Telomerase Detection Research Articles

Simultaneous detection of telomerase and miRNA with graphene oxide-based fluorescent aptasensor in living cells and tissue samples

Telomerase and microRNAs (miRNAs) as important biomarkers are closely related to cancers. Simultaneous detection of telomerase activity and miRNAs would be beneficial to improve the specificity and reliability. Here, we establish a telomerase and miRNA-21 (miR-21) simultaneous sensing platform with graphene oxide-based fluorescent aptasensors (GOFA) including graphene oxide (GO), template strand (TS) primer and fluorophore-labeled telomerase/miR-21 oligonucleotides. Owing to π-π stacking interaction, TS primer and telomerase/miR-21 probes would be loaded onto GO, resulting in fluorescence quenching. However, in the presence of the telomerase or miR-21, the double-stranded oligonucleotides would be away from the GO surface attribute to the hybridization between the extended TS primers and telomerase probe as well as miR-21 and miR-21 probe, leading to obvious fluorescence recovery. We found that GOFA could simultaneously detect telomerase activity and miR-21 with low background signal, high sensitivity and simplified operation. Moreover, GOFA could be used for accurately detecting telomerase activity and miRNA in living cells and cancer patient tissue sample. This sensing platform shows great potential in improving the accuracy in clinical diagnosis of cancer.

Sensitive multicolor visual detection of telomerase activity based on catalytic hairpin assembly and etching of Au nanorods

Detection of telomerase activity is crucial for the telomerase-related early diagnosis of cancer and drug screening. Herein, a multicolor visual telomerase detection method with high sensitivity was developed based on the etching of hexadecyl trimethyl ammonium bromide-stablized Au nanorods (Au NRs) by the oxidation state 3,3′,5,5′-tetramethylbenzidine sulfate (TMB2+). In order to meet the demand of bare-eye inspection, an enzyme-free signal amplification strategy of catalytic hairpin assembly (CHA) was incorporated. After the introduction of telomerase, telomerase extension products specifically triggered cyclic CHA and led to the successive formation of G-quadruplex/hemin DNAzyme, which catalyzed the H2O2-mediated oxidation of TMB. The Au NRs were gradually etched as the concentration of catalysate TMB2+ increased, resulting in the decrease of aspect ratio of the Au NRs. Correspondingly, the longitudinal localized surface plasmon resonance peak of Au NRs was blue-shifted, with the concomitant generation of a series of color transition. Under optimal conditions, a highly sensitive detection toward telomerase was realized down to 15 HeLa cells. Compared to previous colorimetric method for telomerase determination, multiple colors corresponding to telomerase activity was the most attractive virtue of our approach. More strikingly, telomerase-induced cyclic CHA significantly enhanced the accumulation of G-quadruplex/hemin DNAzyme, thus the signal amplification was effectively realized. Our approach exhibited excellent sensitivity and convenient signal readout, which is expected to provide great potential application in cancer diagnosis and therapy.

Quantum dot-based electrochemical biosensor for stripping voltammetric detection of telomerase at the single-cell level

Human telomerase is responsible for the maintenance of chromosome end structures and is a valuable biomarker for malignant growth. However, the accurate measurement of telomerase activity at the single-cell level has remained a great challenge. Here we develop a simple quantum dot (QD)-based electrochemical biosensor for stripping voltammetric detection of telomerase activity at the single-cell level. We designed a thiol-modified capture DNA which may be immobilized on the gold electrode by the gold-sulfur bond. The presence of telomerase enables the addition of the telomere repeats of (TTAGGG)n to the 3′ end of the primer, accompanied by the incorporation of abundant biotins in the extension product with the assistance of the biotin-tagged dATP. The subsequent hybridization of extension product with the capture DNA and the addition of streptavidin-coated QDs induce the assembly of large amounts of QDs onto the electrode via specific biotin-streptavidin binding. After the acidic dissolution of QDs, the released Cd (II) can be simply quantified by anodic stripping voltammetry (ASV). Due to the introduction of large amounts of QDs by telomerase-induced primer extension reaction and the synergistic signal amplification induced by the release of Cd (II) from the QDs, this biosensor can detect telomerase activity at the single-cell level without the involvement of any thermal cycling and extra enzymes for signal amplification. Moreover, this assay exhibits a large dynamic range over four orders of magnitude and it is very simple without the involvement of specific hairpin probe design and complicated labelling, holding great potential in point-of-need testing.

