Digitized Kinetic Analysis Enhances Genotyping Capacity of CRISPR-Based Biosensing.

CRISPR/Cas systems have been widely employed for nucleic acid biosensing and have been further advanced for mutation detection by virtue of the sequence specificity of crRNA. However, existing CRISPR-based genotyping methods are limited by the mismatch tolerance of Cas effectors, necessitating a comprehensive screening of crRNAs to effectively distinguish between wild-type and point-mutated sequences. To circumvent the limitation of conventional CRISPR-based genotyping, here, we introduce Single-Molecule kinetic Analysis via a Real-Time digital CRISPR/Cas12a-assisted assay (SMART-dCRISPR). SMART-dCRISPR leverages the differential kinetics of the signal increase in CRISPR/Cas systems, which is modulated by the complementarity between crRNA and the target sequence. It employs single-molecule digital measurements to discern mutations based on kinetic profiles that could otherwise be obscured by variations in the target concentrations. We applied SMART-dCRISPR to genotype notable mutations in SARS-CoV-2, point mutation (K417N) and deletion (69/70DEL), successfully distinguishing wild-type, Omicron BA.1, and Omicron BA.2 SARS-CoV-2 strains from clinical nasopharyngeal/nasal swab samples. Additionally, we introduced a portable digital real-time sensing device to streamline SMART-dCRISPR and enhance its practicality for point-of-care settings. The combination of a rapid and sensitive isothermal CRISPR-based assay with single-molecule kinetic analysis in a portable format significantly enhances the versatility of CRISPR-based nucleic acid biosensing and genotyping.

Rapid and Portable Quantification of HIV RNA via a Smartphone-enabled Digital CRISPR Device and Deep Learning.

For the 28.2 million people in the world living with HIV/AIDS and receiving antiretroviral therapy, it is crucial to monitor their HIV viral loads with ease. To this end, rapid and portable diagnostic tools that can quantify HIV RNA are critically needed. We report herein a rapid and quantitative digital CRISPR-assisted HIV RNA detection assay that has been implemented within a portable smartphone-based device as a potential solution. Specifically, we first developed a fluorescence-based reverse transcription recombinase polymerase amplification (RT-RPA)-CRISPR assay for isothermally and rapidly detecting HIV RNA at 42 °C in < 30 min. When realized within a commercial stamp-sized digital chip, this assay yields strongly fluorescent digital reaction wells corresponding to HIV RNA. The isothermal reaction condition and the strong fluorescence in the small digital chip unlock compact thermal and optical components in our device, allowing us to engineer a palm-size (70 × 115 × 80 mm) and lightweight (< 0.6 kg) device. Further leveraging the smartphone, we wrote a custom app to control the device, perform the digital assay, and acquire fluorescence images throughout the assay time. We additionally trained and verified a Deep Learning-based algorithm for analyzing fluorescence images and detecting strongly fluorescent digital reaction wells. Using our smartphone-enabled digital CRISPR device, we were able to detect 75 copies of HIV RNA in 15 min and demonstrate the potential of our device toward convenient monitoring of HIV viral loads and combating the HIV/AIDS epidemic.

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.

Sensitive, smartphone-based SARS-CoV-2 detection from clinical saline gargle samples.

Saliva specimens have drawn interest for diagnosing respiratory viral infections due to their ease of collection and decreased risk to healthcare providers. However, rapid and sensitive immunoassays have not yet been satisfactorily demonstrated for such specimens due to their viscosity and low viral loads. Using paper microfluidic chips and a smartphone-based fluorescence microscope, we developed a highly sensitive, low-cost immunofluorescence particulometric SARS-CoV-2 assay from clinical saline gargle samples. We demonstrated the limit of detection of 10 ag/µL. With easy-to-collect saline gargle samples, our clinical sensitivity, specificity, and accuracy were 100%, 86%, and 93%, respectively, for n = 27 human subjects with n = 13 RT-qPCR positives.

Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip.

Respiratory viruses, especially coronaviruses, have resulted in worldwide pandemics in the past couple of decades. Saliva-based paper microfluidic assays represent an opportunity for noninvasive and rapid screening, yet both the sample matrix and test method come with unique challenges. In this work, we demonstrated the rapid and sensitive detection of SARS-CoV-2 from saliva samples, which could be simpler and more comfortable for patients than existing methods. Furthermore, we systematically investigated the components of saliva samples that affected assay performance. Using only a smartphone, an antibody-conjugated particle suspension, and a paper microfluidic chip, we made the assay user-friendly with minimal processing. Unlike the previously established flow rate assays that depended solely on the flow rate or distance, this unique assay analyzes the flow profile to determine infection status. Particle-target immunoagglutination changed the surface tension and subsequently the capillary flow velocity profile. A smartphone camera automatically measured the flow profile using a Python script, which was not affected by ambient light variations. The limit of detection (LOD) was 1 fg/µL SARS-CoV-2 from 1% saliva samples and 10 fg/µL from simulated saline gargle samples (15% saliva and 0.9% saline). This method was highly specific as demonstrated using influenza A/H1N1. The sample-to-answer assay time was <15 min, including <1-min capillary flow time. The overall accuracy was 89% with relatively clean clinical saline gargle samples. Despite some limitations with turbid clinical samples, this method presents a potential solution for rapid mass testing techniques during any infectious disease outbreak as soon as the antibodies become available.

