Highly Sensitive and Robust Detection of Human Telomerase Based on Strand-Displacement Cascade Amplification

Abstract

Telomerase is a well-validated diagnostic target for the early screening of cancers. To address the limitations of existing sensing methods, we herein fabricate a novel strategy based on the delicate integration of strand displacement cascade amplification and three-dimensional DNA nanostructure-assisted signal immobilization. Modified magnetic nanocomposites and electrodes provide suitable reaction interfaces, which facilitate a convenient purification process. The fabricated electrochemical method not only exhibits good sensitivity, selectivity, and stability but also demonstrates great utility for telomerase inhibitor screening and tumor cell discrimination. Besides, validation with human urine samples confirms the concordance with the clinical diagnostic conclusion. Therefore, this method holds significant potential as a point-of-care testing tool for telomerase-related applications.

Introduction

Telomerase is a unique ribonucleoprotein complex with reverse transcriptase activity, which catalyzes the extension of telomeric repetitive nucleotide sequences (TTAGGG) in the presence of primer and dNTPs as raw materials. , It plays a critical role in maintaining the integrity and stability of telomeres. In stem cells and progenitor cells, telomerase is activated to preserve the telomere length and maintain cell stemness. In most normal somatic cells, telomerase activity is nearly undetectable. However, it is overexpressed in most cancer cells to sustain unlimited proliferation. Therefore, telomerase is not only regarded as a biomarker for cancer diagnosis but also acts as a target for cancer treatment. Currently, there are different telomerase assays with distinct advantages and limitations. Telomere Repeat Amplification Protocol (TRAP) and modified versions are the golden standard. However, the protocol is semiquantitative, and it is difficult to provide accurate quantification. Other traditional techniques to detect relative gene expression or protein levels include reverse transcription quantitative PCR (RT-qPCR), Western blotting, quantitative fluorescence in situ hybridization (Q-FISH), and so on. , These methods are limited by indirect detection, complicated operation, or low sensitivity. In addition, a diversity of biosensing approaches have been developed utilizing the techniques including surface-enhanced Raman spectroscopy (SERS), fluorescence, and mass cytometry. For example, Xu et al. prepared DNA-driven nanoparticle self-assembling pyramid to encode Cy5 for intracellular telomerase analysis based on in situ Raman spectroscopy. He et al. achieved cancer cell discrimination by FRET-based ratiometric analysis of telomerase levels. Nevertheless, approaches with standardized procedures are still urgently needed.

Electroanalytical technique converts the information on a target into electric or electrochemical signals, which are widely applied in the fields of bioanalysis and environmental monitoring. − The merits of high sensitivity, simple instrumentation, cost-effective reagents, and portability are appealing for the analysis of telomerase. − To further improve the accuracy and sensitivity, different signal amplification strategies can be integrated. For example, Jia et al. synthesized the composites of platinum nanoparticles/macroporous carbon as electrochemical probes and utilized catalyzed hairpin assembly for amplified detection of telomerase. Liu et al. designed in situ formation of streptavidin-biotin-DNA-biotin networks to enhance the signal of the telomerase-catalyzed elongation reaction.

Toehold-mediated strand displacement amplification (TMSDA) is another powerful isothermal nucleic acid amplification technique that takes advantage of complementary base pairing and strand displacement to amplify target information. Generally, the key element is the design of a toehold domain appended to a double-stranded DNA substrate. The process includes toehold binding, branch migration, and an amplification cycle. TMSDA does not introduce additional enzymes, which exhibits the advantages of exceptional stability and easy modular design. In this study, we have integrated telomerase-catalyzed elongation/displacement and TMSDA. The cascade amplification reactions generate abundant single-stranded probes as reporters to be immobilized at the electrode via controlled triplex nanostructure formation. The entire process is highly efficient and enables accurate quantification of telomerase activity, which is validated using clinical specimens.

Results and Discussion

Confirmation of Nanocomposites

Magnetic Fe₃O₄ nanoparticles (MNPs) are used as the interface for TMSDA, which are also convenient for separation. , All DNA probes are designed and listed in Table S1. MNP1 and MNP2 are first modified with P1 and P3, respectively. Afterward, P2 probes can be immobilized on the surface of MNP1 via hybridization, while P4 and P5 are localized on the surface of MNP2 in the same way (Figure S1a). The successful synthesis of magnetic nanomaterials is confirmed by a scanning electron microscopic (SEM) image. The diameter is about 200 nm, and the sphere structure is well observed (Figure S1b). After the loading of DNA strands, MNPs exhibit an additional absorbance peak at 260 nm, demonstrating the successful modification of DNA strands (Figure S1c).

