Homogeneous Electrochemical Strategy for Human Telomerase

Mar 9, 2015 - units of T7 exonuclease, and a varying amount of STP or telomerase extracts at 25 °C for 1.5 h before the electro- chemical measurement...
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Homogeneous Electrochemical Strategy for Human Telomerase Activity Assay at Single-Cell Level Based on T7 Exonuclease-Aided Target Recycling Amplification Xiaojuan Liu,†,§ Wei Li,†,§ Ting Hou,† Shanshan Dong,† Guanghui Yu,‡ and Feng Li*,† †

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, Shandong 266109, People’s Republic of China ‡ College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong 266109, People’s Republic of China ABSTRACT: As an important biomarker for early cancer diagnostics and a valuable therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Herein, a homogeneous electrochemical strategy based on T7 exonuclease-aided target recycling amplification is proposed for a simple, rapid, and highly sensitive assay of human telomerase activity from crude cancer cell extracts. In this strategy, a 5′ methylene blue (MB)-labeled hairpin (HP) probe is designed, which can hybridize with the telomerase reaction products to initiate the subsequent digestion by T7 exonuclease, and a large amount of MB-labeled mononucleotides are released to result in the significantly amplified electrochemical signal. By taking advantage of the high amplification efficiency of T7-aided target recycling, the present assay enables the detection of telomerase activity at the single-cell level, which is superior or comparable to that of the reported literature. Furthermore, the assay was carried out in a homogeneous solution without complex modification or immobilization procedures, which has the merits of simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensors. With the ability of fast detection, outstanding sensitivity, and excellent selectivity, this strategy offers a convenient and specific method for telomerase activity detection, which exhibits great potential in the practical application in telomerase-based early stage cancer diagnosis.

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So far, several approaches have been developed for the detection of telomerase activity. One of the most frequently used approaches is the telomeric repeat amplification protocol (TRAP),13,14 which is based on the classic polymerase chain reaction (PCR) technique and therefore gives an ultrahigh sensitivity. However, it is susceptible to the same drawbacks of all PCR-based assays, including the risk of carry-over contamination, susceptibility to polymerase inhibition by cellextract, laborious post-PCR processing, and the requirement of expensive equipment and reagents.15−17 To overcome these shortcomings, several PCR-free protocols, based on alternative techniques, have been designed and successfully employed in telomerase activity detection, such as optical or spectral sensors,18−24 nanobiosensors,25−27 surface plasmon resonance (SPR),28,29 electrochemiluminescence (ECL),30−33 electrochemical detection,34−37 enzyme-linked immunosorbent assay (ELISA),38 and so on.39−44 Among them, electrochemical strategies are attractive for telomerase activity detection due to their outstanding advantages of simple operation, rapid

elomeres, which are composed of repeated DNA sequences and protein assemblies, cap and protect the ends of eukaryotic chromosome from undesired degradation, recombination, or end-to-end fusion.1 In human somatic cells, telomeres are progressively shortened with every cell division owing to the end-replication problem.2,3 When telomeres are shortened to a certain length, DNA damage response is activated and signal the cell to arrest its life cycle.4 However, for immortal cells, this process is restrained due to the activation of telomerase.5,6 First identified in 1985 by Greider and Blackburn,7 telomerase is a unique ribonucleoprotein reverse transcriptase that can bind to the telomere ends and add tandem repeats of (TTAGGG)n on them using its endogenous RNA subunit as a template.8 The elongation of the telomeres by telomerase, which compensates for their natural shortening, is strongly associated with cellular immortality and carcinogenesis.9 Previous research indicates that over 85% of human cancers are sustained by the overexpression of telomerase.10,11 Accordingly, telomerase is regarded as one of the most common cancer markers for early diagnosis, prognosis, and understanding the pathogenesis of disease.12 Thus, the rapid, facile, affordable, and sensitive detection of telomerase activity is of significant importance for telomerase-based cancer diagnosis and therapeutics. © XXXX American Chemical Society

Received: January 27, 2015 Accepted: March 9, 2015

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based methods41 or heterogeneous methods,47−49 sophisticated thermal cycling, multiple separations, or washing steps are avoided. Therefore, the strategy we proposed here offers a much more convenient approach for the detection of telomerase activity with high sensitivity and excellent selectivity.

