DNA based signal amplified molecularly imprinted polymer electrochemical sensor for multiplex detection

Abstract

Molecularly imprinted polymers (MIPs) have been widely used as sensitive recognition elements in chemical/biological sensors. Motivated by the urgent need of amplified and multiplexed analysis, the unique advantages of molecular imprinting based electrochemical sensors (MIECSs), herein we first developed a DNA based signal amplified MIECS for multiplex detection of folic acid (FA), folate receptors (FRs), Hg²⁺ and DNA. With folic acid as the template molecule of the molecularly imprinted polymers, FA was detected directly. Due to the specific binding with FA, FRs could then be detected indirectly. Furthermore, with the specific effects of thymine–Hg²⁺–thymine or complementary DNA bases, by the cleavage of exonuclease III (Exo III) towards single strand DNA, Hg²⁺ and DNA were further detected in an amplified way through template molecule cyclic release from the DNA structure. The universal sensor gave satisfactory detection limits for folic acid, folate receptors, Hg²⁺ and DNA at 3 × 10⁻⁸ M, 0.3 ng mL⁻¹, 3.45 pM, 40 nM, respectively. It also demonstrated excellent regenerability, reproducibility, and stability. The proposed strategy may open the road for the development of DNA based multiple target MIP sensors by the combination of the unique characteristics of DNA and MIPs.

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1. Introduction

Biosensors have attracted considerable attention with a blooming development in the field of modern analytical chemistry due to their excellent sensitivities and specificities.¹ Molecular recognition is one of the three necessary components (the other two are signal transduction, and physical readout) in the design and fabrication of biosensors, especially for the development of clinical diagnostic tools and therapeutic modalities.² Initially, biosensor recognition elements were isolated from living systems.³ In recent years, numerous attempts have been made to replace biological receptors with synthetic counterparts as a recognition element in chemo/biosensors. Due to their highly selective recognition properties, easy synthesis and high mechanical/chemical stability,⁴ molecularly imprinted polymers (MIPs) have widely replaced natural receptors as sensitive recognition elements in the biosensors.⁵ Recently, molecularly imprinted electrochemical sensors (MIECS) were fabricated combining the advantages of MIPs and electrochemical measurement systems such as low cost, rapid response, simple instrumentation and operation of miniaturized and automated,^6–8 which have been applied in pharmaceutical analysis,⁹ environmental monitoring and assessment,^10,11 and residue detection of veterinary and pesticides drugs, etc.¹² As we know, in bioanalysis, signal amplification is an essential aspect for sensitive detection of biorecognition events.^13–15 However, although MIECS has showed enormous potential in various biological analysis, few of the previously reported MIECS involved signal amplification strategies, besides the amplification of nanoparticles which often involves unavoidable complicated synthetic process.¹⁶ In addition, multiplexed analysis of several targets is a further goal in biosensing.¹⁷ A universal biosensor capable of detecting different targets is highly desirable and attractive from the economic point of view because many analyses can be performed with the versatile platforms in a single system. Therefore, it is promising to develop novel signal amplified MIECS, esp. for multiplex detection.

Due to its programmability, nanoscale controllability, free of nanomaterials synthesis, biocompatibility and highly amplified efficiency, nucleic acid-based biosensing platforms for the amplified analysis of various analytes have attracted substantial research efforts in the past decades.^18–21 Recently, target DNA recycling as an isothermal signal amplification strategy has attracted considerable attention, because of its striking improvement for the detection sensitivity toward target analytes.^22,23 The target DNA cycling methods usually operated on various nucleases of endonuclease,^24,25 polymerase,²⁶ and exonuclease^27–30 for indirectly amplifying the amounts of target analytes. In DNA nicking endonuclease based signal amplification, by strong recognition for DNA sequences, the target can be reused through repeated cycles of hybridization, cleavage and dissociation, resulting in exponential amplification of the signal.^30–32 In addition, taking advantage of the nicking enzyme, reactions can be performed in an isothermal condition without specialized instrumentation, which holds the potential for routine analysis.³³ However, the signal amplified strategy of DNA cyclic amplification by nicking enzyme applied in MIECS has rarely been reported.

