Hailun He

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 fluorescence assay for rapid detection of RNase H activity based on Tb3+-induced G-quadruplex conjugates

Ribonuclease H (RNase H), a highly conserved damage-repair protein, hydrolyzes RNA in the DNA:RNA hybrid duplex and breaks RNA/DNA junctions. In this study, we demonstrated a unique Tb3+-based fluorescence assay that is low cost, facile, and label-free for assaying RNase H activity and inhibitions by using a G-quadruplex formation strategy. This novel assay method can detect RNase H at the lowest detection limit of 2 U mL−1 under optimal conditions. We demonstrated the utility of the assay by antibiotics screening and identified ethidium bromide (EB) and Gentamycin as RNase H inhibitors. This approach demonstrated that Tb3+ could be used as a functional tool in specific fields in the future.

Real-time monitoring of DNA methyltransferase activity using a hemimethylated smart probe.

A real-time assay for DNA methyltransferase (MTase) activity has been developed. A hemimethylated smart probe is used as the substrate for DNA MTase. Cleavage of the methylated product leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. The method permits real-time monitoring of DNA methylation process and makes it easy to characterize the activity of DNA MTase. It also has the potential to screen suitable inhibitor drugs for DNA MTase.

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 turn-on fluorescence assay of alkaline phosphatase activity using a DNA–silver nanocluster probe

Assays of alkaline phosphatase (ALP) activity play a critical role in clinical diagnostics and drug screening. In this work, we present a novel, sensitive and cost-effective analytical method for the detection of ALP activity based on the design of a G-rich DNA light-up DNA–silver nanocluster (AgNCs) probe and λ exonuclease (exo) cleavage reaction. Upon addition of λ exo, the G-rich DNA was cleaved, resulting in the inhibition of AgNCs to move closer to G-rich DNA sequences and, accordingly, only a low fluorescence signal was observed. Upon treatment of ALP, the 5′-phosphoryl end of pG-rich DNA was hydrolyzed and the λ exo cleavage reaction was impeded. The AgNCs were then enabled to move closer to the G-rich DNA sequences, resulting in an increase in fluorescence. Under optimized conditions, the fluorescence change was determined to be linear with the ALP concentration ranging between 1 U L−1 and 800 U L−1 (detection limit: 1 U L−1). Taken in concert, this strategy may provide a basis for a screening platform for ALP inhibitors.

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 label-free fluorescence method for the detection of uracil DNA glycosylase activity based on G-quadruplex formation

Here, we have developed a novel fluorescence strategy for sensitive detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation by using a label-free DNA hairpin probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the hairpin DNA, resulting in the conformational transition of the hairpin structure. Then, the probe DNA can be recognized quickly by the thioflavin T (ThT) dye resulting in an increase in fluorescence. This strategy could detect UDG activity as low as 0.01 U mL−1. In addition, the strategy was also applied for the detection of UDG activity in HeLa cell lysate. It is simple and of low cost without the requirement of labeling with a fluorophore–quencher pair. Furthermore, UDG activity inhibition by uracil glycosylase inhibitor (UGI) is shown, demonstrating the potential for the discovery and characterization of UDG-targeted drug candidates in pharmaceutical development.