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Label-free Detection of Telomerase Activity in Urine using Telomerase-Responsive Porous Anodic Alumina Nanochannels Xu Liu, Min Wei, Yuanjian Liu, Bingjing Lv, Wei Wei, Yuanjian Zhang, and Songqin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01817 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 17, 2016

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Analytical Chemistry

Label-free Detection of Telomerase Activity in Urine using Telomerase-Responsive Porous Anodic Alumina Nanochannels Xu Liu,a Min Wei,b Yuanjian Liu,a Bingjing Lv,a Wei Wei,a* Yuanjian Zhang,a and Songqin Liua a

Key Laboratory of Environmental Medicine and Engineering, Ministry of Education,

Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China b

College of Food Science and Technology, Henan University of Technology,

Zhengzhou, 450001, China *

Corresponding author. Tel.: 86-25-52090613; Fax: 86-25-52090618.

E-mail: [email protected] (W. Wei)

1

ACS Paragon Plus Environment


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ABSTRACT Telomerase is closely related to cancers, which makes it become one of the most widely known tumor marker. Recently, many methods have been reported for telomerase activity measurement, in which complex label procedures were commonly used. In this paper, a label-free method for detection of telomerase activity in urine based on steric hindrance changes induced by confinement geometry in the porous anodic alumina (PAA) nanochannels was proposed. Telomerase substrate (TS) primer was first assembled on the inside wall of PAA nanochannels by Schiff reaction under mild condition. Then, under the action of telomerase, TS primer was amplified and extended to repeating G-rich sequences (TTAGGG)x, which formed multiplex G-quadruplex in the presence of potassium ions (K+). This configurational change led to the increment of steric hindrance in the nanochannels, resulting in the decrement of anodic current of potassium ferricyanide (K3[Fe(CN)6]). Compared with previously reported methods based on PAA nanochannels (usually one G-quadruplex formed), multiplex repeating G-quadruplex formed on one TS primer in this work. As a result, large current drop (~ 3.6 µA, 36%) was obtained, which gave facility to improve the detection sensitivity. The decreased ratio of anodic current has a linear correlation with the logarithm of HeLa cell number in the range of 10 ~ 5 000 cells, with the detection limit of 7 cells. The method is simple, reliable, and has been successfully applied in the detection of telomerase in urine with good accuracy, selectivity and reproducibility. In addition, the method is nondestructive test compared to blood analysis and pathology tests, which is significant for cancer discovery, development and prognosis.

Keywords: Telomerase; G-quadruplex; Nanochannels; Porous Anodic Alumina; Electrochemistry

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■ INTRODUCTION Telomeres, which located at the end of the eukaryotic chromosome and repeats (TTAGGG)x by the thousands of base composition, is essential to maintain telomere length and ensure the accuracy of genetic information in the replication process.1 Telomerase is a ribonucleoprotein reverse transcriptase that catalyzes the addition of the telomeric repeats (TTAGGG)x onto the end of the human chromosomes, which includes three components: telomerase RNA component (TR/TERC), telomerase associated protein (TP) and telomerase reverse transcriptase (TERT).2 Telomerase activity is inhibited in normal somatic cells, but a large number of data indicate that at least 80% human cancers related to the expression of telomerase.3 A number of studies have indicated that telomerase expression is associated with cell immortalization and tumorigenesis.4 Thus, a simple, nondestructive telomerase activity detection method is necessary for cancer diagnosis, anticancer drugs screening, and cancer therapy evaluation.5 Various methods have been studied to detect the telomerase activity after the discovery of telomerase in 1985. The traditional telomeric repeat amplification protocol (TRAP) was developed as a most widely used method to measure telomerase activity in small samples of cell or tissue extracts, due to its ultra-high sensitivity.5,6 It suffers some drawbacks such as the risk of carry-over contamination, susceptible to polymerase inhibition by clinical extracts and laborious post-PCR processing.7 Although some modified TRAP techniques have been developed to overcome these shortcomings, the practical usefulness is still limited.8 Recently, some assays based on alternative techniques have been developed for detecting telomerase activity,9 including

fluorescence,10-14

chemiluminescence,15-18

colorimetry,19-22

surface

enhanced Raman scattering (SERS),23-25 electrochemical detection,26-31 and so on.32-35 Photostable imaging and multigaps embedded nanoassemblies enhance in situ Raman spectroscopy for intracellular telomerase activity were also reported.36,37 Among these methods, electrochemical strategies were attractive due to their outstanding advantages of simple operation, rapid response, high-sensitivity, high compatibility, and low cost.30,31 The disadvantage was that complex or expensive label procedures were commonly necessary in these methods. Recently, nanopore and nanochannel based sensing has attracted more and more attentions since ionic conductance is a highly sensitive transduction mechanism for 3



