Point-of-Care Assay of Telomerase Activity at Single-Cell Level via

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Point-of-care assay of telomerase activity at single-cell level via gas pressure readout Yanjun Wang, Luzhu Yang, Baoxin Li, Chaoyong James Yang, and Yan Jin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01529 • Publication Date (Web): 07 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017

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Analytical Chemistry

Point-of-care assay of telomerase activity at single-cell level via gas pressure readout Yanjun Wang, a Luzhu Yang, a Baoxin Li, a Chaoyong James Yang b and Yan Jin* a a

Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key

Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China b

State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China

* Corresponding author: Prof. Yan Jin, Email: [email protected], Fax: 86-29-81530727.

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Abstract Detection of telomerase activity at the single-cell level is one of the central challenges in cancer diagnostics and therapy. Herein, we describe a facile and reliable point-ofcare testing (POCT) strategy for detection of telomerase activity via a portable pressure meter. Telomerase primer (TS) was immobilized onto the surface of magnetic beads (MBs), and then was elongated to a long single-stranded DNA by telomerase. The elongated (TTAGGG)n repeat unit hybridized with several short PtNP-functionalized complementary DNA (PtNPs-cDNA), which specifically enriched PtNPs onto the surfaces of magnetic beads (MBs), which were separated using a magnet. Then, nanoparticle-catalyzed gas-generation reaction converted telomerase activity into significant change in gas pressure. Due to the selfamplification of telomerase and enrichment by magnetic separation, the diluted telomerase equivalent to a single HeLa cell was facilely detected. More importantly, the telomerase in the lysate of 1 HeLa cell can be reliably detected by monitoring change in gas pressure, indicating that it is feasible and possible to study differences between individual cells. The difference in relative activity between different kinds of cancer cells was easily and sensitively studied. Study of inhibition of telomerase activity demonstrated that our method has great potential in screening of telomerasetargeted antitumor drugs as well as in clinical diagnosis. Keywords: Point-of-care; Telomerase; Gas pressure; Portable pressure meter

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INTRODUCTION Telomerase is a ribonucleoprotein reverse transcriptase that adds the telomeric repeats (TTAGGG)n to the end of the human chromosomes to achieve cell immortalization.1,2 The activity of telomerase is up-regulated in over 85% of human tumor cells.3 Due to the importance and universality of the telomerase, it is of significant theoretical and practical importance to achieve reliable and sensitive detection of telomerase activity. Currently, the telomeric repeat amplification protocol (TRAP) is the classical method for telomerase activity detection.4,5 As a PCR-based method, TRAP has disadvantages, such as tedious process and producing false signal, which have stimulated the development of alternative approaches, including methods based on fluorescence,6-8 electrochemistry,9,10 electro-chemiluminescence,11 chemiluminescence,12 surface plasmon resonance,13 and colorimetry.14 For example, a fluorescence method based on aggregation-induced emission (AIE) has been established for highly sensitive detection of telomerase in cell extracts and cancer patients’ urine specimens. By the AIE method, the telomerase activity in diluted HeLa extracts equivalent to 5 cells can be detected.15 Ju et al. proposed a smart vesicle kit containing a telomerase substrate and a Cy 5-tagged molecular beacon for in-situ detection of telomerase activity.16 Despite this progress, reliable and point-of-care (POC) detection of telomerase activity is still one of the central challenges for clinical cancer diagnosis. Moreover, most of previous reports detected the mean telomerase activity of a large number of cancer cells, with very little attention to telomerase activity differences of single cancer cells. Li et al. have developed a novel fluorescence method based on stem-loop primer-mediated exponential amplification (SPEA), which can efficiently amplify the telomere repeat sequence. The SPEA method can accurately detect the telomerase 3



