Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band-gap near infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence-dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant noncanonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20-and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van't Hoff transition enthalpy of hybridization on SWCNT CP was -11.9 kJ mol-1, much lower than that of hybridization in solution (-707 kJ mol-1). We used SWCNT solvatochromism to calculate the solvent-exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with a (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNTs with an analyte at 37 degrees C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that the target sequence choice is important as secondary structure formation in the target is negatively correlated with hybridization. Best performing CPs showed detection limits of 11 and 13 nM for DNA and RNA targets, respectively. Finally, we simulated sensing conditions using the saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence-specific hybridization on ssDNA-SWCNT CPs.

DNA-Functionalized Ti3C2Tx MXenes for Selective and Rapid Detection of SARS-CoV-2 Nucleocapsid Gene

Coronavirus disease 2019 (COVID-19) is an emerging human infectious disease caused by severe acute respiratory syndrome 2 (SARS-CoV-2, initially called novel coronavirus 2019-nCoV) virus. Thus, an accurate and specific diagnosis of COVID-19 is urgently needed for effective point-of-care detection and disease management. The reported promise of two-dimensional (2D) transition-metal carbides (Ti3C2Tx MXene) for biosensing owing to a very high surface area, high electrical conductivity, and hydrophilicity informed their selection for inclusion in functional electrodes for SARS-CoV-2 detection. Here, we demonstrate a new and facile functionalization strategy for Ti3C2Tx with probe DNA molecules through noncovalent adsorption, which eliminates expensive labeling steps and achieves sequence-specific recognition. The 2D Ti3C2Tx functionalized with complementary DNA probes shows a sensitive and selective detection of nucleocapsid (N) gene from SARS-CoV-2 through nucleic acid hybridization and chemoresistive transduction. The fabricated sensors are able to detect the SARS-CoV-2 N gene with sensitive and rapid response, a detection limit below 10(5) copies/mL in saliva, and high specificity when tested against SARS-CoV-1 and MERS. We hypothesize that the MXenes' interlayer spacing can serve as molecular sieving channels for hosting organic molecules and ions, which is a key advantage to their use in biomolecular sensing.

Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Label-Free Manner Using Micromechanical Sensors.

Coronavirus (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as a deadly pandemic. The genomic analysis of SARS-CoV-2 is performed using a reverse transcription-polymerase chain reaction (RT-PCR) technique for identifying viral ribonucleic acid (RNA) in infected patients. However, the RT-PCR diagnostic technique is manually laborious and expensive; therefore, it is not readily accessible in every laboratory. Methodological simplification is crucial to combat the ongoing pandemic by introducing quick, efficient, and affordable diagnostic methods. Here, we report how microcantilever sensors offer promising opportunities for rapid COVID-19 detection. Our first attempt was to capture the single-stranded complementary DNA of SARS-CoV-2 through DNA hybridization. Therefore, the microcantilever surface was immobilized with an oligonucleotide probe and detected using complementary target DNA hybridization by a shift in microcantilever resonance frequency. Our results show that microcantilever sensors can discriminate between complementary and noncomplementary target DNA on a micro to nanoscale. Additionally, the microcantilever sensors' aptitude toward partial complementary DNA determines their potential to identify new variants of coronavirus. Therefore, microcantilever sensing could be a vital tool in the effort to extinguish the spreading COVID-19 pandemic.