One-Step Modification of Electrode Surface for Ultrasensitive and

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One-step modification of electrode surface for ultrasensitive and highly selective detection of nucleic acids with practical applications Chao Li, Dan Wu, Xiaolu Hu, Yang Xiang, Yongqian Shu, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01250 • Publication Date (Web): 04 Jul 2016 Downloaded from http://pubs.acs.org on July 4, 2016

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Analytical Chemistry

One-step modification of electrode surface for ultrasensitive and highly selective detection of nucleic acids with practical applications Chao Lia, Dan Wua, Xiaolu Hua, Yang Xianga*, Yongqian Shub*, Genxi Li a, c * a State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China b Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, P. R. China c Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai, 200444, China

Abstract Electrochemistry-based nucleic acid sensors have long been plagued by the limited accessibility of target molecules to the capture probes immobilized on heterogeneous surfaces, which largely hinders their practical application. In this work, we find that dual-thiolated hairpin DNA immobilized on an electrode surface as the capture probe can not only efficiently bind with target molecule as well as the signal probe, but also process impressive protein-repelling ability, which allows us to directly detect as few as attomolar targets (∼ 300 copies in 100 μL sample) with single-base discrimination ability. Meanwhile, the preparation of functional electrode surface becomes simple (one step), fast (30 min) and homogeneous (just one probe modified surface without small molecules co-assembled). These advantages are attributed to the unique probe design, where the stem of the capture probe can act as rigid scaffold to keep it upright, and the loop of the capture probe may provide an enclosed platform for target and signal probe binding. More importantly, through tuning the distance between enzyme and the electrode surface (from 8.5 to 13.6 nm), we find that the performance of the sensor can be favorably controlled. Furthermore, taking advantage of this new binding model, different complex samples including polymerase chain reaction (PCR) product, messenger RNA, and micro RNA can be conveniently analyzed, which may hold great potential for real application. Keywords：：electrochemical sensor; homogenous surface; nucleic acid; stem loop

* E-mail address: [email protected] (Y. Xiang), [email protected] (Y. Shu), and [email protected] (G. Li), Tel: +86 25 83593596. Fax: +86 25 83592510. 1

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1. Introduction Electrochemical nucleic acid (E-NA) sensor has attracted particular attentions owing to the fact that it can provide a simple, portable, and economical platform for nucleic acids sensing. So far, a variety of strategies have been used to improve the performance of E-NA sensors, for instance, employing enzyme,1-4 nanomaterial, 5-8 and DNA nanostructure; 9-11 however, the core of E-NA sensors still lies in the surface-confined capture probe for recognition of their complementary target sequences by hybridization. The immobilization of capture probe onto an electrode surface is still mainly dominated by a two-step assembly strategy, 12-15 which involves self-assembly of thiolated single-stranded DNA (ssDNA) probe on Au surface via thiol-gold chemistry, and passivation with an alkanethiol (e.g., mercaptohexanol, MCH) to backfill unoccupied space and help the probe adopt an upright orientation on the surface. Certainly, such two-component self-assembly monolayer (SAM) may not only largely remove nonspecifically adsorbed DNA but also favor target hybridization, thus pushing detection limits down to picomolar level. Despite these advantages, some long-standing hurdles still remain, which has limited their further application in real sample analysis. First, it is still hard to precisely control the orientation, distribution and packing density of the capture probe,16-18 leading to inevitable interstrand interactions, nonspecific protein adsorption, and steric hindrance on these heterogeneous surfaces.19, 20 Therefore, one often obtains irreproducible detecting signals in practical application. What’s more, the two-step sequential adsorption protocol often takes a long time (several hours) to prepare functional electrodes. Second, large portions of reported E-NA sensors that employ “sandwich-type” assays have nice performances for detecting longer target sequences (> 24 nucleotides, nt);21, 22 however, the short length sequences such as micro RNAs (miRNAs) cannot be efficiently analyzed due to their low melting temperature (Tm). Finally, only a few reports present a whole analytical ability for detection of different kinds of nucleic acids such as genome DNA, messenger RNA (mRNA), and miRNA, etc.23-25 Therefore, better methods have to be developed to meet the requirement of routine analysis. Hairpin structured DNA probe (HSP) has been widely employed as recognition element for E-NA sensor construction.26-28 Typically, target hybridization induced conformation switch of the HSP has become a paradigm for the design of HSP-based E-NA sensor. On the other hand, it has been known that there is different persistence length for ssDNA (∼ 1 nm) and double-stranded DNA (dsDNA, ∼ 50 nm).29 So, HSP may be a chimera with both rigidity stem and flexible loop. Therefore, we propose that re-consideration and more rational utilization of HSP may provide a solution to solve currently existing problems as to develop better E-NA sensors. In this paper, we report a novel approach for constructing an electrochemical sensor that provides a package solution for nucleic acids detection, where a dual-thiol modified hairpin DNA (dSH-HSP) is used to serve as capture probe by forming a Y-shaped DNA duplex upon target binding. With the utilization of electrochemistry and surface plasmon resonance (SPR), we demonstrate for the first 2