response, low cost, and compatibility with micromanufacturing technology.45,46 Over the past decades, much effort has been made toward developing electrochemical methods for the telomerase assay. For example, differential pulse voltammetry (DPV) has been used to detect telomerase activity on the basis of the guanine oxidation signal.47 Electrochemical impedance measurement has been employed on the basis of the principle that the telomerase elongated DNA primer can block the transfer of Fe(CN)63−/Fe(CN)64− electrons on the electrode surface, leading to an increase of the impedance signal.48 Chronocoulometry has been proposed to quantify DNA extended by telomerase based on the electrostatically adsorbed Ru(NH3)63+, which serves as the electrochemical signaling molecule and its number is proportional to the amount of DNA.49 Although impressive progress has been made using different electrochemical methods to detect telomerase activity, most of the electrochemical methods are still laborious and time-consuming due to the heterogeneous assaying processes that often involve the immobilization of the primer on the electrode surface before collecting the DNA extension-induced electrochemical signal. Also, as the recognition of the primer by telomerase occurs on the solution−electrode interface, the spatial hindrance effect of the electrode surface and the loss of configurational freedom usually make these heterogeneous assays suffer from relatively low binding efficiency and enzyme kinetics compared with homogeneous assays.50−55 Therefore, it is highly desirable to develop faster and easier-to-use homogeneous electrochemical strategies for detection of telomerase activity. Homogeneous electrochemistry is an immobilization-free approach, in which the hybridization between the probe DNA and target DNA, as well as the recognition by the enzyme, occurs in the solution phase instead of on the surface of the electrode. Hence, it has the advantages of simplicity, rapid response, and improved recognition efficiency compared with heterogeneous methods.50−55 Using these advantages, numerous homogeneous electrochemical strategies have been developed for the detection of various targets, such as DNA, small biological molecules, and metal ions.50−56 For example, Hsing and colleagues have demonstrated solution-phase electrochemical strategies for sensitive detection of DNA and mercury ion.50−52 Very recently, our group has developed highly sensitive homogeneous electrochemical biosensors for the detection of methyltransferase activity on the basis of releasing redox labels from the redox-labeled oligonucleotide probe.53 Inspired by the different diffusivity between electroactive reporter tagged oligonucleotides and mononucleotides toward a negatively charged electrode surface, herein, we have developed a simple, rapid, and highly sensitive homogeneous electrochemical strategy for the detection of telomerase activity at the single-cell level. This strategy relies on a target recycling amplification through the specific T7 exonuclease catalyzed digestion of methylene blue (MB)-labeled hairpin (HP) DNA probes, which hybridize with the telomerase extended DNA sequences. Taking advantage of the high amplification efficiency of T7-aided target recycling and the excellent electrochemical signals of the released MB molecules, the proposed strategy is capable of detecting the telomerase activity extracted from HeLa cells at the single-cell level. Because in this protocol the results can be read out simply by mixing crude cell extracts with the reaction solution, as compared to the PCR-

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EXPERIMENTAL SECTION Reagents. T7 exonuclease was purchased from New England Biolabs (Ipswich, MA, USA) and used without further purification. Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (HCl), NaCl, EDTA, and MgCl2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tween 20 and ethylene glycol tetraacetic acid (EGTA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The CHAPS lysis buffer was purchased from Millipore (Bedford, MA, USA). All the reagents were of analytical grade and used without further purification. Ultrapure water (18.2 MΩ·cm at 25 °C) obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA) was used throughout the experiments. The deoxynucleotide triphosphates (dNTPs) and all oligonucleotides used in this work were synthesized and HPLC-purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences were as follows: Telomerase substrate (TS) primer: 5′-AAT CCG TCG AGC AGA GTT-3′ Hairpin (HP) probe: 5′-methylene blue−CCC TAA CCC TAA CCG GTT-3′ Synthetic telomerase product (STP): 5′-AAT CCG TCG AGC AGA GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG GTT AGG G-3′ All oligonucleotides were used as provided and diluted in 40 mM Tris−HCl buffer solution (pH 7.9, containing 50 mM NaCl, 10 mM MgCl2, and 1 mM EDTA) to give the stock solutions of 10 μM. HP probe was heated to 95 °C and maintained at this temperature for 10 min and then slowly cooled down to room temperature before use. Cell Culture and Telomerase Extraction. Human cervical carcinoma cells (HeLa) and human normal liver cells (LO-2) were cultured in DMEM medium supplemented with 10% fetal calf serum, and the cells were maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2). Cells were collected in the exponential phase of growth, and 1 × 106 cells were dispensed in a 1.5 mL EP tube, washed twice with ice-cold phosphate buffered saline (PBS) solution, and then resuspended in 100 μL of ice-cold CHAPS lysis buffer (10 mM Tris−HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 5 mM mercaptoethanol, 0.5% CHAPS, 10% glycerol). The lysate was incubated on ice for 30 min and centrifuged at 12 000 rpm for 20 min at 4 °C. The supernatant was collected carefully and transferred to clean microcentrifuge tubes. The collected lysate was used immediately for the telomerase assay or frozen at −80 °C. Telomerase Extension Reaction. Telomerase extracts were diluted in lysis buffer with the respective number of cells; the extracts (5 μL) were added to a 45 μL extension solution containing reaction buffer (20 mM Tris−HCl, pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, 0.1 mg/ mL BSA), 1 mM dNTP, and 0.5 μM TS primer. The extension solution was kept at 37 °C for a certain period of time. Electrochemical Detection of Telomerase Activity. Differential pulse voltammetric (DPV) measurements were performed using a CHI 660E electrochemical analyzer B