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measuring changes in surface charge, confinement geometry and/or ionic concentration induced by surface reactions, which avoided complex label procedures.38-46 A biomimetic potassium ions (K+) responsive nanochannel based on G-quadruplex DNA conformational switching in a synthetic nanopore was reported by Jiang.40 Jiang had also researched a bio-inspired double-gated nanochannel which was dual-responsive to K+ and pH. The two gates of the nanosystem can be turned on and off alternately or simultaneously by switching DNA conformations, which can realize four kinds of ionic transport properties.41 Wu developed a universal strategy for nanopore sensing by employing the combination of aptamer and host-guest interactions.42 As a promising material for nanochannel-based biosensor, porous anodic alumina (PAA) membrane have many advantages of tunable nanopore diameter, well-defined nanochannel array, ease of surface functionalization, and commercial availability. By monitoring of the change in diffusion flux of the indicator molecules, the target molecules that induced the steric hindrance in the nanochannel could be determined. Our group43 reported a K+ detection method based on the conformational switching of DNA aptamer in PAA nanochannels. Xia reported a morpholino-functionalized nanochannel array for label-free DNA analysis and single nucleotide polymorphisms detection.44,45 Burrows reported a nanopore method to detect 8-Oxoguanine in the human telomere repeat sequence.46 In these sensing system, the steric hindrance change in the nanochannel or nanopore was sensitive to the structure switching from single-stranded DNA to G-quadruplex. Herein, a simple, fast, and label-free method to detect telomerase activity based on its induced repeating G-rich sequences (TTAGGG)x, which folded into G-quadruplexes in the presence of K+ and changed the steric hindrance in the PAA nanochannels. The strategy was shown in Scheme 1. The PAA nanochannels were silanized and modified with telomerase substrate (TS) primers through a Schiff reaction, potassium ferricyanide (K3[Fe(CN)6]), indicator molecules, produced highest anodic current in the PAA nanochannels before amplification (route a). When telomerase were introduced into the nanochannels, TS primers were extended with repeating G-rich sequences, which subsequently folded into several G-quadruplexes. Such conformational switching led to a significant increment of the steric hindrance in the nanochannels. As a result, the produced anodic current of K3[Fe(CN)6] decreased obviously (route b). The number of G-quadruplexes were calculated to be about 3 by chronocoulometry (CC) with hexaammineruthenium(III) chloride (RuHex) as 4


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indicator. As a result, larger current drop (~ 3.6 µA, 36%) was obtained compared with previously reported methods based on PAA nanochannels (usually one G-quadruplex formed), which gave facility to improve the detection sensitivity. It was founded that the current decrement ratio (D) was in a linear relation with the logarithm of the HeLa cell number, which could be used to detect telomerase activity. The proposed method is simple, reliable, and has been successfully applied to detect telomerase activity in urine with good accuracy, selectivity and reproducibility, which is significant to the clinical cancer diagnosis and the cancer development evaluation. ■ EXPERIMENT SECTION Chemicals

and

Materials.