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time that this single DNA probe modified surface without co-assembled “helper” molecules can not only efficiently capture target molecules but also exhibit impressive ability to suppress background signal. Meanwhile, we are able to preciously control the distance between the enzyme molecules and electrode surface by adjusting the stem region of dSH-HSP and find it in direct connection with the performance of sensor. Overall, this one-step modification process is particularly suitable for constructing highly reproducible interfaces and allows us to detect kinds of targets in real samples with attomolar sensitivity, which displays the unparalleled ability of E-NA sensor in practical application. 2. Experimental section 2.1 Reagents and Materials. All oligonucleotides (Table S1 of the Supporting Information) were synthesized and purified by Sangon Inc. (Shanghai, China). Ethylenediaminetetraacetic acid (EDTA), mercaptohexanol (MCH), hexaammineruthenium(III) chloride ([Ru(NH3)6]3+), bovine serum albumin, horseradish peroxidase (HRP), tween-20, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and streptavidin-peroxidase polymer were obtained from Sigma. TMB (3,3′,5,5′ tetramethylbenzidine) was purchased from Neogen (Lexington, KY) in the format of a ready-to-use reagent (enhanced K-blue substrate, H2O2 included). The synthesis and modification of gold nanoparticle were according to our previous report.30 The buffer solutions used in this study were as follows: The DNA immobilization buffer contained 10 mM Tris-HCl, 1 mM EDTA, and 100 mM NaCl (pH 7.4). The hybridization buffer was a 10 mM PBS solution containing 1 M NaCl (pH 7.4). Buffers for both electrochemistry and electrode washing were 10 mM PBS with 0.1 M NaCl and 0.05% tween-20. The running buffer was a 10 mM PBS with 100 mM NaCl (pH 8.0). All solutions were prepared with Milli-Q water (18 MΩ cm) from a Millipore system (RNase-free water treated with 0.1% DEPC for RNA detection). 2.2 Synthetic DNA detection. Surface plasmon resonance (SPR) experiments were performed using a four-channel BIAcore X100 instrument (Uppsala, Sweden). All experiments were performed on bare gold chips obtained from BIAcore. For preparation of the kinds of biosensor surface, 1 μM dSH-HSP, 1 μM single strand DNA (ssDNA), 1 μM single thiol group modified HSP (sSH-HSP), 1 mM MCH in running buffer was automatically injected onto an untreated gold surface sensor chips (Sensor Chip Au, GE Healthcare Inc.) to the saturated density in different flow cell, while the heterogeneous surface of ssDNA + MCH was obtained by immersing the nonmodified gold chip into ssDNA (1 μM) for 3 h. The sample was rinsed three times with running buffer and once with nanopure water. Subsequently, the DNA-functionalized chip was in 1 mM MCH solution for 1h. After washing three times with nanopure water, the chip was dried with a stream of nitrogen. The protein was diluted in running buffer. 3



