Detection Of Telomerase Activity Research Articles

A simple colorimetric detection of telomerase exploiting specific cleavage of exonuclease III coupled with telomeric DNA controlled aggregation of nanogold.

Telomerase activity has piqued scientists' interest for the reason that it has the potential to be employed for early-stage cancer detection, anticancer therapy and studies related to cancer progression and metastasis. Several approaches have been developed to detect telomerase activity. However, these approaches were lengthy, challenging to quantify, of limited sensitivity and prone to polymerase chain reaction (PCR)-related artefacts. We herein developed a novel nanoplasmonic sensing platform for colorimetric detection of telomerase activity relying on the telomere elongation of telomerase at the 3' end, structure-specific cleavage activity of exonuclease III that removes mononucleotides from the 3'-hydroxyl termini of double-stranded DNA, and electrostatic interaction of elongated telomeres with plasmonic nanoparticles. Using HeLa cells as a model for colorimetric detection of telomerase activity, this biosensor could detect telomerase activity with a detection limit of ∼100 cells per reaction by visible inspection and ∼5 cells per reaction by spectroscopic measurement and analysis time within about three hours. The proposed sensing method provided a novel tool for simple, rapid, and low-cost detection of telomerase activity, eliminating the necessity for thermal cycling, primers in PCR-based assays, and amplification of telomerase extension products. It exhibits significant potential as a label-free, simple, ultrasensitive strategy for on-site detection of telomerase activity in proteomics and clinical diagnostics.

Circ_0000263 improves radiosensitivity of Hela cells by inhibiting the activity of telomerase protein through miR-338-3p/TERT

Objective: To explore the effect and molecular mechanism of circ_0000263 on HeLa cell activity, apoptosis, telomerase activity, and radiosensitivity. Methods: The Hela cells were divided into si-NC, si-circ, vector, circ_0000263, anti-NC, anti-miR-338-3p, miR-NC, miR-338-3p, si-circ+anti-NC, si-circ+ anti-miR-338-3p, si-circ+vector, si-circ+TERT, sh-NC, sh-circ groups. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of circ_0000263 and miR-338-3p. Cell clone formation array was used to detect cell survival; cell counting kit-8 (CCK-8) to detect cell proliferation; flow cytometry to detect apoptosis; western blot method to detect the expressions of proliferating cell nuclear antigen (PCNA), Cleaved-casp3, telomerase reverse transcriptase (TERT) proteins; double luciferase assay to detect the targeting relationships of circ_0000263 and miR-338-3p, miR-338-3p and TERT; telomere repeat amplification enzyme linked immunosorbent assay (TRAR-ELISA) to detect telomerase activity. Results: Circ_0000263 was highly expressed in Hela cells, miR-338-3p was low expressed, and TERT was highly expressed; circ_0000263 was also highly expressed in Hela cells treated with radiation (P＜0.05). Knockdown of circ_0000263 inhibited the clone formation and cell proliferation ability of HeLa cells, and enhanced the radiosensitivity and apoptosis of HeLa cells. In contrast, knockdown of circ_0000263 decreased PCNA protein expression level and enhanced Cleaved-casp3 protein expression level in HeLa cells (P＜0.05). The apoptosis rate in the si-circ group was (13.19±1.12)%, which was higher than (6.80±0.62)% of si-NC group (P＜0.05). The apoptosis rate in the si-circ+4 Gy group was (24.82±1.57)%, which was higher than (17.00±0.96)% of si-NC+4 Gy group (P＜0.05). Circ_0000263 targeted regulated miR-338-3p, and miR-338-3p targeted regulated TERT. MiR-338-3p was lowly expressed in HeLa cells, and knockdown of circ_0000263 elevated miR-338-3p expression level in HeLa cells. Circ_0000263 regulated TERT expression and inhibited telomerase activity through miR-338-3p. MiR-338-3p/TERT can restore the effect of circ_0000263 on the radiosensitivity of Hela cells. The apoptosis rate in the si-circ+anti-NC group was (27.37±0.89)%, which was higher than (18.22±1.18)% of the si-circ+anti-miR-338-3p group (P＜0.05). The apoptosis rate in the si-circ+vector group was (27.55±0.48)%, which was higher than (20.10±0.68)% of si-circ+TERT group (P＜0.05). After 72 hours of radiation by 4 Gy, the cell survival fraction of si-circ+anti-NC group was 0.41±0.02, which was lower than 0.66±0.03 of the si-circ+anti-miR-338-3p group (P＜0.05); the cell survival fraction of si-circ+vector group was 0.42±0.05, which was lower than 0.70±0.03 of si-circ+TERT group (P＜0.05). Conclusion: Inhibiting the expression of circ_0000263 supresses the proliferation of Hela cells by regulating miR-338-3p/TERT, promotes apoptosis, inhibits telomerase activity, increases the radiosensitivity of cancer cells, and provides a theoretical basis for improving the radiosensitivity of Hela cells.