A facile graphene oxide-based fluorescent nanosensor for the in situ "turn-on" detection of telomerase activity.

A facile and sensitive method for the quantitative detection of telomerase and in situ imaging of intracellular telomerase is developed by using a graphene oxide (GO)-based fluorescent nanosensor. The nanosensor consists of a fluorescent DNA (P1) adsorbed on the GO surface. Here, GO serves not only as a fluorescence quencher but also as a carrier to successfully transport P1 into cancer cells as a signal reporter. P1 is a dye-labeled single-stranded DNA complementary to the telomeric repeated sequence, and initially the combination of P1 and GO exhibits minimal background fluorescence. When telomerase extends its repeat units of TTAGGG on the 3'-end of the primer-DNA, the fluorescence of P1 is subsequently recovered because the telomeric repeated sequence can hybridize with P1 and liberate it from the GO surface. This method enables the determination of telomerase activity down to 10 cells. For the in situ detection of telomerase, upon endocytosis of the P1/GO combinatorial probe into living cancer cells, the intracellular telomerase extends its primer to produce the telomeric repeated sequence and then turns on the fluorescence of P1, which can be directly monitored by confocal laser scanning microscopy. The feasibility of the assay is further investigated by treating with telomerase-related drugs, and the results demonstrate its potential in antitumor drug screening and cancer therapy evaluation.

A simple "mix-and-detection" method for the sensitive detection of telomerase from cancer cells under absolutely isothermal conditions.

We develop a simple "mix-and-detection" method for the sensitive detection of telomerase from cancer cells under absolutely isothermal conditions. This method enables one-step and one-tube detection of telomerase with a detection limit of 3 cells, and it can be further applied for the screening of telomerase inhibitors and the discrimination of cancer cells from normal cells.

Cascaded Electrochemiluminescence Signal Amplifier for the Detection of Telomerase Activity from Tumor Cells and Tissues.

Telomerase is closely linked to the physiological transformation of tumor cells and is commonly overexpressed in most types of tumor cells. Therefore, telomerase has become a potential biomarker for the process of tumorigenesis, progression, prognosis and metastasis. Thus, it is important to develop a simple, accurate and reliable method for detecting telomerase activity. As a high signal-to-noise ratio mode, electrochemiluminescence (ECL) has been widely applied in the field of biomedical analysis. Here, our objective was to construct an improved ECL signal amplifier for the detection of telomerase activity.Methods: A cascaded ECL signal amplifier was constructed to detect telomerase activity with high selectivity via controllable construction of a lysine-based dendric Ru(bpy)32+ polymer (DRP). The sensitivity, specificity and performance index were simultaneously evaluated by standard substance and cell and tissue samples.Results: With this cascaded ECL signal amplifier, high sensitivities of 100, 50, and 100 cells for three tumor cell lines (A549, MCF7 and HepG2 cell lines) were simultaneously achieved, and desirable specificity was also obtained. Furthermore, the excellent performance of this platform was also demonstrated in the detection of telomerase in tumor cells and tissues.Conclusion: This cascaded ECL signal amplifier has the potential to be a technological innovation in the field of telomerase activity detection.

A triple-amplification strategy for sensitive detection of telomerase at the single-cell level

We develop a triple-amplification strategy for sensitive detection of telomerase from cancer cells using telomere-based primer generation-triggered rolling circle amplification in combination with enzyme-assisted cyclic signal amplification. This method can detect telomerase at the single-cell level, and it can be further applied for the screening of telomerase inhibitors for anticancer drug discovery.

