Determination of tuberculosis-related volatile organic biomarker methyl nicotinate in vapor using fluorescent assay based on quantum dots and cobalt-containing porphyrin nanosheets.

Methyl nicotinate (MN) is a representative and typical volatile organic marker of Mycobacterium tuberculosis, and the specific detection of MN in human breath facilitates non-invasive, rapid, and accurate epidemic screening of tuberculosis infection. Herein, we constructed a fluorescent assay consisted of CdTe quantum dots (QD) and cobalt-metalized tetrakis(4-carboxyphenyl) porphyrin (CoTCPP) nanosheets to determine methyl nicotinate (MN) in vapor samples. Red-emission QD (λex=370 nm, λem=658 nm) acts as signal switches whose fluorescence signals can be effectively quenched by CoTCPP nanosheets but restored in the presence of MN. The strategy relied on the distinct binding affinity of cobalt ion and MN. MN restored the fluorescence of QD quenched by CoTCPP in a concentration-dependent manner, which exhibited a well-linear relationship in the range 1-100 µM, and a limit of detection of 0.59 µM. The proposed platform showed sensitivity and selectivity to detect MN in vapor samples with satisfactory RSD below 3.33%. The method is cheap, simple, and relatively rapid (detected within 4 min), which suggests a potential in tuberculosis diagnosis in resource- and professional-lacked areas.

A novel and cost-efficient allele-specific PCR method for multiple SNP genotyping in a single run.

Cost-effective methods for DNA genotyping were needed because single nucleotide polymorphisms (SNPs) were essential biomarkers associated with many diseases. Allele-specific PCR (AS-PCR) has the advantages of mature instruments and high sensitivity. But conventional AS-PCR needs to multiply the number of reactions or primers for multiple targets, which complicates the operation and increases the cost. Herein, we proposed a novel AS-PCR method for multiple SNP genotyping in a single run. Wild-type allele-specific primer (WT primer) was designed for each target gene. The sample and WT primers only needed to undergo multiplexed AS-PCR once simultaneously. After AS-PCR, the concentration of remaining primers varied among the samples of each genotype combination, due to the different matching performance between template and WT primers. The remaining primers then triggered multiplexed molecular beacon-rolling circle amplification, and the molecular beacons labelled with different fluorescent dyes corresponded to different targets. The fluorescence ratios of the sample to the positive control were used as the genotyping indexes. This method was able to detect samples with concentrations as low as 10 fM. We successfully applied the method to the multiple genotyping of 23 hair root samples for ADH1B and ALDH2 genes, obtaining completely consistent results with sequencing. The reagent cost was 0.6 dollar for one sample, showing a good cost performance. This proposed approach had a great application prospect in simultaneously rapid and accurate genotyping of multi-SNPs, and provided a new method for personalized health management.

Detection of Mycobacterium Tuberculosis IS6110 gene fragment by fluorescent biosensor based on FRET between two-dimensional metal-organic framework and quantum dots-labeled DNA probe.

Herein, a universal fluorescent biosensor was developed for detecting Mycobacterium Tuberculosis (MTB) specific insertion sequence IS6110 gene fragment based on Förster resonance energy transfer (FRET) strategy. CdTe quantum dots (QDs), with excellent luminous performance, were used to label single-stranded DNA (ssDNA) as fluorescence donor (QDs-DNA), in which the ssDNA was complementary to the IS6110 gene fragment. A new type of two-dimensional metal-organic framework (Cu-TCPP) was served as an acceptor. The Cu-TCPP exhibited a higher affinity towards ssDNA than double-stranded DNA (dsDNA). In the absence of targets, the fluorescence of QDs-DNA was quenched - due to the π-π stacking interactions between Cu-TCPP and ssDNA. Otherwise, QDs-DNA hybridized with the target to form a double helix and the fluorescence maintained in a target-concentration dependent manner. Excess QDs-DNA would be quenched and produced negligible background signal. The fluorescent sensor possessed a linear range from 0.05 nM to 1.0 nM with a low detection limit of 35 pM. Furthermore, we successfully applied this biosensing system to detect clinical sputum samples. This method displayed high sensitivity, specificity and great potentials in the early diagnosis of Tuberculosis.

A Redox-induced Dual-mode Colorimetric and Fluorometric Method based on N-CDs and MnO2 for Determination of Isoniazid in Tablets and Plasma Samples.

We develop a simple hydrothermal method to prepare a novel nitrogen-doped carbon dots (N-CDs) originated from green carbon source Liu-bao tea and ethylene diamine. The N-CDs emits strong and stable blue fluorescence (Em = 440 nm) under the excitation wavelength of 350 nm with a quantum yield of 35%. And it is used as an excellent fluorescent output for the sensitive and visual dual-mode determination of isoniazid. The fluorescence of N-CDs is "turned off" first by manganese dioxide (MnO2) nanosheets due to inner filter effect, MnO2 nanosheets can also oxidize TMB (3,3',5,5'- tetramethylbenzidine) to blue oxTMB. Isoniazid, however, can reduce MnO2 nanosheets to Mn2+, turning on the fluorescence again. The color of the solution fades from blue to colorless because less TMB can be oxidized. Under the optimal conditions, the dual-mode method has a satisfying linear relationship ranging from 2.0 to 120.0 µM with a limit of detection of 0.7 µM (S/N = 3). And it has been applied successfully to colorimetric and fluorescent determination of isoniazid in tablets and clinical plasma samples, with recoveries ranging from 94.0% to 102.4%. The properties of N-CDs and MnO2 nanosheets were thoroughly characterized using TEM, FT-IR, XPS, AFM and fluorescence spectrophotometer, the quenching mechanism was also discussed.