CHO-labelled

(5′-CHO-AAAAAAAAAAATCCGTCGAGCAGAGTT-3′)

was

TS

primer

synthesized

by

Sangon Biotech Co. Ltd. (Shanghai, China). PAA was purchased from Hefei Puyuan Nanotechnology Co. Ltd. (Anhui, China). (3-Aminopropyl) triethoxysilane (APTES) was obtained from Sigma-Aldrich (Shanghai, China). Benzaldehyde, KCl, and K3[Fe(CN)6] were supplied from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (California, USA). Phenylmethanesulfonyl fluoride

(PMSF),

3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate

(CHAPS), ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid (EDTA), and hexaammineruthenium(III) chloride (RuHex) were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO). All other reagents of certified analytical grade were purchased from Sunshine biotechnology (Nanjing, China). Ultrapure water (18.2 MΩ cm, Barnstead, Thermo Scientific, USA) was used throughout the study. Real urine samples from normal, inflammation and bladder cancer patients were received from Nanjing General Hospital of Nanjing Military Command. The buffer solutions employed in this study were as follows: Tris buffer solution (10 mM Tris-HCl, pH 7.4); Phosphate buffered saline (PBS) solution (pH 7.2 ~ 7.4, 136.89 mM NaCl, 2.67 mM KCl, 8.24 mM Na2HPO4, 1.76 mM NaH2PO4); CHAPS lysis buffer (0.5% (w/v) CHAPS, 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, 10% (v/v) glycerol); Telomerase extension reaction buffer (20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 1 mM EGTA, 63 mM KCl, 0.005% (v/v) Tween 20, 1 mM dNTPs). 5



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Cell Culture and Telomerase Extraction. HeLa cells, A549 cells, MCF-7 cells, and MDA-MB-231 cells were seeded in DMEM supplemented with 10% FBS, penicillin (100 µg/mL), and streptomycin (100 µg/mL) in 5% CO2, 37 °C incubation. All kinds of cells were collected in the exponential phase of growth, and 1 million cells were dispensed in a 1.5 mL eppendorf tube, washed twice with ice-cold PBS solution, and redispersed in 200 µL of ice-cold CHAPS lysis buffer. The cells were incubated for 30 min on ice and then centrifuged for 20 min (12 000 rpm, 4 °C). Without disturbing the pellet, the cleared lysate was carefully transferred to a fresh tube, flash frozen, and stored at -80 °C before use. For extraction of telomerase from urine samples, fresh urine (200 mL) was collected and centrifuged at 1 000 rpm for 10 min at 4 °C and washed with PBS solution. The above samples were centrifuged at 1 800 rpm for 5 min at 4 °C. The precipitate was redispersed in 2 mL of ice-cold lysis buffer and then incubated on ice for 30 min. The mixture was centrifuged at 12 000 rpm for 20 min at 4 °C. The supernatant was transferred and stored at -80 °C before analysis. Modified PAA with TS primer. The functionalization of PAA nanochannels and assembled with aptamer were illustrated in Scheme 1. The morphology and size of PAA membranes were characterized by field-emission scanning electron microscopy (SEM, ZEISS, Germany) with the acceleration voltage of 5 kV. PAA membranes with different pore diameters of 25 ± 5, 55 ± 15, 90 ± 10, 130 ± 20, 180 ± 20 nm were washed by ethanol and ultrapure water in sequence to remove the impurities in the nanochannels. At room temperature, the PAA membranes were first dried by nitrogen and then immersed into 1 mL ethanol solution containing 5% APTES. Amino groups generated on the inner wall of the PAA nanochannels after shaken gently for 12 h.47 After that, the PAA membranes were washed by ethanol again to remove residual silyating reagents and dried by nitrogen. Then, 10 µL of 60 µM TS primer solution was dropped onto the surface of the PAA membrane for 24 h reaction.48 It was important that the PAA membrane was hanged in the air-tight glass bottle with some water at the bottom. The saturated moisture in the bottle prevented the 10 µL TS primer solution from evaporating over 24 h. Immersing the PAA membrane into 1 mL of ultrapure water containing 0.1% benzaldehyde and shaking gently for 12 h to block the remaining amino groups. And then the PAA membranes were washed with ultrapure water to remove the unbound TS primer and residual benzaldehyde. Then, the functionalized PAA membrane with TS primer immobilized on the inner wall was 6