Direct capture and smartphone quantification of airborne SARS-CoV-2 on a paper microfluidic chip.

SARS, a new type of respiratory disease caused by SARS-CoV, was identified in 2003 with significant levels of morbidity and mortality. The recent pandemic of COVID-19, caused by SARS-CoV-2, has generated even greater extents of morbidity and mortality across the entire world. Both SARS-CoV and SARS-CoV-2 spreads through the air in the form of droplets and potentially smaller droplets (aerosols) via exhaling, coughing, and sneezing. Direct detection from such airborne droplets would be ideal for protecting general public from potential exposure before they infect individuals. However, the number of viruses in such droplets and aerosols is too low to be detected directly. A separate air sampler and enough collection time (several hours) are necessary to capture a sufficient number of viruses. In this work, we have demonstrated the direct capture of the airborne droplets on the paper microfluidic chip without the need for any other equipment. 10% human saliva samples were spiked with the known concentration of SARS-CoV-2 and sprayed to generate liquid droplets and aerosols into the air. Antibody-conjugated submicron particle suspension is then added to the paper channel, and a smartphone-based fluorescence microscope isolated and counted the immunoagglutinated particles on the paper chip. The total capture-to-assay time was <30 min, compared to several hours with the other methods. In this manner, SARS-CoV-2 could be detected directly from the air in a handheld and low-cost manner, contributing to slowing the spread of SARS-CoV-2. We can presumably adapt this technology to a wide range of other respiratory viruses.

Environmental Toxicology Assays Using Organ-on-Chip.

Adverse effects of environmental toxicants to human health have traditionally been assayed using in vitro assays. Organ-on-chip (OOC) is a new platform that can bridge the gaps between in vitro assays (or 3D cell culture) and animal tests. Microenvironments, physical and biochemical stimuli, and adequate sensing and biosensing systems can be integrated into OOC devices to better recapitulate the in vivo tissue and organ behavior and metabolism. While OOCs have extensively been studied for drug toxicity screening, their implementation in environmental toxicology assays is minimal and has limitations. In this review, recent attempts of environmental toxicology assays using OOCs, including multiple-organs-on-chip, are summarized and compared with OOC-based drug toxicity screening. Requirements for further improvements are identified and potential solutions are suggested.

Paper-based in vitro tissue chip for delivering programmed mechanical stimuli of local compression and shear flow.

ABSTRACT: Mechanical stimuli play important roles on the growth, development, and behavior of tissue. A simple and novel paper-based in vitro tissue chip was developed that can deliver two types of mechanical stimuli-local compression and shear flow-in a programmed manner. Rat vascular endothelial cells (RVECs) were patterned on collagen-coated nitrocellulose paper to create a tissue chip. Localized compression and shear flow were introduced by simply tapping and bending the paper chip in a programmed manner, utilizing an inexpensive servo motor controlled by an Arduino microcontroller and powered by batteries. All electrical compartments and a paper-based tissue chip were enclosed in a single 3D-printed enclosure, allowing the whole device to be independently placed within an incubator. This simple device effectively simulated in vivo conditions and induced successful RVEC migration in as early as 5 h. The developed device provides an inexpensive and flexible alternative for delivering mechanical stimuli to other in vitro tissue models.

Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips.

The recent Zika virus (ZIKV) outbreak has prompted the need for field-ready diagnostics that are rapid, easy-to-use, handheld, and disposable while providing extreme sensitivity and specificity. To meet this demand, we developed a wax-printed paper microfluidic chip utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). The developed simple and sensitive ZIKV assay was demonstrated using undiluted tap water, human urine, and diluted (10%) human blood plasma. Paper type, pore size, and channel dimension of various paper microfluidic chips were investigated and optimized to ensure proper filtration of direct-use biological samples (tap water, urine, and plasma) during capillary action-driven flow. Once ZIKV RNA has flowed and reached to a detection area of the paper microfluidic chip, it was excised for the addition of an RT-LAMP mixture with a pH indicator, then placed on a hot plate at 68 °C. Visible color changes from successful amplification were observed in 15 minutes and quantified by smartphone imaging. The limit of detection was as low as 1 copy/µL. The developed platform can also be used for identifying other flaviviruses, such as Chikungunya virus (CHIKV) and Dengue virus (DENV), and potentially other quickly transmitted virus pathogens, towards field-based diagnostics.