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primer occurs in the presence of the dNTP mixture, in which TTAGGG repeat units are incessantly added to the 3′ end of the primer. The extended TS telomeric repeat strand can hybridize with multiple HP probes and further open them to form a duplex conformation. This duplex has blunt or recessed 5′ termini and can be recognized by T7 exonuclease, leading to the digestion of the HP probes incorporated in the duplex. This digestion process not only releases the extended telomerase extended DNA strand, which then hybridizes with fresh HP probes to initiate the recycling digestion processes, but also results in the accumulation of MB-labeled mononucleotides. Compared to the hairpin probe or the duplex DNA, the MBlabeled mononucleotide possesses much higher diffusivity toward the negatively charged ITO electrode due to its less negative charge and smaller size, resulting in a distinct increase of the electrochemical signal. As a result, the highly sensitive telomerase activity assay can be realized by monitoring the change of the electrochemical signal. Therefore, the asproposed homogeneous electrochemical strategy based on T7 exonuclease-aided amplification via the sequential recycling of the telomerase extended DNA strand is a promising candidate for highly sensitive detection of the activity of telomerase extracted from cancer cells. Assay of Synthetic Telomerase Product (STP). Before the sensing performance, the feasibility of the proposed strategy was investigated by measuring differential pulse voltammetric (DPV) signals under different conditions. Here, the synthetic telomerase product (STP), corresponding to TS primer extended with eight telomeric repeats (TTAGGG)8, was used as a positive control for the experimental condition optimization and feasibility study of the proposed assay. As shown in Figure 1, in the absence of STP, a very weak

(Shanghai, China). A three-electrode system was employed, with an ITO electrode as the working electrode, an Ag/AgCl as the reference electrode, and a platinum wire as the auxiliary electrode. The ITO electrode was prepared as follows: first, ITO electrode was sequentially sonicated in an Alconox solution (8 g of Alconox per liter of water), propan-2-ol, acetone, and ultrapure water for 15 min each. Then, the ITO electrode was immersed into 1 mM NaOH solution for 5 h at room temperature and sonicated in ultrapure water for 15 min. After these cleaning procedures, a negatively charged working electrode surface was obtained. The experimentation was performed in 50 μL of reaction solution (40 mM Tris−HCl, 50 mM NaCl, 10 mM MgCl2, pH 7.9) containing 2 μM HP, 15 units of T7 exonuclease, and a varying amount of STP or telomerase extracts at 25 °C for 1.5 h before the electrochemical measurements.

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RESULTS AND DISCUSSION Principle of the Assay. The principle of the homogeneous electrochemical strategy for the telomerase activity assay is illustrated in Scheme 1, which is based on a simple system of Scheme 1. Principle of the Homogeneous Electrochemical Strategy for the Detection of Telomerase Activity Based on T7 Exonuclease-Aided Target Recycling Amplification

telomerase substrate (TS) primer, T7 exonuclease, HP DNA probe, and telomerase extracted from crude cancer cells. TS primer contains a nontelomeric repeat sequence and can be recognized by telomerase as a substrate.11 T7 exonuclease is a sequence-independent nuclease that catalyzes the stepwise removal of 5′ mononucleotides from blunt or recessed 5′ termini of double-stranded DNA but has limited activity on single-stranded DNA (ssDNA).22,57 The HP probe carries a small ssDNA tail at its 5′ terminus, which can effectively prevent the digestion by T7 exonuclease and act as a toehold for selective binding with the telomeric repeat strand. Furthermore, the 5′ terminus of the HP probe has been designed to be modified with MB, which serves as an electrochemical signal reporter. In the absence of telomerase, the HP probe remains in the hairpin conformation and gives a very weak electrochemical signal of MB due to electrostatic repulsion between both the negatively charged ITO electrode and the hairpin probe.50−55 Once the telomerase extracted from the cancer cells is added into the solution, elongation of TS

