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The unprecedented demand for rapid diagnostics in response to the COVID‐19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated systems (Cas)‐assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital‐based detection. Thus motivated, the first digital CRISPR/Cas‐assisted assay—coined digitization‐enhanced CRISPR/Cas‐assisted one‐pot virus detection (deCOViD)—is developed and applied toward SARS‐CoV‐2 detection. deCOViD is realized through tuning and discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay into sub‐nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal‐to‐background ratio, broad dynamic range, and high sensitivity—down to 1 genome equivalent (GE) µL−1 of SARS‐CoV‐2 RNA and 20 GE µL−1 of heat‐inactivated SARS‐CoV‐2, which outstrips its benchtop‐based counterpart and represents one of the fastest and most sensitive CRISPR/Cas‐assisted SARS‐CoV‐2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas‐assisted assays and combating the COVID‐19 pandemic and beyond. A digital clustered regularly interspaced short palindromic repeats (CRISPR)/Cas‐assisted nucleic acid detection assay is created by discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay within sub‐nanoliter reaction wells of a commercial microfluidic digital chip, which enables quantitative detection of 1 genome equivalent µL−1 of SARS‐CoV‐2 RNA in <30 min—one of the fastest and most sensitive CRISPR/Cas‐assisted detection to date.

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. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation‐specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid‐biopsy‐based detection of non‐small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low‐volume liquid biopsy specimens from patients with computed tomography (CT)‐scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early‐stage NSCLC. This paper describes the development and analytical/clinical validation of the first mdMSP platform for the simultaneous analysis of four DNA methylation biomarkers for liquid‐biopsy‐based detection of early‐stage NSCLC. The demonstrated analytical sensitivity/specificity and clinical utility indicate the potential of mdMSP platform to be a complementary method for improved CT screening of lung cancer.

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. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%–80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs. Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics but assessing the mRNA packaging characteristics in LNPs is challenging. Here, the authors report that mRNA and lipid contents in LNP formulations can be quantitatively examined by multi-laser cylindrical illumination confocal spectroscopy at the single-nanoparticle level.

Empiric broad‐spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence‐based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet‐based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in‐droplet single‐bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram‐negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50‐specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas‐under‐curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility. Single‐cell measurements of bacterial 16S rRNA are leveraged in picoliter droplets to achieve simultaneous molecular detection of bacteria and phenotypic assessment of antimicrobial susceptibility, unlocking a 30 min sample‐to‐answer diagnostic platform for urinary tract infections.

With a 5-year survival rate of less than 50%, ovarian high-grade serous carcinoma (HGSC) is one of the most highly aggressive gynecological malignancies affecting women today. The high mortality rate of HGSC is largely attributable to delays in diagnosis, as most patients remain undiagnosed until the late stages of -disease. There are currently no recommended screening tests for ovarian cancer and there thus remains an urgent need for new diagnostic methods, particularly those that can detect the disease at early stages when clinical intervention remains effective. While diagnostics for ovarian cancer share many of the same technical hurdles as for other cancer types, the low prevalence of the disease in the general population, coupled with a notable lack of sensitive and specific biomarkers, have made the development of a clinically useful screening strategy particularly challenging. Here, we present a detailed review of the overall landscape of ovarian cancer diagnostics, with emphasis on emerging methods that employ novel protein, genetic, epigenetic and imaging-based biomarkers and/or advanced diagnostic technologies for the noninvasive detection of HGSC, particularly in women at high risk due to germline mutations such as BRCA1/2. Lastly, we discuss the translational potential of these approaches for achieving a clinically implementable solution for screening and diagnostics of early-stage ovarian cancer as a means of ultimately improving patient outcomes in both the general and high-risk populations.