Herein, motivated by the urgent need of signal amplified multiplexed analysis, the unique advantages of molecular imprinting based electrochemical sensor and DNA based cyclic amplification, we first developed a novel MIECS with DNA based cyclic amplification for multiplex detection. Due to the interesting propose for the monitoring of folic acid (FA), we initially developed a molecularly imprinted electrochemical sensor for the assay of FA. The prepared MIPs can then detect the receptor of FA, Folate Receptor (FR), indirectly, because of the specific conjugation of FA and FR. Then based on the specific effects of complementary DNA bases or thymine–Hg²⁺–thymine and the cleavage of the exonuclease III (Exo III), DNA and Hg²⁺ were detected indirectly and amplifiedly through the template molecule released from the DNA structure. Because of MIPs, the sensors possess highly efficient recognition. Furthermore, not only the DNA recognition but also the DNA based signal amplified strategy have been employed in the system, which endows the biosensor with high sensitivity and selectivity besides the common advantages of the traditional MIECS possess.

2. Experimental

2.1. Materials and reagents

All HPLC-purified oligonucleotides were obtained from Sangon Biotechnology Co. Ltd (Shanghai, China). The sequence information of the probes and targets are as shown in Table S1.† The details of materials, reagents and apparatus used in this experiment were displayed in ESI.†

2.2. Fabrication of molecularly imprinted electrode

Preparation of imprinted electrode was according to the literature.³⁴ Briefly, for the preparation of MIPs, each 1.5 mL 40 mM o-phenylenediamine (o-PD, ≥98%), 10 mM poly(N-isopropylacrylamide), amine terminated (PNIPAAm, average M_n = 2500), 10 mM N,N′-methylenebisacrylamide (MBA, ≥98%) and 0.8 mM folic acid (FA) were mixed and dissolved in acetate buffer (0.5 M, pH 5.8) in the electrolytic cell. The resulted solution was deoxygenated by nitrogen gas bubbling for 20 min. The electrochemical pretreated electrode (ESI†) was then immersed in the deoxygenated polymerization solution. Cyclic voltammetry (CV) in 0.0 V to 1.0 V potential range were then performed for 20 cycles at a scanning rate of 0.05 V s⁻¹. Differential pulse voltammetry (DPV) was applied to quantify each concentration of test analyte over a potential range of 0.0 V to 0.6 V at a scan rate of 50 mV s⁻¹ and pulse amplitude of 25 mV. After electrodeposition, the polymers modified gold electrode (denoted as polymers/GE) was washed with deionized water and dried at room temperature for 2 h. To remove the template molecules entrapped in the polymeric matrix, the polymers modified electrodes were rinsed with the methanol–acetic acid (9 : 1, v/v) solution for 20 min at 50 °C followed by subsequent washed with methanol three times, to obtain a molecularly imprinted gold electrode (MIPs/GE). As a control, a modified gold electrode, which was regarded as the non-molecularly imprinted polymers modified gold electrode (NIPs/GE), was prepared under exactly the same procedure but without the addition of the template molecule.

2.3. Detection of FR, Hg²⁺ and target DNA with the MIPs/GE

For the detection of FR, a series of different concentrations of FR was added into 500 μL of 0.05 M Tris–HCl buffer (pH 7.4) solution containing 10 μM FA in a centrifuge tube. After the incubation of the mixture at 37 °C for 1 h in the dark, FR assay solution was obtained. The MIPs/GE was incubated in the FR assay solution for the detection of FR via rebinding the remaining FA to the imprinted cavities.