A triggered DNA nanomachine with enzyme-free for the rapid detection of telomerase activity in a one-step method

BackgroundTelomerase is considered a biomarker for the early diagnosis and clinical treatment of cancer. The rapid and sensitive detection of telomerase activity is crucial to biological research, clinical diagnosis, and drug development. However, the main obstacles facing the current telomerase activity assay are the cumbersome and time-consuming procedure, the easy degradation of the telomerase RNA template and the need for additional proteases. Therefore, it is necessary to construct a new method for the detection of telomerase activity with easy steps, efficient reaction and strong anti-interference ability. ResultsHerein, an efficient, enzyme-free, economical, sensitive, fluorometric detection method for telomerase activity in one-step, named triggered-DNA (T-DNA) nanomachine, was created based on target-triggered DNAzyme-cleavage activity and catalytic molecular beacon (CMB). Telomerase served as a switch and extended few numbers of (TTAGGG)n repeat sequences to initiate the signal amplification in the T-DNA nanomachine, resulting in a strong fluorescent signal. The reaction was a one-step method with a shortened time of 1 h and a constant temperature of 37 °C, without the addition of any protease. It also sensitively distinguished telomerase activity in various cell lines. The T-DNA nanomachine offered a detection limit of 12 HeLa cells μL−1, 9 SK-Hep-1 cells μL−1 and 3 HuH-7 cells μL−1 with a linear correlation detection range of 0.39 × 102–6.25 × 102 HeLa cells μL−1 for telomerase activity. SignificanceIn conclusion, our study demonstrated that the triggered-DNA nanomachine fulfills the requirements for rapid detection of telomerase activity in one-step under isothermal and enzyme-free conditions with excellent specificity, and its simple and stable structure makes it ideal for complex systems. These findings indicated the application prospect of DNA nanomachines in clinical diagnostics and provided new insights into the field of DNA nanomachine-based bioanalysis.

Measuring telomerase activity using TRAP assays.

Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.

HPF1 regulates tendon stem/progenitor cell senescence and tendon repair via PARP1-mediated poly-ADP ribosylation of HuR.

Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear. This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs. Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated β-galactosidase (SA-β-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD. Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence. HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.

DNAzyme-Mediated Biodeposition Coupling Adjustable Cascade Electric Fields for Photoelectrochemical Telomerase Activity Monitoring.

Telomerase, as a specialized reverse transcriptase, plays a vital role in early cancer diagnostics and prognosis; thus, developing efficient sensing technologies is of vital importance. Herein, an innovative "signal-on-off" photoelectrochemical (PEC) sensing platform was developed for ultrasensitive evaluation of telomerase activity based on an electron-transfer tunneling distance regulation strategy and DNAzyme-triggerable biocatalytic precipitation. Concretely, cascade internal electric fields between CuInS2 quantum dots (QDs), graphitic carbon nitride nanosheets (g-C3N4 NSs), and TiO2 nanorod arrays (NRAs) were developed to realize cascade electron extraction and hole transfer. Enabled by such a design, an effective "signal-on" state to gain a progressively enhanced PEC output was designed by suppressing the photogenerated electron-hole pair recombination. With the introduction of hairpin probe H2 and the subsequent extension of the primer sequence driven by the target telomerase, the CuInS2 QDs labeled with hairpin probe H1 were programmatically unfolded, resulting in CuInS2 QDs' close proximity to the working electrode away from the cascade interface, accompanied by the formation of G-quadruplex/hemin complexes. The gradual undermining of tunneling distance and implantation of DNAzyme-initiating biocatalytic precipitation tremendously induced the sluggish migration kinetics of the photoinduced charge, accompanied by the photocurrent intensity decrement, leading to the "signal-off" state. Under optimized conditions, the as-prepared PEC biosensor realizes ultrasensitive detection of telomerase activity from 10 to 105 cell·mL-1 with a detection limitation of 3 cells·mL-1. As a proof of concept, this well-designed method provides new insights into signal amplification for telomerase activity evaluation and also presents promising potential for further development in drug screening, healthcare diagnostics, and biological assays.