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. Addressing this issue necessitates not only discovering new antimicrobials but also deepening the understanding of bacterial responses to these agents, which can lead to new markers for rapid antimicrobial susceptibility testing (AST). Such advancements can enhance treatment outcomes and promote antimicrobial stewardship. In this study, single‐cell techniques, including live‐cell imaging, flow cytometry, and digital polymerase chain reaction (PCR) are utilized, to investigate the lysis dynamics and molecular features of N. gonorrhoeae upon exposure to β‐lactam antimicrobials. Distinct patterns of bacterial lysis and DNA fragmentation are uncovered in susceptible strains. Leveraging these discoveries, a microfluidic dual‐digital PCR approach that combines single‐cell and single‐molecule analyses, facilitate rapid and efficient phenotypic molecular AST for N. gonorrhoeae against β‐lactams is developed. This proof‐of‐concept validation demonstrates the effectiveness of the method in accessing antimicrobial susceptibility across a range of bacterial strains, contributing valuable insights for advancing the battle against AMR. Utilizing DNA fragmentation and abrupt bacterial lysis induced by β‐lactam antimicrobials, a microfluidic dual‐digital PCR that integrates single‐cell and single‐molecule analyses for rapid phenotypic molecular antimicrobial susceptibility testing of Neisseria gonorrhoeae to β‐lactams is developed.

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs. Brain myelinating oligodendrocytes are rare and difficult to isolate, which has limited data on their development. Here the authors develop a reporter for scalable purification of human pluripotent stem cell derived oligodendrocyte lineage cells, and use this to map differentiation using single cell RNA-sequencing,

The ability to accurately measure aberrant DNA methylation levels is integral to the understanding of DNA methylation biology. It is well-established that in cancer, the largest, and thus, most biologically important absolute gains of DNA methylation levels occur at CpG sites with low native levels while the largest losses occur at CpG sites with high native levels. Conventional wisdom assumes that the observed association between the degree of the alterations and the native levels are largely due to the limitations of change within the DNA methylation scale. Here, we present evidence that this association is largely caused by alterations occurring as a global rate of change relative to the native level. We show that DNA methylation alterations can be accurately compared by calculating the rate of change relative to the native level. Most importantly, this approach enables the identification of more biologically significant DNA methylation alterations. The authors show that, when looking at differential methylation, assessing relative changes rather than absolute changes finds more biologically relevant targets.

Gold standard quantitative nucleic acid tests for diagnosis of viral diseases are currently limited to implementation in laboratories outside of the clinic. An instrument for conducting nucleic acid testing at the point-of-care (POC) that is easily operable by the clinician would reduce the required number of visits to the clinic and improve patient retention for proper treatment. Here we present a droplet magnetofluidic (DM) platform, which leverages functionalized magnetic particles to miniaturize and automate laboratory assays for use in the clinic at the POC. Our novel thermoformed disposable cartridge coupled to a portable multiaxial magnetofluidic instrument enables real-time PCR assays for quantitative and sensitive detection of nucleic acids from crude biosamples. Instead of laborious benchtop sample purification techniques followed by elution and spiking into PCR buffer, the user simply injects the biosample of interest into a cartridge with magnetic particles and loads the cartridge into the instrument. We demonstrate the utility of our platform with hepatitis C virus (HCV) RNA viral load quantitation from blood serum in approximately 1 hour. Clinical serum samples (n = 18) were directly processed on cartridges with no false positives and a limit of detection of 45 IU per 10 µl sample injection.

Bloodstream infections (BSI) caused by pathogenic bacteria are a global health threat, highlighting the need for improved diagnostic methods. Current methods, such as mass spectrometry, depend on time-consuming blood cultures, which hinder timely diagnosis. Nucleic acid amplification tests (NAATs) offer potential for sensitive bacteria detection but are bottlenecked by the inability to isolate bacteria at low concentrations from blood. This study introduces a facile dual filter workflow for isolating low concentrations of bacteria from blood without blood culture. The workflow, which involves blood dilution and osmolysis, blood cell filtration, chemical and enzymatic lysis, and filter-based bacteria capture, can be completed in ~ 30 min without special equipment. It readily combines with downstream broad-based bacteria detection via PCR and high-resolution melt (HRM), as well as pheno-molecular antimicrobial susceptibility testing (AST) via PCR. When coupled with species-specific PCR, ~ 10 CFU of bacteria in 0.5 mL of blood could be detected. The small sample volume is particularly attractive because it is comparable to those obtained from fingerstick collections or pediatric patients. With further optimization, testing with a wider range of bacteria, and integration with rapid point-of-care testing, this simple dual filter workflow can be a key driver for advancing blood culture-free bacterial BSI diagnosis, especially in resource-limited settings.