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Ultrasensitive Aptamer-Based Multiplexed Electrochemical Detection by Coupling Distinguishable Signal Tags with Catalytic Recycling of DNase I Dianping Tang,* Juan Tang, Qunfang Li, Biling Su, and Guonan Chen* Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, P.R. China
bS Supporting Information ABSTRACT: This work reports an aptamer-based, disposable, and multiplexed sensing platform for simultaneous electrochemical determination of small molecules, employing adenosine triphosphate (ATP) and cocaine as the model target analytes. The multiplexed sensing strategy is based on target-induced release of distinguishable redox tag-conjugated aptamers from a magnetic graphene platform. The electronic signal of the aptasensors could be further amplified by coupling DNase I with catalytic recycling of self-produced reactants. The assay was based on the change in the current at the various peak potentials in the presence of the corresponding signal tags. Experimental results revealed that the multiplexed electrochemical aptasensor enabled the simultaneous monitoring of ATP and cocaine in a single run with wide working ranges and low detection limits (LODs: 0.1 pM for ATP and 1.5 pM for cocaine). This concept offers promise for rapid, simple, and cost-effective analysis of biological samples.
T
he increasing interest in proteomics and clinical diagnostics has promoted the development of efficient tools for sensitive, selective, rapid, and cost-effective analysis of biomolecules. The assay is generally performed using certain affinity ligands including aptamers and antibodies that specifically interact with the biomolecules and thus mediate a target-responsive signal transduction cascade.1 Over the past decade, although significant progress has been made worldwide in the single-analyte assay, it is often insufficient to diagnose and follow the disease status by analyzing one target analyte. Simultaneous determination of multiplexed analytes with ultrahigh sensitivity is of great interest. Magneto-controlled molecular electronics and bioelectronics have become new tools for monitoring of biomolecules in clinical diagnostics and treatment.24 Batch-type magnetic separators have been fabricated on a single chip for trapping and directed sequential elution of magnetic particles in flowing fluids. Aptamers are synthetic oligonucleotides that can bind a wide range of target molecules (proteins, drugs, amino acids, etc.) with high specificity and selectivity.58 Recently, our group found that graphene nanosheets could strongly bind single-stranded DNA, such as aptamers, as a result of hydrophobic and π-stacking interactions between the nucleobases and graphene.9,10 The interaction could protect the release of aptamers from the surface of graphene nanosheets. Upon addition of the target analytes, however, the functionalized aptamers formed a stable and rigid structure and were released from the graphene surface. We reasoned that, if such assays could be extended to detect species other than the fluoresence assay, it might be generally useful for r 2011 American Chemical Society
other types of highly sensitive and selective assays. Electrochemistry holds great potential as the next-generation detection strategy because of its high sensitivity, simple instrumentation, and excellent compatibility with miniaturization technologies. Up to date, however, there is still no report focusing on the electrochemical detection of biomolecules based on the targetinduced release of the aptamers from the graphene nanosheets. One of the bottlenecks for developing “point of care” electrochemical detection is the readout method. Herein, we design a novel magneto-controlled multiplexed electronic detection of biomolecules based on the target-induced release of the aptamers conjugated with distinguishable redox signal tags from the magnetic graphene sensing platform. The electrochemical signals were simultaneously obtained at various peak potentials due to the presence of different redox tags (Scheme 1). Another important issue for successful development of aptamer-based multiplexed assays is signal amplification and noise reduction. A common resort is to exploit signal-transduction labels including ligand-conjugated enzymes and nanolabels.1113 The labels can be usually transformed into readily detectable electro-active species through enzymatic conversion of certain substrates or chemical decomposition of the metal or its insoluble salts. However, in these homogeneous assays, one aptamer can bind with only one target molecule. The 1:1 binding ratio Received: July 23, 2011 Accepted: August 30, 2011 Published: August 30, 2011 7255
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Scheme 1. Schematic Illustration of Multiplexed Aptamer-Based Electrochemical Assay Using Target-Induced Release of Redox Tag-Conjugated Aptamers from Magnetic Graphene Platform and DNase I-Based Catalytic Recycling of the Analytea
a
Th: thionine; Fc: ferrocene.