For the detection of Hg²⁺, first, folate acid-linked DNA probe 1 (detailed sequence are in the ESI†) (FA-P1) was prepared as follows. The coupling reactions for probe 1 with FA adopted the succinimide coupling (EDC-NHS) method^35–38 to form amides between the carboxyl groups of FA and the primary amine groups of P1. Briefly, 250 μL folate (10.0 mM) were mixed in 250 μL 0.05 M Tris–HCl buffer (pH 7.4) containing 5.0 mM sulfo-NHS and 1.0 mM EDC and then incubated at room temperature for 30 min in the dark. Afterwards, 0.5 mL probe 1 solution (10 μM) was added and incubated for another 2 h in the dark. At last, the mixed solution (FA-P1) was dialyzed using a membrane with molecular weight cutoff of 1000 Da against phosphate buffer solution (PBS, pH 7.4) for the removal of excessive folate acid. For the assay of Hg²⁺, 20 μL Hg²⁺ with varying concentrations was added into the obtained FA-P1 solutions in a centrifuge tube and incubated for 40 min at 37 °C. Then by the addition of 200 U mL⁻¹ Exo III individually, which was widely recognized as an enzyme with a high exodeoxyribonuclease activity on DNA duplexes in the direction from 3′ to 5′ end and a limited activity for DNA duplexes with a protruding 3′ end or single stranded DNA, the aforementioned mixture solutions were hydrolyzed for ∼30 min and FA was released from the FA-P1.^37,38 The resulting solution was denoted as Hg²⁺ assay solution. Then, the MIPs/GE was immersed in Hg²⁺ assay solution for the detection.

Similar procedures are followed for the assay of target DNA. At first, the coupling reactions for probe 2 with FA to form FA-P2 adopted a method similar to that described earlier for the FA-P1. Subsequently, 20 μL of varying concentrations of target DNA was added into the centrifuge tube containing the previously obtained FA-P2 solutions with sufficient hybridization for 40 min at 37 °C. Afterwards, 200 U mL⁻¹ Exo III was added to the centrifuge tube; the resulting solution was named as target DNA assay solution.

3. Results and discussion

3.1. Sensor fabrication strategy

The fabrication and assay procedure of the universal electrochemical sensing platform based on molecularly imprinted polymers and DNA based cyclic amplification is depicted in Scheme 1. Part I of Scheme 1 is the traditional preparation of MIPs modified electrode and its direct detection of FA. When the obtained MIPs/GE was immersed in the FA-containing solution, the imprinted sites of the MIPs/GE were blocked by FA rebinded in the imprinted cavities, which impeded the electron transfer of the probe, along with the drop of electrochemical signal of the MIPs/GE. Thus the sensor could be applied to analyze FA directly as expected. Then the MIPs electrochemical sensor was developed for the indirect detection of folate receptor, Hg²⁺ and DNA, as shown in the part II of Scheme 1. As shown in section A, in the presence of FR, the specific FA–FR interaction in a 1 : 1 ratio with strong affinity,³⁹ resulted in the decrease in the amount of the free FA in the assay solution. When the MIPs/GE was immersed in the reacted solution, FA in FA–FR complex was prevented from rebinding in the imprinted cavities of the prepared MIPs/GE. The amount of FR is closely related with the number of cavities or the electron tunnels corresponding to the electrochemical signal. Thus the as-prepared MIPs allow us to indirectly detect FR.

Scheme 1 Fabrication process of the electrochemical sensor based on MIPs/GE for the determination of FA, FR, Hg²⁺, and target DNA.

For the detection of Hg²⁺, one T-rich DNA sequence with FA modified on 3′ end (probe 1) was designed first. As shown in section B, in the presence of Hg²⁺, because of its interaction with the T bases, T–Hg²⁺–T base pairs would be formed,^37,38 folding the free folate-linked T-rich probe into a duplex structure. This conformational switch converted the T-rich probe into an Exo III-preferred substrate and therefore activated the enzyme, resulting in the stepwise removal of mononucleotides from the 3′ terminus of the T-rich probe and the deforming of duplex structure. Ultimately, the FA tag was released into the Hg²⁺ assay solution. The releasing FA would rebind in the imprinted cavities when the MIPs/GE was incubated in the solution, resulting in the decrease of the number of the electron tunnels and the electrochemical response. Moreover, because Hg²⁺ could be released from the T–Hg²⁺–T base pair during the Exo III-assisted digestion process,⁴⁰ a new T-rich probe would be folded, starting a new cycle of digestion. In this way, Hg²⁺ could cyclically shuffle between the substrate solution and the T-rich probes, serving as a “catalytic trigger”, which can amplify the released FA amount by cyclic digestion of a large amount of T-rich probes.