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The dynamic light scattering (DLS) measurements were collected using a Malvern Nano ZSP system equipped with a red laser (633 nm, He-Ne) and a scatting angle of 90° (fixed without changing possibility). Electrochemical measurements were made using an electrochemical analyzer (CHI 660d, CH Instruments, Austin, TX) with connecting to a conventional Au disk working electrode (3 mm diameter), a Ag/AgCl reference electrode, and a Pt wire auxiliary electrode. Cyclic voltammetry was carried out at a scan rate of 100 mV/s. The chronocoulometric plots (charge vs square root of time) of the DNA recognition surface were obtained in 10 mM Tris-HCl (pH 7.4) buffer containing 50 μM [Ru(NH3)6]3+. Amperometric detection was fixed at -100 mV and the electrocatalytic reduction current was measured at 100 s after the HRP redox reaction reached the steady state. Electrochemical impedance spectra were recorded in 5 mM [Fe(CN)6]3−/4 − with 1 M KNO3. The experimental parameters were as follows: bias potential, 0.225 V; amplitude, 5 mV; frequency range, 0.1-10 kHz. Gold electrodes (3 mm in diameter) were cleaned following the reported protocol. 31 In parallel, dSH-HSP solution (1 μM) was reduced in 10 mM TCEP for 1 h to cleave disulfide bonds. After that, 5 μL of dSH-HSP were dropped to the cleaned gold electrode and incubated 30 min at room temperature. The resulting electrodes were rinsed with washing buffer and dried lightly with N2 before hybridization. Of note, single thiol groups labeled hairpin structured probes solution (sSH-HSP) undergone the same modification process. As for two-component modification, 5 μL of SH-ssDNA probes were first dropped to the cleaned gold electrodes and incubated 2 h at room temperature and then the electrodes were immersed in the MCH solution (1 mM) for 2 h. The resulting electrodes were rinsed with washing buffer and dried lightly with N2 before hybridization. 50 μL of different concentrations of the DNA target were mixed with the biotinylated signal probe (50 μL, 100 nM) in the hybridization buffer. The electrode was incubated in the 100 μL solution in the 2-mL microcentrifuge tube for hybridization. After 40 min incubation at 37 °C, the electrode was rinsed with washing buffer and then incubated with 4 μL of streptavidin-peroxidase polymer (1 μg mL-1) for 15 min at room temperature. The sensor was then thoroughly rinsed with washing buffer and subjected to electrochemical measurements. For regeneration, the electrodes were treated by rinsing with Milli-Q filtered water for ∼3 min, followed by incubation in PBS buffer for ∼1-2 min to allow for the reformation of the stem-loop structure. 2.3 PCR product detection. E.coli genomic DNA was isolated by using a Bacteria Genomic DNA Isolation Kit (SK8725, Sangon, Shanghai) and was amplified using a Bio-Rad PCR cycler (PTC-100). A pair of asymmetric primers (primer 1/primer 2 = 100:1) was employed in order to generate the ssDNA target.32 The amplification protocol consisted of 2 min at 95 °C followed by 30 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s. Prior to the detection, the PCR products were diluted to 10 times with hybridization buffer. As for electrochemical detection, 5 μL of capture probe (middle) was first dropped to the 4


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cleaned gold electrode and incubated 0.5 h at room temperature. After that, the electrode was washed with buffer and dried lightly with N2. Then, 50 μL of diluted PCR product was mixed with the biotinylated signal probe (50 μL, 100 nM) in the hybridization buffer. The electrode was incubated in the 100 μL solution in the 2-mL microcentrifuge tube for hybridization. After 40 min incubation at 37 °C, the electrode was incubated with 4 μL of streptavidin-peroxidase polymer (1 μg mL-1) for 15 min at room temperature. The sensor was then thoroughly rinsed with washing buffer and subjected to electrochemical measurements. 2.4 RNA detection. Total RNA was isolated from cells using TRIzol purification kit (Invitrogen, America). Prior to the detection, the total RNA samples were diluted with hybridization buffer. For mRNA detection, reacting time is extended to 1.5 h for ensuring efficient hybridization. Others were similar experimental procedure as described above. One-array chip analysis for microRNA was performed by OE biotech Inc. (Shanghai, China).

3. Result and discussion 3.1 Sensor design As shown in Scheme 1, a synthetic DNA strand (18 nt) is firstly used for a proof-of-concept detection. Initially, dSH-HSP was robustly coupled onto gold substrates using Au-S chemistry. The signal DNA that was partially complementary with the loop of capture probe (7 base pairs, bp) could not bind to the electrode surface because the Tm (16.3 °C, calculated by Mfold) was below the operating temperature (i.e., 37 °C).33 However, once introduction of the target, perfect complementary Y-shaped structure was formed on the loop of the capture probe because the Tm was above the working temperature. Consequently, streptavidin-labeled horseradish peroxidases (SA-HRP), an enzyme catalyzing thousands of reduction reactions of H2O2, could be specifically connected with the biotin modified signal probes via streptavidin-biotin affinity binding, leading to significantly amplification of the electrochemical current signal in the presence of the co-substrate, 3, 3’, 5, 5’ tetramethylbenzidine (TMB). 3.2 Modification characteristics. The probe adsorption process is firstly monitored in real time by a surface-sensitive method, surface plasmon resonance (SPR). Fig. 1a shows that most of dSH-HSPs can adsorb onto the gold surface within 10 min, which are much faster than SH-ssDNA. In addition, nonthiolated HSP exhibits much less nonspecific adsorption (∼ 12.3 times) compared with nonthiolated ssDNA (inset of Fig. 1a). Also of note, SPR provides a surface density estimate of 3.3 × 1012 cm-2 for dSH-HSP, and the surface coverage of SH-ssDNA is approximately 7.4 × 1012 cm-2. These differences may be attributed to the higher molecular rigidity and fewer exposed bases of the 5