Digital CRISPR/Cas12a-based platform for precise quantification of telomerase activity

Precise quantification of telomerase activity is of great significance for telomerase-based early cancer diagnosis, as well as the screening of prodrugs. Telomeric repeat amplification protocol (TRAP) has been most widely used for assessing telomerase activity, which is limited by false-positive results, complicated operations, and long detection time. Herein, we develop a droplet digital hyperbranched amplification enhanced CRISPR/Cas12a-based telomerase detection platform (ddHECTelo) for precise quantitative detection of telomerase activity. By combining with the droplet digital platform and hyperbranched amplification, the ddHECTelo allows precise quantification of telomerase activity with single-molecule sensitivity. A polydisperse droplets digital system was employed, so that the reaction mixtures could be partitioned into picoliter-scale polydisperse droplets in several seconds to avoid the pre-amplification, without the need for a sophisticated microfluidic chip. This is the first digital isothermal platform for sensitive and precise quantification of telomerase activity, which shows great potential in telomerase-based early cancer diagnosis. It could also be used to analyze the telomerase activity in fundamental biomedical research.

An Activatable Nanoscintillator Probe for Detecting Telomerase Activity and Screening Inhibitors In Vivo

Telomerase represents an essential molecular machinery for tumor occurrence and progression and a potential therapeutic target for cancer treatment. Sensitive and reliable analysis of telomerase activity is of significant importance for the diagnosis and treatment of cancer. In this study, we developed a telomerase-activated nanoscintillator probe for deep-tissue and background-free imaging of telomerase activity and screening telomerase inhibitors in tumor-bearing living mice models. The probe was constructed by modifying lanthanide-doped nanoscintillators with aptamer-containing DNA anchor strands which hybridized with quencher labelled–oligonucleotide strands and telomerase primers. The X-ray-induced fluorescence of the probe was quenched originally but turned on upon telomerase-catalyzed extension of the primer. Benefiting from exceptional tissue penetrating properties and negligible autofluorescence of X-ray excitation, this probe enabled direct detection of telomerase activity in vivo via fluorescence imaging. Furthermore, with the direct, readable fluorescent signals, the probe enabled the screening of telomerase inhibitors in living cells and whole-animal models in the native states of telomerase. This strategy would inspire the development of low autofluorescence and deep tissue bioimaging probes for disease diagnosis and drug development in high-level living settings.

A SERS Biosensor Based on Functionalized Au-SiNCA Integrated with a Dual Signal Amplification Strategy for Sensitive Detection of Telomerase Activity During EMT in Laryngeal Carcinoma.

This paper aims to construct a surface-enhanced Raman spectroscopy (SERS) biosensor based on functionalized Au-Si nanocone arrays (Au-SiNCA) using a dual signal amplification strategy (SDA-CHA) to evaluate telomerase activity during epithelial-mesenchymal transition (EMT) in laryngeal carcinoma (LC). A SERS biosensor based on functionalized Au-SiNCA was designed with an integrated dual-signal amplification strategy to achieve ultrasensitive detection of telomerase activity during EMT in LC patients. Labeled probes (Au-AgNRs@4-MBA@H1) and capture substrates (Au-SiNCA@H2) were prepared by modifying hairpin DNA and Raman signal molecules. Using this scheme, telomerase activity in peripheral mononuclear cells (PMNC) could be successfully detected with a limit of detection (LOD) as low as 10-6 IU/mL. In addition, biological experiments using BLM treatment of TU686 effectively mimicked the EMT process. The results of this scheme were highly consistent with the ELISA scheme, confirming its accuracy. This scheme provides a reproducible, selective, and ultrasensitive assay for telomerase activity, which is expected to be a potential tool for the early screening of LC in future clinical applications.

CuS quantum dots activated DNAzyme for ratiometric electrochemical detection of telomerase activity.