If the probe was designed as the complementary sequence with another target DNA, for example, a selected human immunodeficiency virus (HIV) DNA sequence, the proposed method could be a parallel DNA sensor shown as section C. Briefly, with the DNA complementary base pairs, probe 2 formed a duplex structure by the hybridization with target DNA sequences. In the presence of Exo III, the obtained duplex probe was digested from the 3′ terminus with the stepwise removal of mononucleotides, resulting in the release of linked FA and target DNA into the assay solution. Similarly, it also can trigger the cyclically amplified determination of target DNA.

3.2. Characterization of the MIPs/GE

In this paper, the electrochemical sensors were based on molecularly imprinted polymers electrode. Cyclic voltammogram of electropolymerisation for preparation of MIPs/GE was shown as Fig. S1† and the morphology characterizations of MIPs/GE or NIPs/GE were investigated from the scanning electron microscopy (SEM) images shown as Fig. 1. The SEM of NIPs/GE shows smooth surface (Fig. 1B). However, a layer of rough film was formed on the MIPs/GE (Fig. 1A), which could be ascribed to the electropolymerization of polymers in the presence of FA. Considering EIS is a powerful tool for probing the features of a surface-modified electrode, EIS measurements were also used to characterize the surface change of the modified electrode (Fig. S2†). According to the EIS results shown in Fig. S2,† the modification of the MIPs/GE was successfully performed and the prepared MIPs/GE had the capacity to selectively adsorb template FA.

Fig. 1 SEM images of MIPs/GE (A) and NIPs/GE (B).

CV (Fig. 2A) and DPV (Fig. 2B) were also performed to further investigate the change in electrochemical response of the polymer modified GE, and MIPs/GE before and after template removal. There is almost no redox peak after the deposition of the polymers onto the GE surface in solutions containing the [Fe(CN)₆]^3−/4− probe (curve a of Fig. 2A and B). This may be due to the poor conductivity of the polymers. However, an obviously intense quasi-reversible redox response records on the MIPs/GE (curve b of Fig. 2A and B). After the incubation of the MIPs/GE (c of Fig. 2A and B) in phosphate buffer solution (PBS) containing 8 × 10⁻⁶ M FA solution, the current decreases significantly, implying that the rebinding FA blocked the diffusion of [Fe(CN)₆]^3−/4−.

Fig. 2 (A) Cyclic voltammograms and (B) differential pulse voltammetry in 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1 : 1) and 0.1 M KCl on the polymers/GE (a), the MIPs/GE before (b) and after (c) incubating in PBS (pH = 7.4) containing 8 × 10⁻⁶ M FA solution.