Functionalized paper microzone plate for colorimetry and up-conversion fluorescence dual-mode detection of telomerase based on elongation and capturing amplification

As an important cancer biomarker and therapeutic target, telomerase has attracted special attention especially concerning its detection and monitoring. Here, we established a portable cellulose paper substrate for the detection of telomerase based on colorimetry and up-conversion fluorescence dual-mode methods The sterile cellulose paper substrate functionalized with telomerase substrate oligonucleotide were used as the capturing substrate, on which the telomerase extended telomeric repeats, and the corresponding telomeric repeat complementary oligonucleotide labelled with methylene blue or the up-conversion nanoparticles (UCNPs) were employed as the colorimetric or up-conversion fluorescence reporting nanolabels. The extended telomeric repeats subsequently facilitate capturing of the more reporting nanolabels via hybridization between telomeric repeat and its complementary oligonucleotide, inducing a significant amplified signal in the color and up-conversion intensity. Colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye, and the low background up-conversion fluorescence increases the sensitivity of this method. Moreover, the developed cellulose paper device is more stable and easily stores the tests results compared with majority solution-phase systems.

Visual, Label-Free Telomerase Activity Monitor via Enzymatic Etching of Gold Nanorods

Early diagnosis and life-long surveillance are clinically important to improve the long-term survival of cancer patients. Telomerase activity is a valuable biomarker for cancer diagnosis, but its measurement often used complex label procedures. Herein, we designed a novel, simple, visual and label-free method for telomerase detection by using enzymatic etching of gold nanorods (GNRs). First, repeating (TTAGGG)x sequences were extented on telomerase substrate (TS) primer. It formed G-quadruplex under the help of Hemin and K+. Second, the obtained horseradish peroxidase mimicking hemin/G-quadruplex catalyzed the H2O2-mediated etching of GNRs to the short GNRs, even to gold nanoparticles (GNPs), generating a series of distinct color changes due to their plasmon-related optical response. Thus, this enzymatic reaction can be easily coupled to telomerase activity, allowing for the detection of telomerase activity based on vivid colors. This can be differentiated sensitively by naked eyes because human eyes are more sensitive to color variations rather than the optical density variations. As a result, telomerase activity can be quantitatively detected ranging from 200 to 15000 HeLa cells mL-1. The detection limit was 90 HeLa cells mL-1 (S/N = 3). Importantly, the application of this method in bladder cancer samples was in agreement with the clinical results. Thus, this method was considerably suitable for point-of-care diagnostics in resource-constrained regions because of the easy readout of results without the use of sophisticated apparatus.

A PCR-free colorimetric strategy for visualized assay of telomerase activity

A simple yet powerful polymerase chain reaction (PCR)-free strategy for visualized assay of human telomerase activity was reported in this work. Gold nanoparticles (AuNPs) based colorimetric strategy was applied with well-designed enzyme-aided cyclic amplification. Briefly, the detection relies on the elongated primers of telomerase substrate (TS) induced by telomerase, which open the hairpin DNA and hybridize with linker DNA, the trigger of AuNPs aggregation. Nicking endonuclease was added in the sensing system, which cleaved linker DNA after hybridization and released complimentary strand for cyclic hybridization with linker DNA, resulted in high sensitivity for the detection of telomerase. Down to 25 HeLa cells with high expression of telomerase could be recognized. The proposed strategy provides a good platform for the determination of telomerase activity, differentiation of cancer cell lines from normal cell line and screening of telomerase-targeted anticancer drugs.

Convertible DNA ends-based silver nanoprobes for colorimetric detection human telomerase activity

Human telomerase is an endogenous ribonucleoprotein that is over-expressed in most types of malignant cancer cells. Sensitive and specific detection of telomerase activity is crucial for better understanding its role in cancer cells and further exploring its function in cancer diagnosis. Here, we develop convertible DNA ends-based silver nanoprobes for sensitive and specific colorimetric detection telomerase activity. Silver nanoprobes are constructed by modifying telomerase binding substrates (TS) that are pre-hybridized with complementary sequences onto silver nanoparticles (AgNPs), via the coordination between consecutive cytosines in TS strand and AgNPs. This forms blunt-end terminated, double-stranded DNA on the surface of AgNPs. Under the action of telomerase, TS on the silver nanoprobes are elongated with telomeric repeats, converting DNA stiff blunt ends to flexible single-stranded dangling ends. The dangling ends enhance the stability of nanoprobes and relieve their salt-induced aggregation, and the solution shows a yellow color. When telomerase is inactive, the blunt end-terminated nanoprobes cannot resist salt-induced aggregation, resulting in a gray color of solution. Based on telomerase-regulated DNA “blunt-dangling” ends conversion-induced AgNPs' dispersity and color change, colorimetric detection of the endogenous telomerase with AgNPs is realized. The detection limit is equivalent to 1 cell/μL of telomerase activity, and extracts from cancer cells and normal cells are visually distinguished through color difference. The proposed strategy will offer a new approach for reliable, convenient quantification of telomerase activity in biochemical research and clinical diagnosis.

