A new spectrophotometric method for uric acid detection based on copper doped mimic peroxidase.

Cascade reactions catalyzed by natural uricase and mimic peroxidase (MPOD) have been applied for uric acid (UA) detection. However, the optimal catalytic activity of MPOD is mostly in acidic conditions (pH 2-5), mismatching the optimal catalytic alkaline environment of uricase. In this paper, using CuSO4 and urea as raw materials, a MPOD with high catalytic activity in alkaline environment was synthesized by hydrothermal method. Then, based on coupling reaction of uricase/UA/MPOD/guaiacol (GA) system, a novel spectrophotometric method was established to detect 5-60 µmol/L UA (limit of detection = 3.14 µmol/L (S/N = 3)) and accurately quantified serum UA (275.6 ± 39.9 µmol/L, n = 5) with 95-105% of standard addition recovery. The results were consistent with commercial UA kit (p > 0.05). The MPOD could replace natural POD to reduce the cost of UA detection due to simple preparation and cheap raw materials, and is expected to achieve the specific detection of some substances, like glucose and cholesterol, combined with glucose oxidase and cholesterol oxidase.

Synthesis optimization of rich-urea carbon-dots and application in the determination of H2S in rich- and barren-liquids of desulphurizing solutions.

In the research of carbon dots (CDs) containing various nitrogen sources, it was first found that urea/citric acid-CDs showed a selective discolouration reaction with sulphide ions. Therefore, by optimizing various synthesis and detection conditions of the CDs determining sulfur ions, such as the raw material ratio, temperature, time, pH, and oxidation atmosphere in the CD synthesis, a discolour CD-probe method for trace-level sulphide ions was developed. The method is environmentally friendly, shows two linear-response ranges in 0.050-1.0 mg L-1 (A = -0.0827c + 0.8366) and 1.0-15 mg L-1 S2- (A = -0.0209c + 0.7587) and can be used for the high and low concentration quantification of sulphide in various wastewaters. Subsequently, in order to realize the separation and detection of sulphide ions in wastewaters or rich- and barren-liquids containing N-methyldiethanolamine and other substances in desulphurizing solutions, an automatic pretreatment system was also established.

[Microassay of High-Risk Human Papillomavirus Genotype Based on R6G-ddATP/SNaPshot-Gel Fluorescence Method].

OBJECTIVE: R6G-ddATP was used as a dideoxy fluorescence substrate to establish the single base end extension (SNaPShot)-gel fluorescence method for the rapid detection of the genotypes of three high-risk human papillomaviruses (HR-HPV) ( HPV18, HPV33 and HPV35) genotypes. METHODS: HPV quality control products were used as as samples, and R6G-ddATP dideoxy fluorescence reagent was used as substrate. Firstly, HPV was amplified by using universal primers to obtain the first round of amplified products, which were purified and used as templates for subsequent SNaPShot reactions. Then, specific one-step extension primers were used to perform SNaPShot reaction to generate R6G-fluorescence-labeled DNA extension products. The product was subjected to agarose gel electrophoresis, the results of which were observed under a Gel Imager, and the HPV genotyping was done with different one-step extension primers. Each sample was tested three times and the results were compared with DNA sequencing results. RESULTS: The preferred annealing temperature for SNaPShot reaction is 55 ℃. Three HPV genotypes were examined by R6G-ddATP/SNaPShot gel fluorescence assay under optimal conditions, and the results were consistent with DNA sequencing results. CONCLUSION: The R6G-ddATP/SNaPShot-gel fluorescence method for the micro-detection methods of three HR-HPV genotypes was successfully established and can be used for rapid detection of HPV genotypes.

[Microanalysis of Acetaldehyde Dehydrogenase 2 Genotype Based on SNaPShot-FCA].

OBJECTIVE: To establish SNaPShot-fluorescence capillary analysis (SNaPShot-FCA) assay for rapid detection of the genotype of aldehyde dehydrogenase 2 gene (ALDH2) rs671 locus. METHODS: The genomic DNA was extracted from peripheral blood cells. Using R6G-ddATP and cy5-ddGTP as fluorescent substrates, the ALDH2 gene was amplified by SNaPShot to generate DNA products with different fluorescent dyes at the 3' end. FCA was used to detect the products separated by agarose gel electrophoresis and recovered by gel recovery kit, and the genetype of ALDH2 polymorphism was analyzed by fluorescence spectrum. The samples were tested three times repeatedly and compared with the results of DNA sequencing. RESULTS: The optimal concentrations of R6G-ddATP and cy5-ddGTP were 1.4 µmol/L and 8.0 µmol/L, respectively. 106 samples were tested for ALDH2 genotype by SNaPShot-FCA under optimal conditions, including 67 of wild type (GG), 38 of hybrid type (AG), and 1 of mutant type (AA), which were consistent with the sequencing results. CONCLUSION: This study successfully established the SNaPShot-FCA for the micro-detection of ALDH2 genotype for the rapid screening and identification of ALDH2 gene.

Development of a fluorescent capillary biosensor based on self-assembled AuNP/LDH for micro-volume intracellular pyruvate.