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activity in the crude lysate of a single cell.17 This paper reports a method to evaluate the telomerase activity in single cancer cell. The method is sufficiently simple for point-of-care testing (POCT), and can detect differences in telomerase activity among single cells. Currently, POCT is attracting increasing attention in disease-related target detection due to the simple detection process and miniaturization of the devices.18,19 To achieve reliable and facile assay, the signal strategy plays a crucial role in the development of POCT. Although colorimetric methods are among the most common techniques in bio-analysis, for quantitative assays, a bulky instrument is needed, thereby limiting applications in POCT. To overcome these limitations, common physical parameters, such as distance20or time,21 have been creatively used as the signal output. For example, Lu et al. reported a portable personal glucometer (PGM)-based sensing platform for simple and rapid quantitative monitoring of telomerase activity without a bulky instrument and complicated procedure. Unfortunately, the detection limit of the method is insufficient to detect low telomerase activity, for example in a HeLa extract with concentration of 80 cells mL-1.22 Moreover, Lu’s method detects the mean telomerase activity in a cell population, which hardly reflects the differences between individual cells. In this work, we used a portable pressure meter to develop a facile and reliable POC assay for telomerase activity. Gas pressure is a very familiar physical parameter, and pressure measurements are widely used in industry.23 However, bio-analysis based on measurement of gas pressure is very rare. Yang’s group has creatively used platinum nanoparticles (PtNPs) to catalyze the decomposition of H2O2 and generate O2 for immunoassay.24 The decomposition of H2O2 to H2O and O2 is environment-friendly 4


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and produces no toxic products, thereby offering a promising POC protocol. In our case, the (TTAGGG)n repeat unit is added to the TS primer by telomerase, resulting in the specific binding of PtNP-labeled cDNA onto magnetic beads (MBs). After magnetic separation and washing, the MBs are mixed with H2O2 solution, and a significant increase in gas pressure is obtained due to the PtNP-catalyzed decomposition of H2O2. Therefore, telomerase activity can be facilely and ultrasensitively detected by a portable pressure meter. Due to the high sensitivity, it is possible to directly measure the telomerase activity in a single cancer cell. No POC assay has been previously reported for direct measurement of telomerase activity using a portable pressure meter. Thus, this new assay offers a promising bio-sensing platform for diagnostic applications. EXPERIMENTAL SECTION Chemicals and Reagents. The thiol-labeled complementary DNA (5’-SHTTTTTTTTTTCCCTAACCCTAA-3’, cDNA) and the aldehyde-labeled telomerase primer DNA (5’-CHO-TTTTTTTTTTTTTTTTTTTTAATCCGTCGAGCAGAGTT3’, TS) were synthesized by Sangon Biotech Co. (Shanghai, China). Chloroplatinic acid (H2PtCl6) was purchased from Aladdin (Shanghai, China). Amine groupmodified magnetic beads (MBs) were obtained from Shaanxi Lifegen Co., Ltd. (Xi’an, China). 3-[(3-cholamidopropyl) dimethylamino]-1-propanesulfonic acid (CHAPS), βmercaptoethanol, glycerol, phenylmethylsulfonyl ﬂuoride (PMSF), ethylene glycolbis (β-aminoethyl ether) -N,N,N′,N′-tetraacetic acid (EGTA), Tween 20 and tris(hydroxymethyl) aminomethane (Tris) were purchased from Sigma-Aldrich (Shanghai, China). Diethyl pyrocarbonate (DEPC) was purchased from Sangon Biotechnology Co. (Shanghai, China). Phosphate buffered saline (PBS) consisted of 5



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0.1 M NaH2PO4, 0.1 M Na2HPO4, 0.1 M KCl, and 10 mM MgCl2 (pH 7.4). PBS-T consisted of 0.1 M NaH2PO4, 0.1 M Na2HPO4, 0.1 M KCl, 10 mM MgCl2, and 0.02% Tween-20 (pH 7.4). All metal salts were purchased from Xi’an chemical reagent Co., Ltd. (Xi’an, China). All other reagents were of analytical reagent grade. Ultrapure water obtained from a Millipore filtration system was used throughout all experiments.

