Cross-kingdom pathogen detection via duplex universal PCR and high-resolution melt.

Infectious diseases caused by pathogenic bacteria and fungi continuously pose a significant threat worldwide. The occurrence of polymicrobial infections, including polybacterial, polyfungal or bacteria-fungal co-infections further complicates diagnosis and treatment. Current diagnostic methods, heavily reliant on culture methods, are slow and often inefficient.

Multiplex digital profiling of DNA methylation heterogeneity for sensitive and cost-effective cancer detection in low-volume liquid biopsies.

Molecular alterations in cancerous tissues exhibit intercellular genetic and epigenetic heterogeneity, complicating the performance of diagnostic assays, particularly for early cancer detection. Conventional liquid biopsy methods have limited sensitivity and/or ability to assess epigenetic heterogeneity of rare epiallelic variants cost-effectively. We report an approach, named REM-DREAMing (Ratiometric-Encoded Multiplex Discrimination of Rare EpiAlleles by Melt), which leverages a digital microfluidic platform that incorporates a ratiometric fluorescence multiplex detection scheme and precise digital high-resolution melt analysis to enable low-cost, parallelized analysis of heterogeneous methylation patterns on a molecule-by-molecule basis for the detection of cancer in liquid biopsies.

Exploiting β-Lactams-Induced Lysis and DNA Fragmentation for Rapid Molecular Antimicrobial Susceptibility Testing of Neisseria Gonorrhoeae via Dual-Digital PCR.

The evolution of antimicrobial resistance (AMR) presents substantial challenges to global medical health systems. Neisseria gonorrhoeae (N. gonorrhoeae), in particular, has developed resistance to all currently available antimicrobials.

Enhanced CRISPR/Cas-Based Immunoassay through Magnetic Proximity Extension and Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated systems have recently emerged as a focal point for developing next-generation molecular diagnosis, particularly for nucleic acid detection. However, the detection of proteins is equally critical across diverse applications in biology, medicine, and the food industry, especially for diagnosing and prognosing diseases like cancer, Alzheimer's and cardiovascular conditions. Despite recent efforts to adapt CRISPR/Cas systems for protein detection with immunoassays, these methods typically achieved sensitivity only in the femtomolar to picomolar range, underscoring the need for enhanced detection capabilities.

Automatic wild bird repellent system that is based on deep-learning-based wild bird detection and integrated with a laser rotation mechanism.

Wild bird repulsion is critical in agriculture because it helps avoid agricultural food losses and mitigates the risk of avian influenza. Wild birds transmit avian influenza in poultry farms and thus cause large economic losses. In this study, we developed an automatic wild bird repellent system that is based on deep-learning-based wild bird detection and integrated with a laser rotation mechanism.

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).

Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles.

Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level.

Cancer Methylation Biomarker Detection in an Automated, Portable, Multichannel Magnetofluidic Platform.

Early detection of cancer is critical to improving clinical outcomes, especially in territories with limited healthcare resources. DNA methylation biomarkers have shown promise in early cancer detection, but typical workflows require highly trained personnel and specialized equipment for manual and lengthy processing, limiting use in resource-constrained areas. As a potential solution, we introduce the Automated Cartridge-based Cancer Early Screening System (ACCESS), a compact, portable, multiplexed, automated platform that performs droplet magnetofluidic- and methylation-specific qPCR-based assays for the detection of DNA methylation cancer biomarkers.

Automated and miniaturized screening of antibiotic combinations robotic-printed combinatorial droplet platform.

Antimicrobial resistance (AMR) has become a global health crisis in need of novel solutions. To this end, antibiotic combination therapies, which combine multiple antibiotics for treatment, have attracted significant attention as a potential approach for combating AMR. To facilitate advances in antibiotic combination therapies, most notably in investigating antibiotic interactions and identifying synergistic antibiotic combinations however, there remains a need for automated high-throughput platforms that can create and examine antibiotic combinations on-demand, at scale, and with minimal reagent consumption.

Improving bacteria identification from digital melt assay via oligonucleotide-based temperature calibration.

Background: Bacterial infections, especially polymicrobial infections, remain a threat to global health and require advances in diagnostic technologies for timely and accurate identification of all causative species. Digital melt - microfluidic chip-based digital PCR combined with high resolution melt (HRM) - is an emerging method for identification and quantification of polymicrobial bacterial infections. Despite advances in recent years, existing digital melt instrumentation often delivers nonuniform temperatures across digital chips, resulting in nonuniform digital melt curves for individual bacterial species.

Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics.

Harnessing Variabilities in Digital Melt Curves for Accurate Identification of Bacteria.

