Hanchun Chen

Fluorescence detection of adenosine triphosphate using smart probe

A novel fluorescent probe for adenosine triphosphate (ATP) assay based on DNA ligation is proposed in this article. This approach uses a novel smart probe, T4 DNA ligase, and two short oligonucleotides. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction and the ligation product restores the fluorescence of the smart probe. This method is very sensitive with a 0.5-nM limit of detection. Compared with current assay methods, the strategy is simpler, cheaper, and 40 times more sensitive.

Simultaneous detection of kinase and phosphatase activities of polynucleotide kinase using molecular beacon probes.

Phosphorylation and dephosphorylation of DNA by polynucleotide kinase (PNK) has an important role in DNA damage repair, replication, and recombination. Traditionally, it is assayed by denaturing gel electrophoresis and autoradiography, which are tedious and not sensitive. We report on the development of a sensitive and simple method for PNK assay based on DNA ligation using a molecular beacon. Enzyme activity of PNK is measured down to a limit of 0.002 unit/ml. The method not only provides a universal platform for simultaneous monitoring of kinase and phosphatase activities, but also shows great potential in biological research, drug discovery, and clinical diagnostics.

A facile label-free G-quadruplex based fluorescent aptasensor method for rapid detection of ATP

The present work demonstrates a simple, rapid and label-free ATP detection method using a fluorescent aptasensor that is based on G-quadruplex formation. In the absence of ATP, the Thioflavin T (ThT) dye binds to the G-rich ATP aptamer and forms an ATP aptamer/ThT G-quadruplex complex, which results in high fluorescence intensity. Upon addition of ATP, the ATP aptamer/ThT complex will be replaced by the formation of an ATP aptamer/ATP complex. During this process, separation of the ThT dye from the ATP aptamer/ThT complex decreases the fluorescence intensity of the reaction mixture dramatically. This fluorescence aptasensor is highly sensitive and rapid, with a detection limit of 18nM and a total reaction time of only 10min. Furthermore, this method is cost-effective and simple, removing the requirement for labeling the detection reagents with a fluorophore-quencher pair.

Label-free highly sensitive detection of telomerase activity in cancer cell by chemiluminescence imaging

We have developed a new methodology for label-free highly sensitive telomerase activity assay using chemiluminescence imaging. This method can detect the telomerase activity from as little as 10 cultured cancer cells without PCR. Furthermore, telomerase inhibition is shown, demonstrating the potential for screening of telomerase inhibitors as anticancer drug agents.

Label-free monitoring of DNA methyltransferase activity based on terminal deoxynucleotidyl transferase using a thioflavin T probe.

We have developed a new methodology for fluorescence turn-on detection of DNA methyltransferase (MTase) activity based on terminal deoxynucleotidyl transferase (TdT) using a thioflavin T probe. This method is highly selective and sensitive. The fluorescence intensity was direct proportion to Dam MTase concentration in the range from 0.1 to 8.0 U/mL with a detection limit of 0.1 U/mL. And because no labeling with a fluorophore-quencher pair was required, it is simple and low cost. We envision that our novel fluorescent detection method for Dam MTase activity could be applied as a useful tool in biomedical research.

Label-Free G-Quadruplex Aptamer Fluorescence Assay for Ochratoxin A Using a Thioflavin T Probe

Ochratoxin A (OTA) is one of the most common mycotoxins contaminating feed and foodstuffs. Therefore, a great deal of concern is associated with AFB1 toxicity. In this work, a fast and sensitive fluorescence aptamer biosensor has been proposed for the OTA assay. In the absence of OTA, the OTA aptamer can form a G-quadruplex structure with thioflavin T (ThT) dye, which results in increased fluorescence. After joining OTA, OTA aptamer combines with OTA and the G-quadruplex can be formed. Only faint fluorescence was finally observed when ThT weakly reacts with the quadruplex. Through this test method, the entire reaction and analysis process of OTA can be completed in 10 min. Under optimal experimental conditions (600 nM OTA-APT, 7 μM ThT, and 3 min incubation time), this proposed assay has a good limit of detection (LOD) of 0.4 ng/mL and shows a good linear relationship within the range of 1.2–200 ng/mL under the best experimental conditions. This method has a high specificity for OTA relative to Ochratoxin B (23%) and Aflatoxin B1 (13%). In addition, the quantitative determination of this method in real samples has been validated using a sample of red wine supplemented with a range of OTA concentrations (1.2 ng/mL, 12 ng/mL, and 40 ng/mL) with recoveries of 96.5% to 107%.

A Novel Detection Method of Human Serum Albumin Based on the Poly(Thymine)-Templated Copper Nanoparticles

In this work, we developed a facile fluorescence method for quantitative detection of human serum albumin (HSA) based on the inhibition of poly(thymine) (poly T)-templated copper nanoparticles (CuNPs) in the presence of HSA. Under normal circumstances, poly T-templated CuNPs can display strong fluorescence with excitation/emission peaks at 340/610 nm. However, in the presence of HSA, it will absorb cupric ion, which will prevent the formation of CuNPs. As a result, the fluorescence intensity will become obviously lower in the presence of HSA. The analyte HSA concentration had a proportional linear relationship with the fluorescence intensity of CuNPs. The detection limit for HSA was 8.2 × 10−8 mol·L−1. Furthermore, it was also successfully employed to determine HSA in biological samples. Thus, this method has potential applications in point-of-care medical diagnosis and biomedical research.

A sensitive detection method of carcinoembryonic antigen based on dsDNA-templated copper nanoparticles

As a tumor marker, carcinoembryonic antigen (CEA) is an effective indicator for the evaluation of the response to treatments of cancers. Therefore, it is necessary to find some sensitive and effective methods for CEA detection. Herein, we describe a facile method of CEA determination based on dsDNA-templated copper nanoparticles (CuNPs) coupled with an aptamer. In this fluorescence bioassay, the dsDNA residue contains a CEA-specific aptamer that seizes the formation of fluorescent CuNPs in the presence of CEA, resulting in weak fluorescence emission. In the absence of CEA, the dsDNA-templated CuNPs can generate strong fluorescence with emission peaks at 565 nm. This assay enables a sensitive and precise determination of CEA with a detection limit of 0.0065 ng mL−1 (S/N = 3) and functions successfully in biological samples.

Targeting ubiquitin specific protease 7 in cancer: A deubiquitinase with great prospects: Regulatory roles of USP7

Deubiquitinase (DUB)‐mediated cleavage of ubiquitin chain balances ubiquitination and deubiquitination for determining protein fate. USP7 is one of the best characterized DUBs and functionally important. Numerous proteins have been identified as potential substrates and binding partners of USP7; those play crucial roles in diverse array of cellular and biological processes including tumour suppression, cell cycle, DNA repair, chromatin remodelling, and epigenetic regulation. This review aims at summarizing the current knowledge of this wide association of USP7 with many cellular processes that enlightens the possibility of abnormal USP7 activity in promoting oncogenesis and the importance of identification of specific inhibitors.