Figure 1. Differential pulse voltammograms under different conditions: (a) HP only; (b) HP and T7 exonuclease; (c) HP, T7 exonuclease, and STP; the figures on the right are the corresponding schematic illustrations. The concentrations of HP, T7 exonuclease, and STP were 2 μM, 15 U, and 100 pM, respectively.

electrochemical signal was observed in the buffer solution of HP probes (curve a) due to the electrostatic repulsion between the negatively charged ITO electrode and the MB-labeled HP probe. After incubating HP probe with T7 exonuclease at 25 °C for 90 min, a slightly increased electrochemical signal was obtained (curve b), which might be explained by the residual cleavage activity of T7 exonuclease toward the HP probe. However, when the STP was added, the electrochemical signal showed a significant enhancement (curve c). This suggests that the HP probe can hybridize with the STP and then be cleaved by T7 exonuclease, leading to the release of MB-labeled C

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Figure 2. DPV peak current observed under (A) different T7 exonuclease concentrations: 5, 7.5, 10, 12.5, 15, 17.5, and 20 U; (B) different reaction time of T7 exonuclease: 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, and 2.5 h. The error bars represent the standard deviation of three measurements.

Figure 3. (A) Differential pulse voltammograms corresponding to the analysis of STP with different concentrations: (a) 0, (b) 0.1 pM, (c) 0.5 pM, (d) 1 pM, (e) 5 pM, (f) 10 pM, (g) 50 pM, (h) 100 pM, (i) 1 nM, (j) 5 nM, and (k) 10 nM. (B) DPV peak current plotted against STP concentration. Inset: The linear plot of DPV peak current versus the logarithm of STP concentration ranging from 0.1 pM to 10 nM. The error bars represented the standard deviation of three repetitive measurements.

monoucleotides and STP strand. The recycling of the STP for repeated hybridization to excessive HP probes followed by T7 exonuclease-mediated digestion resulted in a significantly enhanced electrochemical signal. It should be noted that the electrochemical signal observed in curve b was relatively weak and could be ignored compared to the significantly larger signal in curve c. Thus, the aforementioned results clearly demonstrated the feasibility of our strategy for the telomerase activity assay. In order to obtain the best performance, some experimental conditions were optimized. Since the concentration and the reaction time of T7 exonuclease have significant influence on the amplification reaction, the systems with different T7 exonuclease concentrations and different reaction time were investigated, respectively. As shown in Figure 2A, when other conditions were kept unchanged, the DPV signal increased dramatically as the concentration of T7 exonuclease increased from 5 to 15 U. Further increasing the concentration of T7 exonuclease led to little change in the electrochemical signal. Thus, 15 U was chosen as the optimal T7 exonuclease concentration. Furthermore, when the T7 exonuclease digestion reaction time was carried out from 0.5 to 2.5 h, the electrochemical signals were analyzed, and the results are

shown in Figure 2B. Obviously, the electrochemical signals increase significantly with the extension of the T7 digestion time up to 1.5 h. Afterward, there was a slight increase in the electrochemical signal with the reaction time further extended from 1.5 to 2.5 h. Hence, 1.5 h was chosen as the optimal reaction time for T7 exonuclease. Under the optimized experimental conditions, the STP was employed as a model telomerase extended product to evaluate the sensitivity of the proposed strategy. When STP with different concentrations was added to the reaction solution, the electrochemical response was recorded by DPV. As shown in Figure 3A, the DPV peak current was gradually increased as the concentration of STP increased. This is in accordance with the fact that STP with a higher concentration can hybridize with more HP probes, which are subsequently digested by T7 exonuclease to release more MB-labeled mononucleatides, leading to a significant increase of the current via recycling signal amplification. As shown in Figure 3B, the DPV peak current was highly dependent on the concentration of STP. A directly measured detection limit of 0.1 pM could be readily achieved without any background subtraction. The inset of Figure 3B revealed a good linear correlation between the DPV peak current and the logarithm of the STP concentration D

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Figure 4. (A) Differential pulse voltammograms under different conditions: (a) HP, TS, and dNTP; (b) HP, TS, T7 exonuclease, and dNTP; (c) HP, TS, T7, and active telomerase extracted from 100 HeLa cells. (B) DPV peak current obtained under different telomerase extension reaction time: 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, and 2.5 h. The concentrations of HP, TS, T7 exonuclease, and dNTP were 2 μM, 0.5 μM, 15 U, and 1 mM, respectively. The error bars represent the standard deviation of three repetitive measurements.