Novel plasma telomerase detection method to improve cancer diagnostic assessment.

BackgroundThe activity levels of telomerase and its mRNA have been found to be more diagnostically sensitive than cytological results in many cancerous tissues and correlate well with the clinical disease stage. Currently, there are several methods of detecting telomerase in tissues and in blood. The most commonly used method is a conventional quantitative real-time polymerase chain reaction (PCR) which is time and labor exhausting.MethodsWe have developed a simple and innovative blood test method that allows us to diagnose cancer and relapsed cancer in a cost- and time -effective manner. We had evaluated our novel method in two populations: 1) in vivo in three mice with pancreatic ductal adenocarcinoma (PDAC) versus one control mouse and 2) clinically in 30 cancer patients versus 10 individuals without cancer. We compared our novel method with the old conventional method. At least one sample was obtained from each patient included in the study.ResultsThe novel method substantially increased the sensitivity (from 37% to 77%, p<0.001) and negative predictive value (from 32% to 56%, p = 0.005) of the telomerase test for all cancer patients (those who were substantially treated and those who were not). There was no significant difference in telomerase activity between cancer patients and healthy volunteers using the conventional method (p = 0.13), whereas there was a significant difference using the novel method (p = 0.001).ConclusionConventional method shows no significant difference in telomerase activity between cancer patients and healthy volunteers (p = 0.13), whereas there was a significant difference using the novel method (p = 0.001).

Reliable Förster Resonance Energy Transfer Probe Based on Structure-Switching DNA for Ratiometric Sensing of Telomerase in Living Cells.

In situ detection and monitoring of telomerase is of great importance as it is a relatively specific cancer target. However, the complexity of the biological system makes it difficult for the nanoprobe to keep absolutely stable and have a low background in living cells. This study designs a probe termed Förster resonance energy transfer (FRET) nanoflare to achieve ratiometric fluorescent detection of intracellular telomerase with higher specificity, which can effectively resist the disturbance from DNase I and GSH, etc. The probe is composed of a gold nanoparticle (AuNP) which is functioned with telomerase primer sequences (TS) and flares fluorescently labeled donors and acceptors at two terminals. In the presence of telomerase, flares are displaced from the primer sequences and form hairpin structures, so that the donors and acceptors are brought into close proximity, resulting in high FRET efficiency. The probe exhibits good performance for efficiently distinguishing tumor cells from normal cells and monitoring the change of intracellular telomerase activity during treatment with telomerase-related drugs, showing great potential for cancer diagnosis and estimating therapeutic effect.

Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

A programmed terminal extension strategy to light up multiple beacons for DNA and cellular telomerase detection.

Here we develop a new method for the sensitive detection of DNA and cellular telomerase using an enzyme-triggered terminal extension strategy that the produced strand can light up multiple beacons on the surface of gold nanoparticles.

Integration of intracellular telomerase monitoring by electrochemiluminescence technology and targeted cancer therapy by reactive oxygen species.