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obtained and stored in Tris buffer solution at 4 °C. Telomerase Amplification Reaction. An appropriate volume of telomerase extracted from a certain number of cells was diluted in CHAPS lysis buffer and then added into the telomerase extention reaction buffer containing dNTPs and TS primer. The mixture was pipetted to the PAA surface and incubated at 37 °C for 5h. For inhibition of telomerase, BIBR 1532 or curcumin was performed in the presence of 3 000 cells. For negative control experiments, telomerase extracts were heat-treated at 95 °C for 10 min. Electrochemical Measurements. All electrochemical measurements were performed with a homemade nanodevice (Scheme 1). A platinum plate was selected as a working electrode and it was placed on a conductive copper pedestal. Then, the PAA membrane was put on the platinum plate. After that, an insulating block (poly(methyl methacrylate), PMMA) was placed onto the PAA membrane, which containing a cell for electrolyte. A silicone O-ring between the PAA membrane and the PMMA cell to prevent liquid leakage. A CHI 660C electrochemical workstation was used for all electrochemical measurements (Chenhua Instrument, Shanghai, China). A platinum electrode as counter electrode and a saturated calomel electrode (SCE) as a reference electrode, which formed a three-electrode electrochemical system with the platinum plate under the PAA membrane. The steady-state anodic current of 5 mM K3[Fe(CN)6]

was used to illustrate the steric hindrance changes in

PAA nanochannels with and without the telomerase treatments. Before the electrochemical measurement, the system were standing for a few minutes in order to keep K3[Fe(CN)6] concentration homogeneous inside and outside the nanochannels. All currents were read at 600 s in order to keep the data consistent and valid. CC was measured in Tris buffer solution containing 50 µM RuHex. The condition of CC measurements was as follows: initial potential = -0.5 V, final potential = 0.2 V, number of steps = 2, pulse width = 0.25 s, sample interval = 0.002 s, sensitivity (C or A/V) = 10×e–5 A/V. Quantification of Extension Repeating G-quadruplex Number on Inner Wall by CC. The modification of PAA membrane and extension of TS primer was operated as previously described. The number of amplified repeating sequences (x in (TTAGGG)x) were quantified by CC with RuHex as indicator.49,50 Detailed deduced processes were presented as the follows: 7



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Q=

భ/మ శ ஼బ

ଶ௡ி஺஽బ

గ భ/మ

Γ଴ =

‫ ݐ‬ଵ/ଶ + ܳௗ௟ + ݊‫ܣܨ‬Γ଴

(ொ್೐೑೚ೝ೐ ିொ೏೗ ) ௡ி஺ ௭

Γ஽ே஺ = Γ଴ (௠)ܰ஺ ΔΓ =

ொೌ೑೟೐ೝ ିொ್೐೑೚ೝ೐ ௡ி஺

(TTAGGG)୶ =

୼୻(୫/଺)ேಲ ୻ವಿಲ

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(1) (2) (3) (4) (5)

The parameters were listed as follows: D0, diffusion coefficient of RuHex; C0, primary concentration of RuHex; t, time of electrolysis; n, number of electrons per molecule for reduction (n = 3); F, Faraday constant (C/equiv); A, electrode area (cm2); Qdl, capacitive charge (C); Γ0, amount of redox marker, RuHex (mol/cm2); ΓDNA, probe surface density (molecules/cm2); m, the number of bases in the TS primer (m = 27); ∆Γ, elongated products per electrode area (mol/cm2); z, charge of the redox molecule (z = 3); NA, Avogadro’s number (molecules/mol); Qbefore, charge before amplification; Qafter, charge after amplification, and (TTAGGG)x, average amplification time per hexanucleotide per primer molecule. ■ RESULT AND DISCUSSION Conformational Switching of the Primer in the Nanochannels. A nanochannel-based electrochemical method was applied to study the relationship between the conformational switching of various aptamers and the concentration of their target molecules. The consumption of K3[Fe(CN)6] at the working electrode decreased the concentration of K3[Fe(CN)6] inside the nanochannels, which facilitated the diffusion of K3[Fe(CN)6] molecules in the bulk electrolyte into the nanochannels. Thus, a flux of K3[Fe(CN)6] formed, which reflects the steric hindrance in the nanochannels and was indicated by the steady-state anodic current of [Fe(CN)6]3-. Here, “D” was introduced to represent the current drop ratio, which was defined as D = 1 - I/I0, where I0 and I were the steady-state current for PAA/TS primer and PAA/TS primer/telomerase, respectively. As shown in Figure 1, it can be seen that the steady-state anodic current decreased obviously when TS primer modified PAA was incubated with 3 000 HeLa cells for PAA with various pore diameters. This indicated that the repeating G-rich sequences were extended on the TS primer under the action 8