Principle of Telomerase-Triggered TMSDA

The working principle of the proposed electrochemical method is shown in Figure a. A primer can be attached on the P1/P2 duplex at the MNP1 interface. In the presence of telomerase, it is extended, and the produced strand displaces two P2 probes in succession. The released P2 acts as the trigger for subsequent TMSDA. Briefly, the duplex formed by P3/P4/P5 contains a toehold, which recognizes P2 and the gradual displacement reaction releases P4, creating a new toehold. The further introduced P6 strand hybridizes with this region and simultaneously releases P2 and P5. Free P2 can be recycled to generate more free P5 strands on the surface of MNP2. After magnetic separation, multiple P5 strands in the supernatant can be purified. The upstream and downstream strand displacement reactions are first predicted by NUPACK (Figures S2–S4). The formed products are also evidenced by the observation of the expected bands in the polyacrylamide gel electrophoresis images (Figure b). On the other hand, three-dimensional tetrahedral DNA (TDNA) is formed (Figure c), which is immobilized on the surface of the gold electrode. TDNA is able to capture P5 via triplex-forming oligonucleotide (TFO) assembly at neutral pH conditions. Methylene blue (MB) labeled at the 5′ terminal of P5, thus provides an intense electrochemical response, which is directly related with initial telomerase activity.

1.

(a) Illustration of the working strategy for the analysis of telomerase activity. (b) PAGE analysis of the strand displacement reaction: (1) P3, (2) P4, (3) P5, (4) P3/P4/P5, (5) P3/P4/P5+P2, (6) P2/P3/P5+P6, and (7) P2. (c) PAGE analysis of the TDNA assembly process: (1) T1, (2) T1+T2, (3) T1+T2+T3, (4) T1+T2+T3+T4.

Characterization of the Fabricated Electrochemical Interface

The electrochemical features of the gold electrodes during different modification steps are characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV), respectively. As shown in Figure a, a pair of well-defined current peaks are observed in the case of the bare gold electrode, indicating good conductivity of the substrate electrode. After the immobilization of TDNA, the negatively charged DNA scaffold repels the electrochemical species, [Fe­(CN)₆]^3–/4–. Therefore, the peak currents are significantly decreased. Meanwhile, the difference between oxidation and reduction peaks increases. The further loaded TFO strand (P5) makes the interface more negative, and the repellent effect is enhanced, which is evidenced by the further decreased CV peaks (Figure b). In the Nyquist diagrams, the changes are in good accordance with the CV conclusion (Figure c). During the stepwise modification of TDNA and P5, the diameter of the semicircle domains becomes larger, which indicates the increase of charge transfer resistance (R _ct). Detailed values are listed in Figure d. The modified MB at the end of P5 contributes to the significant SWV peak at −0.3 V, which reveals the assembly process well (Figure e). The P5 attachment and detachment can be monitored by MB signals. As shown in Figure f, the TDNA-modified electrode without MB exhibits a limited primary current at −0.3 V. After P5 incubation at neutral pH conditions, the peak intensity increases significantly. After pH adjustment, the TFO strand can be disassociated, and the peak current is recovered to its initial state. After additional three cycles of pH adjustment and P5 incubation, stable high and low peak intensities are recorded, demonstrating good regeneration capability.

2.

(a) Cyclic voltammograms of the bare electrode and TDNA-modified electrode before and after incubation with TFO. (b) Comparison of differences between oxidation and reduction peaks. (c) Nyquist diagrams of the bare electrode, TDNA-modified electrode before and after incubation with TFO. (d) Comparison of corresponding R _ct values. (e) Square wave voltammograms of the bare electrode and TDNA-modified electrode before and after incubation with TFO. (f) Corresponding peak current intensities for TFO assembly in different cycles of regenerated electrodes. Statistical analysis is performed using one-way ANOVA. Statistical significances are defined as **p < 0.01, ****p < 0.0001.

Analytical Performance

The SWV technique is utilized for the quantitative analysis of telomerase activity. To achieve the best analytical performance, some critical experimental parameters are optimized, including 0.75 μM TDNA for electrode modification and 0.6 μM P6 and 90 min for the strand displacement reaction (Figure S5). Under these conditions, SWV curves reflecting a series of concentrations of telomerase are overlaid. As shown in Figure a, with the increase of telomerase activity, the SWV peak becomes larger, as expected. The detailed relationship between the telomerase concentration and peak current variation (ΔI_p) is depicted in Figure b. A linear range is established from 10 to 10000 cells (Figure c). The limit of detection (LOD) is estimated to be 1 cell/mL, which is superior to most existing assays (Table S2).

3.

