Biosensors and Bioelectronics 62 (2014) 52–58

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Functionalized graphene as sensitive electrochemical label in target-dependent linkage of split aptasensor for dual detection Lingyan Feng a,b, Zhijun Zhang a,b, Jinsong Ren a, Xiaogang Qu a,n a Laboratory of Chemical Biology, Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China b University of Chinese Academy of Sciences, Beijing 100039, China

art ic l e i nf o

a b s t r a c t

Article history: Received 13 March 2014 Received in revised form 29 May 2014 Accepted 3 June 2014 Available online 11 June 2014

A new type of electrochemical aptasensor was reported here for highly sensitive detection of adenosine triphosphate (ATP) and adenosine deaminase (ADA) activity using functionalized graphene as efficient electrochemical label. The specific binding of ATP and its aptamer could link the split aptamers modified graphene and magnetic beads together. After ADA catalysis and magnetic separation, graphene material anchored on electrode surface would efficiently facilitate electron transfer, thus produce detectable electrochemical signals. The detection limits for ATP and ADA activity were 13.6 nM and 0.01 unit/mL (
1.2 nM), respectively. Our work would supply new horizons for the diagnostic applications of graphene-based materials in biomedicine and biosensors. & 2014 Elsevier B.V. All rights reserved.

Keywords: graphene split aptamer ATP adensine deaminase activity dual detection

1. Introduction Graphene, which structure is one-atom-thick planar sheet of carbon atoms, has received worldwide research interest owing to the extraordinary physical and chemical properties since its discovery in 2004 (Novoselov et al., 2004). In the biomedical application, graphene and its derivatives have been utilized to design new types of biosensors and diagnostic platforms (Song et al., 2010; Liu et al., 2008; Yang et al., 2010). For developing electrochemical sensors, for instance, they can be used to modify the electrode surface as magnified materials for produced signals or nanocarriers for loading more redox probes and/or biomolecular recognition elements, which would further provide an efficient and biocompatible platform for biomolecule immobilization (Huang et al., 2012; Wang et al., 2010; Li et al., 2008; Feng et al., 2011). In the field-effect transistors (FETs) devices, semiconducting graphene has also utilized as the electron channel between two electrodes of source and drain (Myung et al., 2011). Very recently, a new type of graphene electrode in which graphene sheet immobilized on alkylthiol-modified gold electrode was reported to exhibit high activity for electrochemical electron transfer reactions to metal electrode (Wang et al., 2012; Yan et al., 2013; Zhang et al., 2013). However, until now, the direct application of this unique n

Corresponding author: Tel./fax: þ86 431 85262656. E-mail address: [email protected] (X. Qu).

http://dx.doi.org/10.1016/j.bios.2014.06.008 0956-5663/& 2014 Elsevier B.V. All rights reserved.

label in electrochemical biosensors for highly sensitive detection has only shown a limited success. Aptamers are specific nucleic acids selected from random sequence libraries (Ellington and Szostak, 1990; Lubin and Plaxco, 2010a,b; Qiu et al., 2013; Tan and Donovan, 2013). Small molecules triggered linkage of split-aptamer fragments has been introduced to construct biosensors (Liu et al., 2010), DNA tweezers (Elbaz et al., 2009), DNA-templated reaction (Sharma and Heemstra, 2011), etc. The 27-mer adenosine triphosphate (ATP) aptamer was divided into two different sequences. In the absence of ATP, the two fragments did not interact with each other. (Stojanovic et al., 2001; Zuo et al., 2009; Liu et al., 2010; Elbaz et al., 2009; Sharma and Heemstra, 2011). Since ATP provides the support for the metabolism in biological tissues, the detection of ATP is of clinical importance in the biomedical field (Liu et al., 2010). On the other hand, adenosine deaminase (ADA), which can efficiently catalyse the deamination of adenosine (deoxyadenosine) into inosine (deoxyinosine), is a key hydrolytic enzyme in purine metabolism and is ubiquitously distributed in almost all tissues. Both of ATP and ADA expression levels play important roles in many diseases, such as tuberculosis, acute leukaemia, severe combined immunodeficiency (SCID), etc. (Aldrich et al., 2000). Therefore, it is essential to detect ATP molecule and ADA enzyme activity in one system. Previous reports mainly focus on the individual target monitor based on fluorescence, colorimetric assays, high-performance liquid chromatography (HPLC) (Xing et al., 2012; Paul et al.,

L. Feng et al. / Biosensors and Bioelectronics 62 (2014) 52–58

53

The ATP split aptamer sequences were all synthesized by Sangon (Shanghai, China). Their sequences were as below: Aptamer-1 (Apt-1):5′-NH2-(CH2)6-ACC TGG GGG ATA AT-3′ Aptamer-2 (Apt-2):5′-TGC GGA GGA AGG TAA AAA AAA AA(CH2)6-NH2-3′.