limits the signal amplification and thus the sensitivity of the assays. Inspiringly, the emergence of nuclease cleavage provides exciting new possibilities for achieving high sensitivity.1416 A very important point using nuclease cleavage for signal amplification is to prevent DNA from nuclease cleavage, owing to the steric-hindrance effect; thus, an efficient substrate (e.g., nanostructures) should be necessary for protecting nucleases from binding onto the immobilized DNA. Magnetic graphene nanosheets (MGPs) with high magnetocrystalline anisotropy, high electrical conductivity, and high surface-to-volume ratio, have emerged as an exciting two-dimensional nanomaterial, showing great promise for the construction of nanoscale device. In this work, we expect that the synthesized MGPs can protect aptamers from nuclease cleavage. To realize our design, we fabricated a magneto-controlled graphene sensing platform for a multiplexed aptamer-based electronic assay using nuclease cleavage (DNase I in this case) and distinguishable signal tags (thionine and ferrocene in this case) (Scheme 1). Ferrocene-labeled adenosine triphosphate (ATP) aptamer (50 -Fc-(CH2)3-ACCTG GGGGA GTATT GCGGA GGAAG GT-30 , Fc-P1) and thionine-labeled cocaine aptamer (50 -Th-(CH2)3-GACAA GGAAA ATCCT TCAAT GAAGT GGGTC-30 , Th-P2) were utilized as our models (see experimental details in the Supporting Information). Due to the strong noncovalent binding of MGPs with nucleobases and aromatic compounds, Fc-P1 and Th-P2 are initially bound onto the surface of MGPs. Attraction of the functional MGPs to the probe with an external magnet activates the electrical contact between the immobilized biomolecules and the base electrode, and the sensor’s circuit is switched on. Fc and Th tages exhibit two strongly well-resolved voltammetric peaks at the distinct potentials, respectively. Positioning the magnet above the cell retracts the functional MGPs from the probe, and the electrochemical behavior of the functional MGPs is switched off. In the presence of the analytes (i.e., ATP and cocaine), the analytes react with the corresponding aptamers (i.e., Fc-P1 and Th-P2) and disturb the interaction between the aptamers and MGPs. Such interactions enable the release of the aptamers from the MGPs. Meanwhile, the released ATP/Fc-P1 and cocaine/ThP2 complexes can be cleaved by the DNase I, and the analyte is delivered from the analyteaptamer complex, which can reattack other aptamers on the MGPs. Following that, the cycle starts anew, resulting in the successive release of distinguishable signal
Figure 1. QCM response of (a) Fc-P1-MGP-Th-P2 probe, (b) after incubation of the MGP probe with Fc-P1, Th-P2, and DNase I, (c) after incubation of the Fc-P1-MGP-Th-P2 probe with DNase I, (d) after incubation of the Fc-P1-MGP-Th-P2 probe with 10 nM ATP and 10 nM cocaine, and (e) after incubation of the Fc-P1-MGP-Th-P2 probe with 10 nM ATP, 10 nM cocaine, and 50 U mL1 DNase I.
tag-based aptamers from the MGP probes, resulting in the dramatic decrease of the electrochemical signal of the functional MGP probes. By monitoring the change in the electrochemical signal at the various peak potentials, we can quantitatively determine the concentration of the corresponding analyte. Since the strong noncovalent binding of graphene nanosheets with the aptamers was demonstrated by AFM and gel electrophoresis in our provious reports,9,10 we utilized quartz crystal microbalance (QCM) to investigate the validity of the method herein. Figure 1a shows the QCM response (frequency vs time) of the MGP substrate after incubation with Fc-P1 and Th-P2 in the absence of DNase I. The frequency initially increased with the increase of time and then tended to level off after 8 min. Compared with the frequency of the bare MGPs, the increase in the frequency was attributed to the interaction between the functional aptamers and the MGPs. When the prepared MGPs were incubated with the mixture containing Fc-P1, Th-P2, and DNase I, however, no significant change in the frequency was observed (Figure 1b) in contrast with that of the bare MGPs. The reason might be the fact that the free aptamers (i.e., Fc-P1 and Th-P2) in the incubation solution were digested by the DNase I. For comparison, we initially prepared the Fc-P1-MGP-Th-P2 7256
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Figure 2. SWV curves of (a) Fc-P1-MGP-Th-P2 probe, (b) after incubation of probe “a” with 10 nM ATP and 50 U mL1 DNase I, (c) after incubation of probe “a” with 10 nM cocaine and 50 U mL1 DNase I, and (d) after incubation of probe “a” with 10 nM ATP, 10 nM cocaine, and 50 U mL1 DNase I in pH 7.4 PBS (vs. Ag/AgCl).
