Bisintercalating DNA redox reporters for real-time electrochemical qLAMP.

The electrochemical detection methods have emerged as a potential alternative to the bench-top optical systems in monitoring nucleic acid amplification. DNA intercalating redox reporters play a crucial role in such monitoring schemes. Here, a series of bisintercalating redox probes have been tailor-made to meet specific requirements of electrochemical quantitative loop-mediated isothermal amplification (qLAMP). The probes composed of two naphthoquinone-imidazole (NQIM) derivatives as signal motifs that are covalently bridged by different linkers (R). They are bis-NQIM-R; R = Alkane (Ak), ethylene glycol (EG) and phenyl (Ph). The linkers allow the probes to be fine-tuned for securing ideal redox reporter. DNA binding studies via electrochemical and fluorescence techniques demonstrate that the bis-NQIM-R probes possess better ds-DNA bisintercalating ability compared to their mono-analogs. The bis-NQIM-Ph was implemented in a real-time electrochemical qLAMP, for which a prototype custom-made device that can perform fully automated multiplexed analyses is devised. A single copy of Salmonella DNA was quantified in just 10 min and the performance is comparable to the benchtop fluorescence method. Thus, the bisintercalating redox reporters incorporated electrochemical detection schemes hold great promise in qLAMP.

Designing anthraquinone-pyrrole redox intercalating probes for electrochemical gene detection.

The real-time quantitative electrochemical monitoring of nucleic acid amplification through PCR is a promising renowned methodology to detect pathogenic DNAs. In this work, anthraquinone-pyrrole derivatives based redox intercalating probes (AP probes: AP1, AP2) have been designed, synthesized, characterized and successfully demonstrated in real-time like quantitative PCR. The rationally designed AP probes exhibited excellent DNA binding abilities and electrochemical behaviors. The binding parameters such as binding constant, binding site size and diffusion coefficient were estimated which were comparable to literature reports. Besides, the AP probes are highly stable under PCR thermal conditions and did not inhibit PCR. Therefore, a real-time like quantification of DNA amplification was demonstrated to quantify the initial copy number of target genes. The probe AP2 has excellent ability to detect ~10(3) copies of target tpc DNA with good sensitivity. The AP probes are metal-free, easily synthesizable, non-toxic, thermally stable and feasible for miniaturized PCR chips.

A radiate microstructure MALDI chip for sample concentration and detection.

Although matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis is an important tool for analyzing and characterizing biomolecules of varying complexity, the sensitivity of MALDI-TOFMS is dependent on proper preparation of the sample, a process that is oftentimes problematic and requires considerable expertise. In this study, we have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The sample/matrix mixture was deposited onto the central space of the well on the chip and allowed to dry. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Optimizing processes of several preparation factors were also performed to ensure matrix homogeneity in our chip. Analysis of the samples with MALDI-TOFMS showed that the signals from samples on our chip were significantly greater than those on the unmodified plate. The feasibility of using this chip to detect peptides and phosphopeptides was also demonstrated.

Droplet-based Microtexture Biochip System for Triglycerides and Methanol Measurement.

A droplet-based microfluidic system was developed for biochemical assays of triglycerides and methanol as potential applications in medical rapid diagnostics and food safety. We present a novel platform to manipulate biology reaction droplets with features of self-moving, self-mixing, and self-positioning toward the detection spot. The driving force comes from the gradient of surface tension force generated by the hydrophobic microtexture PDMS surface. We also demonstrate the experiments in water, and blood droplets running uphill to overcome the gravity. These findings support the ongoing works to develop the multifunctional biosensors in medicine and food safety.