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of telomerase, which decreased the flux of [Fe(CN)6]3- sharply. Compared with our previously reported work, in which less than 1.00 µA (< 20%) current drop were obtained in the PAA nanochannel when G-quadruplex was formed in the presence of K+ or ATP, 3.2 µA (~ 30%) current drop were obtained when 3 000 HeLa cells were used in this method(Figure 1G). The reason was that multiple repeating G-rich sequences were extended on one DNA primer by telomerase. The large current drop ratio gave facility to improve the detection sensitivity. CC method was used to calculate the repeat number of extension by using adsorbed RuHex as indicator. RuHex binded electrostatically to the phosphate backbone of DNA immobilized on the electrode giving rise to an electrochemical signal whose intensity was proportional to the base pairs. As shown in Figure 2, the charge increased obviously after the amplification. The number of G-quadruplexes were calculated to be about 3 according to the previously mentioned method. Impacts of Pore Diameter, DNA Concentration, and Incubation Time. To achieve optimal performance of the present biosensing device, various factors influence the steric hindrance in nanochannels such as pore diameter of nanochannels, DNA concentration, and telomerase amplification time were optimized. The pore diameter had considerable effect on the current drop ratio. Figure 1A-E illustrated the SEM images of the top and cross-sectional views with different pore diameter of PAA membrane. It can be seen that the pore diameter was uniform in the nanochannels. Figure 1F-J showed the current drop ratio in various pore diameter of PAA. It was founded that the current drop ratio for 25 nm PAA was 13% (Figure 1F), while it increased to 30% with 55 nm PAA (Figure 1G). It was reported that the size of telomerase is about 13 nm and the human telomerase is only functional as a dimer.50,51,52 So, the reason for the smaller D values in 25 nm PAA is that telomerase entered into the 25 nm pores is more difficult than into 50 nm ones. On the other hand, telomerase activity always decreased in the confined cavity. However, the current was getting lower and lower from 55 nm to 90, 130 and 180 nm (Figure 1H-J). It should be noted that there was different nature of the observed current drop in the smaller and larger pores. The possible reason for the D became smaller and smaller with the increasing large pores was that more effective primer were anchored on 55 nm PAA due to its relatively large specific surface area. On the other hand, the formed G-quadruplex had smaller and smaller effects on the free transport area in nanochannels with the increasing large pores. Hence, 55 nm PAA was used as 9



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optimized membrane in the experiments.

DNA concentration also exerted important effect on the currents. Figure 3A illustrated the effect of DNA concentration on the value of D. It was clearly that the D increased quickly as the DNA concentration increased from 10 µM and then leveled off at 60 µM. Figure 3B showed the relationship between the amplification time and the current drop ratio. The current drop also increased sharply at first and reached a platform at 5 h as the amplification time went on. Thus, 60 µM DNA and 5 h amplification time were used in the subsequent research. Detection of Telomerase Activity. Under optimum detection conditions, the telomerase activity of HeLa cell extracts was determined by the proposed method. As shown in Figure 4A, when the HeLa cell number increased from 0 to 5 000 cells, the steady-state current dropped obviously because an increasing steric hindrance happened in the nanochannels. This suggested that the degree of conformational switching in the nanochannels was strongly depended on the number of HeLa cells. In brief, the larger number of HeLa cell led to the more G-quadruplex formation in the nanochannels. As shown in Figure 4B, current drop ratio leveled off when the number of HeLa cell was higher than 5 000. The inset plot showed a linear relationship between D and the logarithm of HeLa cell number ranging from 10 cells to 5 000 cells (using logarithmic x-axis scaling). The linear equation was D = - 3.8784 + 10.6004 log N (N means the number of HeLa cell), and the correlation coefficient was 0.9945. The detection limit was 7 cells at a signal-to-noise ratio of 3. The result of this method was almost comparable to that obtained from sensitive strategies based on multiple telomeric hemin/G-quadruplex triggered polyaniline deposition (1 cells),53 the chemiluminescence assay,15 the SERS signal amplification assay,23 and HCR enzyme-free amplification (2 cells).28 It was superior to the most reported assays such as