2.2. Apparatus and characterization

Scheme 1. Schematic illustration of the biosensor using pyrene-functionalized graphene as a novel electrochemical label for ATP and ADA activity detection. The following steps are crucial for the functionalized graphene mediated electrochemical response: first, the ATP binding links split aptamers modified PBTA/CCG and MNPs together; second, ADA treatment catalyzes ATP into ITP, then releases the linked nanomaterials; finally magnet removes the remaining MNPs on alkylmodified electrode, and the ones with PBTA/CCG adsorption show efficient currents.

2005). Compared to them, electrochemical biosensor is easier to operate, more sensitive, and suitable for automation and field analysis (Zhang et al., 2010). Hence, greater efforts are urgently needed to develop new electrochemical strategy that can effectively detect ATP and ADA activity using split-aptamer fragments. Here, we explore a unique electrochemical biosensor using pyrene-functionalized graphene as a novel electrochemical label for dual detection of ATP and ADA activity. As shown in Scheme 1, in the presence of ATP, the “sandwich-type” construction is mediated to form between split-aptamers modified graphene materials and magnetic beads. Graphene labels can be collected by magnetic separation and then transferred controllably on an insulating self-assembled monolayer (SAM) modified electrode. After treatment with ADA, the collected sandwiched complex would release graphene and facilitate magnetic beads removal from electrode. The redox response of electroactive species in solution can be mediated by graphene on surface with a strong current signal generated. The signal intensity is direct related to the amount of graphene labels anchored on surface, which is related to the ATP concentrations. In addition, ADA activity would also be measured when ATP concentration was fixed. The developed strategy shows highly sensitive responses to ATP and ADA activity, with the detection limits of 13.6 nM and 0.01 unit/mL, respectively, which are more sensitive than previously reported methods. Our work would provide a novel idea for the dual detection of ATP and ADA activity with highly sensitivity and selectivity as well as the applications of functionalized graphene materials in biosensor fields.

2. Experimental Section 2.1. Reagents Graphite was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). 1-pyrene butyric acid (PBTA), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino) ethanesulfonic acid (MES), Tween 20 solution (5% in ethanol solution), and all proteins, including ADA, bovine serum albumin (BSA), human serum albumin (HSA) and lysozyme, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hydrazine solution (85%) and ammonia solution (25 wt%) were provided by Beijing Chemicals Inc. (Beijing, China). Ultra-pure water (18.2 MΩ, Milli-Q, Millipore) was used to prepare all aqueous solutions.

