Dual-probe ligation without PCR for fluorescent sandwich assay of EGFR nucleotide variants in magnetic gene capture platform.

A simple, rapid, and highly efficient fluorescent detection technique without PCR through dual-probe ligation with the genetic capture of magnetic beads and reported probe was developed for determination of epidermal growth factor receptor (EGFR) gene exon 19 deletions. The EGFR exon 19 deletion mutation makes up 48% of all mutations associated with anti-tyrosine kinase inhibition sensitivity, and thus, the EGFR nucleotide variant is very important in clinical diagnosis. In this approach, the dual-probe ligation was designed to target exon 19 deletion. The magnetic genetic captured system was then applied to capture the successful dual-probe ligation. Thereafter, a reporter probe which is coupled with 6-fluorescein amidite (6-FAM) was introduced to hybridize with dual-probe ligation product on the surface of streptavidin magnetic beads, and finally, the supernatant was taken for fluorescence measurements for distinguishing mutant types from wild types. After optimization (the RSD of the fluorescent intensity was less than 4.5% (n = 3) under the optimal condition), 20 blind DNA samples from the population were analyzed by this technique and further confirmed by direct sequencing. The results of our assay matched to those from direct sequencing data, evidencing that the developed method is accurate and successful. These 20 blind DNA samples were diagnosed as wild and then spiked with different percentages of the mutant gene to quantify the ratio of the wild and mutant genes. This strategy was also successfully applied to quantify the ratio of the wild and mutant genes with good linearity at the λex/λem of 480 nm/520 nm (r = 0.996), and the limit of detection reached 1.0% mutant type. This simple fluorescent detection of nucleotide variants shows its potential to be considered a tool in biological and clinical diagnosis. Importantly, this strategy offers a universal detection capability for any kind of mutation (point, deletion, insertion, or substitution) in a gene of interest.

One Single Tube Reaction of Aptasensor-Based Magnetic Sensing System for Selective Fluorescent Detection of VEGF in Plasma.

In this study, a simple, easy and convenient fluorescent sensing system for the detection of the vascular endothelial growth factor (VEGF) based on VEGF aptamers, aptamer-complementary fluorescence-labeled probe and streptavidin magnetic beads was developed in one single tube. The VEGF is the most important biomarker in cancer, and it is investigated that the serum VEGF level varied according to the different types and courses of cancers. Hence, efficient quantification of VEGF is able to improve the accuracy of cancer diagnoses and the precision of disease surveillance. In this research, the VEGF aptamer was designed to be able to bind with the VEGF by forming G-quadruplex secondary structures; then, the magnetic beads would capture the non-binding aptamers due to non-steric interference; and finally, the fluorescence-labeled probes were hybridized with the aptamers captured by the magnetic beads. Therefore, the fluorescent intensity in the supernatant would specifically reflect the present VEGF. After an overall optimization, the optimal conditions for the detection of VEGF were as followed, KCl, 50 µM; pH 7.0; aptamer, 0.1 µM; and magnetic beads, 10 µL (4 µg/µL). The VEGF could be well quantified within a range of 0.2-2.0 ng/mL in plasma, and the calibration curve possessed a good linearity (y = 1.0391x + 0.5471, r = 0.998). The detection limit (LOD) was calculated to be 0.0445 ng/mL according to the formula (LOD = 3.3 × σ/S). The specificity of this method was also investigated under the appearance of many other serum proteins, and the data showed good specificity in this aptasensor-based magnetic sensing system. This strategy provided a simple, sensitive and selective biosensing platform for the detection of serum VEGF. Finally, it was expected that this detection technique can be used to promote more clinical applications.

Copper nanoclusters on specific-primer PCR fragments with magnetic capture for the label-free fluorescent sensing of the T315I single nucleotide variant in the BCR-ABL1 gene.

In this study, a simple and facile procedure using the all or none formation of double-stranded DNA-templated copper nanoclusters on specific-primer PCR fragments was designed to fluorescently identify the T315I single nucleotide variant on the BCR-ABL1 gene. Chronic myeloid leukaemia (CML), a disease caused by the BCR-ABL1 fusion of tyrosine kinase, is well known for the T315I mutation that causes tyrosine kinase inhibitors (TKIs) to be resisted due to the alternative structure of the drug-binding site. Therefore, it is an important single nucleotide variant for clinical detection. In this study, only specific functional primers and the digestion of the wild genotype from the T315I mutation site with specific restriction enzymes were designed, and the different digested products could then be captured using magnetic beads. The final products would allow for fluorescent sensing via the all or none formation of double-stranded DNA-templated copper nanoclusters for the detection of the T315I mutation. This study has been successfully applied for identifying wild and mutant homozygotes and the mutant/wild heterozygote of the T315I mutation. It is expected that this analytical system can serve as a tool for the clinical diagnosis of T315I mutations and be applied to real samples of CML patients in the future.

A chemometric experimental design with three-step stacking capillary electrophoresis for analysis of five tobacco-specific nitrosamines in cigarette products.