Figure 5. (A) Differential pulse voltammograms corresponding to the analysis of telomerase extracts from different of HeLa cells: (a) 0, (b) 2, (c) 5, (d) 10, (e) 20, (f) 40, (g) 100, (h) 200, (i) 500, and (j) 1000. (B) DPV peak current plotted against the number of HeLa cells. Inset: the linear plot of the DPV peak current versus the logarithm of the number of HeLa cells. The error bars represent the standard deviation of three repetitive measurements.

ranging from 0.1 to 100 pM. The correlation equation is ip = 233.7 + 16.8 log CSTP (R2 = 0.9869), where ip is the DPV peak current and CSTP is the concentration of STP. These results suggest that the as-proposed homogeneous electrochemical strategy was efficient for sensitive detection of the telomerase extended product STP. Thus, the proposed strategy can be used for sensing telomerase activity extracted from cancer cells. Detection of Telomerase Activity. To verify the capability of the proposed strategy for sensing the activity of telomerase extracted from cancer cells, the HeLa cell, which is a telomerase-positive human cervical carcinoma, was used as a model cancer cell. In the experiment, the crude telomerase extracts were incubated with the TS primer and dNTP at 37 °C for 1.5 h, followed by incubation with the HP probe and T7 exonuclease at 25 °C for 1.5 h. As shown in Figure 4A, a readily detectable DPV peak at around −0.32 V was observed in the presence of T7 exonuclease and telomerase extracted from 100 HeLa cells (curve c). This peak could be attributed to the electrochemical oxidation of the released MB-labeled mononucleotides, indicating that the tandem repeats (TTAGGG)n were successfully added to the TS primer by telomerase, and

the recycling signal amplification process was indeed realized. However, in the absence of the telomerase extracts, with T7 exonuclease added (curve b) or not (curve a), the MB signals were both negligible. These results demonstrated the feasibility of the designed homogeneous electrochemical strategy for the detection of telomerase activity from cell extracts. The telomerase extension time is a key factor to determine the number of the produced TTAGGG repeat units, which highly influences the sensitivity of the telomerase activity detection. Therefore, it is necessary to optimize the telomerase extension time. Here, DPV was used to monitor the electrochemical signals of the HP probes and T7 exonuclease in the presence of the telomerase products after incubating TS primer, dNTP, and telomerase extracts under different periods of time. As shown in Figure 4B, a higher DPV signal was observed when a longer incubation time was adopted, and a plateau was reached after 1.5 h. Thus, 1.5 h was selected as the optimal telomerase extension time for the subsequent experiments. To investigate the sensitivity of the proposed strategy for the detection of telomerase activity from the cell extracts, HeLa cell E

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Table 1. Comparison of the Analytical Performance for the Detection of Telomerase Activity by the As-Proposed Method and Those Reported in the Literature strategy

detection modes

detection limit

reference

Au nanoparticle-based ECL assay ECL sensor based on porphyrin-functionalized graphene DNAzyme-based assay exonuclease-assisted target recycling amplification assay exponential isothermal amplification of telomere repeat assay TRAP assay gold shell coated magnetic nanobeads-based assay Au film-based assay heterogeneous electrochemical assay heterogeneous electrocatalytic assay nanoparticle-mediated signal amplification heterogeneous electrochemical biosensor based on structure-switching DNA probe homogeneous electrochemical assay based on T7-aided target recycle amplification

ECL ECL fluorescence fluorescence fluorescence radioactive signal colorimetry and SERS spectroscopy SPR chronocoulometry amperometry amperometry differential pulse voltammetry differential pulse voltammetry

148 HL-60 cells 10 HeLa cells/mL 200 HeLa cells 5 HeLa cells 1 HeLa cell 1 293 cell 10 SKBR3 cells/mL 18 293T cells/μL 5 HeLa cells 100 A549 cells/mL 1 MCF-7 cell 100 HeLa cells/mL 1 HeLa cell