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dSH-HSP (Fig. S1). Electrochemistry itself is easy to perform and sensitive to changes in the surface properties. Therefore, we have used cyclic voltammetry (CV) to investigate the electron transfer reactivity of TMB at the dSH-HSP modified surfaces (Fig. 1b). We find that two pairs of well-defined redox peaks in the CV, indicating that TMB can easily penetrate the SAM and facilely mediate electron exchange with the underlying electrode. In addition to CV, electrochemical impedance spectroscopy (EIS) as a highly sensitive method to provide quantitative information about the surface properties through charge transfer resistance (Rct) has also been employed to characterize the electrode surface. Fig. 1c depicts the Rct values obtained from 20 independent tests about the three kinds of probes immobilized electrodes, indicating a highly reproducible surface obtained by dSH-HSP modification. 3.3 Electrochemical characteristics. Electrophoresis results and different amperometric responses have been studied to demonstrate the formation of Y-shaped structure and the feasibility of the proposed method (Fig. 2a). Then, just proofing our hypothesis, we find that MCH passivation is an essential step for the fabrication of ssDNA probe-based sensors, while the HSP modified surface without occupation by small molecules can also obtain comparably large amperometric signal (Fig. 2b). We have also compared the signal responses obtained at electrode immobilized with single thiol group modified HSP (sSH-HSP) or dSH-HSP, and the results are shown in Fig. S3. Although both of them work well at a 5 nM concentration of target, the suppression of background with sSH-HSP surface is far behind the dSH-HSP modified surface. This is attributed to the limited rigidity of sSH-HSP which tolerates the adsorption of HRP to the gold surface. Using an assay based on Ru(NH3)63+ adsorption,34 hybridization efficiency can be determined, and the results indicate that dSH-HSP based surface can dramatically improve the efficiency and provide better access for incoming target molecules (Fig. S4). These effects are attributed to the unique interfacial probe design that the stem of the probe can act as rigid scaffold to keep it upright, and the loop provides an enclosed platform for target and signal probe binding, which efficiently avoids the interstrand entanglement, probe-to-target steric hindrance, and the interaction between nitrogen atoms of bases and the Au surface. The influence of the distance between enzyme molecules and the electrode surface has been hardly studied previously, because it is hard to preciously control the orientation of hybrid probes. However, it becomes easy to study the factor in this assay through prolonging the stem region of the dSH-HSP. As shown in Fig. 2c, increasing the stem from 5 bp to 15 bp (the distance between enzyme and electrode surface is from 8.5 nm to 11.9 nm), a nearly 2.9-fold signal enhancement is observed, but further increment of the distance will decrease the electrochemical signal. This indicates that proper space supported by DNA probes, matching the size of HRP molecule (40.04 × 66.81 × 116.36 Å), is benefit to enzyme-based signal amplification. On the other hand, the increase of the background signal may be due to the inadequate occupation of the probes, which can be solved by improving the 6