probes, and then, the as-prepared probes were incubated with the DNase I. As seen from Figure 1c, the steady-state frequency of the probes after incubation was the same as that of the Fc-P1MGP-Th-P2 probes (Figure 1a). The results indicated that the DNase I could not cleave the immobilized aptamers on the MGPs; that is, the synthesized MGPs could protect the functional aptamers from the cleavage of DNase I. Meanwhile, we also found that the freqency decreased with the concentration increase of the corresponding analyte at the absence DNase I (Figure 1d), which was mainly abscribed to the release of the functional aptamers from the MGPs. Furthermore, the shift in frequency was greatly enhanced in the presence of DNase I (Figure 1e). The reason might be the fact that the functional aptamers were released from the MGPs one by one and digested by the DNase I in the presence of the targets. On the basis of these results, we might make the conclusion that the MGPs could be utilized for simultaneous detection of ATP and cocaine by coupling target-induced release of the aptamers from the MGPs with DNase I for the signal amplification. Following that, we used square wave voltammetry (SWV) to evaluate the electrochemical properties of the magnetic graphene sensing platform for simultaneous detection of ATP and cocaine in 0.1 M, pH 7.4 phosphate buffered saline (PBS) solution with a conventional three-electrode system in the sequential injection mode. Initially, Fc-P1 and Th-P2 were incubated for 60 min with the MGPs to form the Fc-P1-MGP-Th-P2 complex, and then, the ATP/DNase I and cocaine/DNase I were added and incubated in turn for another 60 min (Note: 60 min as a model was chosen as the incubation time in order to ensure the adequate reaction between DNase I, aptamers, and MGPs). Figure 2 shows the SWV responses of the Fc-P1-MGP-Th-P2 probe in pH 7.4 PBS after incubation with various incubation solutions. As shown in Figure 2a, the prepared Fc-P1-MGP-Th-P2 probe exhibited two well-resolved peaks at 180 mV and 290 mV in pH 7.4 PBS before incubation, and the peak separation (ΔEp) was 470 mV. Two peaks were mainly derived from the labeled thionine and ferrocene, respectively. After the Fc-P1-MGP-Th-P2 probe was incubated with 10 nM ATP and 50 U mL1 DNase I, the peak current was decreased only at the corresponding Fc tags
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Figure 3. SWV curves of (a) Fc-P1-MGP-Th-P2 probe, (b) after incubation of probe “a” with 100 pM ATP and 100 pM cocaine, and (c) after incubation of probe “a” with 100 pM ATP, 100 pM cocaine, and 10 U mL1 DNase I.
(Figure 2b). The results indicated the dissociation of the Fc-P1ATP complexes from the MGPs. Similarly, when the Fc-P1MGP-Th-P2 probe was incubated with 10 nM cocaine and 50 U mL1 DNase I, a remarkable decrease was obtained only at the corresponding Th tags (Figure 2c). Furthermore, we applied a mixture containing 10 nM ATP, 10 nM cocaine, and 50 U mL1 DNase I as incubation solution, and as expected, two distinct and well-resolved voltammetric peaks, which correspond to Fc and Th, respectively, were observed (Figure 2d). This demonstrates the capability of our strategy for simultaneous electrochemical determination of ATP and cocaine. For the successful development of a multianalyte assay, signal amplification is crucial. To verify the advantage of the DNase I, the method was applied for the detection of ATP and cocaine with and without the aid of DNase I under the same protocols. The assay was based on the shift in the current relative to zero analyte. As indicated from Figure 3, the shift in the peak current was significantly higher in the presence of DNase I than that without DNase I (100 pM ATP or cocaine as an example). The amplified electrochemical signal using the DNase I was 300400% of the use of 1:1 binding strategy toward two analytes with the same concentration. Meanwhile, we also observed that the assay using DNase I exhibited low and smooth current changes at the low levels. The results indicated that the use of DNase I not only displayed high sensitivity but also favored the determination of the analytes at very low level. The sensitivity and dynamic range of the multiplexed magnetic graphene sensing platform was evaluated with ATP and cocaine standards based on target-induced release of redox tagconjugated aptamers from the MGPs and catalytic recycling of DNase I. A square wave voltammetric (SWV) measurement of the Fc-P1-MGP-Th-P2 probes was carried out in pH 7.4 PBS after incubation with 200 μL of ATP or cocaine with various concentrations and DNase I (20 μL, 50 U mL1) for 60 min. Figure 4a represents the SWV curves of the multiplexed magnetic graphene sensing platform toward ATP and cocaine with different concentrations. The peak currents decreased with the increase of ATP and cocaine concentrations. Both calibration plots displayed a good linear relationship between the SWV peak currents and the analyte concentration in the ranges of 1.0 pM to 500 μM for ATP (Figure 4b) and 10 pM to 400 μM for cocaine (Figure 4c). The correlation coefficients were 0.9961 and 0.9924 7257
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Figure 4. (a) SWV responses and (b,c) calibration plots of the multiplexed magnetic graphene sensing platform in pH 7.4 PBS toward (b) cocaine and (c) ATP standards with various concentrations.