31 33 23 22 41 14 24 29 49 37 34 36 this work

extracts were serially diluted with lysis buffer and used as a source for telomerase. Since the telomerization is controlled by the content of telomerase in the cell lysate samples, the electrochemical signal of the biosensing platform should relate to the concentration of cancer cells. As expected, it is observed in Figure 5A that the electrochemical signal increased as the number of HeLa cells increased from 2 to 1000, which clearly indicated that more telomerase could recognize and elongate more TS primers, resulting in more hybridized sites for HP probe and a high DPV signal. The relationship between the DPV peak current and the number of HeLa cells is shown in Figure 5B. Obviously, the telomerase activity in the HeLa cell extracts equivalent to 2−1000 cells could be detected by the proposed strategy. When the DPV peak current values were plotted against the logarithm of the number of HeLa cells, the resulting standard curve showed a linear relationship in the range of 2−200 cells with the correlation equation of ip = 75.9 log N − 12.5 (R2 = 0.9946), where N is the number of cells (inset of Figure 5B). The detection limit was estimated to be as low as 1 HeLa cell, which is better than that of the vast majority of the reported methods and comparable to that of the recently reported exponential isothermal amplification of the telomerase repeat assay (Table 1). To validate the specificity of the proposed homogeneous electrochemical strategy for telomerase activity detection, the cell extracts equivalent to 0 cell, 100 HeLa cells with and without heat treatment, and 100 human normal liver cells (LO2) were tested. As shown in Figure 6, the blank sample without cell extract gave negligible DPV signal, whereas the HeLa cell extracts without heat treatment showed a significantly amplified signal, indicating its positive telomerase activity. When the telomerase extracted from HeLa cells was pretreated by heat, a weak electrochemical signal was observed due to the loss of telomerase activity. As is well-known, telomerase is a ribonucleoprotein reverse transcriptase endogenous to human cells that requires an internal RNA component to act as a template,11 so its activity is sensitive to heat, which can destroy the essential RNA template and the reverse transcriptase protein of telomerase. Furthermore, weak electrochemical signal was also observed on the test of LO-2 cells due to the lack of telomerase activity in normal cells. Therefore, the asproposed strategy exhibits excellent selectivity for telomerase activity detection and has great potential to be applied in clinical tests.

Figure 6. Comparison of the DPV peak currents of the biosensing platform in the presence of cell extracts equivalent to 0 cell (blank), 100 HeLa cells with and without heat treatment, and 100 LO-2 cells. The error bars represented the standard deviation of three repetitive measurements.

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CONCLUSIONS In summary, a novel, simple, rapid, and highly sensitive homogeneous electrochemical strategy based on T7 exonuclease-aided target recycling amplification was proposed to detect the activity of telomerase from crude cell extracts. By taking advantage of the high amplification efficiency of T7 exonuclease-aided target recycling, highly sensitive detection of telomerase activity is realized with a detection limit as low as 1 HeLa cell, which is superior or comparable to that of the reported literature. Furthermore, it is a fast and easy-to-use immobilization-free method that was carried out in the homogeneous solution; thus, complex modification or immobilization procedures are avoided. Therefore, this strategy holds great promise as a simple and highly sensitive method for telomerase activity detection and shows great potential in the practical application in telomerase-based early stage cancer diagnosis.

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AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: 86-532-86080855. E-mail: [email protected]. Author Contributions §

X.L. and W.L. contributed equally to this work.