UV absorbance spectra were carried out on a JASCO V-550 UVvis spectrophotometer with a Peltier temperature control accessory. Protein concentration was determined by the UV absorbance value at 280 nm. Fluorescence measurements were realized on a JASCO FP-6500 spectrofluorometer with 1.0 cm path length cell. BRUKER Vertex 70 FT-IR spectrometer was used for the FT-IR characterization of materials, and all samples were firstly ground with dried KBr power. AFM images were carried using Nanoscope V multimode atomic force microscope (Veeco Instruments, USA). Scanning electron microscopic (SEM) measurements were recorded using a Hitachi S-4800 Instrument (Japan). TGA result was calculated with a Pyres 1 TGA apparatus (Perkin Elmer, MA), with 10 °C/min heating rate ranging from 50 °C to 900 °C, N2 atmosphere. 2.3. Preparation of GO, PBTA/ chemically converted graphene (CCG) and CCG Graphene oxide (GO) was synthesized from graphite by a modified Hummers method. PBTA/CCG was prepared by following the procedure: 0.5 mM PBTA (5.767 mg) dissolved in 100 mL 0.5 M NaOH solution was mixed with 5.72 mL 7 mg/mL GO, added H2O to 40 mL. The whole solution after 1 h ultrasonication was stirred at 40 °C overnight. Then 53.6 μL hydrazine solution and 600 μL ammonia solution were added to the solution and agitated vigorously at 95 °C for 0.5 h. The product solution was subsequently filtered through a Nylon membrane (0.22 μm pores), rinsed with water and dried under vacuum at room temperature. CCG solution without PBTA stabilizer was obtained by the similar reduction process. 2.4. Functionalization of PBTA/CCG with Apt-1 20 mL Tween 20 (5% in ethanol solution), 1600 mL PBTA/CCG were mixed in 10 mM PBS solution with a final concentration of 200 mg/mL. After 1 h stirring in room temperature, EDC and sulfoNHS (200 mM and 50 mM in 400 mL 10 mM MES buffer, respectively) were added to activate PBTA/CCG 0.5 h in 37 °C. Apt-1 DNA was added into the solution with 10 mM final concentration. The whole suspension was stirred at 37 °C overnight. Then the solution was centrifuged to separate graphene aggregations at 13000 rpm three times for 30 min. Then the PBTA/CCG in supernatant was collected and filtered through a centrifugal tube with a molecular weight cutoff (MWCO:1000, Spectrum Laboratories, Inc. US). The resulting Apt-1-PBTA/CCG complex was suspended in PBS solution and then stored at 4 °C. The DNA unlinked on graphene surface was collected and the absorbance at 260 nm was measured to calculate the linked efficiency on PBTA/CCG. 2.5. Preparation of Apt-2-MNPs bioconjugates The Apt-2 modified Fe3O4 magnetic nanoparticles (MNPs) were also obtained using EDC/NHS method as above, which was described briefly as follows: 4 mg MNPs dispersed in 100 mL DMSO/H2O solution was first mixed with 20 mL Tween 20 (5% in ethanol solution) at room temperature. Then EDC and sulfo-NHS

54

L. Feng et al. / Biosensors and Bioelectronics 62 (2014) 52–58

(200 mM and 50 mM in 400 mL 10 mM MES buffer, respectively) were added to activate MNPs sample for 0.5 h in 37 °C. Apt-2 DNA with a final 10 mM concentration was added into above solution and then was stirred at 37 °C overnight. The Apt 2-MNPs sediments were collected under magnetic field then rinsed three times with PBS solution to remove unmodified DNA. Subsequently, the Apt-2-MNPs was recollected and suspended in PBS, finally stored at 4 °C. The DNA unlinked on MNPs was collected and the absorbance at 260 nm was measured to calculate the linked efficiency on MNPs surface. 2.6. Electrochemical treatment and modification Gold disk electrodes (Φ¼ 2 mm, CH Instruments, Austin, TX) were polished with 0.3, 0.05 μm deagglomerated γ-alumina (BUEHLER, UAS) suspensions followed by sonication, then four steps of electrochemical cleaning was carried out in 0.5 M NaOH, 0.5 M H2SO4, 0.1 M H2SO4/0.01 M KCl, 0.05 M H2SO4 solutions. The electrodes were dried under mild N2 stream, then immersed in 6-mercaptohexanol (MCH) solution (1 mL 50 mM) for 36 h to produce a dense self-assembly monolayer (SAM). The produced SAM-modified gold electrode were then rinsed to remove the unmodified MCH on surface, finally whole modified electrodes were stocked in PBS solution (10 mM) for further use. 2.7. Electrode modification

2.8. Electrochemical measurements Cyclic voltammetry (CV) results were carried out on a CHI 660B electrochemistry workstation (CHI, USA) with K3[Fe(CN)6] (10 mM, 1 M KCl) as electrolyte and a conventional three-electrode system (platinum wire as the auxiliary electrode, Ag/AgCl as the reference electrode and the modified electrode used as working electrode). The scanning potential was between  0.2 V and 0.7 V, while differential pulse voltammetry (DPV) was scanned from 0.7 V to  0.2 V (vs. Ag/AgCl) with 0.05 V modulation amplitude, 0.001 V step potential and 0.004 V/s scan rate. Electrochemical impedance spectroscopy (EIS) was performed with 10 mM PBS containing K3[Fe(CN)6]/K4[Fe(CN)6] (10 mM, 1:1), 1 M KCl mixture as supporting electrolyte, and the frequency range was recorded within 10  2–105 Hz, with 5 mV amplitude of the applied sine wave potential.

