Novel Homogeneous Label-Free Electrochemical Aptasensor Based

Sep 2, 2013 - We first developed a label-free and immobilization-free homogeneous electrochemical aptasensor, which combined a smart functional DNA ...
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Novel Homogeneous Label-Free Electrochemical Aptasensor Based on Functional DNA Hairpin for Target Detection De-Wen Zhang,† Ji Nie,† Fang-Ting Zhang,† Li Xu,‡ Ying-Lin Zhou,*,† and Xin-Xiang Zhang*,† †

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry, Peking University, Beijing 100871, China ‡ Beijing Institute of Microchemistry, Beijing 100091, China S Supporting Information *

ABSTRACT: We first developed a label-free and immobilizationfree homogeneous electrochemical aptasensor, which combined a smart functional DNA hairpin and a designed miniaturized electrochemical device. Cocaine was chosen as a model target. The anticocaine aptamer and peroxidase-mimicking DNAzyme were integrated into one single-stranded DNA hairpin. Both aptamer and G-quadruplex were elaborately blocked by the stem region. The conformation switching induced by the affinity interaction between aptamer and cocaine released G-quadruplex part and turned on DNAzyme activity. The designed electrochemical device, constructed by a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode, was applied to the detection of homogeneous DNAzyme catalytic activity at the microliter level. The aptasensor realized the quantification of cocaine ranging from 1 to 500 μM with high specificity. The clever combination of the functional DNA hairpin and the novel device achieved an absolutely label-free electrochemical aptasensor, which showed excellent performance like low cost, easy operation, rapid detection, and high repeatability.

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The combination of two key techniques provides the feasibility for the construction of a homogeneous label-free electrochemical aptasensor. One is the design of a functional DNA hairpin consisting of aptamer and DNAzyme as recognition and signal readout element, respectively;21−25 the other is the setup of a microliter electrochemical device combined with ultramicroelectrode using for homogeneous enzyme electrochemical biosensor reported in our previous work.26 DNAzyme is catalytic nucleic acid, which was selected by SELEX (systematic evolution of ligands by exponential enrichment).27 Some G-quadruplex sequence can interact with hemin to form a G-quadruplex/hemin DNAzyme and perform peroxidase activity similar to that of horseradish peroxidase (HRP).28−32 The HRP-mimicking DNAzyme could be used as catalytic tag to amplify colorimetric33,34 or electrochemical signal.35−38 As aptamer and DNAzyme are both single-stranded oligonucleotides, they can be integrated into one single-stranded DNA hairpin as both target recognition element and signal transduction element without extra labeling or modification. The catalytic activity of DNAzyme could be controlled by conformation switching of

lectrochemical aptasensors, which combine the speed, portability, high sensitivity, and low cost of electrochemical transducers and the high affinity and specificity of aptamer, have attracted much attention during the last 10 years.1−5 Up to now, electrochemical aptasensors have been mainly constructed in heterogeneous format.6,7 Most of them require labeling redox-active units,8−10 enzymes,11,12 or nanoparticles13 as signal transduction elements. Some label-free aptasensors have also been developed based on electrochemical impedance spectrometry,14−17 square wave voltammetry,18 and chronocoulometry.19 However, the modification of aptamers with linking group was still needed for the immobilization of DNA onto the electrode surface, which undoubtedly increases the cost. The immobilization of DNA is a key step in the construction of a heterogeneous electrochemical aptasensor with the advantages, such as recycle and the low consumption. But it was time-consuming and labor-intensive. Absolutely label-free homogeneous electrochemical aptasensors would be advantaged in some ways, such as low cost, easy operation, and time saving, and would be complementary to the heterogeneous electrochemical aptasensors, although heterogeneous electrochemical aptasensors may have the capability for the analysis in the cases that homogeneous aptasensors cannot achieve.20 And the homogeneous reaction could increase the reliability and reproducibility of electrochemical sensor without the immobilization step. © 2013 American Chemical Society

Received: July 25, 2013 Accepted: September 2, 2013 Published: September 2, 2013 9378

