Gold Nanoparticle-Labeled CRISPR-Cas13a Assay for the Sensitive Solid-State Nanopore Molecular Counting.

Rapid fluorescent nucleic acid sensing with ultra-thin gold nanosheets

Moving toward rapid and low-cost point-of-care molecular diagnostics with a repurposed 3D printer and RPA

Electrochemical biosensors utilizing hybridization chain reaction (E-HCR) have witnessed substantial advancement over the past two decades, yet achieving simultaneous rapid and ultrasensitive detection remains a challenge with current strategies. Herein, we report a portable, wireless E-HCR biosensor that leverages S9.6 antibody-mediated bivalent capture for ultrasensitive nucleic acid detection, achieving a record-fast assay completion time. The detection mechanism involves target nucleic acid-triggered opening of a hairpin probe, followed by hybridization with a preassembled HCR/HRP amplifier. The resulting target/hairpin/HCR/HRP complex contains two segments of DNA/RNA heteroduplex, enabling efficient capture by an S9.6 antibody-modified screen-printed carbon electrode (SPCE) through bivalent S9.6 antibody-heteroduplex interactions. The bivalent capture strategy demonstrates a 1.6-fold enhancement over single-site S9.6 antibody-heteroduplex binding and a 3.1-fold improvement in capture efficiency compared to monovalent hybridization. This one-pot strategy offers three unique advantages. First, the integration of bivalent capture, homogeneous hybridization, and preformed HCR/HRP amplifiers enables the heterogeneous E-HCR assay to be completed within 34 min, significantly faster than conventional methods. Second, optimization of HCR amplifier and background signal suppression achieves a high signal-to-noise ratio, facilitating ultrasensitive nucleic acid detection. Third, the biosensor features wireless signal output and utilizes low-cost SPCE, making it suitable for point-of-care applications. Collectively, these unique merits enable the bivalent capture biosensor to achieve portable, one-pot, rapid, and ultrasensitive nucleic acid detection, addressing limitations in current E-HCR biosensing platforms.

Effect of single nanoparticle-nanopore interaction strength on ionic current modulation

Pathogenic pathogens invade the human body through various pathways, causing damage to host cells, tissues, and their functions, ultimately leading to the development of diseases and posing a threat to human health. The rapid and accurate detection of pathogenic pathogens in humans is crucial and pressing. Nucleic acid detection offers advantages such as higher sensitivity, accuracy, and specificity compared to antibody and antigen detection methods. However, conventional nucleic acid testing is time-consuming, labor-intensive, and requires sophisticated equipment and specialized medical personnel. Therefore, this review focuses on advanced nucleic acid testing systems that aim to address the issues of testing time, portability, degree of automation, and cross-contamination. These systems include extraction-free rapid nucleic acid testing, fully automated extraction, amplification, and detection, as well as fully enclosed testing and commercial nucleic acid testing equipment. Additionally, the biochemical methods used for extraction, amplification, and detection in nucleic acid testing are briefly described. We hope that this review will inspire further research and the development of more suitable extraction-free reagents and fully automated testing devices for rapid, point-of-care diagnostics.

Microfluidic-assisted integrated nucleic acid test strips for POCT

In-situ fixation (ISF): A rapid, reusable, and high-throughput nucleic acid extraction method for plant molecular analysis.

Nucleic acid detection plays a vital role in both biomedical research and clinical medicine. The temperature circulation changes of the widely used polymerase chain reaction technique are time-consuming and technically challenging for system development. Recombinase polymerase amplification (RPA) is an isothermal method for rapid nucleic acid detection. However, current RPA amplicon detection methods are complicated and expensive and easily generate false positives, restricting the promotion of RPA techniques. In this work, a hyperspectral interferometric amplicon-complex quantitation method is presented, combined with asymmetric dipole complex strategy optical scattering analysis. GelRed dye was utilized to form amplicon-complex particles, and the Fourier domain spectrum computation contributed to complex scattering quantitation. With this method, a supporting microfluidic chip and automatic system were developed to achieve integrated, rapid, quantitative, and miniscule nucleic acid detection. The Plasmodium falciparumdhfr gene was utilized as an example for targeted nucleic acid quantitation and single nucleotide polymorphism detection. The total reaction time was decreased to merely 20 min, and the limit of detection was only 3.17 ng/μL. The minimum measurable concentration of target was 1.68 copies/μL, 31.67 times more sensitive than turbidity detection, and the single reaction chamber was only 9.33 μL. No scattering increase occurred for template-free control, and thus, false positives caused by primer dimers and nonspecific products could be avoided. The experimental results prove that the provided method and system can detect single-base mutations in the dhfr gene and is a reasonable technique for rapid, automatic, and low-cost nucleic acid detection.