Notes

The authors declare no competing financial interest. F

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(30) Zhang, H. R.; Wu, M. S.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2014, 86, 3834−3840. (31) Zhang, H. R.; Wang, Y. Z.; Wu, M. S.; Feng, Q. M.; Shi, H. W.; Chen, H. Y.; Xu, J. J. Chem. Commun. 2014, 50, 12575−12577. (32) Zhou, X. M.; Xing, D.; Zhu, D. B.; Jia, L. Anal. Chem. 2009, 81, 255−261. (33) Wu, L. W.; Wang, J. S.; Feng, L. Y.; Ren, J. S.; Wei, W. L.; Qu, X. G. Adv. Mater. 2012, 24, 661. (34) Wu, L.; Wang, J. S.; Ren, J. S.; Qu, X. G. Adv. Funct. Mater. 2014, 24, 2727−2733. (35) Liu, J.; Lu, C. Y.; Zhou, H.; Xu, J. J.; Wang, Z. H.; Chen, H. Y. Chem. Commun. 2013, 49, 6602−6604. (36) Yi, Z.; Wang, H. B.; Chen, K.; Gao, Q.; Tang, H.; Yu, R. Q.; Chu, X. Biosens. Bioelectron. 2014, 53, 310−315. (37) Zhang, Z. J.; Wu, L.; Wang, J. S.; Ren, J. S.; Qu, X. G. Chem. Commun. 2013, 49, 9986−9988. (38) Kha, H.; Zhou, W.; Chen, K.; Karan-Tamir, B.; Miguel, T. S.; Zeni, L.; Kearns, K.; Mladenovic, A.; Rasnow, B.; Robinson, M.; Wahl, R. C. Anal. Biochem. 2004, 331, 230−234. (39) Zhu, X.; Xu, H. F.; Lin, R. L.; Yang, G. D.; Lin, Z. Y.; Chen, G. N. Chem. Commun. 2014, 50, 7897−7899. (40) Jou, A. F. J.; Lu, C. H.; Ou, Y. C.; Wang, S. S.; Hsu, S. L.; Willner, I.; Ho, J. A. A. Chem. Sci. 2015, 6, 659−665. (41) Tian, L. L.; Weizmann, Y. J. Am. Chem. Soc. 2013, 135, 1661− 1664. (42) Zhao, Y. X.; Qi, L.; Chen, F.; Zhao, Y.; Fan, C. H. Biosens. Bioelectron. 2013, 41, 764−770. (43) Wang, L. J.; Zhang, Y.; Zhang, C. Y. Anal. Chem. 2013, 85, 11509−11517. (44) Zhang, Y.; Wang, L. J.; Zhang, C. Y. Chem. Commun. 2014, 50, 1909−1911. (45) Liu, X. J.; Chen, M. Q.; Hou, T.; Wang, X. Z.; Liu, S. F.; Li, F. Electrochim. Acta 2013, 113, 514−518. (46) Hou, T.; Liu, X. J.; Wang, X. Z.; Jiang, A. W.; Liu, S. F.; Li, F. Sens. Actuators, B: Chem. 2014, 190, 384−388. (47) Eskiocak, U.; Ozkan-Ariksoysal, D.; Ozsoz, M.; Oktem, H. A. Anal. Chem. 2007, 79, 8807−8811. (48) Yang, W. Q.; Zhu, X.; Liu, Q. D.; Lin, Z. Y.; Qiu, B.; Chen, G. N. Chem. Commun. 2011, 47, 3129−3131. (49) Sato, S.; Takenaka, S. Anal. Chem. 2012, 84, 1772−1775. (50) Luo, X. T.; Lee, T. M. H.; Hsing, I. M. Anal. Chem. 2008, 80, 7341−7346. (51) Xuan, F.; Luo, X. T.; Hsing, I. M. Anal. Chem. 2012, 84, 5216− 5220. (52) Xuan, F.; Luo, X. T.; Hsing, I. M. Anal. Chem. 2013, 85, 4586− 4593. (53) Wang, X. Z.; Liu, X. L.; Hou, T.; Li, W.; Li, F. Sens. Actuators, B 2015, 208, 575−580. (54) Liu, S. F.; Wang, Y.; Zhang, C. X.; Lin, Y.; Li, F. Chem. Commun. 2013, 49, 2335−2337. (55) Liu, S. F.; Lin, Y.; Wang, L.; Liu, T.; Cheng, C. B.; Wei, W. J.; Tang, B. Anal. Chem. 2014, 86, 4008−4015. (56) Zhuang, J. Y.; Tang, D. P.; Lai, W. Q.; Chen, G. N.; Yang, H. H. Anal. Chem. 2014, 86, 8400−8407. (57) Kerr, C.; Sadowski, P. D. J. Biol. Chem. 1972, 247, 305−310.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21375072, 21405089, 21445002, and 21175076), Scientific Research Award Fund for Excellent Middle-aged and Young Scientists of Shandong Province (No. BS2014CL004), and the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (Nos. 631311 and 631404).