Apparatus. Transmission electron microscopy (TEM) images were collected on a Tecnai G2 F20 Field Transmitance Electron Microscropy (FEI, Japan). Gel electrophoresis was performed on a vertical electrophoresis system (Bio-Rad Laboratories Inc., America). The Molecular Imager system was purchased from Shanghai Peiqing Science & Technology. Co. Ltd (Shanghai, China).The pressure meter was purchased from PASSTECH (Xiamen, China). The IX73 Inversion fluorescence microscope was purchased from Olympus. Preparation of Platinum Nanoparticles (PtNPs). PtNPs were prepared according to the procedure reported elsewhere.10 A 1.63 mL aliquot of 19.3 mM H2PtCl6 was added to 14.1 mL of an aqueous solution containing 11.1 mg sodium citrate under vigorous stirring; then 1.75 mL of aqueous sodium borohydride (NaBH4) (7.3 mg) solution was added dropwise. The solution was kept stirring for another 30 min. Preparation of DNA-modified PtNPs. The synthesized PtNPs was centrifuged to remove excess electrolyte. Then, the PtNPs were incubated with thiol-labeled cDNA using a PtNPs:DNA concentration ratio of 1:10 for 24 h.25 The PtNPs/cDNA were obtained by centrifugation, washed with PBS and stored at 4 °C. Preparation of DNA-modified Magnetic Beads. The procedure for the preparation of DNA-modified magnetic beads followed a previous method.26 First, after being rinsed three times with PBS, the MBs were resuspended in 240 µL of PBS. Second, 6


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2.4 µL of the telomerase primer (10 µM) was added to the MBs solution, followed by shaking for 2 h at 30 °C. The resulting MBs/TS then were rinsed two times with PBST, two times with PBS, resuspended in a final volume of 240 µL PBS and stored at 4 °C. Cell Culture. CCRF-CEM cells, AGS cells, HL-7702 cells, HeLa cells were cultured according to our previously reported work.6 The breast cancer cells (MDA-MB-231) were cultivated in DMEM medium supplemented with 10% FBS and 100 U mL−1 of penicillin–streptomycin. Then the cells were maintained at 37 °C under a humidified atmosphere (95% air and 5% CO2). Extraction of Telomerase. The extraction process of telomerase is described in our published work.6 Briefly, when in exponential phase of growth, the cells were collected, counted, and then dissociated by CHAPS buffer to extract the telomerase. The resulting extracts were stored at -80 °C. Telomerase Extension. Telomerase extracts were diluted in lysis buffer. After being rinsed two times with 1 × TRAP buffer (20 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, BSA 0.1 mg mL−1), the MBs/TS were resuspended in 20 µL of 1 × TRAP buffer. Then, the desired volume of the extracts and 0.4 µL of dNTP (10 mM) were added to the aforementioned solution. Last, the solution was incubated at 30 °C for 4 h. For negative control experiments, telomerase extracts were heat-treated at 95 °C for 10 min. Detection of the Mean Telomerase Activity. The changes in pressure were recorded on a pressure meter. When the telomerase extension was completed, the MBs were rinsed two times with PBS-T and two times with PBS, then mixed with PtNPs-DNA. The mixture was diluted to a final volume of 20 µL with 100 mM PBS (pH 7.4). After 7



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incubation for 1.5 h at 35 °C, the unreacted PtNPs-DNA was twice separated from the final reaction solution by a controllable magnetic field. After repeated washing, 100 µL of 30% H2O2 was added and reacted with the above mixture for a given time prior to pressure measurement with a pressure meter. Direct Detection of the Telomerase Activity in Crude Lysate of Cancer Cells. First, the cell suspension was progressively diluted. Then, with the assistance of an inversion fluorescence microscope, different numbers of cells were collected using a capillary. Finally, 1 µL CHAPS buffer was added to the collected cells to extract the telomerase. The crude cell lysate was used as the source of telomerase to perform TS elongation and gas pressure measurements. Telomeric Repeat Amplification Protocol (TRAP). The process of TRAP is described in our previous work.27 Briefly, the telomerase was extracted from different kinds of cells, and each type was mixed with TS and dNTPs. After the elongation reaction was complete, the product was analyzed by PCR amplification and gel electrophoresis. Detection of Gas Pressure. The gas pressure was measured by a portable gas pressure meter which is composed of a 1602E LCD monitor, a BMP085 pressure sensor, and a needle with 0.7 mm inner diameter. Gas pressure value is digital displayed. After magnetic washing, MBs were re-dispersed into 100 µL 30% H2O2 solution. Then, the wells of 96-well plate were sealed by rubber seal immediately. The needle of gas pressure meter was inserted through the rubber seal to detect gas pressure. The gas pressure was read at once. RESULTS AND DISCUSSION Characterization of PtNPs. In this work, PtNPs were synthesized according to the proposed method,10 and characterized by transmission electron microscopy (TEM). 8


