Nucleic Acid Target Sensing Using a Vibrating Sharp-Tip Capillary and Digital Droplet Loop-Mediated Isothermal Amplification (ddLAMP).

Nucleic acid tests are key tools for the detection and diagnosis of many diseases. In many cases, the amplification of the nucleic acids is required to reach a detectable level. To make nucleic acid amplification tests more accessible to a point-of-care (POC) setting, isothermal amplification can be performed with a simple heating source. Although these tests are being performed in bulk reactions, the quantification is not as accurate as it would be with digital amplification. Here, we introduce the use of the vibrating sharp-tip capillary for a simple and portable system for tunable on-demand droplet generation. Because of the large range of droplet sizes possible and the tunability of the vibrating sharp-tip capillary, a high dynamic range (~2 to 6000 copies/µL) digital droplet loop-mediated isothermal amplification (ddLAMP) system has been developed. It was also noted that by changing the type of capillary on the vibrating sharp-tip capillary, the same mechanism can be used for simple and portable DNA fragmentation. With the incorporation of these elements, the present work paves the way for achieving digital nucleic acid tests in a POC setting with limited resources.

A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing.

Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. In recent years, CRISPR-Cas12a has shown great potential in improving the performance of DNA biosensing. However, the nonspecific trans-cleavage activity of Cas12a complicates the multiplexing capability of Cas12a biosensing. We report a 3D-printed composable microfluidic plate (cPlate) device that utilizes miniaturized wells and microfluidic loading for a multiplexed CRISPR-Cas12a assay. The device easily combines loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure the maximum performance of the device, the concentration of Cas12a and detection probe was optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates sensitive detection to the fg mL- 1 level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1 h, making it suitable for low-resource settings.

Recent Advances in Digital Biosensing Technology.

Digital biosensing assays demonstrate remarkable advantages over conventional biosensing systems because of their ability to achieve single-molecule detection and absolute quantification. Unlike traditional low-abundance biomarking screening, digital-based biosensing systems reduce sample volumes significantly to the fL-nL level, which vastly reduces overall reagent consumption, improves reaction time and throughput, and enables high sensitivity and single target detection. This review presents the current technology for compartmentalizing reactions and their applications in detecting proteins and nucleic acids. We also analyze existing challenges and future opportunities associated with digital biosensing and research opportunities for developing integrated digital biosensing systems.

A portable droplet generation system for ultra-wide dynamic range digital PCR based on a vibrating sharp-tip capillary.

Monodisperse droplet has been widely used as a versatile tool in different disciplines including biosensing. Existing methods still struggle to balance the droplet generation performance with system simplicity. Here we introduce a novel droplet generation scheme based on the acoustic streaming generated from a vibrating sharp-tip capillary. The unique fluid pattern enables efficient droplet generation without any external pressure sources. This method achieved real-time modulation of droplet size over an ultra-wide range (6.77-661 µm), high throughput (up to 5000 droplets/s), and good monodispersity (<4%) with a power consumption below 60 mW. This method has enabled a multi-volume digital PCR with a dynamic range of ~6 orders of magnitude and multiplexing capability. It has also enabled a simple protocol to produce cell-laden alginate microcapsules in variable sizes with excellent biocompatibility. Overall, the present method combines high performance with small footprint and portability, which will be especially valuable for droplet applications requiring variable droplet size and performed in resource-limited settings.