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REFERENCES

(1) Xu, Y. Chem. Soc. Rev. 2011, 40, 2719−2740. (2) Olovnikov, A. M. J. Theor. Biol. 1973, 41, 181−190. (3) Cesare, A. J.; Reddel, R. R. Nat. Rev. Genet. 2010, 11, 319−330. (4) Martinez, P.; Blasco, M. A. Aging Cell 2010, 9, 653−666. (5) Bryan, T. M.; Cech, T. R. Curr. Opin. Cell. Biol. 1999, 11, 318− 324. (6) Zglinicki, T. V. Cancer Lett. 2001, 168, 111−116. (7) Greider, C. W.; Blackburn, E. H. Cell 1985, 43, 405−413. (8) Moyzis, R. K.; Buckingham, J. M.; Cram, L. S.; Dani, M.; Deaven, L. L.; Jones, M. D.; Meyne, J.; Ratcliff, R. L.; Wu, J. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6622−6626. (9) Lange, T. D. Science 2009, 326, 948−952. (10) Riou, J. F.; Guittat, L.; Mailliet, P.; Laoui, A.; Renou, E.; Petitgenet, O.; Megnin-Chanet, F.; Helene, C.; Mergny, J. L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2672−2677. (11) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science 1994, 266, 2011−2015. (12) Zhou, X. M.; Xing, D. Chem. Soc. Rev. 2012, 41, 4643−4656. (13) Piatyszek, M.; Kim, N.; Weinrich, S.; Hiyama, K.; Hiyama, E.; Wright, W.; Shay, J. Methods Cell Sci. 1995, 17, 1−15. (14) Kim, N. W.; Wu, F. Nucleic Acids Res. 1997, 25, 2595−2597. (15) Cian, A. D.; Cristofari, G.; Reichenbach, P.; Lemos, E. D.; Monchaud, D.; Teulade-Fichou, M. P.; Shin-Ya, K.; Lacroix, L.; Lingner, J.; Mergny, J. L. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17347−17352. (16) Krupp, G.; Kuhne, K.; Tamm, S.; Klapper, W.; Heidron, K.; Rott, A.; Parwaresch, R. Nucleic Acids Res. 1997, 25, 919−921. (17) Xiao, Y.; Dane, K. Y.; Uzawa, T.; Csordas, A.; Qian, J.; Soh, H. T.; Daugherty, P. S.; Lagally, E. T.; Heeger, A. J.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 15299−15307. (18) Wang, J. S.; Wu, L.; Ren, J. S.; Qu, X. G. Small 2012, 8, 259− 264. (19) Wang, J. S.; Wu, L.; Ren, J. S.; Qu, X. G. Nanoscale 2014, 6, 1661−1666. (20) Sharon, E.; Golub, E.; Niazov, A.; Balogh, D.; Willner, I. Anal. Chem. 2014, 86, 3153−3158. (21) Quach, Q. H.; Jung, J.; Kim, H.; Chung, B. H. Chem. Commun. 2013, 49, 6596−6598. (22) Wang, H. B.; Wu, S.; Chu, X.; Yu, R. Q. Chem. Commun. 2012, 48, 5916−5918. (23) Tian, T.; Peng, S.; Xiao, H.; Zhang, X. E.; Guo, S.; Wang, S. R.; Zhou, X.; Liu, S. M.; Zhou, X. Chem. Commun. 2013, 49, 2652−2654. (24) Zong, S. F.; Wang, Z. Y.; Chen, H.; Hu, G. H.; Liu, M.; Chen, P.; Cui, Y. P. Nanoscale 2014, 6, 1808−1816. (25) Qian, R. C.; Ding, L.; Yan, L. W.; Lin, M. F.; Ju, H. X. Anal. Chem. 2014, 86, 8642−8648. (26) Qian, R. C.; Ding, L.; Ju, H. X. J. Am. Chem. Soc. 2013, 135, 13282−13285. (27) Wang, Y. M.; Lu, M. H.; Zhu, J. H.; Tian, S. F. J. Mater. Chem. B 2014, 2, 5847−5853. (28) Maesawa, C.; Inaba, T.; Sato, H.; Iijima, S.; Ishida, K.; Terashima, M.; Sato, R.; Suzuki, M.; Yashima, A.; Ogasawara, S.; Oikawa, H.; Sato, N.; Saito, K.; Masuda, T. Nucleic Acids Res. 2003, 31, e4. (29) Sharon, E.; Freeman, R.; Riskin, M.; Gil, N.; Tzfati, Y.; Willner, I. Anal. Chem. 2010, 82, 8390−8397. G

DOI: 10.1021/acs.analchem.5b00355 Anal. Chem. XXXX, XXX, XXX−XXX