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As shown in Figure 1A and 1B, the size of PtNPs is around 4 nm, and the dispersity is satisfactory. And the lattice spacing is 0.20 nm, which is consistent with that of Pt (100) planes.10 The DNA-modification may affect the condition of PtNPs. It was found from Figure 1C that the morphology of PtNPs changed when cDNA was modified on the surface of PtNPs. Then, the catalytic ability of PtNPs for H2O2 decomposition was tested. As shown in Figure 1D, the final pressure gradually increased with increasing concentration of H2O2. The linear relation between pressure and the concentration of PtNPs was excellent (Figure 1E). The Kcat of PtNPs was 1.29×109 s-1 (compared to the Kcat of catalase = 2.31×107 s-1), demonstrating that the catalytic rate of the synthesized PtNPs is greater than that of catalase.24 These results indicated that our synthesized PtNPs possess excellent catalytic efficiency for the decomposition reaction of H2O2.

Figure 1. (A and B) TEM images of PtNPs. (C) TEM image of PtNPs/cDNA. (D) Pressure-change profiles for the decomposition reaction of H2O2. (E) Pressure-change profiles for the H2O2 decomposition reaction at different PtNPs concentrations. 9



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Proof of Principle. The POC strategy for detection of telomerase activity is depicted in Scheme 1. The 5’-aldehyde-functionalized TS was conjugated onto the surface of the amine-modified magnetic beads (MBs) via a Schiff reaction. The 5’-thiolated cDNA was covalently coupled to the surface of PtNPs via Pt-S self-assembly. When TS was incubated with telomerase and a mixture of dNTPs, TTAGGG repeat units were continuously synthesized at the 3’ end of the TS to form a long single-stranded DNA. Thus, one elongated TS could be hybridized with multiple cDNAs providing plenty of PtNPs attached onto the MBs surfaces. To remove free PtNPs, MBs were magnetically collected and washed three times with PBS buffer. Then, samples were mixed with 100 µL 30% H2O2 solution. The wells of 96-well plate were sealed by rubber seal immediately. The gas pressure is measured as soon as the needle of gas pressure inserted into the wells of 96-well plate. Therefore, telomerase activity could be sensitively and portably detected via change in gas pressure.

Scheme 1. Schematic illustration of gas pressure-based POC assay for sensitive detection of telomerase activity. 10


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To verify the feasibility of this POC strategy, the gas pressure was recorded under different conditions. As shown in Figure 2, upon the addition of cell extract from 500 HeLa cells, the pressure was extremely high and reached almost 75 kPa. The result indicated that the TS on the surface of MBs may be effectively elongated in the presence of the telomerase equivalent to 500 HeLa cells, bringing PtNPs catalyst onto the surface of MBs. Then, H2O2 was decomposed to generate plenty of O2. The preliminary result was as expected. To further exclude other possibilities of nonspecific interaction, negative control experiments were designed. First, the cell extract from 500 HeLa cells was heated at 95 °C for 10 mins, and then incubated with MBs/TS in 1×TRAP buffer. As clearly shown in Figure 2, the change in pressure was negligible upon the addition of heat-treated cell extract. That is, in the presence of heat-inactivated telomerase, the TS cannot be elongated, and the PtNPs cannot be integrated onto the surface of MBs, demonstrating that the change in gas pressure was dependent on the activity of telomerase. Second, as a universal cancer biomarker, the activity of telomerase is expressed in over 85% of cancer cells. Therefore, as a control experiment, the cell extract from normal cells was tested. As shown in Figure 2, the addition of normal cell extract generated little change in gas pressure, indicating that there was no telomerase or low telomerase activity in normal cells. Thus, the output of gas pressure was related to the telomerase activity. Third, because the CHAPS solution is essential in the extraction of telomerase, the effect of CHAPS solution on the gas pressure was studied. We can observe in Figure 2 that the pressure only slightly changed with the addition of CHAPS solution, and that the CHAPS solution had no influence on the decomposition of H2O2. Fourth, because PtNPs/cDNA are crucial for the decomposition of H2O2 to generate oxygen, we studied the non-specific catalytic effect of PtNPs/cDNA. As shown in Figure 2, the change in gas pressure was 11