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concentration of modification. For better understanding the reasons behind the low background, we employ SPR to interrogate the protein adsorption ability of Au surfaces modified by sSH-HSP, dSH-HSP and SH-ssDNA + MCH. As shown in Fig. 3, these results demonstrate that dSH-HSP-surface displays higher protein-repelling ability compared with sSH-HSP and SH-ssDNA modified surface. These effects are due to the reduced number of contacting points between protein molecules and DNA probe that adopts a more compact structure and less room for interaction between protein molecules and hydrophobic Au interface separated by the rigid DNA structure. Consistently, it provides a rational explanation for the differential background signal response obtained from the electrochemical assays. 3.4 Performance characteristics. Fig. 4a depicts the current (I)-versus-time (t) decay curve at 100 mV for different concentrations of target DNA, which reaches a plateau within 100 s. The amperometric signal is logarithmically related to the target DNA concentration (Fig. 4b), spanning a response region of at least 10 orders of magnitude. In addition, we are able to distinguish concentrations as low as 5 aM (∼ 300 molecules in 100 μL) above background without the need of extra amplification. In contrast, when testing the same target by traditional “sandwich-type” structure assay, the linear dynamic range is approximately five orders of magnitude, with a higher limit of detection around 1-5 pM, demonstrating that our sensor is much more sensitive than that fabricated with the traditional method. 3.5 Practical application. Having confirmed the excellent electrochemical performance for synthetic DNA, we have examined whether our sensor can work well in the routine analysis of different kinds of real samples. The PCR product is one of the most common analytes in biological laboratory, so we firstly select a 250-base PCR amplicon from the uidA gene of Escherichia coli (E. coli) as a model target. Considering that the possible steric effects caused by the longer amplicon may reduce hybridization efficiency, we have designed three kinds binding fashions to test the sensors’ performance. However, different from the linear DNA-based sensor, we observe that there is no obvious difference for all of the three plans, which makes it easy to design probes (Fig. 5a) .35 Basically, electrophoresis is the most commonly used technique to detect PCR-amplified products. Therefore, we have compared the performance of our sensor versus electrophoretic analysis coupled with asymmetric PCR amplification (Fig. 5b). We find that this sensor can easily identify as few as 5 fetograms E. coli DNA (∼one copy in the 25 μL PCR system), with the increase of sensitivity by 3 orders of magnitude compared with the conventional electrophoresis. Compared with the genomic PCR amplicon, mRNA is a more attractive target given that most mRNAs exist as linear sequences and therefore denaturation is not required. However, the conventional methods to detect mRNA such as Northern blots, quantitative PCR (qPCR) and microarrays are cumbersome, time-consuming, 7



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and labor-intensive, which may not be suitable for resource-limited environments. Therefore, we have examined whether our sensor can be used to sensitively and selectively detect mRNA extracted from cell lines. The selected model target here is bcr-abl gene fusion and has an 8500 nt long transcript that is specific to chronic myeloid leukemia (CML).36 As shown in Fig. 5c, we are able to detect as low as 0.2 ng of total RNA when challenged with purified total RNA from k562 cell line for 90 min, which contains the most common form of the bcr-abl gene fusion. The detection limit observed corresponds to mRNA from tens of cells within 90 min (Fig. S6), which is comparable to a commercially available PCR assay designed to specifically detect CML (Fig. 5d) 37. Moreover, when challenged with other cell lines such as Hela, MCF-7, and A549 that do not contain the fused gene complementary to the probe, we are not able to obtain obvious electrochemical responses (Fig. S7). By the way, it should be mentioned that the “sandwich-type” sensor becomes useless for detection of such a long molecule and no detectable signal can be achieved even with a higher total RNA concentration (10 ng, Fig. S8). In view of the biological significance of miRNA, the analytical ability for miRNA with high similarity is also of importance. Having demonstrated the excellent performance of small synthetic DNA fragments, we might expect the specificity to be superior to commercial glass microarray chip that is often problematic by cross-hybridization of related miRNAs. Indeed, a spiking experiment involving eight related sequences (a family of human let-7 sequences) shows increased specificity of electrochemistry-based detection compared with microarray-based detection, even for single base-pair mismatches (Fig. 5e, f).

4. Conclusion In summary, we have developed a novel E-NA sensor for detection of nucleic acid with attomolar detection limit and an excellent selectivity. The unique recognition fashion of the probes on the electrode interfaces can overcome several shortcomings of the traditional approaches of modifying probes on heterogeneous surfaces. First, the dSH-HSP can be rapidly and robustly assembled on Au surfaces in a single step and keep proper upright orientation conformation without co-assembled alkanethiol. Second, the adjustable stem length can preciously separate target and signal probe from the surface, thereby providing a solution-phase like environments for efficient hybridization. Third, the ultrasensitive detection limit and one-base mismatch discrimination ability make us easily analyze kinds of targets such as genomic DNA, mRNA and miRNA without employing extra and complex amplification steps, which is particularly suitable for routine detection of laboratory. In addition, such probe-binding design might be valuable for other solid-supported NA sensors, including microarray, SPR, and microcantilever, etc. Given these advantages, we believe this strategy may become a new paradigm in the design of biosensors and significantly help the development of the field of chip-based biomolecular detection.