Rapid and precise nucleic acid testing at the point-of-care (POC) is essential for effective screening and management of infectious diseases. Current polymerase-based molecular diagnostics often suffer from potential cross-contamination issues, particularly in POC settings. Here, we introduce DECODE, a contamination-free nucleic acid detection platform integrating digital microfluidics (DMF) for nucleic acid extraction and a digital CRISPR amplification-free assay for pathogen detection. The digital CRISPR assay demonstrates sensitivity, detecting target DNA and RNA in the reaction mixture at concentrations of 10 and 5 copies/μL, respectively. Leveraging DMF-extracted samples enhances the performance of the digital CRISPR amplification-free assay. DECODE offers a sample-to-result workflow of 75 min using compact devices. Validation studies using clinical samples confirm DECODE's robust performance, achieving 100% sensitivity and specificity in detecting HPV18 from cervical epithelial cells and influenza A from nasal swabs. DECODE represents a versatile, contamination-free detection platform poised to enhance integrated public health surveillance efforts.

Spherical nucleic acids (SNAs) have been used to construct various nanobiosensors with gold nanoparticles (AuNPs) as nuclei. The SNAs play a critical role in biosensing due to their various physical and chemical properties, programmability, and specificity recognition ability. In this study, CRISPR-responsive self-assembled spherical nucleic acid (CRISPR-rsSNA) detection probes were constructed by conjugating fluorescein-labeled probes to the surface of AuNPs to improve the sensing performance. Also, the mechanism of ssDNA and the role of different fluorescent groups in the self-assembly process of CRISPR-rsSNA were explored. Then, CRISPR-rsSNA and reverse transcription-recombinase polymerase amplification (RT-RPA) were combined to develop an ultrasensitive fluorescence-detection strategy for norovirus. In the presence of the virus, the target RNA sequence of the virus was transformed and amplified by RT-RPA. The resulting dsDNA activated the trans-cleavage activity of CRISPR cas12a, resulting in disintegrating the outer nucleic acid structure of the CRISPR-rsSNA at a diffusible rate, which released reporter molecules. Norovirus was quantitated by fluorescence detection. This strategy facilitated the detection of the norovirus at the attomolar level. An RT-RPA kit for norovirus detected would be developed based on this method. The proposed method would be used for the detection of different viruses just by changing the target RNA and crRNA of the CRISPR cas12a system which provided a foundation for high-throughput detection of various substances.

Rapid, low-cost, and sensitive nucleic acid detection and quantification assays enabled by microfluidic paper-based analytical devices (μPADs) hold great promise for point-of-care disease diagnostics and field-based molecular tests. Through the capillary action in μPAD, flexible manipulation of nucleic acid samples can be achieved without the need for external pumps or power supplies, making it possible to fabricate highly integrated sample-to-answer devices that streamline the nucleic acid extraction, separation, concentration, amplification, and detection. To detect minute amounts of genetic materials from clinical and biological samples, it is also critical to develop sensitive signal readouts that generate physically detectable signals for in-device nucleic acid detection and/or quantification. In this review, we will focus on μPAD approaches for the facile manipulation of nucleic acids and emerging signal transduction strategies allowing sensitive and specific nucleic acid detection in μPAD. Graphical abstract ᅟ.

Comparison of point-of-care-compatible lysis methods for bacteria and viruses

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

Gold nanoparticles (GNPs) are widely used in many fields due to their compatibility with various biosystems and their ability to be easily functionalized with biomolecules. However, current citrate-reduced GNPs are prone to aggregate and unstable at high ionic strength solution, which greatly limits their applications. Thus, new preparation methods of the GNPs, which can maintain the stability under the extreme conditions, are still urgently needed. Here, we describe a new GNP synthesis strategy by using trisodium citrate to reduce hydrogen tetrachloroaurate (III) (HAuCl4) in the presence of single-strand DNA (ssDNA) at 37 °C. Under the assistance of the ssDNA, the GNPs can be prepared at physiological temperature and still maintain the uniform shape and dispersity. More importantly, the stability of the GNPs against salt-induced aggregation was improved greatly. With their high stability and in situ functionalized ssDNA, these GNPs have great potential in physiological systems and could be used for nanom...

Polymerase chain reaction (PCR) is the most commonly used molecular biology technique to amplify nucleic acid (DNA and RNA) in vitro. This technique is highly temperature sensitive and thermal management has an important role in PCR operation in reaching the required temperature set points at each step of the process (denaturing, annealing and elongation). In this work, an innovative microfluidic PCR thermal cycling device is designed to increase the heating∕cooling thermal cycling speed while maintaining a uniform temperature distribution throughout the substrate containing the aqueous nucleic acid sample. The device design is incorporating the jet impingement and micro-channel thermal management technologies utilizing a properly arranged configuration filled with a porous medium. Porous Inserts are attractive choices in heat transfer augmentation. They provide a very large surface area for a given volume which is a key parameter in heat transfer processes. Various effective parameters that are relevant in optimizing this flexible thermal cycler are investigated such as thermal cycler configuration, thickness of inlet and exit fluid channels, fluid flow rate and velocity, the porous matrix material and properties, and utilization of thermal grease. An optimized case is established based on the effects of the cited parameters on the temperature ramp, temperature distribution and the required power for circulating the fluid in the thermal cycler. The results indicate that the heating∕cooling temperature ramp (temperature change per heating∕cooling cycling time) of the proposed device is considerably higher (150.82◻C∕s) than those in literature. In addition, the proposed PCR offers a very uniform temperature in the substrate while utilizing a low power.