Telomerase activity detection has attracted much attention concerning its importance for early cancer diagnosis. Here, we established a ratiometric electrochemical biosensor for telomerase detection based on CuS quantum dots (CuS QDs) dependent DNAzyme-regulated dual signals. The telomerase substrate probe was used as the linker to combine the DNA fabricated magnetic beads and CuS QDs. In this way, telomerase extended the substrate probe with repeated sequence to from hairpin structure, releasing CuS QDs as an input to DNAzyme modified electrode. DNAzyme was cleaved with high current of ferrocene (Fc) and low current of methylene blue (MB). On the basis of the obtained ratiometric signals, telomerase activity detection was achieved in the range of 1.0×10-12-1.0×10-6 IU/L, with the limit of detection down to 2.75×10-14 IU/L. Moreover, telomerase activity from HeLa extracts was also tested to verify the clinical application.

A visualized automatic particle counting strategy for single‐cell level telomerase activity quantification

AbstractThe accurate evaluation of telomerase activity, a typical cancer biomarker, is vital for early cancer screening. In this study, we developed a dark‐field microscopy (DFM) visual single‐particle detection scheme to detect telomerase activity based on automatic counting gold nanoparticles (AuNPs). This method started with attaching the telomerase substrate (TS) primer to the magnetic beads (MBs) through streptavidin‐biotin interaction. In the presence of telomerase and dNTPs, the TS primer was expanded with (TTAGGG)n repeat units to form the telomerase extension product (MBs‐telomerase extension product), which could be hybridized with the complementary DNA (cDNA) modified with AuNPs through Au‐S bonds (AuNPs‐SH‐cDNA). After magnetic separation and DNA double‐strand unwinding, AuNPs were collected from the supernatant, and the telomerase activity was quantitatively measured by visually counting bright spots based on DFM. This strategy achieved a limit of detection as low as 1 HeLa cell and distinguished telomerase activity among different cell lines, thus verifying its excellent sensitivity and specificity. Further, two common telomerase inhibitors (BIBR1532 and curcumin) were screened with the consistent IC50 values with other methods, respectively. It is worth mentioning that this strategy can clearly identify bladder cancer among various urinary diseases. Consequently, the visualized automatic particle counting strategy is potential as a powerful tool in early and noninvasive diagnosis of bladder cancer.

A dual-amplification strategy-intergated SERS biosensor for ultrasensitive hepatocellular carcinoma-related telomerase activity detection.

Telomerase has been considered as a biomarker for early diagnosis and prognosis assessment of hepatocellular carcinoma (HCC), while the highly sensitive and specific methods remain challenging. To detect telomerase, a novel surface-enhanced Raman scattering (SERS) biosensor was constructed using the dual DNA-catalyzed amplification strategy composed of strand displacement amplification (SDA) and catalytic hairpin assembly (CHA). This strategy relies on the extension reaction of telomerase primer induced by telomerase, forming long-stranded DNAs with repetitive sequence to catalyze the follow-up SDA event. Subsequently, the SDA products can trigger the CHA reaction between the SERS probes (Au-Ag nanocages (Au-AgNCs) modified with hairpin DNA1 and Raman reporters) and capture substrate (Au@SiO2 array labeled with hairpin DNA2), resulting in the formation of numerous "hot spots" to significantly enhance the SERS signal. Results are promising that the established biosensor presented excellent reproducibility, specificity and sensitivity. Moreover, ELISA was applied as the golden standard to verify the application of the proposed biosensor in real samples and the results confirmed the satisfactory accuracy of our method. Therefore, the proposed SERS biosensor has the potential to be an ideal tool for the early screening of HCC.

A Telomerase-Activated Magnetic Resonance Imaging Probe for Consecutively Monitoring Tumor Growth Kinetics and In Situ Screening Inhibitors.

Telomerase has long been considered as a biomarker for cancer diagnosis and a therapeutic target for drug discovery. Detecting telomerase activity in vivo could provide more direct information of tumor progression and response to drug treatment, which, however, is hampered by the lack of an effective probe that can generate an output signal without a tissue penetration depth limit. In this study, using the principle of distance-dependent magnetic resonance tuning, we constructed a telomerase-activated magnetic resonance imaging probe (TAMP) by connecting superparamagnetic ferroferric oxide nanoparticles (SPFONs) and paramagnetic Gd-DOTA (Gd(III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) complexes via telomerase-responsive DNA motifs. Upon telomerase-catalyzed extension of the primer in TAMP, Gd-DOTA-conjugated oligonucleotides can be liberated from the surface of SPFONs through a DNA strand displacement reaction, restoring the T1 signal of the Gd-DOTA for a direct readout of the telomerase activity. Here we show that, by tracking telomerase activity, this probe provides consistent monitoring of tumor growth kinetics during progression and in response to drug treatment and enables in situ screening of telomerase inhibitors in whole-animal models. This study provides an alternative toolkit for cancer diagnosis, treatment response assessment, and anticancer drug screening.