2.9. SEM measurements

20 mL ATP in different concentrations was mixed with 10 mL Apt-1-PBTA/CCG and 3 mL Apt-2-MNPs for 0.5 h in 37 °C. The complex was separated under magnetic field rinsing with PBS solution for three times, then dispersed in PBS solution and dropped on the surface of MCH/AuE. In the progress of ATP detection, 10 mL 1 unit/mL ADA solution was dropped on the electrode to active for 1 h at 25 °C. The magnet was carefully put beside the electrode to absorb the released MNPs while washing by PBS solutions for three times. In the progress of ADA activity

2.5 d

2.0 1.5 c

1.0

b

0.5

Apt-1-PBTA/CCG and Apt-2-MNPs solutions were dispersed on clean Si surface for SEM measurements. 20 mL 10 mM ATP, 10 mL Apt-1-PBTA/CCG and 3 mL Apt-2-MNPs for 0.5 h in 37 °C. The complex was separated under magnetic field rinsing by PBS solution for three times, then dispersed on Si surface. The last sample for SEM measurement was obtained through activation with 10 mL 1 unit/mL ADA in above solution then separated under magnetic field.

Fluorescence Intensity

3.0

UV Absorbance

detection, ATP concentration was fixed as 10 mM, the other progresses were as similar as ATP detection except the different ADA units active on the electrode. Finally the modified electrodes were transferred into new electrolyte solution for next measurements.

a

0.0

350 300 250 200 PBTA

150 100 50

PBTA/CCG

0 -50

200 300 400 500 600 700 800

300

Wavelength/nm

400

500

600

700

Wavelength/nm

100 CCG

%

75 50

PBTA/CCG

25 PBTA 0 0

200

400

600

Temperature

800

2.50 1.25 0.00

nm

0

100

200

300

400

500

Fig. 1. (A) Absorption spectra of (a) GO; (b) CCG; (c) PBTA and (d) PBTA/CCG solutions. Inset: Images (from left to right) of water dispersions of GO, CCG and PBTA/CCG. (B) Fluorescence excitation and emission spectra of PBTA (black line) and PBTA /CCG (red line) in water. (C) TGA curves of CCG, PBTA/CCG, and PBTA composites. (D) AFM image for PBTA/CCG. The bottom image: height of the crossed area.

L. Feng et al. / Biosensors and Bioelectronics 62 (2014) 52–58

3. Results and discussion 3.1. Fabrication and characterization of PBTA/CCG 1-pyrenebutyric acid (PBTA) was adopted here to functionalize chemically converted graphene (CCG) noncovalently, which could introduce more carboxylic groups on graphene surface that being as uniformly distributed sites for the aptamer modification while preserve its well conductivity (Xu et al., 2008). The preparation of PBTA-stabilized CCG, named PBTA/CCG, was described as shown in the Experimental Section. The absorption peak of GO at 230 nm changed into 270 nm after the reduction process, and the PBTA spectrum contained three strong peaks at 239.7, 274.3 and 339.9 nm, which red-shifted to 246.8, 277.7 and 343.2 nm, respectively (Fig. 1A). The PBTA fluorescence was obviously quenched by graphene materials, which indicated that the electron transfer occurred from the excited state of PBTA to CCG, and it was also in agreement with the similar electron transfer from porphyrin to graphene, which previously reported by us (Fig.1B) (Feng et al., 2012). FT-IR analysis confirmed the existence of carboxylic groups at PBTA/CCG surface (Fig. S1). The peak around 1702 cm  1 in PBTA molecule was ascribed to ν(C ¼ O) vibration in –COOH, while in PBTA /CCG composite it shifted to 1722 cm  1, which might due to the π–π stacking and hydrophobic forces between PBTA and CCG materials (Feng et al., 2012). Compared with that of CCG without PBTA stabilizer, the carboxylic groups at the surface made the whole composite more stable in aqueous solution, which was black and stable for several months at room temperature (Fig. 1A inset). The amount of PBTA assembled on CCG was evaluated through thermogravimetric analysis (TGA) under N2 atmosphere (Fig. 1C). The thermal decomposition near 542 °C corresponded to the carbonization of the material. 20.5% of the mass loss between PBTA/CCG and CCG accounted for the PBTA present in the composite sample (Feng et al., 2011). All the above spectroscopic and TGA results showed that there existed strong π–π stacking and hydrophobic interactions between PBTA and CCG. In addition, AFM data (Fig. 1D) demonstrated that PBTA/CCG sample well-separated in single sheet with average topographic height of
2.1 nm, which meant that PBTA covered both sides of CCG with offset face-to-face orientation through π–π interaction, as previously reported (Xu et al., 2008; Feng et al., 2012). The PBTA/CCG size was about 280.5 nm based on the statistical analysis of 105 sheets by AFM images (Fig. S2). 3.2. Cyclic voltammetry measurements of electrochemical biosensor The split aptamer sequences of ATP are 14-mer Apt-1 (of sequence 5′-NH2-(CH2)6-ACC TGG GGG ATA AT-3′) and 23-mer Apt-2 (of sequence 5′-TGC GGA GGA AGG TAA AAA AAA AA(CH2)6-NH2-3′), which have been covalently linked on PBTA/CCG