Cancer therapies based on reactive oxygen species (ROS) have emerged as promising clinical treatments. Electrochemiluminescence (ECL) technology has also attracted considerable attention in the field of clinical diagnosis. However, studies about the integration of ECL diagnosis and ROS cancer therapy are very rare. Here we introduce a novel strategy that employs ECL technology and ROS to fill the above vacancy. Briefly, an ITO electrode was electrodeposited with polyluminol-Pt NPs composite films and modified with aptamer DNA to capture HL-60 cancer cells with high specificity. After that, mesoporous silica nanoparticles (MSNs) filled with phorbol 12-myristate 13-acetate (PMA) were closed by the telomerase primer DNA (T-primer DNA) and aptamer. After aptamer on MSN@PMA recognized and combined with the HL-60 cancer cells with high specificity, T-primer DNA on MSN@PMA could be moved away from the MSN@PMA surface after extension by telomerase in the HL-60 cancer cells and PMA was released to induce the production of ROS by the HL-60 cancer cells. After that, the polyluminol-Pt NPs composite films could react with hydrogen peroxide (a major ROS) and generate an ECL signal. Thus the intracellular telomerase activity of the HL-60 cancer cells could be detected in situ. Besides, ROS could induce apoptosis in the HL-60 cancer cells with high efficacy by causing oxidative damage to the lipids, protein, and DNA. Above all, the designed platform could not only detect intracellular telomerase activity instead of that of extracted telomerase, but could also kill targeted tumors by ECL technology and ROS.

Assessing Telomerase Activities in Mammalian Cells Using the Quantitative PCR-Based Telomeric Repeat Amplification Protocol (qTRAP).

Telomerase expression and activity appear elevated in >80% of human cancers. The activity of the telomerase may serve as a diagnostic marker for malignancy, and an indicator of the proliferative potential of somatic and stem cells. The telomeric repeat amplification protocol (TRAP) is a sensitive and accurate PCR-based assay for telomerase detection and measurement. Here, we describe a quantitative PCR-based TRAP assay (qTRAP) that is more convenient and amenable to high-throughput applications compared to traditional gel-based TRAP assays. qTRAP can not only facilitate drug screening processes for compounds that regulate telomerase activities but also enable the measurement of total telomerase activities of cultured cells or clinical specimens; the latter should prove particularly valuable to investigators of malignancies and diseases that are associated with telomerase and telomere dysfunction.

Nanoscaled Porphyrinic Metal-Organic Frameworks for Electrochemical Detection of Telomerase Activity via Telomerase Triggered Conformation Switch.

In this work, we designed a nanoscaled porphyrinic metal-organic framework (PorMOF) with iron porphyrin as linker and zirconium ion as node for electrochemical detection of telomerase activity. The as-prepared PorMOF was characterized with scanning electron microscopy, powder X-ray diffraction, and spectroscopic techniques and demonstrated excellent electrocatalytic activity toward O2 reduction. Sequentially, the functionalization of PorMOF with streptavidin results in a water-stable electrochemical tracer for detection of telomerase. Upon the telomerase-triggered extension, the assistant DNA 1 (aDNA1)-assistant DNA 2 (aDNA2) duplex could switch into a hairpin structure, and thus, the aDNA2 was released and then hybridized with the capture DNA. Therefore, the PorMOF@SA tracer could be introduced on the electrode surface via biotin-streptavidin recognition, leading to the strong electrochemical signal for readout. The developed approach displayed desirable dynamic range and limitation of detection down to 30 HeLa cells mL-1. The telomerase activity was 2.2 × 10-11 IU in a single HeLa cell with good reproducibility and stability. The nanoscaled porphyrinic MOF provided a powerful platform for electrochemical signal transduction and had a promising application in the determination of various biomolecules.

Correction to Ratiometric Fluorescent Bioprobe for Highly Reproducible Detection of Telomerase in Bloody Urines of Bladder Cancer Patients

Correction to Ratiometric Fluorescent Bioprobe for Highly Reproducible Detection of Telomerase in Bloody Urines of Bladder Cancer Patients

Scissor‐Like Chiral Metamolecules for Probing Intracellular Telomerase Activity

A DNA‐driven gold (Au) heterodimer for intracellular telomerase detection is fabricated. The highly biocompatible and intracellularly stable probe shows an active chiroptical property in the visible region, due to the scissor‐like configuration formed by prolate nanoparticles. Importantly, the telomerase activity is specifically quantified using circular dichroism intensity in situ after internalization of the heterodimer into cancer cells. Moreover, the results clearly illustrate that this method has a remarkable linear range from 0.8 × 10−12 to 32 × 10−12 IU, and the limit of detection for telomerase activity is 1.7 × 10−15 IU in a single HeLa cell. This strategy paves the way for chirality‐based ultrasensitive detection of intracellular cancer markers.