Tobacco-specific nitrosamines (TSNAs) as carcinogens endanger our health and life from cigarette products. However, the safe range of TSNAs levels in commercial cigarette products has not yet been established. For the purpose of safety and supervision, a three-step stacking approach including field amplified sample injection (FASI), sweeping, and analyte focusing by micelle collapse (AFMC), was developed for the simultaneous determination of five TSNAs levels in cigarette products. This approach also involved aspects of chemometric experimental design, including fractional factorial design and central composite design. After the multilevel optimization of the experimental design, the five TSNAs were well separated. The LOD (S/N = 3) values of the N´-nitrosonornicotine (NNN), N´-nitrosoanatabine (NAT), N´-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the FASI-sweeping-AFMC CE approach were 1.000 ng/mL, 0.500 ng/mL, 0.125 ng/mL, 1.000 ng/mL, and 0.500 ng/mL respectively. The results of relative standard deviation (RSD) and relative error (RE) were all less than 3.35%, demonstrating good precision and accuracy. Finally, this novel approach was further applied to monitor three commercial cigarette products, and a range of 250.1-336.6 ng/g for NNN, 481.6-526.7 ng/g for NAT, 82.2-247.6 ng/g for NAB, 167.7-473.7 ng/g for NNAL, and 39.4-246.7 ng/g for NNK could be observed among these. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of five TSNAs levels in cigarette products and could serve as a tool for assays of quality control of nitrosamines.

High-Performance Liquid Chromatographic Analysis of Sarcosine as a Fluorescent Levofloxacin Derivative.

Sarcosine, N-methyl glycine, could be used as a biomarker for the diagnosis of prostate cancer. It exists in biosamples at low levels; therefore, sensitive methods are necessary for its detection. In this study, we developed a sensitive and selective method for the analysis of sarcosine, based on derivatizing sarcosine with a fluorescent reagent levofloxacin acyl chloride. The resulting derivative is highly responsive to a fluorimetric detector (λex = 290 nm, λem = 460 nm). The sarcosine derivative can be separated from its molecular isomers (α-l-alanine, α-d-alanine and ß-alanine) on a hexyl-phenyl column by gradient elution using sodium acetate buffer (pH 3.8; 50 mM) and tetrahydrofuran as the mobile phase. The method showed a determination range of sarcosine in water over 44.5-1780.0 ng/mL (0.5-20.0 µM) and the limit of detection at 8.9 ng/mL (0.1 µM) (S/N = 3 with 20 µL injected). Intra- and inter-day precision (as % relative standard deviation) and accuracy (as % relative error) were all below 4.8%. Application of the method to the analysis of sarcosine in human urine proved feasible.

Effect of Di(2-ethylhexyl)phthalate on Helicobacter pylori-Induced Apoptosis in AGS Cells.

Plastic products are wildly used in human life. Di(2-ethylhexyl)phthalate (DEHP) is an essential additive in plastic manufacturing and is used as plasticizer for many products including plastic food packaging. DEHP is a teratogenic compound and can cause potent reproductive toxicity. DEHP can also cause liver damage, peroxisome proliferation, and carcinogenesis. DEHP is also strongly associated with peptic ulcers and gastric cancer; however, the underlying effect and mechanism of DEHP on the gastrointestinal tract are not entirely clear. The oral infection route of H. pylori parallels the major ingestion route of DEHP into the human body. Therefore, we wanted to study the effect of DEHP and H. pylori exposure on the human gastric epithelial cell line, AGS (gastric adenocarcinoma). The viability of the AGS cell line was significantly lower in 80 µ M-DEHP and H. pylori (MOI = 100 : 1) coexposure than DEHP or H. pylori alone. DEHP and H. pylori coexposure also induced caspase-3 activation, and increased Bax/Bcl-2 ratio and DNA fragmentation in AGS cells. These results indicate that DEHP can enhance H. pylori cytotoxicity and induce gastric epithelial cell apoptosis. Therefore, it is possible that DEHP and H. pylori coexposure might enhance the disruption of the gastric mucosa integrity and potentially promote the pathogenesis of gastric carcinogenesis.

Simultaneous determination of methotrexate and its eight metabolites in human whole blood by capillary zone electrophoresis.

A simple and selective capillary electrophoretic method was established for the simultaneous determination of methotrexate (MTX), 7-hydroxymethotrexate (7-OHMTX), 2,4-diamino-N10-methylpteroic acid (DAMPA), and polyglutamate derivatives [MTX-(Glu)n, n=2-7] in whole blood. After extraction, those analytes were separated by fused-silica capillary and a running buffer containing glycine (1.2 M, pH 9.3). The quantitative ranges were 10-50 microM for each analyte. The intra- and inter-day RSD and RE values were all less than 6 and 11%, respectively. The limits of detection (S/N= 3, injection 5 s) were found to be 1 microM for MTX, 7-OHMTX, MTX-(Glu)2, MTX-(Glu)3, and MTX-(Glu)4; 3 microM for MTX-(Glu)5 and MTX-(Glu)6; 5 microM for MTX-(Glu)7, and 8 microM for DAMPA. All recoveries were greater than 94%. This method was applied to blood MTX monitoring in a patient with acute lymphoblastic leukemia.

Highly sensitive analysis of iodide anion in seaweed as pentafluorophenoxyethyl derivative by capillary gas chromatography.

A simple and sensitive gas chromatography (GC) method is described for the trace analysis of iodide anion (iodide) in processed seaweed as an organic derivative. The method is based on the derivatization of aqueous iodide extracted from seaweed with 2-(pentafluorophenoxy)ethyl 2-(piperidino)ethanesulfonate in toluene using tetra-n-hexylammonium bromide as a phase-transfer catalyst. The resulting pentafluorophenoxyethyl iodide is highly responsive to an electron-capture detector (ECD) and was analyzed by GC-ECD, giving a low detection limit of approximately 2.7 nM (2.7 fmol/microL injected). Interferences of some common anions in the analysis of iodide were studied and proved to be minimal. Application of the method to the analysis of iodide in processed seaweed was performed.