Digital PCR combined with high resolution melt (HRM) is an emerging method for identifying pathogenic bacteria with single cell resolution via species-specific digital melt curves. Currently, the development of such digital PCR-HRM assays entails first identifying PCR primers to target hypervariable gene regions within the target bacteria panel, next performing bulk-based PCR-HRM to examine whether the resulting species-specific melt curves possess sufficient interspecies variability (i.e.

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.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition.

Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays.

Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported.

Sensitive and Quantitative Point-of-Care HIV Viral Load Quantification from Blood Using a Power-Free Plasma Separation and Portable Magnetofluidic Polymerase Chain Reaction Instrument.

Point-of-care (POC) HIV viral load (VL) tests are needed to enhance access to HIV VL testing in low- and middle-income countries (LMICs) and to enable HIV VL self-testing at home, which in turn have the potential to enhance the global management of the disease. While methods based on real-time reverse transcription-polymerase chain reaction (RT-PCR) are highly sensitive and quantitatively accurate, they often require bulky and expensive instruments, making applications at the POC challenging. On the other hand, although methods based on isothermal amplification techniques could be performed using low-cost instruments, they have shown limited quantitative accuracies, i.

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.

Payload distribution and capacity of mRNA lipid nanoparticles.

Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level.

Highly Sensitive Serum Protein Analysis Using Magnetic Bead-Based Proximity Extension Assay.

Many protein biomarkers are present in biofluids at a very low level but may play critical roles in important biological processes. The fact that these low-abundance proteins remain largely unexplored underscores the importance of developing new tools for highly sensitive protein detection. Although digital enzyme-linked immunosorbent assay (ELISA) has demonstrated ultrahigh sensitivity compared with conventional ELISA, the requirement of specialized instruments limits the accessibility and prevents the widespread implementation.

Facile and scalable tubing-free sample loading for droplet microfluidics.

Droplet microfluidics has in recent years found a wide range of analytical and bioanalytical applications. In droplet microfluidics, the samples that are discretized into droplets within the devices are predominantly loaded through tubings, but such tubing-based sample loading has drawbacks such as limited scalability for processing many samples, difficulty for automation, and sample wastage. While advances in autosamplers have alleviated some of these drawbacks, sample loading that can instead obviate tubings offers a potentially promising alternative but has been underexplored.

Emerging platforms for high-throughput enzymatic bioassays.

Enzymes have essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in vitro enzymatic bioassays have been developed for use in industrial and research fields, such as cell biology, enzyme engineering, drug screening, and biofuel production. Of note, many of these require the use of high-throughput platforms.

Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification.

Polymerase chain reaction (PCR)-based diagnostic testing is the gold standard method for pathogen identification (ID) with recent developments enabling automated PCR tests for point-of-care (POC) use. However, multiplexed identification of several pathogens in PCR assays typically requires optics for an equivalent number of fluorescence channels, increasing instrumentation's complexity and cost. In this study, we first developed ratiometric PCR that surpassed one target per color barrier to allow multiplexed identification while minimizing optical components for affordable POC use.

A Portable Droplet Magnetofluidic Device for Point-of-Care Detection of Multidrug-Resistant .

is an emerging multidrug-resistant fungal pathogen that can cause severe and deadly infections. To date, has spurred outbreaks in healthcare settings in thirty-three countries across five continents. To control and potentially prevent its spread, there is an urgent need for point-of-care (POC) diagnostics that can rapidly screen patients, close patient contacts, and surveil environmental sources.

Filtration-assisted magnetofluidic cartridge platform for HIV RNA detection from blood.

The ability to detect and quantify HIV RNA in blood is essential to sensitive detection of infections and monitoring viremia throughout treatment. Current options for point-of-care HIV diagnosis ( lateral flow rapid tests) lack sensitivity for early detection and are unable to quantify viral load. HIV RNA diagnostics typically require extensive pre-processing of blood to isolate plasma and extract nucleic acids, in addition to expensive equipment for conducting nucleic acid amplification and fluorescence detection.

Bridging the gap between development of point-of-care nucleic acid testing and patient care for sexually transmitted infections.

The incidence rates of sexually transmitted infections (STIs), including the four major curable STIs - chlamydia, gonorrhea, trichomoniasis and, syphilis - continue to increase globally, causing medical cost burden and morbidity especially in low and middle-income countries (LMIC). There have seen significant advances in diagnostic testing, but commercial antigen-based point-of-care tests (POCTs) are often insufficiently sensitive and specific, while near-point-of-care (POC) instruments that can perform sensitive and specific nucleic acid amplification tests (NAATs) are technically complex and expensive, especially for LMIC. Thus, there remains a critical need for NAAT-based STI POCTs that can improve diagnosis and curb the ongoing epidemic.