3.3. Detection of FA and FR

Based on the current response of [Fe(CN)₆]^3−/4− on the MIPs/GE and under the discussed optimized conditions (Fig. S3†), the sensitivity of the sensor towards FA was investigated (Fig. S4†). As shown in Fig. S4A,† the current response of [Fe(CN)₆]^3−/4− on the MIPs/GE decreased accordingly with the increase in the concentration of template FA, which could be ascribed to the increasing number of imprinted sites in the film on the MIPs/GE occupied by the rebinding FA molecules. A linear relationship between the anodic peak current and the logarithm of FA concentration was obtained covering the concentration range from 0 M to 4 × 10⁻⁴ M (Fig. S4B†); the linear regression equation was I (μA) = −7.2070 × 10⁻⁶ − 2.3144 × 10⁻⁶ lg c, with a correlation coefficient of 0.9934 and a detection limit of 3 × 10⁻⁸ M. To investigate the selectivity of the MIPs sensor for FA assay, the MIPs sensor was incubated in 500 μL Tris–HCl buffer (pH 7.4) solutions containing 4 × 10⁻⁴ M of different potential interfering substances for 30 min, (i.e. vitamins B₁, B₂, B₆, vitamin C, tryptophan, cysteine and histidine) using the same method. The specificity of the sensor for the detection of FA was also studied as exhibited in Fig. S4C.† It is observed that the different potential interfering substances had no noteworthy effect on the current response. This indicates that the proposed approach had a good selectivity for the determination of folic acid. In addition, the obtained sensor was simply rinsed with room temperature water for 30 s to test its regenerability. As shown in Fig. S4D,† the initial DPV current of [Fe(CN)₆]^3−/4− on the MIPs/GE was regarded as 100%. When the MIPs/GE was incubated in FA solution (8 × 10⁻⁶ M), the current response decreased to 52%. However, with the extraction of FA, MIPs/GE was regenerated again, and the DPV current of [Fe(CN)₆]^3−/4− increased to 86%. In spite of the minor loss of current, it also shows nice regeneration of the sensor. Furthermore, we have studied 8 group imprinted sensors to test the reproducibility of the proposed sensor, in which each group was prepared in the same condition. The RSD% of the response of 8 different sensors was less than 6%. At the same time, the stability of the sensor was examined by recording the remaining amount of signal response after successive cycling of the MIPs/GE incubated in FA solution (8 × 10⁻⁶ M) for 10 cycles. As shown in the insert of Fig. S4D,† the result was an average from 3 different sensors. It was also noteworthy that the response of [Fe(CN)₆]^3−/4− was slightly reduced, highlighting the sensor's satisfactory reproducibility, reusability and stability. Since our assay worked very well for FA, it would also be applied for its receptor, FR, a glycosylphophatidylinositol-linked membrane glycoprotein, which is a type of tumor-associated antigen and over-expressed in many human tumors, including ovarian carcinomas, choroid plexus carcinomas, and ependymomas,⁴¹ esp. over expressed in a relatively high percentage in breast cancer patients.⁴² The high affinity between FR and FA allows them work as a bridge for the detection of FR with FA conjugation methods. Prior to the detection of FR, the effect of the specifically binding time of FR with FA on the current response was studied (Fig. S3†). Then an indirect detection of FR by the MIPs/GE was studied. Fig. 3A gave the DPV response of MIPs/GE as varying concentrations of FR in [Fe(CN)₆]^3−/4− solutions. It is observed that DPV peak currents increase in response to the concentrations of FR which is in accordance with what we anticipated. With the concentration of FR increasing, more FR was conjugated with FA resulting in less remaining FA in the assay solution and bringing more electron tunnels. The linear increasing range was 1.0–20.0 ng mL⁻¹ (Fig. 3B) and detection limit was 0.3 ng mL⁻¹. We also studied the specificity of the sensor for the detection of FR, as exhibited in Fig. 3C. The representative common and potential interference proteins (including BHb, CVH, BSA, LZM, HRP, Th, CEA, and IgG at 0.1 mg mL⁻¹ each) were replaced 20 ng mL⁻¹ FR, under the same experimental conditions to compare the current response in the investigation of the specificity. It is obvious that the presence of the common interference proteins have no significant effect on the current intensity, illustrating that the proposed approach has high selectivity. The stability and regenerability of the sensor for the detection of FR were also studied by incubating the MIPs/GE in 500 μL of 0.05 M Tris–HCl buffer (pH 7.4) solution containing the mixed reaction solution of 10 μM FA and 10 ng mL⁻¹ FR. Following a successive cycling of the sensor for 10 cycles, the signal response slightly reduced. The current response decreased to about 78% relative to the 100% initial DPV current. After the extraction of FA, MIPs/GE was regenerated, and the DPV current increased to 94%. This demonstrates a satisfactory stability and regeneration of the sensor. The RSD% of the response of 8 different imprinted sensors was less than 6.31%, showing a satisfying reproducibility of the proposed sensor.

Fig. 3 (A) DPV of FR: (a–h) 0, 0.1, 0.5, 1.0, 3.0, 6.0, 10; 20 ng mL⁻¹ in Tris–HCl buffer, (B) corresponding calibration curve of current vs. the concentration of FR, and (C) selectivity of FR analysis in Tris–HCl buffer (20 ng mL⁻¹ FR or 0.1 mg mL⁻¹ other substances as example).