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Scheme 1. Principle of Homogeneous Label-Free Electrochemical Aptasensor Based on Aptamer−DNAzyme Hairpin for Cocaine Detection

oligonucleotides were dissolved in 0.1 M pH 7.4 phosphate buffer (PB) containing 5 mM KCl and 5 mM MgCl2. Hemin (5 mM) was dissolved in dimethyl sulfoxide (DMSO) and then diluted to 100 μM in PB containing 0.01% Triton X-100. All samples and buffer solutions were prepared using ultrapure water from a Milli-Q water purification system (Bedford, MA, U.S.A.). Electrochemical Measurements. A CHI 660C electrochemical workstation (Shanghai Chenhua Instruments Co., Shanghai, China) was employed to accomplish the electrochemical experiments. All measurements were carried out in the designed electrochemical device. The carbon fiber ultramicroelectrode was fabricated as our previous work.26 As shown in Scheme 1, the analyte solution with fixed volume was in the micropipet tip. The carbon fiber ultramicroelectrode as a working electrode (WE) was inserted into the micropipet tip. The micropipet tip was placed in a homemade glass cell containing buffer. An Ag/AgCl reference electrode (RE) and a Pt counter electrode (CE) were assembled in the cell to form a full three-electrode system. Electrochemical Assay of Cocaine Using Hairpin. The CocAD in the buffer was heated to 95 °C, and slowly cooled down (0.02 °C/s) to 20 °C by TC-512 Gradient PCR (TECHNE, U.K.). Then it was held at 20 °C by a block heater. Cocaine and hemin were incubated with CocAD at 20 °C for 1 h. HQ and H2O2 were added to a final concentration 1 mM, respectively. A 20 μL solution was used for detection by the miniaturized electrochemical device.

the functional hairpin structure, which directly correlated to the target triggering event. In our previous work, we had constructed a simple and easily operated miniaturized microliter electrochemical device using a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode.26 This novel electrochemical device set the electrochemical reaction in a micropipet tip containing an ultramicroelectrode, which greatly reduced the reaction solutions to several microliters. As the volume was very small, the sample consumption was very low and the disposable consumption became affordable. At the same time, the carbon fiber ultramicroelectrode enhanced mass transport, which could monitor the redox substrate and product nearly in real time. As it had been successfully applied to the detection of homogeneous HRP catalytic activity, it can also be applicable to detecting the homogeneous HRP-mimicking DNAzyme activity. In this work, a homogeneous label-free electrochemical aptasensor requiring only a single-stranded DNA hairpincoupled aptamer and DNAzyme was established (Scheme 1). The triggering of target cocaine resulted in the opening of hairpin and the release of DNAzyme sequence which performed catalytic activity with hemin. Our designed electrochemical device was chosen as the detector toward generated DNAzyme. As low as 1 μM cocaine was detectable with high specificity. During the whole process, no need to immobilize DNA to the electrode surface, no need to modify DNA with linking group. A simple and rapid “mix-and measure” strategy was demonstrated, which will bring a new developmental direction for the construction of homogeneous label-free electrochemical aptasensors.

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RESULTS AND DISCUSSION Electrochemical Measurement of HRP-Mimicking DNAzyme. In this work, it was first demonstrated to detect the catalytic activity of DNAzyme by the miniaturized electrochemical device which had been successfully used for the detection of homogeneous HRP catalytic activity. Using HQ as an electron mediator, the cyclic voltammograms (CVs) in the absence or presence of EAD2 (a G-quadruplex sequence) were measured. For the columnar ultramicroelectrode, the quasi-steady state current accords with the equation iqss = ((2nFAD0C0*)/(r0 ln T)), where C0* is the concentration of the electroactive species (mol/L). The oxidation quasi-steady state current is proportional to the concentration of the substrate HQ; the reduction quasi-steady state current is corresponding to the product benzoquinone (BQ). In the presence of DNAzyme and H2O2, HQ is chemically reduced to BQ, which leads to the decrease of HQ concentration accompanying with the increase of BQ in solution. Therefore along the catalytic reaction, the oxidation current decreases and the reduction current increases. Because of the enhanced mass transport of

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EXPERIMENTAL SECTION Chemicals. The DNA oligonucleotides were purchased from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) and have the following sequences: 5′-CTGGGAGGGAGGGAGGGA-3′, EAD2, and 5′-CTGGGAGGGAGGGAGGGATGTCGAGGGAGACAAGGAAAATCCTTCAATGAAGTGGGTCGACATCCC-3′, CocAD. (The bold portion is the DNAzyme sequence EAD2, and the italic portion is the aptamer of cocaine) Cocaine hydrochloride and pethidine hydrochloride were obtained from National Institute for the Control of Pharmaceutical and Biological Products. KCl, MgCl2, hemin, and ferrocenecarboxylic acid were purchased from SigmaAldrich (St. Louis, MO, U.S.A.). Hydrogen peroxide (H2O2) was purchased from Beijing Chemical Reagent Company (Beijing, China). Hydroquinone (HQ) was purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). The 9379

