 nM in 20 mM Tris-HCl buffer (pH 7.4, containing 100 mM NaCl and 2 mM MgCl 2 ) and mixed with 0.09 mg/mL MGN for 30 min prior to the addition of target T1. Then T1 was added to the prepared DNA1-MGN solution. The fi nal T1 concentration in detection solu-tion ranged from 50 pM to 50 nM. After allowing hybridization for about 15 min at 37 ° C, the mixture was exposed to external mag-netization for 2 h. The fi shed and enriched T1 molecules on the MGN were collected. Then a solution of 100 nM P1 was added to the collected MGN and incubated for 1.5 h at room temperature to complete the hybridization. Finally, the fl uorescence of the mixture was detected with a fl uorescence analyzer.

Characterization : We characterized the MGN by transmission electron microscopy (TEM, JEOL 3010, operated at 120 to 200 kV) and Raman spectroscopy (Horiba Jobin Yvon LabRAM-010 Raman microscope with 632 nm He-Ne laser excitation). All fl uorescence spectra were collected with a Hitachi F-7000 spectrophotometer equipped with a xenon lamp excitation source. Dynamic light scat-tering (DLS) was measured on the Malvern Zetasizer Nano ZS90 (Malvern Instruments, Ltd., Worcestershire, UK).

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

This work was fi nancially supported by the Research Fund for the Program on National Key Scientifi c Instruments and Equipment Development of China (No. 2011YQ0301241402), the National Natural Science Foundation of China (No. 21105025), and the Research Fund for the Doctoral Program of Higher Education of China (No. 20110161120010).

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Received: August 13, 2012 Revised: November 5, 2012Published online: December 3, 2012

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