(a) Square wave voltammograms for the detection of telomerase with a series of concentrations. (b) Calibration curve reflecting the relationship between peak current and telomerase concentration. (c) Linear range. (d) Concentration-dependent decline of telomerase activity after the introduction of MST-312. (e) Selectivity investigation of the telomerase assay with potential interfering proteins. (f) SWV responses in the cases of telomerase extracted from heat-treated HeLa, HeLa, L02, MCF-7, and HepG2 cells. Statistical analysis is performed using one-way ANOVA. Statistical significance is defined as ****p < 0.0001.

Telomerase inhibitors may be used for potential therapeutic applications. To check the ability of this method to screen telomerase inhibitors, we introduced N,N′-1,3-phenylenebis-[2,3-dihydroxybenzamide] (MST-312) as a model. A series of standard concentrations of MST-312 solutions are prepared to disturb the telomerase-catalyzed reaction. According to the measured SWV peak current intensities, the relative activities of telomerase are calculated. As depicted in Figure d, with the increase of MST-312, the relative activity becomes decreased. The IC₅₀ of MST-312 is estimated to be 0.68 μM, which is consistent with those in previous reports. The result indicates that the electrochemical method is robust for screening telomerase inhibitors. To check the anti-interference ability, several common proteins, including bovine serum albumin (BSA), trypsin, lysozyme, and single-stranded DNA-binding protein (SSB), are introduced. None of these potentially interfering proteins can trigger the extension reaction and subsequent strand displacement reaction, which are demonstrated by the barely unchanged peak intensities (Figure e). After heat treatment, telomerase can be inactivated. We have also compared telomerase activities extracted from different cell lines. As shown in Figure f, heat-treated HeLa cells are comparable with the control, while untreated HeLa cells can generate a high ΔI_p value. In addition, telomerase extracted from L02, MCF-7, and HepG-2 cells can exhibit different electrochemical responses, which stand for different expression levels. Therefore, this electrochemical method can be utilized for the analysis of diverse cell lines.

Human Sample Analysis

We further investigated the efficacy of this electrochemical method for identifying urine samples collected from healthy donors and bladder cancer (BC) patients (Figure a). A total of 24 samples were previously collected. Among them, 12 were diagnosed as BC. After pretreatment, the two groups of samples were tested by conducting electrochemical detection. The ΔI_p values of healthy donors and BC patients are presented in Figure b and c, respectively. The electrochemical variations of healthy individuals show statistically significantly lower intensities compared with those of BC patients (Figure d). The receiver operating characteristic (ROC) curve analysis is further established to quantitatively assess the analytical performance in clinical samples (Figure e). An impressive AUC value of 0.986 indicates superior sensitivity and specificity for diagnosing BC.

4.

(a) Illustration of the process for extracting telomerase from clinical urine samples, strand displacement amplification, and electrochemical detection. (b) Heat map of the electrochemical response of telomerase across samples from 12 healthy individuals. (c) Heat map of the electrochemical response of telomerase across samples from 12 BC patients. (d) Box plot of SWV responses for the analysis of the two groups of real samples. (e) ROC curve analysis of the electrochemical method for the detection of 24 clinical samples. Statistical analysis is performed using Student’s t test. Statistical significance is defined as ****p < 0.0001.

Experimental Section

Materials and Instruments

Ferric chloride, sodium citrate dihydrate, urea, glutaraldehyde, polyacrylamide, CHAPS, BSA, trypsin, lysozyme, SSB, phenylmethylsulfonyl fluoride (PMSF), MST-312, and tris­(2-carboxyethyl)-phosphine hydrochloride (TCEP) were ordered from Sigma (USA). Other reagents were of analytical grade and used without further treatment. The cell lines of HeLa, L02, MCF-7, and HepG2 were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human urine samples were collected from the People’s Hospital of Suzhou New District. All oligonucleotides were purchased from Sangon Biotech (Shanghai, China). Sequences and modifications were listed in Table S1. Double-distilled water was purified by a Millipore system. SEM image was taken by using a Hitachi S4800 scanning electron microscope (Hitachi, Japan). UV–vis absorption spectra were measured using a Thermo Scientific NanoDrop One^C spectrophotometer (USA). Gel electrophoresis images were taken by a GelDoc XR+Imaging System (Bio-Rad, USA).