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Author information Corresponding Authors *E-mail: [email protected] (Y. Xiang), [email protected] (Y. Shu), and [email protected] (G. Li), Tel: +86 25 83593596. Fax: +86 25 83592510. Notes The authors declare no competing financial interest.

Acknowledgements This work is supported by the National Natural Science Foundation of China (Grant No. 21235003), and Fundamental Research Funds for the Central Universities (Grant No. 021414380001).

Supporting information 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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(34) Wong, E. L. S.; Chow, E.; Gooding, J. J., Langmuir 2005, 21, 6957-6965. (35) Zhang, J.; Lao, R.; Song, S.; Yan, Z.; Fan, C., Anal. Chem. 2008, 80, 9029-9033. (36) van Rhee, F.; Hochhaus, A.; Lin, F.; Melo, J.; Goldman, J.; Cross, N., Blood 1996, 87, 5213-5217. (37) Jobbagy, Z.; van Atta, R.; Murphy, K. M.; Eshleman, J. R.; Gocke, C. D., J. Mol. Diagn. 2007, 9, 220-227.

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Scheme 1. Schematic illustration of the “sandwich-type” based E-NA sensor (a) and the dSH-HSP based E-NA sensor (b).

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Figure 1. Characterization of the surface modification. (a) Self-assembly processes of SH-ssDNA (black line) and dSH-HSP (red line) monitored in real-time using surface plasmon resonance (SPR). Inset: Adsorption of nonthiolated-HSP and ssDNA on the Au surface. (b) Cyclic voltammograms for the redox reaction of the TMB substrate at gold electrodes modified with dSH-HSP (solid line) and MCH (dash line). Scan rate: 100 mV/s. (c) The Rct for 20 electrodes modified with different probes. The concentrations of all modified probes are 1 μM.

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Figure 2. Electrochemical characterization of the sensor. (a) Proof of the feasibility of the proposed electrochemical method. Amperometric responses of target (5 nM) + signal probe + enzyme (i), signal probe + enzyme (ii), target + enzyme (iii), only enzyme (iv), and target + reporter probe without enzyme (v). Inset demonstrates the formation of Y-shaped structure using electrophoresis. Line M: maker, line 1: HSP+target+signal probe, line 2: HSP+signal probe, line 3: HSP+target, line 4: signal probe, line 5: target, line 6: HSP. (b) Comparison of the electrochemical performance of dSH-HSP and ssDNA probe-based sensors with or without MCH passivation (1 mM for 2 h). Target DNA concentration: 5 nM. (c) Amperometric responses for dSH-HSPs with different lengths of stems modified surfaces. Error bars represent standard deviations for measurements taken from three independent experiments.

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Figure 3. SPR angle-time curves for different probes modified interfaces with injecting 1 mg mL-1 HRP.

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Figure 4. Electrochemical performances of dSH-HSP based sensor for synthetic target. (a) Amperometric curves for the detection of synthetic target DNA at a series of concentrations (from top to bottom: 50 nM, 5 nM, 50 pM, 50 fM, 0.5 fM, 5 aM, and 0). (b) Logarithmic plot of amperometric current versus target DNA dSH-HSP and SH-ssDNA probe-based sensors. Insert shows the low concentration range of target DNA. Error bars represent standard deviations for measurements taken from six independent experiments.

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Figure 5. Practical application of the proposed sensor. (a) Comparison of the electrochemical performances of the three probe-binding fashions with two target concentrations. (b) Electrochemical detection of PCR amplicon. The DNA template is the genomic DNA extracted from E. coli and serially diluted by 10 times. Inset: The electrophoretic analysis of corresponding PCR products. The PCR reaction was performed for 30 cycles. (c) Amperometric curves for the detection of bcr-abl transcript at a series of concentrations (from top to bottom: 2 μg, 0.2 μg, 20 ng, 2 ng, 0.2 ng and 0 ng). (d) Comparison of PCR and electrochemistry-based analysis of K562 cells. The grey squares reflect ΔCt values extracted from reported reference. These data points are compared to current values collected from dSH-HSP based sensor (black squares). (e, f) Heat maps comparing the specificity of the electrochemical assay with that of fluorescent microarray. Significant signals are marked as positive: positive signals for specific target are regarded as ‘true-positive’, and positive signals from non-targeted samples are classed as ‘false-positive’.

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Graphic abstract for TOC only

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One-Step Modification of Electrode Surface for Ultrasensitive and

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