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

Abstract

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

Electrochemical Detection and Point-of-Care Testing for Circulating Tumor Cells: Current Techniques and Future Potentials

“…The most important thing is that the output signal of POCT is very simple and easy to get, usually including color, distance, pressure, temperature, etc. [131][132][133][134][135].…”

“…Wang et al immobilized telomerase primer (TS) on the surface of magnetic bead (MB), and the primer could be extended under the action of telomerase, and formed a single-stranded DNA containing (TTAGGG) repeating unit, the repeating unit can specifically bind to cDNA-nanoparticles (PtNPs), using the H 2 O 2 catalytic ability of PtNPs, adding substrate H 2 O 2 , the gas is generated and the pressure changed. Using a portable pressure gauge to detect the change of the pressure could achieve the detection of single cell telomerase activity ( Figure 6C) [134].…”

“…(C) Use a portable pressure gauge to detect the pressure, to detect the telomerase activity of a single cells. Reprinted with permission from ref[134]. Copyright 2017, American Chemical Society.…”

<p>A smartphone-assisted pressure-measuring-based diagnosis system for acute myocardial infarction diagnosis</p>

“…[23][24][25][26] Different from traditional detection methods that are based on light, color, electrical activity, magnetic force, heat, or distance, the developed assay transforms molecular signal into pressure signal by enzyme-or catalyst (nanoparticles)-linked immunosorbent assay. Furthermore, a similar system was further applied for analysis at the single-cell level, 27,28 drug detection, and analysis of disease biomarkers. 29 PASS has shown high sensitivity, high reliability, and good portability in the reported works, which can be attributed to pressure sensor that is highly sensitive to pressure variations caused by the immunity assay.…”

A DNA Nanoflower‐Assisted Separation‐Free Nucleic Acid Detection Platform with a Commercial Pregnancy Test Strip

“…[4] These efforts have led to a variety of repurposed tests that are portable and cost-effective, require small sample volumes and offer excellent reliability. [8] However, these tests have additional requirements, such as the use of special substrates for signal outputs and/or additional steps to separate reaction products for detection. Taking PTS repurposing as an example, it is an antibody-based sandwich test strip to detect hCG and works by the mechanism shown in Figure S1.…”