and Fe3O4 magnetic nanoparticles (MNPs) through the wellknown EDC/NHS method (Feng et al., 2011, 2012). The calculated efficiencies were 6.97  10  7 mol/g and 5.5  10  8 mol/g for Apt-1 and Apt-2, respectively. In the presence of target ATP, Apt-1-PBTA/ CCG and Apt-2-MNPs could be formed into “sandwich-type” complex through the specific binding between ATP and its aptamer DNA (Liu et al., 2010). After magnetic separation, the ATP-triggered complex could be efficiently collected and then well transferred onto gold electrode (AuE) modified with an insulating 6-mercaptohexanol SAM. Treatment of the ATP-aptamer structure with ADA could transform ATP into inosine triphosphate (ITP), which was comparably much less bound to the aptamer sequence (Elbaz et al., 2009; Zhang et al., 2012). Further, PBTA/CCG would be released from the composite and then assembled on the insulating AuE surface through hydrophobic interactions between graphene surface and densely alkyl chains. Typical cyclic voltammetry (CV) curves of K3[Fe(CN)6]/K4[Fe(CN)6] at different surfaces were shown in Fig. 2A, and the oxidation and reduction peaks were negligible at only SAM modified AuE (curve a). After PBTA/CCG assembled on surface and MNPs removal in the presence of 10 mM ATP, a pair of well-defined redox peaks was observed (curve b). Graphene on surface could mediate electron transfer between the electrode and redox species in solution, thus triggering a strong redox current. Diffusion-controlled progress was confirmed with a linear relationship between the electrochemical peak current and the square root of different scan rates (Fig. S3A) (Feng et al., 2009). As shown in Fig. S3B, PBTA/CCG modified electrode was stable enough, even after one thousand scanning rounds, there was still no obvious desquamation. However, in the absence of ATP, the redox current was nearly unchangeable (curve c). GO solution was also used as control, and it could be seen that the pair of redox peaks decreased obviously (curve d), further demonstrating that PBTA/CCG could well facilitate the electron transfer working as electrochemical labels on surface. 3.3. Impedance and differential pulse voltammetry responses The same progress was also measured by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) methods, which were reported more sensitive than CV technique (Feng et al., 2012, 2009). EIS results were shown in Fig. S4. Upon modifying the PBTA/CCG on the surface, the electrontransfer resistance (Ret) value was about 292.6 Ω, which was lower than the values of only SAM-modified (3889.4 Ω) and GO-modified (4783.6 Ω) electrodes. As shown in Fig. 2B, a small DPV peak was observed around 0.19 V (vs. Ag/AgCl) on the SAM-modified electrode. After graphene composite was assembled on the surface in the presence of 10 mM ATP, a strong DPV peak appeared at 0.278 V (vs. Ag/AgCl). In addition, the modified surface produced negligible current in the presence of GO. These results further confirmed that

140

150

b

100

d

50

c

0

b

120

Current/µA

Current/µA

55

a

-50

100 80 60 40 a

20

-100

c

0

-150 -0.2

0.0

0.2

0.4

E/V vs. Ag/AgCl

0.6

d 0.0

0.2

0.4

0.6

E/V vs. Ag/AgCl

Fig. 2. (A) CV and (B) DPV curves of MCH modified AuE (a), electrochemical responses in the presence of (b) 10 mM and (c) 0 mM ATP, (d) 40 mg/mL GO modified electrodes.