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negligible when MBs/TS was incubated with PtNPs/cDNA in the absence of telomerase, indicating that PtNPs/cDNA cannot be non-specifically adsorbed onto the MBs. Lastly, because MBs are dispensable in this work, we investigated whether MBs have the catalytic activity to the decomposition of H2O2. The concentration of MB and H2O2 is the same as they used in the proof-of-principle. As shown in Figure S1 and Figure 2, the pressure value is small and remains unchanged with the prolonging of reaction time, indicating that the 0.4 µg/µL MBs will not catalyze the decomposition of H2O2, suggesting that MBs will not cause non-specific pressure output. The above results demonstrated that the change in pressure can reliably reflect the activity of telomerase, and that our pressure-based POC strategy is feasible for the reliable and sensitive detection of telomerase activity.

Figure 2. Pressure change under different conditions. The number of cells is 500. Characterization of the MBs/TS@PtNPs/cDNA. To verify the conjugation of MBs/TS@PtNPs/cDNA, we have tried to take TEM images of MBs and MBs/TS@PtNPs/cDNA. However, we cannot see any change in the roughness of MBs from TEM images (Figure S2). This could be because of a huge difference in size between MB and PtNPs. Because the size of purchased MBs is around 1.5 µm (Figure 3A), and the size of PtNPs is around 4 nm, it is difficult to simultaneously 12


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observe clear TEM images of MB and PtNPs under the same view. However, as shown in Figure 3B, the elementary mapping results clearly exhibit the signals of Fe and Pt, respectively, which more clearly proved the successful conjugation of the MBs/TS@PtNPs/cDNA.

Figure 3. (A) TEM image of MBs/TS@PtNPs/cDNA. (B) TEM elementary mapping of the MBs/TS@PtNPs/cDNA (corresponding to red section of A). Optimization of Experimental Condition. In order to achieve the best performance, several possible factors were optimized. The TS are linked to the surfaces of the magnetic beads, and only the combination of TS and telomerase can trigger the elongation reaction. If the concentration of TS on the MBs surface is too high, there may be steric hindrance between TS and telomerase, which would lead to decreased efficiency of the elongation reaction. In contrast, low TS concentration would also has a negative effect on the experimental result. Therefore, we investigated the influence of TS concentration on the experimental results. As shown in Figure S3, when the TS concentration was 100 nM, the pressure of the system was the largest, so we selected 100 nM as the TS concentration in the experiment. Secondly, the effect of elongation 13



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time on the readout signal was studied. It was observed that the pressure increased as the elongation time increased within the range of 1 h to 4 h, with a plateau beyond 4 h. Thus, the elongation time was set at 4 h (Figure S4). Third, because the concentration of catalyst plays a vital role in the sensitivity, the effect of PtNPs/cDNA concentration was carefully studied. As shown in Figure S5, the optimal concentration of PtNPs/cDNA was 0.32 µM. Another important factor is the hybridization time. Figure S6 demonstrates that the pressure was the largest when the hybridization time was 1.5 h, which was chosen for subsequent reactions. Evaluation of Repeatability. In this work, the reliability depends on the repeatability and stability of the pressure reading. Many factors can affect the stability of pressure, and these factors also affect the sensitivity of detecting telomerase activity. Thus, the repeatability of pressure measurements was investigated. A mixture of H2O2 /PtNPs was added to 48 wells of a 96-well plate, and the pressure was measured by a portable pressure meter after 1 h. As shown in Figure 4, the pressure of each of the 48 wells was in the range of 95.7±3.2% kPa, indicating that the repeatability was satisfactory, and that O2 gas generations is a reliable way to detect telomerase activity. Moreover, any leaks will affect the precision and accuracy of the POC assay. As shown in Table S1, the pressure fell by 3.3% within 3 h. For detecting telomerase activity, the gas pressure would be measured within one hour. Thus, the system seal is sufficient to ensure reliability and accuracy.