3.4. Detection of Hg²⁺

Mercury was one of the most well-known heavy metal ions toxicants in the world. The exposure to mercury even at very low concentration would result in digestive, kidney, and especially neurological diseases.^43,44 Therefore, the probe of Hg²⁺ ions was a focus of many research endeavors. Among the methods for the detection of Hg²⁺, the Hg²⁺ sensors based on thymine–Hg²⁺–thymine (T–Hg²⁺–T) have attracted intense attention in recent years due to the unique binding specificity and stability of T–Hg²⁺–T coordination chemistry.^45,46 Here, based on the T–Hg²⁺–T, the FA labeled DNA was switched to a duplex structure and by the digestion of Exo III, the duplex could be deformed releasing FA tag for the detection of the amount of Hg²⁺ indirectly. We used agarose gel electrophoresis to confirm the degradation process of DNA probe by Exo III (Fig. S5†). The results proved that degradation of the duplex DNA by Exo III had occurred because the digested DNA probes would migrate out of the gel, which provided immediate evidence for the postulated mechanism of our Exo III-mediated conformational switch converted the T-rich probe for assay. To achieve an optimal electrochemical signal, the effect of incubation time between the sensor and Hg²⁺, and the effects of Exo III concentration and digestion time on the electrochemical responses were conducted (Fig. S3†).

Then, the indirect Hg²⁺ assay based on the MIPs/GE strategy was investigated using DPV. As exhibited in Fig. 4A, the DPV response of MIPs electrode towards [Fe(CN)₆]^3−/4− decreased dynamically with the increasing concentrations of Hg²⁺ between 10⁻¹⁰ M and 10⁻⁴ M. The resulting calibration curve was depicted in Fig. 4B with a detection limit of 3.45 pM, which was evaluated using a signal three-fold the background noise. To our knowledge, such a low detection limit, which should be ascribed to the Exo III-assisted autocatalytic target recycling, the specific T–Hg²⁺–T coordination chemistry^47,48 and the high recognition effect of MIPs, is lower than the toxic level of Hg²⁺, as defined by the United States Environmental Protection Agency (USEPA) in drinkable water (10 nM) and better than that of other previous electrochemical Hg²⁺ sensors (shown in Table S2†). This demonstrates that the sensor may hold a great promise for environmental monitoring of mercury exposure. Thus, a signal-amplified and immobilization-free electrochemical Hg²⁺ sensor platform with ultrahigh sensitivity and simple operation was presented. As other natural or synthetic bases selectively bind other metal ions (e.g., Ag⁺ by cytosine), alternative sensing devices may be envisaged.

Fig. 4 (A) DPV of Hg²⁺: (a–g) 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴ M in Tris–HCl buffer, (B) corresponding calibration curve of current vs. the concentration of Hg²⁺, and (C) selectivity of Hg²⁺ analysis in Tris–HCl buffer (10⁻⁴ M Hg²⁺ or equal concentration of other ions as example).

Besides the detection sensitivity, selectivity is another critical factor to evaluate the performance of the proposed sensing system. The selectivity of the sensor was studied by substituting Hg²⁺ with various metal ions, such as Ca²⁺, Mg²⁺, Mn²⁺, Pb²⁺, Fe²⁺, Cu²⁺, Co²⁺, Zn²⁺, Ni²⁺, Ag⁺, K⁺, and Na⁺, each at 10⁻⁴ M. As exhibited in Fig. 4C, the resulting MIPs/GE shows remarkable current change for Hg²⁺, and the current hardly changes towards other metal ions. This indicates that the electrochemical sensor has a promising specificity toward the indirect detection of Hg²⁺. Similarly, the stability and regenerability of the sensor for the detection of Hg²⁺ were also studied. At first, the MIPs/GE was incubated in 5 μM Hg²⁺ assay solution. The current response decreases to about 52% relative to the 100% initial DPV current. After the extraction of FA, MIPs/GE was regenerated, and the DPV current increased to 84%. This demonstrates a satisfactory stability and regeneration of the sensor. The RSD% of the response of 8 different imprinted sensors was less than 6.52% showing a satisfactory reproducibility of the proposed sensor.