56

L. Feng et al. / Biosensors and Bioelectronics 62 (2014) 52–58

140

140 120

100 80

Current/µA

Current/µA

120

ATP

60 40

100 80 60 40 20

20

0

0 0.0

0.2

0.4

0

0.6

2

E/V vs.Ag/AgCl

8

10

TTP

UTP

100

Current/ µA

120

Current/µA

6

120

140

100 80 60 40

R2=0.991

80 60 40 20

20 0 -1.5

4

ATP/µM

0 -1.0

-0.5

0.0

0.5

1.0

Log(CATP/(µM))

ATP

CTP

GTP

Fig. 3. (A) DPV responses and (B) current intensities in the presence of different ATP concentrations from 0 to 10 mM. (C) A linear relationship between DPV peaks and the logarithm of ATP concentration from 0.05 to 10 μM. (D) Specificity of the assay for ATP. The concentration of ATP was 1 mM, and four nonspecific molecules were 10 mM.

graphene label could be selectively collected by magnetic separation and triggered the detectable electrochemical signals, which would offer great potential for highly sensitive and selective detection of ATP and ADA activity. The DPV responses have been carefully investigated at different ATP concentrations (Fig. 3A). In the absence of ATP, the current intensity was about 9.5 mA around 0.195 V (vs. Ag/AgCl). It was observed that the DPV peaks dynamically increased with increasing the ATP concentration within the range from 0 mM to 10 mM (Fig. 3B). When ATP concentration was 10 mM, the current increased to 125.8 mA, which was 13.2-fold of the original signal. Also, the reduction peak potential shifted from 0.195 V to 0.278 V (vs. Ag/AgCl) with increasing ATP concentrations (Fig. 3A). A linear dependence relation was obtained between the peak current and the logarithm of ATP concentration from 0 to 1 mM. The detection limit was calculated to be 13.6 nM at 3s with a correlation coefficient R2 of 0.991 (n¼3) (Fig. 3C), which was much better than the values previously reported by fluorescence assay (Song et al., 2009) and electrochemiluminescence aptasensor (Liu et al., 2010), with the detection limit of 0.1 mM and 0.64 mM, respectively. The detection limit was also comparable with that of 15 nM of an electrochemical sensor recently reported (Wang et al., 2012). More results compared with other optical, electrochemical, and transistor sensors etc. were summarized in Table S1.

selectivity for ATP. A strong DPV signal was obtained for ATP after magnetic separation and ADA treatment in the progress of detection (Fig. S5A). The current peaks were all quite low when either or both of the treatments not applied as shown in Fig. S5B-D. It could be seen that both the magnetic separation and enzyme treatment were important for the electrochemical response due to the poor MNPs conductivity, therefore the removal of MNPs was necessary from SAM-modified electrode while still keeping the graphene labels on the insulating surface (Nie et al., 2012, 2009). 3.5. SEM characterization Scanning electron microscopy (SEM) was utilized to characterize the materials morphology after varying treatment processes, as shown in Fig. S5. Apt-1-PBTA/CCG showed wrinkling paper-like structure and close-packed Apt-2-MNPs were found on the surface with a diameter about 200 nm (Fig. S6A-B). After 10 mM ATP incubation and following magnetic separation, the complex collected was formed the agglomerate of PBTA/CCG and MNPs on the surface, indicating the high efficiency of ATP linking and magnetic separation for the collected grapheme composite (Fig. S6C). However, when ADA treatment was applied while MNPs were removed, only PBTA/CCG with a few MNPs assembled on the surface because of the dissociation of ATP-bound complex (Fig. S6D).

3.4. Selectivity of the electrochemical biosensor 3.6. ADA sensitivity and selectivity To further confirm the selectivity for ATP compared to different analogues, four control agents, cytidine triphosphate (CTP), guanosine triphosphate (GTP), thymine triphosphate (TTP) and uridine triphosphate (UTP) were further measured. As seen in Fig. 3D, the changes in electrochemical response induced by the nonspecific binding were much lower than that of the response induced by the specific binding of ATP, even at 10-fold higher concentration than ATP. These results indicated that this novel electrochemical platform would provide superior sensitivity and

The DPV peak was directly related to the amount of PBTA/CCG on surface, which was only released after ADA treatment, thus the sensor we designed here could also be used for ADA activity detection. The progress of measurement was as similar as previous ATP detection, only keeping the ATP concentration fixed at 10 mM. It was observed that the DPV peak currents increased with increasing ADA active units, which would release more PBTA/CCG on surface, thus facilitated the whole electron transfer (Fig. 4A).

