Kefeng Wu

Label-free fluorescence turn-on detection of uracil DNA glycosylase activity based on G-quadruplex formation.

We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research.

An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

MicroRNAs (miRNAs) act as biomarkers for the diagnosis of a variety of cancers. Since the currently used methods for miRNA detection have limitations, simple, sensitive, and cost-effective methods for the detection of miRNA are required. This work demonstrates a facile, quencher-free, fluorescence-based analytical method for cost-effective and sensitive detection of miRNA using a super 2-aminopurine (2-AP)-labeled hairpin probe (HP) and exonuclease I activity. Specifically, the fluorescence of 2-AP is strongly quenched when it is incorporated within DNA. In the presence of a target miRNA, HP attains an open conformation by hybridizing with the target miRNA to form a double-stranded structure with a protruding 3′-terminus. Next, the digestion of the protruding 3′-terminus is triggered by exonuclease I, during which 2-AP is released free in solution from the DNA, thereby increasing fluorescence. This method is highly sensitive, with a detection limit of 0.5 nM—10 times lower than a previously reported quencher-free fluorescence method. Furthermore, this method has potential applications in clinical diagnosis and biomedical research.

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.

A fluorescence-based assay for T4 polynucleotide kinase/phosphatase activity based on a terminal transferase-aided photoinduced electron transfer strategy

We have developed a new assay for the detection of T4 polynucleotide kinase/phosphatase activity (T4 PNKP) based on a terminal transferase-aided photoinduced electron transfer (PIET) strategy. The method is highly sensitive and the T4 PNKP detection limit was estimated to be 0.01 U mL−1, which is superior or comparable to previously reported assays. Furthermore, the proposed method was also applied to assay the inhibition of T4 PNKP activity. This approach may offer potential applications in drug screening, clinical diagnostics and some other related 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%.

Label-free and nicking enzyme-assisted fluorescence signal amplification for RNase H determination based on a G-quadruplexe/thioflavin T complex

In this paper, we describe a novel, label-free and nicking enzyme-assisted fluorescence signal amplification strategy that demonstrates to be cost efficient, sensitive, and unique for assaying the RNase H activity and inhibition based on G-quadruplex formation using a thioflavin T (ThT) dye. This novel assay method is able to detect RNase H with a detection limit of 0.03 U /mL and further exhibits a good linearity R2 = 0.9923 at a concentration range of 0.03-1 U/mL under optimized conditions. Moreover, the inhibition effect of gentamycin on the RNase H activity is also studied. This strategy provides a potential tool for the biochemical enzyme analysis and inhibitor screening.

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.

A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition

Adenosine deaminase (ADA), able to catalyze the irreversible deamination of adenosine into inosine, can be found in almost all tissues and plays an important role in several diseases. In this work, we developed a label-free fluorescence method for the detection of adenosine deaminase activity and inhibition. In the presence of ADA, ATP has been shown to be hydrolyzed. The ATP aptamer was shown to form a G-quadruplex/thioflavin T (ThT) complex with ThT and exhibited an obvious fluorescence signal. However, the ATP aptamer could bind with ATP and exhibited a low fluorescence signal because of the absence of ADA. This assay showed high sensitivity to ADA with a detection limit of 1 U/L based on an SNR of 3 and got a good linear relationship within the range of 1–100 U/L with R2 = 0.9909. The LOD is lower than ADA cutoff value (4 U/L) in the clinical requirement and more sensitive than most of the reported methods. This technique exhibited high selectivity for ADA against hoGG I, UDG, RNase H and λexo. Moreover, this strategy was successfully applied for assaying the inhibition of ADA using erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and, as such, demonstrated great potential for the future use in the diagnosis of ADA-relevant diseases, particularly in advanced drug development.