Highly sensitive monitoring of telomerase activity in living cells based on rapidly triggered cascade amplification reaction.

Telomerase is an important potential biomarker for the study of tumor progression. Herein, we designed a cascade-amplification-reaction-based nanoprobe for intracellular telomerase detection based on the integration of rolling circle amplification (RCA) and catalytic hairpin assembly (CHA) onto MnO2 nanosheets. Firstly, MnO2 nanosheets rapidly delivered and released signal amplification units into cells, and very short telomerase extension products formed RCA circular templates and initiated the exponential RCA, producing enriched telomere sequence amplification products. Then the amplification products specifically triggered the CHA process and numerous H1/H2 complexes were formed, realizing the exponential amplification of fluorescence signals. The detection limit is as low as 1 LoVo cell for telomerase activity in cell extract. We further designed a microfluidic chip with six independent cell culture regions for in situ fluorescence imaging. Simultaneous detection of six types of cells was realized on the chip, and only 1-2 µL of cell suspension and reagents are needed. Our detection method features faster response speed and stronger fluorescence signal. Telomerase in living cells showed strong fluorescence signal within 1.5 h, and tumor cells were effectively distinguished from normal cells. Telomerase activities of different types of tumor cells and activity changes were both monitored conveniently. These results demonstrate that this method holds the potential for the sensitive detection of low abundance biomarkers in living cells, and will contribute to cancer diagnosis, cancer treatment and telomerase-related drug screening.

A pH-responsive colorimetric detection of human telomerase RNA based on a three-dimensional DNA amplifier.

Human telomerase RNA (hTR), one of the essential components of telomerase, serves as a reverse template to add repeated segments of (TTAGGG)n to the 3' end of telomere DNA for maintaining the length of telomere DNA, endowing cells indefinite proliferation capability. Expression level of hTR displays a close relationship with tumor grade. Inspired by the mechanism of urease hydrolyzing urea to release ammonia and elevate the pH value of the sample solution, we developed a facile and novel pH-responsive colorimetric strategy for hTR detection by incorporating catalyzed hairpin assembly (CHA) onto the magnetic beads (MBs). The CHA process was initiated by target hTR and recycled via toehold binding and branch migration, thereby abundant urease being anchored on the surface of MBs. After separated by an external magnetic field, the assembled urease catalyzed the hydrolysis of urea to release a large amount of ammonia, which gave rise to a remarkable pH signal. Thus, quantification of hTR was achieved by measuring the solution pH via a hand-held pH meter or visualizing the solution color with the assistance of the pH indicator phenol red. The proposed sensing platform exhibits excellent performance toward hTR with a detection limit as low as 41 pM and a remarkable sequence selectivity, being able to differentiate a single mismatch in the target DNA. The pH-responsive colorimetric sensing platform contributes to introducing pH-related portable strategies into the detections of numerous universal biomarkers such as nucleic acids and proteins.

Electrochemical sensing platform based on kelp-derived hierarchical meso-macroporous carbons.

In this paper, kelp (Laminaria japonica), as a kind of abundant biomass, is used as the precursor for the preparation of kelp-derived hierarchical meso-macroporous carbons (K-dHMMCs) through the carbonization under nitrogen (N2) atmosphere at high temperature. The K-dHMMCs exhibits the unique structure with high specific surface area of 416.02 m2 g-1, large pore volume of 0.24 cm3 g-1, the hierarchical meso-macroporous size distribution centered at 2, 12 and 82 nm and high density of defective sites, enabling K-dHMMCs attractive for the electrocatalysis. Drop-casting K-dHMMCs on the glassy carbon (GC) surface allows the construction of K-dHMMCs based electrochemical sensing platform, which shows electrocatalytic activities towards many electroactive molecules, such as potassium ferricyanide, nicotinamide adenine dinucleotide (NADH), hydrogen peroxide (H2O2), dopamine (DA), uric acid (UA), ascorbic acid (AA), epinephrine (EP), l-tyrosine (Tyr) and acetaminophen (APAP). Especially, the K-dHMMCs modified GC (K-dHMMCs/GC) electrode exhibits higher sensitivity, wider linear range, and lower detection limit than both carbon nanotubes modified GC (CNTs/GC) and GC electrodes for H2O2 detection, which makes the K-dHMMCs/GC electrode to be able to determine the H2O2 levels in human urine sample and monitor the H2O2 released from human cancer cells. These results demonstrate that K-dHMMCs/GC possesses a great potential for conventional electrochemical sensing applications.