L. Feng et al. / Biosensors and Bioelectronics 62 (2014) 52–58

57

140

120 120

80

ADA

Current/µA

Current/µA

100

60 40 20

80 60 40 20

0 -0.2

100

0.0

0.2

0.4

0.0

0.6

0.2

0.4

0.6

0.8

1.0

[ADA]/unit

E/V vs.Ag/AgCl 80

120 100

60 50 40

R2=0.954

80 60 40 20

30 0.00

Current/µA

Current/µA

70

0.02

0.04

0.06

0.08

0.10

0 ADA

[ADA]/unit

ADA 0 70 C

BSA

HSA Lysozyme

Fig. 4. (A) DPV responses and (B) current intensities in the presence of different ADA active units from 0.01 to 1 unit/mL. (C) A linear relationship between DPV peaks and ADA active units from 0.01 to 0.1 unit/mL. (D) Specificity of the assay for ADA activity. The concentration of ADA was 0.1 unit/mL, and four nonspecific proteins were 1 mM.

The current increased to 117.2 mA when 1 unit/mL ADA was used. The electrochemical signal was shown in Fig. 4B when treating with different ADA units, and there was a linear relationship from 0.01 to 0.1 unit/mL with R2 value of 0.954 (n ¼ 3). The direct detection of ADA was 0.01 unit/mL, about 1.2 nM (Fig. 4C). This result was 20-fold lower than previous report using the binding of G-quadruplex DNA with hemin as electroactive probe (0.2 unit/ mL) (Zhang et al., 2010), and also lower than the fluorescent biosensor using graphene as quencher (0.0129 unit/mL) (Xing et al., 2012), indicating that our platform had highly sensitive response to ADA activity using functionalized graphene as electrochemical labels. We also compared with more previously reported ADA detection results, and summarized in Table S2. The superiority of our present work was ascribed to effective PBTA/CCG accumulation after magnetic separation and highly efficient signal enhancement by graphene label since the graphene material on insulting layer was capable to mediate the redox reactions of numerous indicator molecules in solution (Nie et al. 2012, 2009). To further demonstrate the sensor selectivity for ADA, different other proteins, including 1 mM bovine serum albumin (BSA), human serum albumin (HSA), lysozyme and 0.1 unit/mL ADA after inactivation at 70 °C, were used for the control experiments. All DPV currents were much lower than ADA, as shown in Fig. 4D. The inactivated ADA could not catalyze the deamination of ATP into ITP, thus the formed complex was not opened efficiently (Elbaz et al., 2009), and the PBTA/CCG remaining on surface was quite few after magnetic separation. The controls further demonstrated that the sensor showed high selectivity for ADA, and could distinguish the active enzyme with the one inactivated.

4. Conclusion In summary, using functionalized graphene as efficient electrochemical label, we construct a novel electrochemical biosensor to realize dual detection of ATP and ADA activity. Through specific binding of ATP and its aptamers, the sandwich-type complex could

be formed between the split aptamers modified PBTA/CCG and MNPs. After ADA catalysis, the MNPs were removed under magnetic field, and the graphene materials were anchored on the electrode surface through the strong hydrophobic interaction between conjugated graphene surface and alkyl chains. The functionalized graphene would well facilitate electron transfer, thus produced amplified electrochemical signals. More importantly, the developed strategy shows highly sensitive responses to ATP and ADA activity, with the detection limits of 13.6 nM and 0.01 unit/ mL, respectively, which are more sensitive than previously reported methods. Our work will shed lights on new diagnostic applications of graphene, such as the design of biodevices for detection of other biomolecules including DNA, even cancer cells.

Acknowledgements Financial support was provided by 973 Project (2011CB936004, 2012CB720602), and NSFC (21210002, 91213302).

Appendix A. Suplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.008.