for ATP and cocaine (n = 30), respectively. The low detection limits (LODs) for ATP and cocaine were found at 0.1 pM and 1.5 pM estimated at the 3sB criterion. In contrast, the LOD for both analytes was 1.0 nM when the 1:1 binding strategy without amplification was used. Although the system has not yet been optimized for maximum efficiency, the assay senstivity was 25 orders of magnitude higher than those of target-induced displacement or conformational changes of aptamers.1720 To investigate the reproducibility of the multiplexed magnetic graphene sensing platform, we repeatedly assayed both analytes with three different concentrations, using identical batch Fc-P1MGP-Th-P2 probes. Experimental results revealed that the coefficients of variation (CVs) of the intra-assay between six runs were 7.1%, 8.2%, and 7.4% for 10 pM, 10 nM, and 10 μM ATP and 9.27%, 6.3%, and 8.8% for 50 pM, 50 nM, and 50 μM cocaine, respectively, whereas the CVs of the interassay with various batches were 9.7%, 8.6%, and 9.1% for ATP, and 7.1%, 8.9%, and 8.4% for cocaine toward the above-mentioned analyte. The low CVs indicated that the multiplexed magnetic graphene sensing platform could be regenerated and used repeatedly and further verified the possibility of batch preparation. When the FcP1-MGP-Th-P2 probes were not in use, they were stored in pH 7.4 PBS at 4 °C. No obvious change was achieved after storage for 28 days but a 10% decrease of its initial currents was noticed after 60 days. The specificity of the multiplexed magnetic graphene sensing platform was evaluated by challenging it against other biomolecules, and the results were listed in Figure 5. With an amount of nontarget molecules, such as cytochrome C and thrombin, cytosine triphosphate (CTP), guanidine triphosphate (GTP), and uridine triphosphate (UTP), no apparent change in the current was observed compared with that of the blank test. However, the presence of the target analytes resulted in the dramatic decrease in the current, indicating the high specificity of the multiplexed magnetic graphene sensing platform. Importantly, the magnetic graphene sensing platform could be used for the repeated detection of biomolecules through the rebinding of Fc-P1 and Th-P2 aptamers. In summary, we for the first time demonstrate the ability of magnetic graphene nanosheets as sensing platform for simultaneous electrochemical detection of proteins and small molecules in this work. Compared with other strategies, the method is sensitive, rapid, simple, and reusable. Significantly, the assay does not require sophisticated fabrication and is well suited for highthroughput biomedical sensing and application in both clinical and biodefense areas.
Figure 5. Selectivity of the multiplexed magnetic graphene sensing platform toward cocaine, ATP, UTP, GTP, thrombin, and cytochrome C.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail:
[email protected] (D.T.),
[email protected] (G.C.).
’ ACKNOWLEDGMENT Support by the National Natural Science Foundation of China (Nos. 21075019, 41176079, and 20735002), the Research Fund for the Doctoral Program of Higher Education of China (No. 20103514120003), the Award Program for Minjiang Scholar Professorship (No. XRC-0929), the National Science Foundation of Fujian Province (No. 2011J06003), and the “973” National Basic Research Program of China (No. 2010CB732403) is gratefully acknowledged. ’ REFERENCES (1) Reiter, L.; Rinner, O.; Picotti, P.; Huttenhain, R.; Back, M.; Bursniak, M.; Hengartner, M.; Aebersold, R. Nat. Methods 2011, 8, 430–435. 7258
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