Herein, a fluorescent capillary biosensor was developed for quantifying micro-volume intracellular pyruvate (PA), in which AuNPs and lactate dehydrogenase (LDH) were modified on the inner surface of an amination capillary (20 µL) via a self-assembly technique. The PA concentration was quantified by the change in the value of the fluorescence of NADH after sucking a mixed solution of the sample and NADH into the biosensor. This study investigated factors including the degree of protonation of the amino groups on the surface of the capillary, the AuNP concentration and time for self-assembly, the activity concentration and time for the LDH self-assembly, the flow rate and acidity for LDH immobilization, pH, temperature, and reaction time for the NADH/PA/LDH reaction system. Under the optimized conditions, the linear response range of the biosensor towards PA was 2.5-120 µmol L-1, in which the determination limit and detection limit were 2.5 and 0.75 µmol L-1, respectively. The biosensor could be reused more than 41 times when its relative standard deviation (RSD) was controlled at less than 1.5%. At room temperature (approximately 25 °C), the intracellular PA in the erythrocyte of a healthy person was measured using the biosensor, and the PA content was observed to be 241.76 ± 68.05 µmol L-1 (n = 8). The standard addition recovery was 95-106%. Employment of the AuNPs in the PA biosensor not only improved the affinity of the immobilized LDH towards PA and its stability, but also significantly enhanced the service life of the PA biosensor.

On-line monitoring of a recirculating-flow fluorescent capillary system for exploring the interaction mechanism of carbon dots/metal ions.

The interaction mechanism between carbon dots (CDs) and metal ions is essential for optimizing their design, synthesis, and application. However, it must be accurately distinguished and quantified because of CDs' complex structure, composition, and coexisting various response mechanisms or products. Herein, a recirculating-flow fluorescence capillary analysis (RF-FCA) system was developed to online monitor the fluorescence kinetics of CDs interacting with metal ions. The fluorescence kinetics of purification and dissociation of CDs/metal ion complexes were easy to monitor online by integrating immobilized CDs and RF-FCA. Here, CDs derived from citric acid and ethylenediamine were used as a model system. We found that the fluorescence of CDs is quenched by Cu(II) and Hg(II) only through the formation of a coordination complex, by Cr(VI) only through the inner filtering effect, and by Fe(III) through the above two mechanisms. Then the kinetics of the competitive interaction between metal ions were used to address the difference of binding sites on CDs with metal ions, wherein Hg(II) was bound to other sites of CDs besides the same sites of CDs with Fe(III) and Cu(II). Finally, from the fluorescence kinetics of fluorescent molecules in the CD structure with metal ions, the difference was due to the presence of two fluorescent centers in the carbon core and molecular state in the CDs. Therefore, the RF-FCA system can distinguish and quantify the interaction mechanism between metal ions and CDs effectively and accurately and be a potential detection or performance characterization method.

Enzyme-free fluorescence determination of uric acid and trace Hg(II) in serum using Si/N doped carbon dots.

A new fluorescence probe method for the detection of Hg(II) in serum was established, which has the detection limit of 3.57 nM and quantification limit of 5 nM, based on the electrostatic induced agglomeration quenching and complexation between Hg(II) and silicon-nitrogen-doped carbon nanodots (Si/N-CDs). Furthermore, the fluorescence probe also showed the satisfactory results in the determination of Hg(II) in human serum. Subsequently, take advantage of the uric acid (UA) to recover the fluorescence of the Si/N-CDs-Hg(II) complex probe, another enzyme-free ways to determine UA was developed. The complex probe can selectively detect the UA content in the 0.5-30 µM range, and its detection limit can reach 0.14 µM, which has successfully detected the UA in total serum, and the results were no significant difference comparing with the controls.

A flow injection fluorescence "turn-on" sensor for the determination of metformin hydrochloride based on the inner filter effect of nitrogen-doped carbon dots/gold nanoparticles double-probe.

In this paper, an ultrasensitive and rapid "turn-on" fluorescence sensor, integrating flow-injection (FI) with nitrogen-doped carbon dots/gold nanoparticles (N-CDs/AuNPs) double-probe is established for the determination of metformin hydrochloride (MET) in biological fluids. The sensing strategy involves the weak inner filter effect between AuNPs and N-CDs due to aggregation products of MET with AuNPs. Unfortunately, the degree of AuNPs aggregation is difficult to control through manual assays, resulting in intolerable measurement error that limits further applications. However, the proposed method overcomes the above problem, and significantly lowers the consumption of expensive reagents (AuNPs: about 60 µL per test). Under optimal conditions, the fluorescence intensity at 400 nm excitation and 505 nm emission wavelengths display a linear correlation with MET concentration (5-100 µg L-1) and the limit of detection is 2.32 µg L-1 (3.3 S/k). The advantages of the presented method include high sensitivity, rapid speed (60 sample h-1), good accuracy and precision (RSD ≤ 2.1%, n = 11) and low cost. Since MET is the first-line hypoglycemic agent in patients with type II diabetes, this method can preliminarily determine MET content in urine samples, giving satisfactory results.