Extended Enrichment for Ultrasensitive Detection of Low-Frequency Mutations by Long Blocker Displacement Amplification.

Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

Recent research trends in voltammetric sensing platforms for hormones and their applications to human serum analyses.

Hormones are essential chemical messengers that modulate body homeostasis and regulate appetite, mood, reproduction, and metabolism in the human body. Its dysregulation in the body can cause diabetes, Alzheimer's disease, and autism. The detection of hormone levels in biological fluids, including serum samples, could facilitate the precaution, diagnosis, and cure of diseases. Voltammetric sensing devices exhibiting significant sensitivity, selectivity, as well as facile, cost-effective fabrication, are one of the promising methods for detecting hormone levels. Thus, this review briefly highlights recent advances toward developing voltammetric sensors for a wide spectrum of amino acid-derived, peptide, and lipid-derived hormones alongside their applications to the analyses of human serum samples. Furthermore, several challenging issues and the prospects of voltammetric hormone-sensing devices in clinical diagnostic applications are discussed.

A serotonin voltammetric biosensor composed of carbon nanocomposites and DNA aptamer.

A sensitive and selective voltammetric biosensor composed of layer-by-layer (LbL) self-assembly of positively charged poly(diallyldimethylammonium)-wrapped oxidized single-walled carbon nanotubes (PDDA-oSWCNTs), negatively charged serotonin (5-hydroxytryptamine, 5-HT)-specific aptamer, and tyrosinase on Au nanoparticles deposited screen printed carbon electrode was developed for measurement of 5-HT. Surface characteristics of 5-HT biosensor were explored using scanning electron microscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. The respective effects of 5-HT-specific aptamer and oSWCNTs on the detection of 5-HT were investigated by differential pulse voltammetry (DPV). The peak current at the potential of 0.29 V (vs. Ag/AgCl) increased with respect to 5-HT concentration resulting in two dynamic ranges from 0.05 to 0.5 and 1 to 20 µM with a limit of detection of 2 nM from the LbL biosensor in buffer solution, which were better than those without the LbL of aptamer and oSWCNTs. The developed biosensor was applied to the direct determination of 5-HT concentrations in undiluted healthy control and Internet gaming disorder serum samples. The results were verified by comparison with those from liquid chromatography-mass spectrometric analyses.

Nanocomposites of poly(l-methionine), carbon nanotube-graphene complexes and Au nanoparticles on screen printed carbon electrodes for electrochemical analyses of dopamine and uric acid in human urine solutions.

A sensitive electrochemical sensor featuring novel composites of gold and carbon nanocomplexes alongside a polymerized amino acid was developed for the determination of uric acid (UA) and dopamine (DA) concentrations in both buffer and human urine sample solutions. The sensor was fabricated by electropolymerization of l-methionine (l-Met) followed by coating of carbon nanotube-graphene complexes and electrodeposition of gold nanoparticles on a screen printed carbon electrode surface. The electrode surfaces were characterized by field emission scanning electron microscopy and energy dispersive spectroscopy, and the electrochemical properties were investigated by cyclic voltammetry and differential pulse voltammetry. Linear ranges of 0.05-3 µM and 1-35 µM with limits of detection of 0.0029 and 0.034 µM were achieved for DA and UA, respectively. In addition, the developed sensor was applied for the analysis of native UA and DA concentrations in undiluted and diluted human urine samples. The UA analysis results were compared to those obtained using high performance liquid chromatography and a fluorometric assay kit while the DA analysis results were compared to those obtained using liquid chromatography-tandem mass spectrometry.

Layer-by-layer electrochemical biosensors configuring xanthine oxidase and carbon nanotubes/graphene complexes for hypoxanthine and uric acid in human serum solutions.

A selective biosensing platform for the determination of hypoxanthine (Hx) and uric acid (UA) concentrations in both buffer and human serum sample solutions was developed. The biosensor features the layer-by-layer (LbL) self-assembly of negatively charged xanthine oxidase (XOD) and positively charged poly(diallyldimethyl ammonium chloride) (PDDA) wrapped oxidized multi-walled carbon nanotubes and graphene (CNTs-G) complexes (PDDA-CNTs-G) on screen printed carbon electrode (SPCE) surfaces. Catalytic responses of the XOD modified biosensor with the chosen optimum number of layers for LbL assembly on SPCE towards Hx in buffer solutions were first investigated using both cyclic and square wave voltammetries. The peak current at around 0.08 V (vs. Ag/AgCl) associated with the production of UA increased as a function of the Hx concentration due to the surface selective catalytic reaction of XOD and Hx. A linear dynamic range of 5-50 µM Hx with a detection limit of 4.40 µM was obtained and the sensor was further applied to the analysis of Hx in normal human serum solutions in addition to myocardial infarction (MI) patient serum sample solutions from a local hospital. Since untreated serum solutions contain a certain amount of UA, a XOD free SPCE biosensor consisted of only PDDA-CNTs-G was also employed to evaluate the native concentration of UA in the serum and further assist the determination of Hx concentration when using the developed LbL biosensor. Our sensing results for the real biological fluidic solutions were finally validated by comparing to those using liquid chromatography-mass spectrometry(LC-MS).