the

dual-functional

electrochemical

biosensor

(500

cells),27

electrochemiluminescence sensor based on multifunctional Au nanoparticles (148 cells),16 the structure-switching DNA assay (100 cells),54 the DNAzymebased fluorescence assay (200 cells),55 and the quantum dots based biosensor assay (185 cells).56 Detection of Telomerase activity from various cell lines. The proposed sensor could be used to detect telomerase activity extracted from other cancer cell lines. In 10


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this case, four cancer cell lines including HeLa, A549, MCF-7 and MDA-MB-231 cells were selected as examples. Results showed that all of these cell lines were positive for telomerase activity, whereas the heat-treated HeLa cells was negative and used as control (Figure 5A). It was also found that telomerase activity in the HeLa and A549 cells was higher than that in MCF-7 and MDA-MB-231 cells. These results were consistent with the previously reported results, demonstrating the reliability of the assay.19 Selectivity and Reproducibility. Selectivity of the proposed method were studied by using HeLa (3 000 cells), bovine serum albumin (BSA, 100 µg), glucose oxidase (GOD, 100 µg), and horse radish peroxidase (HRP, 100 µg) as controls. As shown in Figure 5B, the current drop for HeLa cells was 30%, while all current drops were less than 5% for BSA, GOD or HRP, which were similar to the heated HeLa cells. Thus, this proposed method has high selectivity for detection of telomerase. When the cell number increased from 100 to 5 000, the relative standard derivations were in the range of 1.25 ~ 2.60% (n = 3), indicating that the method had good reproducibility. Therefore, these results indicated that the proposed strategy held great potential for clinical applications. Detection of Inhibition Efficiency of BIBR 1532 and Curcumin. Telomerase activity is usually reactivated in most cancer cells but not in neighboring normal cells. Activation of telomerase and telomere stabilization are important necessary steps in tumorigenesis. Thus, two kinds of compounds BIBR 1532 and curcumin were selected as model inhibitors to verify the proposed method. Currently, BIBR 1532 is the most effective inhibitor act on hTERT, an important target for cancer therapy. Several auxiliary proteins such as TEP-1, p23 and hsp90 were also involved in telomerase activity regulation. Curcumin can control the positioning of telomerase by separating Hsp90-p23 and hTERT. Therefore, curcumin provides an important way to adjust the activity of telomerase. The inhibition efficiency (%) was evaluated as follows: Inhibition (%) =

Iଶ − Iଷ × 100% Iଵ − Iଶ

where I1, I2, I3 were the steady-state current of [Fe(CN)6]3- throughout PAA without telomerase, with telomerase, with telomerase and inhibitors, respectively. As shown in Figure 6A and B, the inhibition efficiencies increased gradually with the increasing concentration of inhibitors and then leveled off at certain 11



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concentration. The derived IC50 value for BIBR 1532 and curcumin were 300 ± 27 nM, 12.8 ± 1.4 µM, respectively, which were in accordance with the previously reported results.57, 58 Hence, the assay can be used for rapid evaluation and screening of the telomerase inhibitors, which has a potential to be applied in discovery of new anticancer drugs. Clinical Applicability. To certify the applicability and reliability of the proposed strategy in clinical application, 7 human urine samples including 1 normal, 1 inflammation and 5 bladder cancer patients were pretreatment for detection (Table 1). A threshold of 10.75%, the mean value of D from 50 blank samples plus 3 times the relative standard deviation, was proposed to evaluate the development of the bladder cancer. The current drop ratio in normal individual or inflammation patient was 4.0% and 5.7%, respectively. Both of them were significantly lower than the threshold value. It was also founded that the current drop ratio was more than 18.9% for all the bladder cancer urine samples, which was higher than the proposed threshold value. On the other hand, the higher D values corresponds to the more serious bladder cancers patient, indicating that the results were consistently matched with the bladder cancers’ developments. Therefore, this sensitive, reliable and noninvasive method has great potential to be developed for cancer development evaluation. ■ CONCLUSIONS In summary, a simple label-free strategy for detection of telomerase was constructed via nanochannel-based electrochemical platform. The TS primer was assembled on the inside wall of PAA channels. Telomerase would extend the repeat sequences (TTAGGG)x on the TS primer, which folded into multiple G-quadruplexes in the presence of K+. Multiple G-quadruplexes increased the steric hindrance in the nanochannels sharply and resulted in almost 3.6 µA current drop, which increased the telomerase detection sensitivity. The method could detect telomerase activity in real urine samples from normal, inflammation or bladder cancer patients. According to the proposed threshold value, the judgments for bladder cancer were in coincidence with clinical diagnosis. In conclusion, the proposed label-free method for detection of telomerase activity was simple, sensitive and reliable, which was nondestructive compared to blood analysis and pathology tests and is significant for cancer discovery, development and prognosis. 12