References Aldrich, M.B., Blackburn, M.R., Kellems, R.E., 2000. Biochem. Biophys. Res. Commun. 272, 311–315. Elbaz, J., Moshe, M., Willner, I., 2009. Angew. Chem. 121, 3892–3895. Ellington, A.D., Szostak, J.W., 1990. Nature 346, 818–822. Feng, L., Chen, Y., Ren, J., Qu, X., 2011. Biomaterials 32, 2930–2937. Feng, L., Li, X., Peng, Y., Geng, J., Ren, J., Qu, X., 2009. Chem. Phys. Lett. 480, 309–312. Feng, L., Wu, L., Wang, J., Ren, J., Miyoshi, D., Sugimoto, N., Qu, X., 2012. Adv. Mater. 24, 125–131. Li, X.L., Zhang, G.Y., Bai, X.D., Sun, X.M., Wang, X.R., Wang, E., Dai, H.J., 2008. Nat. Nanotechnol. 3, 538–542.

58

L. Feng et al. / Biosensors and Bioelectronics 62 (2014) 52–58

Liu, Z., Robinson, J.T., Sun, X., Dai, H., 2008. J. Am. Chem. Soc. 130, 10876–10877. Liu, Z., Zhang, W., Hu, L., Li, H., Zhu, S., Xu, G., 2010. Chem. Eur. J 16, 13356–13359. Lubin, A.A., Plaxco, K.W., 2010a. Acc. Chem. Res. 43, 496–505. Lubin, A.A., Plaxco, K.W., 2010b. Acc. Chem. Res. 43, 631–641. Myung, S., Solanki, A., Kim, C., Park, J., Kim, K.S., Lee, K.B., 2011. Adv. Mater. 23, 2221–2225. Nie, H., Liu, S., Yu, R., Jiang, J., 2009. Angew. Chem. Int. Ed. 48, 9862–9866. Nie, H., Yang, Z., Huang, S., Wu, Z., Wang, H., Yu, R., Jiang, J., 2012. Small 8, 1407– 1414. Novoselov, K.S., Geim, A.K., Morozov, S.V., Jiang, D., Zhang, Y., Dubonos, S.V., Grigorieva, I.V., Firsov, A.A., 2004. Science 306, 666–669. Paul, M.K., Grover, V., Mukhopadhyay, A.K., 2005. J. Chromatogr., B. 822, 146–153. Qiu, L., Wu, C., You, M., Han, D., Chen, T., Zhu, G., Jiang, J., Yu, R., Tan, W., 2013. J. Am. Chem. Soc. 135, 12952–12955. Sharma, A.K., Heemstra, J.M., 2011. J. Am. Chem. Soc. 133, 12426–12429. Song, Y., Qu, K., Zhao, C., Ren, J., Qu, X., 2010. Adv. Mater. 22, 2206–2210.

Song, Y., Zhao, C., Ren, J., Qu, X., 2009. Chem. Commun., 1975–1977. Stojanovic, M.N., de Prada, P., Landry, D.W., 2001. J. Am. Chem. Soc. 123, 4928–4931. Tan, W., Donovan, M.J., Jiang, J., 2013. Chem. Rev. 113, 2842–2862. Wang, L., Xu, M., Han, L., Zhou, M., Zhu, C., Dong, S., 2012. Anal. Chem. 84, 7301– 7307. Xing, X., Liu, X., He, Y., Luo, Q., Tang, H., Pang, D., 2012. Biosens. Bioelectron. 37, 61– 67. Xu, Y., Bai, H., Lu, G., Li, C., Shi, G., 2008. J. Am. Chem. Soc. 130, 5856–5857. Yang, K., Zhang, S., Zhang, G., Sun, X., Lee, S., Liu, Z., 2010. Nano Lett. 10, 3318–3323. Yan, G., Wang, Y., He, X., Wang, K., Liu, J., Du, Y., 2013. Biosens. Bioelectron. 44, 57– 63. Zhang, B., Fan, L., Zhong, H., Liu, Y., Chen, S., 2013. J. Am. Chem. Soc. 135, 10073– 10080. Zhang, K., Zhu, X., Wang, J., Xu, L., Li, G., 2010. Anal. Chem. 82, 3207–3211. Zhang, M., Guo, S., Li, Y., Zuo, P., Ye, B., 2012. Chem. Commun. 48, 5488–5490. Zuo, X., Xiao, Y., Plaxco, K.W., 2009. J. Am. Chem. Soc. 131, 6944–6945.