In addition, the practicality of the proposed strategy was also investigated by the detection of Hg²⁺ in the processed river samples and the detection procedure was identical to that described in the aforementioned experiment for Hg²⁺ detection in clean Tris–HCl buffer solution. As shown in Table S3,† the results are in good agreement with the found values.

3.5. Detection of DNA

The highly sensitive and selective detection of nucleic acid is a current focus of research in the field of clinical diagnostics and gene therapy.⁴⁹ The system was further extended to the indirect detection of DNA. As shown in Fig. 5A, the electrochemical current decreases when the target DNA concentration increases in the range of 0 μM to 5 μM. The resulting calibration curve is outlined in Fig. 5B with an achieved detection limit of 40 nM. In addition, the important aspect related to the specificity of the sensing platform and the possibility to discriminate bases mismatches, was studied. Fig. 5C shows the relative current response of the sensor with two different mismatched targets DNA (i.e. Ta and Tb) replacing target DNA. As the resulting MIPs/GE shows remarkable current change for target DNA, but no significant change for the two different mismatched targets DNA, the single-base-mismatched DNA strands could be directly discriminated from the perfectly complementary targets, indicating the remarkably high specificity of this assay.

Fig. 5 (A) DPV of target DNA: (a–g) 0.1, 0.25, 0.5, 1.0, 1.5, 2.5, 5.0 μM in Tris–HCl buffer, (B) corresponding calibration curve of current vs. the concentration of target DNA, and (C) selectivity of target DNA analysis in Tris–HCl buffer (5 μM target DNA or equal concentration of other sequences as example).

Similarly, the stability and regenerability of the sensor for the detection of target DNA were also studied. When the MIPs/GE was incubated in 5 μM target DNA assay solution. The current response decreases to about 53.3% relative to the 100% initial DPV current. After the extraction of FA, MIPs/GE was regenerated, and the DPV current increases to 83%. This demonstrates a satisfactory stability and regeneration of the sensor. The RSD% of the response of 8 different imprinted sensors is less than 6.71% showing a satisfactory reproducibility of the proposed sensor. In addition, the practicality of the proposed strategy was also investigated by the detection of target DNA in 100-fold diluted human serum samples and the detection procedure was the same as that described in the aforementioned experiment for target DNA detection in clean Tris–HCl buffer solution. From Table S4,† although the serum samples were complex, the detection results obtained are satisfying, suggesting that this strategy could be applied to the detection in complex samples.

4. Conclusions

In summary, the proposed electrochemical sensor was fabricated based on molecularly imprinted polymers electrode and DNA based cyclic amplification for multiplexed detection of folic acid, folate receptor, Hg²⁺ and DNA. It is an ingenious strategy that combines classical MIPs electrochemical biosensoring with programmable unique DNA structure. In addition, the electrochemical sensor demonstrates excellent regenerability, reproducibility and stability owing to the advantages of MIPs. We hope this mode may open up the combination of MIPs with DNA based cyclic amplification for other various analytes biosensors, such as the recognition of aptamer-protein.

Acknowledgements

This work was financially supported by the projects from Natural Science Foundation of Anhui (1408085QB41, 1408085MB40), the Natural Science Research Project of Education Department of Anhui Province (KJ2013A247, KJ2016A888), National Natural Science Foundation of China (21271136), the funded project of Anhui Province Cultivate Outstanding Talent (2014SQR01), the Opening Project of Anhui Key Laboratory of Spin Electron and Nanomaterials (2013YKF20, 2011YKF03), the Program of Innovative Research Team of Anhui Provincial Education Department (Photoelectric information material new energy device), the Program of Innovative Research Team of Suzhou University (2013kytd02), and the College Students' Innovative Entrepreneurial Training Plan Program of Anhui Province (201510379133, 201410379067).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra05797g

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