Preparation of DNA-Modified Magnetic Nanoparticles

Pristine Fe₃O₄ nanoparticles were first prepared via a hydrothermal method. Generally, 40 mL of solution containing ferric chloride (0.5 g), sodium citrate dihydrate (1.5 g), and urea (0.5 g) was prepared. Polyacrylamide solution (0.5 g in 40 mL) was then slowly added to the above mixture with vigorous stirring. After being transferred into a 100 mL Teflon-lined autoclave, the temperature was increased to 200 °C for 12 h. Next, the obtained product of Fe₃O₄ nanoparticles was isolated using a magnet, followed by washing with absolute distilled water and ethanol. Subsequently, 1 mg of Fe₃O₄ nanoparticles and 200 μL of glutaraldehyde solution with the concentration of 2.5% were spiked into 300 μL of PBS (pH = 7.4). After incubation for 4 h, glutaraldehyde-activated nanoparticles were isolated using a magnet and washed three times. The redispersed nanoparticles were then added to 200 μL of streptavidin solution (12 μg/mL). The mixture was treated with ultrasonic processing for 30 min. Afterward, the solution was shaken and incubated at 4 °C for 9 h. The magnetically purified nanoparticles were then blended with biotin-modified DNA strands (5 μM) for 1 h to achieve the conjugation. Briefly, MNP1 was modified with P1 and P2, while MNP2 was modified with the P3/P4/P5 duplex.

Preparation of TDNA-Modified Electrode

DNA strands of T1, T2, T3, and T4 were dissolved in PBS buffer solution containing 100 mM NaCl (pH = 7.5). The four strands were blended with the final concentrations of 0.75 μM. The mixture was heated to 95 °C, which was then slowly cooled to room temperature for the assembly of TDNA. The gold electrode was first incubated with piranha solution (98% H₂SO₄:30% H₂O₂ = 3:1) for 10 min. After that, it was polished with 1.0, 0.3, and 0.05 μm-sized alumina slurries, followed by sequential sonication in ethanol and ultrapure water for 10 min. The electrode was further electrochemically activated in 0.5 M H₂SO₄ via CV scans for 20 cycles. After careful rinsing, TDNA was applied to the surface of the gold electrode for 8 h.

Telomerase-Catalyzed Strand Displacement Reactions

All cells were cultured in DMEM containing 10% FBS. The cells were collected after trypsin-assisted detachment, and they were washed and resuspended in 10 mM Tris-HCl buffer (pH 7.4) containing 1 mM MgCl₂, 1 mM EGTA, 0.1 mM PMSF, 0.5% CHAPS (w/v), and 10% glycerol (v/v). The solutions were placed on ice for 30 min and then pipetted for several times. Next, the resulted solutions were centrifuged for 30 min at 4 °C (12000 rpm). Telomerase in the supernatant could be diluted with the extension solution (20 mM Tris-HCl, 1 mM EGTA, 63 mM KCl, 1.5 mM MgCl₂, 10 mM dNTPs, 0.05% Tween 20, pH 8.3). These diluted solutions were further mixed with primer (1 μM), MNP1 (0.1 mg/mL), and MNP2 (0.5 mg/mL) at 37 °C for 90 min. Subsequently, these nanomaterials were removed by magnetic separation. The TDNA modified electrode was incubated with the supernatant for 60 min.

Electrochemical Measurements

EIS, CV, and SWV experiments were conducted using a CHI 660D electrochemical workstation (CH Instruments, China). A three-electrode system was integrated, consisting of a reference electrode, a counter electrode, and a TDNA-modified working electrode. The electrolyte for EIS and CV was 5 mM [Fe­(CN)₆]^3–/4– containing 1 M KNO₃. The parameters included 0.208 V of biasing potential, 5 mV of amplitude, 0.1–100 000 Hz of frequency range, and 100 mV/s of CV scan rate. The electrolyte for SWV was 20 mm Tris-HCl buffer containing 100 mm NaCl and 50 mM MgCl₂ (pH 7.5). The parameters included a 50 mV/s scan rate and a 4 mV step potential.

Regeneration of the Electrode Sensing Interface

After electrochemical detection of telomerase, the gold electrode was incubated with an alkaline environment to remove the strands of P5, which were previously attached at the TDNA scaffold. Generally, the electrode was first rinsed with pure water and then immersed in 500 μL of Tris-HCl (10 mM, 100 mM NaCl, 50 mM MgCl₂, pH 10) at 37 °C for 60 min. Subsequently, the electrode was further rinsed with pure water before the additional tests.

Conclusions

In summary, we have delicately designed telomerase-initiated strand displacement reactions on two independent MNPs. The produced single-stranded DNA can be captured on the edges of TDNA on the electrode via the TFO assembly. This electrochemical biosensor not only exhibits excellent sensitivity due to the cascade amplification but also achieves satisfactory electrode regeneration for additional tests. Good stability, specificity, and reproducibility are demonstrated. Moreover, the proposed method can not only screen potential inhibitors of telomerase but also discriminate expression levels of telomerase extracted from different cells. The performance for identifying BC patients’ samples is quite satisfactory, verifying great potential utility for clinical applications.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.6c00090.

• Illustration and characterizations of MNP conjugation; predictions of secondary structures of the reactions; optimizations of TDNA concentration, P6 concentration, and reaction time; DNA sequences; comparison of telomerase activity assays (PDF)

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.