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Figure 4. Stability of gas pressure detection. The concentrations of H2O2 and PtNPs are 10 mM and 5.5 nM, respectively. The total number of wells is 48. Sensitivity of POC Assay. As a versatile cancer biomarker, sensitive detection of telomerase activity is vital in cancer prognostics, diagnostics and therapeutics. Under the optimal conditions, the relationship between gas pressure and cell number was investigated to evaluate the sensitivity of the POC strategy. Figure 5A depicts the change in gas pressure corresponding to different numbers of cells. The pressure increased with increasing mean telomerase activity equivalent to 1 to 1000 HeLa cells. As shown in Figure 5B, the pressure change exhibited a linear relation with the logarithm of cell number. Furthermore, the mean telomerase activity equivalent to 1 HeLa cell was sensitively detected. The cell extracts equivalent to 1 HeLa cell was added in the 20 µL TRAP buffer to elongate telomerase substrate TS, suggesting that the detection limit of the mean telomerase activity is 50 cells/mL. However, although these results are exciting, they did not reveal the difference of telomerase activity between single cells. Thus, we again directly explored the telomerase activity in the lysates of different numbers of cells. It is clearly from Figure 6 shows that pressure increased as the cell number increased. The telomerase in 1 HeLa cell and 3 HeLa 15



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cells caused a 14% and 38% change in gas pressure, respectively. Comparing the gas pressure in Figure 5 and Figure 6 shows a difference in telomerase activity between diluted cell extract and directly lysed HeLa cells. The gas pressure induced by directly lysed HeLa cells (Figure 6) was about 50% lower than that of the diluted cell extract with the same number of cells. Since the gas pressure is an average value, the telomerase extraction from a collection of cells (1×106) balances the difference in telomerase activity between single cells. Moreover, telomerase was concentrated when it was extracted from a large number of HeLa cells, leading to a higher mean telomerase activity compared to that of the directly prepared cell lysate. In order to further verify the reliability of pressure from the direct lysate of 1 HeLa cell, we investigated the influence of telomerase inhibitor (aloe-emodin derivative 3, AED3) on the pressure resulting from the direct lysate of 1 HeLa cell. As shown in Figure 7A, the pressure can reach about 4.4 kPa with the addition of the lysate of 1 HeLa cell. However, once AED3 was added, the pressure decreased significantly, and the pressure was consistent with the result without HeLa cells, thus demonstrating that AED3 can inhibit telomerase activity, leading to a decrease of gas pressure. The result also demonstrated that the pressure induced by the lysate of 1 HeLa cell is reliable. We also studied the effect of H2O2 decomposition time on the sensitivity of single-cell detection. Figure 7B clearly shows that, when a HeLa cell extract was added, the pressure gradually increased with increasing reaction time and was close to 20 kPa after 3 h. These results also show that the pressure change induced by a HeLa cell extract is reliable. All these results demonstrated that the POC assay can detect the telomerase activity in a single cell.

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Figure 5. (A) POC detection of telomerase activity in a diluted cell extract of HeLa cells. (B) Linear response of pressure change to the logarithm of the cell number from 1 to 500.

Figure 6. （A）Detection of telomerase activity from cell lysate of HeLa cells via portable pressure meter. （B）Linear response of pressure change to the number of HeLa cell. The numbers of cells were 1, 3, 5, 7, 10, 15, 20 and 30.