A label- and enzyme-free fluorescence assay based on thioflavin T–induced G-quadruplexes for the detection of telomerase activity

A label- and enzyme-free fluorescence assay based on thioflavin T–induced G-quadruplexes is developed to sensitively and specifically detect telomerase activity. Thioflavin T has a dual role as an efficient inducer and fluorescent probe, and the incorporation of thioflavin T into the thioflavin T–induced G-quadruplexes results in an intense fluorescence enhancement. In the presence of thioflavin T and K+, G-quadruplexes are formed by elongation of the telomerase substrate primer that is catalyzed by telomerase extracted from cancer cells. Thus, the telomerase activity in cancer cell extracts can be evaluated by measuring the thioflavin T fluorescence. More importantly, thioflavin T can specifically recognize and bind to G-quadruplexes, whereas it cannot recognize single- and double-stranded DNAs, which leads to the thioflavin T–based fluorescence assay exhibiting a reduced background and improved signal-to-noise ratio. As a result, the proposed assay has the linear range from 5 to 200 HeLa cells and the detection limit is 34 HeLa cells, which holds great potential for use in the detection of telomerase activity and the diagnosis of cancer.

Amplification-free detection of telomerase activity at the single-cell level via Cas12a-lighting-up single microbeads (Cas12a-LSMBs).

Telomerase overexpresses in almost all cancer cells and has been deemed a universal biomarker for cancer diagnosis and therapy. However, simple and ultrasensitive detection of telomerase activity in single-cells is still a huge challenge. Herein, we wish to report Cas12a-lighting up single microbeads (Cas12a-LSMBs) for ultrasensitive detection of telomerase activity without nucleic acid amplification. In this platform, single-strand DNA reporter (ssDNA reporter)-functionalized single-microbeads (functionalized-SMBs) are employed as a reactor for the trans-cleavage of telomerase-activated CRISPR/Cas12a as well as a reporting unit for fluorescence signal enrichment and visualization. Due to the space-confined effect and signal enrichment mechanism on the surface of the functionalized SMBs, the Cas12a-LSMBs can accurately detect telomerase activity in crude cell lysates with high specificity. Importantly, we have demonstrated that the Cas12a-LSMBs are a reliable and practical tool to detect telomerase activity in single cells and investigate cellular heterogeneity of telomerase activity from cell-to-cell variations.

Background-filtered telomerase activity assay with cyclic DNA cleavage amplification.

Overexpression of telomerase incites the abnormal proliferation of cancer cells. Thus, it has been regarded as a cancer biomarker and a potential therapeutic target. Existing assays suggest a promising sensing scheme to detect telomerase activity. However, they are complicated in terms of assay preparation and implementation. We herein report a Quenching-Exempt invader Signal Amplification Test, termed 'QUEST'. The assay leverages on a high turnover, specific cleaving enzyme, flap endonuclease I (FEN1), and graphene oxide (GO) for background (BG) filtering. In response to the target, FEN1 significantly boosts the signal with invader signal amplification. To distinguish the target signal, GO filters out the BG. It captures residual reporter invader probes (RP) to quench undesired signals. QUEST is straightforward without any pre-preparatory steps and washing/separation. Its probe design is simple and cost-effective. With QUEST, we investigated telomerase activities in various cell lines. Notably, we discriminated cancer cell lines from normal cell lines. In addition, a candidate inhibitor for telomerase was screened, which showed the promising potential of QUEST in real applications.