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■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Phone: 86-25-52090613. Fax: 86-25-52090618.

Notes The authors declare no competing financial interest. ■ ACKNOWLEDGEMENTS Research reported in this publication was supported by National Natural Science Foundation of China (Grant Nos. 21475020, 21375014), Fundamental Research Funds for the Central Universities and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (1107047002). ■ ASSOCIATED CONTENT Supporting Information Functionalization of the inner walls of PAA nanochannels with TS primer and the SEM images for top and bottom of PAA with various pore diameters were listed in supporting information.

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Figure captions Scheme 1. Schematic illustration of the telomerase activity detection strategy via nanochannel-based electrochemical platform. Figure 1. SEM images for the top (top) and cross-sectional (bottom) views of PAA membranes with various pore diameters: (A) 25 nm, (B) 55 nm, (C) 90 nm, (D) 130 nm, and (E) 180 nm. The scale bar is 200 nm. Panels F, G, H, I, and J show time-course curves of steady-state currents of Fe[(CN)6]3- flowing through the PAA modified membrane before (black line) and after (red line) amplification with various pore diameter. 3 000 HeLa cells were used. Figure 2. Chronocoulometry for the TS primer immobilized PAA (a) in the absence of RuHex, (b) before and (c) after amplification in the presence of RuHex. 3 000 HeLa cells were used. Figure 3. The impact of (A) DNA concentration and (B) amplification time on the current drop ratio. 3 000 HeLa cells were used. Error bars showed the standard deviation of three experiments. Figure 4. (A) Time-course curves of current for different number of HeLa cells (a) 0, (b) 10, (c) 50, (d) 100, (e) 200, (f) 1 000, (g) 2 500, (h) 5 000, (i) 7 500, and (j) 10 000, respectively. (B) Calibration curves of the current drop ratio versus different number of HeLa cells. Insets: linear plots of calibration curves versus the logarithm of HeLa cell number ranging from 10 cells to 5 000 cells (using logarithmic x-axis scaling). The pore diameter of the PAA nanochannels was 55 nm. Figure 5. (A) Telomerase activity detection extracts from various cell lines. (B) Selectivity of the biosensor. 3 000 cells were used for each cell line. Heated inactive HeLa cells were used as negative control. Error bars show the standard deviation of three experiments. Figure 6. The inhibition effect of (A) BIBR 1532 and (B) curcumin on telomerase activity. 3 000 HeLa cells were used. Error bars showed the standard deviation of three experiments. Table 1. Comparison of the results obtained by the proposed method and clinical 17



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diagnosis.

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Scheme 1.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Table 1. Comparison of the results by clinical diagnosis and the proposed method. No. Patient ID Clinical diagnosis D in the method/% Judgment 1 — normal 4.015 Negative 2 1003729557 inflammation 5.719 Negative 3 1003424591 bladder cancer 42.284 Positive 4 1007045669 bladder cancer 25.229 Positive 5 1007677148 bladder cancer 21.653 Positive 6 1007924814 bladder cancer 18.997 Positive 7 1007922178 bladder cancer 30.873 Positive a

Threshold of 10.75% (the mean value of D in 50 blank samples plus 3 times the standard deviation) was proposed to evaluate the positive or negative of bladder cancer.

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For TOC only

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Label-Free Detection of Telomerase Activity in ... - ACS Publications

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