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Figure 7. (A) Inhibition effect of AED3 on telomerase activity of a single HeLa cell. (B) Linear response of pressure change to decomposition reaction time of H2O2 from 1 h to 3.0 h. Error bars show the standard deviations of three experiments. Specificity of the POC Assay. To test the specificity of the proposed strategy, we investigated the telomerase activity of four different kinds of cell lines, including HeLa cells, CCRF-CEM cells, AGS cells, MDA-MB-231 cells and HL-7702 cells. As shown in Figure 8A, for the same number of cells (500 of each type), the HeLa cell extract caused the largest change in gas pressure, demonstrating that the telomerase activity in HeLa cells is higher than that in CCRF-CEM, AGS cells and MDA-MB231 cells. HL-7702 normal cells acted as a negative control. The pressure change induced by the extract of 500 HL-7702 cells was tiny and approximately equal to that of the inactive telomerase, indicating that the telomerase activity from normal cells is hardly detectable. Furthermore, the direct detection of telomerase activity from the lysates of 5 cells and 10 cells was performed (Figure 8B), further demonstrating that the POC method can evaluate differences in telomerase activity. Therefore, this POC strategy can reliably discriminate the difference in telomerase activity of different cells. To validate the result of Figure 8A, we used the TRAP method to investigate the telomerase activity of different kinds of cell. As shown in Figure 8C, lanes 1, 2, 5 and 6 represent HeLa cells, CCRF-CEM cells, MDA-MB-231 cells and AGS cells, respectively, and lanes 3 and 4 represent HL-7702 normal cells and inactivated the HeLa cell extracts, respectively. We can see the obvious TS elongated product bands in lanes 1, 2, 5 and 6, but there was no band in lanes 3 and 4, demonstrating that telomerase activity is expressed in the four kinds tumor cells, but not in the HL-7702 normal cells. And compared with the lanes 2, 5 and 6, there was a more obvious band in lane 1, indicating that telomerase activity in HeLa cells is higher than that in the 18


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other three tumor cells. These results indicate that the proposed strategy can be used to detect telomerase activity in different kinds of cancer cells.

Figure 8. Telomerase activity of different kinds of cells. (A) The number of cells is 500. (B) The numbers of cells are 5 and 10, respectively. (C) Comparison of telomerase activity of different kinds of cells using conventional TRAP assay. Error bars were calculated from three replicate measurements. The biggest error bar was ±1.2%. Evaluation of Inhibition of Telomerase Activity. Researches have confirmed that the unfolded single-stranded telomeric overhang is required for the expression of telomerase activity. However, the fold of the telomere DNA into a G-quadruplex structure can inhibit the telomerase activity. To further test the applicability of this POC strategy, we used aloe-emodin derivative 3 (AED3) as a G-quadruplex ligand to study the inhibition of telomerase activity. Aloe-emodin derivative 3, which was designed and synthesized by our group,28 has been demonstrated to have Gquadruplex binding ligand properties.6 As shown in Figure 9, the change in gas pressure gradually decreased with increasing of AED3 concentration, demonstrating that the proposed method is capable of reliable detection of telomerase activity, as well as evaluation of potential inhibitors of telomerase. 19



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Figure 9. Telomerase activity with different concentrations of AED3. The concentrations of AED3 were 0, 1, 2, 5, 10, 15, 20 µM. The number of cells was 500. Error bars were calculated from three replicate measurements. The biggest error bar was ±2.8%. CONCLUSION In summary, a reliable, facile and ultra-sensitive POC strategy was developed for detection of telomerase activity at the single-cell level via a low-cost and portable pressure meter. Taking advantage of self-amplification of telomerase, the highefficiency of gas generation and enrichment by magnetic separation, the telomerase activity in 1 HeLa cell can be reliably detected. The pressure-based POC assay is facile and cost-effective and can avoid bulky apparatus and complex experiment procedures. The gas pressure of each sample can be rapidly measured. Moreover, the differences of telomerase activity in different cell lines were evaluated. The study on the inhibition demonstrated the feasibility of screening telomerase inhibitors. Compared with previous methods, this is the first POC assay that can detect telomerase activity at the single-cell level, and it holds great importance for studying

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the difference between individual cells. Therefore, it offers a promising platform for ultra-sensitive bioanalysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Effect of MBs on the catalytic decomposition of H2O2, TEM images of MBs and MBs/TS@PtNPs/cDNA, Effect of TS concentration, elongation time, PtNPs/cDNA concentration and hybridization time on the change of gas pressure respectively, Stability of gas pressure measurement (PDF) AUTHOR INFORMATION

Corresponding Author * Tel: 86-29-81530726; Fax: 86-29-81530727; E-mail: [email protected] Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT

We acknowledge financial support through grants from the National Natural Science Foundation of China (No.21375086), the Fundamental Research Funds for the Central Universities (GK201701002), and the Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28). 21



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for TOC only

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Point-of-Care Assay of Telomerase Activity at Single-Cell Level via

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