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consisted of anticocaine aptamer and DNAzyme was shown in Scheme 1. After annealing treatment, it formed hairpin structure chiefly. The stem domain contained partial aptamer sequence, partial EAD2 sequence and corresponding complementary bases of the above two. It blocked the specific functions of both aptamer and DNAzyme via base paring. In the presence of cocaine, the affinity interaction of cocaine and aptamer triggered the conformation switching of CocAD hairpin. The stem domain was opened and the G-quardruplex was formed. It performed HRP-mimicking activity with hemin. Figure 2 showed the triggering event of cocaine. Two

ultramicroelectrode, it can realize nearly real-time monitoring the concentration change of HQ and BQ. The CVs in PB containing 1 mM HQ and 1 mM H2O2 and different concentrations of EAD2 were measured at 3 min after mixing the solutions. As shown in Figure 1a, the oxidation current was

Figure 1. CVs of 1 mM HQ and 1 mM H2O2 after reaction for 3 min in PB in the absence of (a) hemin and DNAzyme, in the presence of (b) 4 μM hemin, (c) 500 nM EAD2 and 4 μM hemin, (d) 1 μM EAD2 and 4 μM hemin, (e) 2 μM EAD2 and 4 μM hemin, and (f) 4 μM EAD2 and 4 μM hemin. Scan rate: 100 mV/s. Figure 2. CVs of 1 mM HQ and 1 mM H2O2 after reaction for 9 min in PB in the presence of 2 μM CocAD and 4 μM hemin which were incubated with (a) buffer, (b) 1 mM pethidine, and (c) 500 μM cocaine for 1 h. Scan rate: 100 mV/s.

much higher than the reduction current in the absence of EAD2 and hemin since HQ occupied the majority of redox species. In the presence of 4 μM hemin only, the change of reduction current or oxidation current was not obvious (Figure 1b), indicating the catalytic activity of hemin alone was too weak to catalyze the reaction between HQ and H2O2. With the addition of 0.5 μM EAD2, whatever the oxidation current or the reduction current changed obviously (Figure 1c), because of the much higher catalytic activity of DNAzyme than hemin alone. Increased EAD2 in the buffer further increased the reduction peak intensity and decreased the oxidation peak intensity (Figure 1d−f). The results showed that the reduction peak occupied the majority in the presence of 4 μM DNAzyme, indicating most of HQ in solution was chemically reduced to BQ. There was a good relationship between the current change and the concentration of EAD2. Therefore, our designed electrochemical device was conveniently and economically available for the detection of homogeneous HRP-mimicking DNAzyme catalytic reaction and can be further applied for the homogeneous biosensor using DNAzyme as signal reporter. We also checked the possibility of hemin/G-quadruplex catalyzed reaction of HQ by oxygen without H2O2, which was shown in Supporting Information Figure S1. At a standard atmospheric pressure and 20 °C, the water solubility of oxygen is 9.1 mg/L (284 μM). In the presence of 5 μM EAD2/hemin, the oxidation quasi-steady current in 200 μM HQ and dissolved oxygen (the buffer was not treated by highly pure nitrogen to remove the dissolved oxygen) decreased 0.41 nA (from Supporting Information Figure S1a, S1b) after reaction for 11 min. In the presence of 5 μM EAD2/hemin and 200 μM H2O2, the oxidation quasi-steady current decreased 5.5 nA (from Supporting Information Figure S1a−S1c) after reaction for only 1 min. Therefore, the EAD2/hemin had a much lower catalytic effect for the oxidation of HQ by oxygen than H2O2. Using dissolved oxygen to replace H2O2 was not applicable for the homogeneous DNAzyme activity detection. Performance of Homogeneous Label-Free Electrochemical Aptasensor. The functional DNA hairpin (CocAD)