Entropy-Driven Self-Assembly of Single Quantum Dot Sensor for Catalytic Imaging of Telomerase in Living Cells

Telomerase is a highly valuable cancer diagnosis biomarker and a promising cancer therapy target. So far, most telomerase assays are limited by the involvement of tedious procedures, multiple enzymes, and complicated reaction schemes. Sensitive monitoring of low-abundant telomerase in living cells remains a challenge. Herein, we demonstrate an entropy-driven catalytic assembly of quantum dot (QD) sensors for accurate detection and imaging of telomerase activity in living cells. In this sensor, target telomerase specifically catalyzes extension of telomerase primer, and the extended primer subsequently acts as a catalyst to continuously initiate entropy-driven catalytic reaction, generating a large number of fluorophore- and biotin-labeled DNAs that can be self-assembled on the QD surface to induce an efficient Föster resonance energy transfer signal. The proposed sensor requires a single step for both recognition and amplification of the telomerase signal, eliminating the use of either protein enzymes or laborious procedures. Taking advantage of the inherent superiority of single-molecule fluorescence detection and high amplification efficiency of the entropy-driven reaction, this sensor demonstrates single-cell sensitivity for the in vitro assay. Moreover, it is capable of screening the telomerase inhibitor, discriminating different tumor cells from normal ones, and even real-time imaging telomerase in living cells, providing a novel platform for telomerase-associated cancer diagnosis and drug screening.

Multifunctional semiconducting carbon nitrides enabling sequential fluorescent sensing of telomerase activity and internal self-checking

Improving the detection reliability of biosensors is essential but challenging. The dual-signal self-checking function is useful for the development of reliable biosensors; however, it inevitably increases the complexity of the sensing system. Thus, biosensors with intrinsic self-checking functions that do not require additional chemicals, specific sensor assemblies, and/or instruments are highly desirable. Herein, integrating the ability of fluorescence detection and photodegradation by carbon nitride nanosheets (CNNS), a biosensor with a self-checking function for sensitive telomerase activity detection was constructed. Acriflavine (AF) was screened from various dyes to form the most effective CNNS-AF donor-acceptor (D-A) interfaces by experiments and density functional theory (DFT) calculations, considering spectral match, electrostatic interaction, and non-covalent stacking. As a result, the proposed fluorescence biosensor not only showed highly sensitive detection of telomerase activity but also had an internal self-checking function by photodegradation of AF via continuous excitation and measurement of recovered fluorescence. This study sheds light on CNNS-based D-A interfaces with synergistic fluorescence detection and photocatalysis-induced self-checking properties for future clinical diagnosis with high sensitivity and reliability.

Label-free palindromic DNA nanospheres as naked-eye colorimetric assay platform for detection of telomerase activity

A versatile naked-eye colorimetric assay platform is a desirable format for rapid analysis of disease-specific biomarkers, but is constrained by a complicated labeling process and false-positive signal readout. Target-triggered label-free palindromic DNA nanospheres (P-DNANS) were developed for the first time for the visual detection of telomerase activity. Telomerase recognition and extension trigger a hybridization event that yields Y-shaped double helices as core motifs. Y-shaped DNAs were linked by their palindromic sticky ends and P-DNANS were subsequently assembled with SYBR Green I (SG) in the helix. The SG-induced aggregation of gold nanoparticles is inhibited, and thus the red color can be observed by the naked eye. This label-free platform offers a cost-effective method for the cancer diagnosis and identification of breast cancer phenotypes.

Sensitive and amplification-free detection of telomerase activity by self-extension of telomerase and trans-cleavage of CRISPR/Cas12a

Telomerase is a well-known cancer biomarker, and the reliable and sensitive detection of telomerase activity is of vital significance for cancer diagnosis and treatment. Despite many attempts, the sensitive methods for telomerase activity assay mainly depend on the various nucleic acid amplification, which requires complex primers/probes design and multiple enzymes. In addition, the multiple experimental procedures may introduce false-positive results from cross-contamination of amplification products challenging the reliability of telomerase-based diagnostics detection. Herein, we present a reliable, amplification-free, and sensitive strategy for the detection of telomerase activity based on the self-extension of telomerase and trans-cleavage of CRISPR/Cas12a. In this work, crRNA/Cas12a complexes can specifically target the self-extending telomere-repetitive DNA and activate the typical CRISPR/Cas12a-based fluorescent output system, which allows the detection of telomerase activity in very few cancer cells (5 HeLa cells) without complex primer design and any nucleic acid amplification. Furthermore, the proposed method holds high reliability and specificity in distinguishing tumor cells from normal cells. With these unique features, including high sensitivity and reliability, excellent specificity, and simplicity, this method highlights a great potential for telomerase-related research and broadens the CRISPR-Cas12a-based sensing toolbox.