micromolar CocAD, 4 μM hemin, and 500 μM cocaine were incubated for 1 h. The CV was measured after mixing the above solution with HQ and H2O2 for 9 min. The oxidation current observably decreased accompanying with the obvious increase of the reduction current (Figure 2c) comparison with that performed without cocaine (Figure 2a). It clearly indicated that cocaine led to the opening of hairpin and the release of the DNAzyme activity. The specificity of the aptasensor was investigated using 1 mM pethidine to replace cocaine to make sure the triggering event was due to the interaction between cocaine and aptamer (Figure 2b). For the CV was almost the same to the background, it demonstrated the high specificity of the aptasensor. Unlike HRP, whose enzymatic reaction rate of HRP was so fast that 1 mM HQ and 1 mM H2O2 in the presence of 1 μg/ mL HRP (about 25 nM) could react completely in the first minute (the CV curves did not change with time),26 obtaining a steady CV curve in the presence of DNAzyme required more time because of the much lower enzymatic reaction rate. To obtain a sensitive signal, the effect of catalytic reaction time on the oxidation current change at different concentrations of cocaine was investigated (Figure 3). The slope of the curve at the given cocaine concentration indicated the enzymatic reaction rate, which increased with the increase of cocaine concentration, indicating more DNAzyme was release from the hairpin structure. A very small time-dependent current change in the absence of cocaine (Figure 3a) was observed. It indicated that the CocAD did not form the hairpin completely and tiny released G-quadruplex formed DNAzyme with hemin. The equilibrium between the ideally blocked hairpin with stable stem duplex and imperfect blocked hairpin resulted in the background of system. To generate remarkable current change 9380

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was reached. The calibration curve of cocaine ranged from 1 to 500 μM, and the lowest detectable limit was 1 μM. To verify the repeatability, the samples of every concentration were repeated three times independently. The homogeneous incubation and catalytic reaction greatly reduced the operational difficulty and time consumption as the DNA needn’t to be immobilized onto the sensing interface. It further improved the repeatability of the aptasensor. Although the DNA was not reusable, the disposable DNA consumption was affordable because of small volume (20 μL) and low concentration (2 μM) of DNA without modification. The ultramicroelectrode played an important role as the detector in the performance of aptasensor. As mentioned above, the carbon fiber ultramicroelectrode performed well in this sensing strategy. The properties such as contaminantresistance and easy cleaning guaranteed that the carbon fiber ultramicroelectrode could be repeatedly used before mechanically broken. It further ensured the repeatability of the device. The Pt ultramicroelectrode was also tried in this work. Although it was more durable, the nonspecific adsorption would deform the CV curves after measurements for several times. Thus, the carbon fiber ultramicroelectrode was a better choice at present. Different electron mediator was also investigated. Supporting Information Figure S2 showed the CV responses of the aptasensor in different cocaine concentrations using 5 mM ferrocenecarboxylic acid after catalytic reaction for 5 min. There was no obvious difference for the lowest detectable limit of cocaine by different electron mediators. But the product of the catalytic reaction between ferrocenecarboxylic acid and H2O2 was unstable, which might limit the detection time.

Figure 3. Time-dependent oxidation steady-state current changes upon the interaction of CocAD with different concentrations of cocaine: (a) 0, (b) 1, (c) 10, (d) 100, and (e) 1000 μM.

especially at low target concentration, 9 min was chosen as the catalytic reaction time. The CV responses of the aptasensor at different concentrations of cocaine after catalytic reaction for 9 min were measured (Figure 4A). The calibration curve was obtained

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CONCLUSIONS In summary, a novel homogeneous electrochemical aptasensor was developed for cocaine detection, which was label-free and immobilization-free. It cleverly combined aptamer-DNAzyme hairpin and a miniaturized electrochemical device. The release of DNAzyme triggered by cocaine could be sensitively detected by our designed electrochemical device in a cheap and effective mode. The detection can be finished in a short period of time without immobilization operation, which is a necessary step in heterogeneous electrochemical aptasensors. The small volume in microliters decreased the sample consumption. In addition, the homogeneous detection and reusable carbon fiber ultramicroelectrode improved the reproducibility of quantification. This simple, low cost, and robust electrochemical assay could potentially benefit on-site monitoring applications.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 4. (A) CV responses of the aptasensor to cocaine at different concentrations: (a) 0, (b) 1, (c) 4, (d) 20, (e) 100, and (f) 500 μM in PB containing 1 mM HQ and 1 mM H2O2. (B) The calibration curve of the oxidation steady-state current changes to different concentrations of cocaine, N = 3.

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

using the oxidation steady-state current changes (Figure 4B). The current changes increased with the concentration of the added cocaine from 0 to 500 μM, which agreed with the principle that more target triggered more hairpin to form DNAzyme. The signal reached a plateau when 500 μM cocaine or higher concentration was used. It indicated that the saturation of the interaction between CocAD and cocaine

*E-mail: [email protected]. Tel: +86-10-62754112 Fax: +8610-62751708. *E-mail: [email protected]. Tel: +86-10-62754680 Fax: +86-1062754680. Notes

The authors declare no competing financial interest. 9381

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No 21275009 and 20805002) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, MOE. China.

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