Toward Decentralizing Antibiotic Susceptibility Testing via Ready-to-Use Microwell Array and Resazurin-Aided Colorimetric Readout.

In the face of the global threat from drug-resistant superbugs, there remains an unmet need for simple and accessible diagnostic tools that can perform important antibiotic susceptibility testing against pathogenic bacteria and guide antibiotic treatments outside of centralized clinical laboratories. As a potential solution to this important problem, we report herein the development of a microwell array-based resazurin-aided colorimetric antibiotic susceptibility test (marcAST). At the core of marcAST is a ready-to-use microwell array device that is preassembled with custom titers of various antibiotics and splits bacterial samples upon a simple syringe injection step to initiate AST against all antibiotics. We also employ resazurin, which changes from blue to pink in the presence of growing bacteria, to accelerate and enable colorimetric readout in our AST. Even with its simplicity, marcAST can accurately measure the minimum inhibitory concentrations of reference bacterial strains against common antibiotics and categorize the antibiotic susceptibilities of clinically isolated bacteria. With more characterization and refinement, we envision that marcAST can become a potentially useful tool for performing AST without trained personnel, laborious procedures, or bulky instruments, thereby decentralizing this important test for combating drug-resistant superbugs.

Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine.

The development of accelerated methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) for infectious diseases is necessary to facilitate evidence-based antibiotic therapy and reduce clinical overreliance on broad-spectrum antibiotics. Towards this end, droplet-based microfluidics has unlocked remarkably rapid diagnostic assays with single-cell and single-molecule resolution. Yet, droplet platforms invariably rely on testing purified bacterial samples that have been clinically isolated after lengthy (>16 h) plating. While plating-based clinical isolation is important for enriching and separating out bacteria from background in clinical samples and also facilitating buffer exchange, it creates a diagnostic bottleneck that ultimately precludes droplet-based methods from achieving significantly accelerated times-to-result. To alleviate this bottleneck, we have developed facile syringe filter-enabled strategies for bacterial separation, enrichment, and buffer exchange from urine samples. By selecting appropriately sized filter membranes, we separated bacterial cells from background particulates in urine samples and achieved up to 91% bacterial recovery after such 1-step filtration. When interfaced with droplet-based detection of bacterial cells, 1-step filtration improved the limit of detection for bacterial ID and quantification by over an order of magnitude. We also developed a facile buffer exchange strategy to prepare bacteria in urine samples for droplet-based AST that achieved up to 10-fold bacterial enrichment during buffer exchange. Our filtration strategies, can be easily integrated into droplet workflows, enable clinical isolation-free sample-to-answer ID and AST, and significantly accelerate the turnaround of standard infectious disease diagnostic workflows.

Highly Efficient Real-Time Droplet Analysis Platform for High-Throughput Interrogation of DNA Sequences by Melt.

Droplet microfluidic platforms have greatly enhanced the throughput and sensitivity of single-molecule and single-cell analyses. However, real-time analyses of individual droplets remain challenging. Most droplet microfluidic platforms have fundamental drawbacks that undermine their utility toward applications that rely on real-time monitoring to identify rare variants, such as bacterial persistence, drug discovery, antibody production, epigenetic biomarker analyses, etc. We present a platform for high-density droplet trapping and real-time analysis with 100% loading and trapping efficiency at a packing density of 110,000 droplets per in2. To demonstrate real-time analysis capabilities, we perform digital PCR and parallelized digital high-resolution melt curve acquisition on droplets to discriminate methylation levels of a tumor suppressor gene, CDO1, on a molecule-by-molecule basis. We hope that this platform, which is compatible with a large range of droplet sizes and generation technologies, may facilitate high-throughput real-time analyses on a molecule-by-molecule or cell-by-cell basis of heterogeneous populations.

Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens.

Point-of-care (POC) diagnostics for infectious diseases have the potential to improve patient care and antibiotic stewardship. Nucleic acid hybridization is at the core of many amplification-free molecular diagnostics and detection probe configuration is key to diagnostic performance. Modified nucleic acids such as peptide nucleic acid (PNA) offer advantages compared to conventional DNA probes allowing for faster hybridization, better stability and minimal sample preparation for direct detection of pathogens. Probes with tethered fluorophore and quencher allow for solution-based assays and eliminate the need for washing steps thereby facilitating integration into microfluidic devices. Here, we compared the sensitivity and specificity of double stranded PNA probes (dsPNA) and PNA molecular beacons targeting E. coli and P. aeruginosa for direct detection of bacterial pathogens. In bulk fluid assays, the dsPNAs had an overall higher fluorescent signal and better sensitivity and specificity than the PNA beacons for pathogen detection. We further designed and tested an expanded panel of dsPNA probes for detection of a wide variety of pathogenic bacteria including probes for universal detection of eubacteria, Enterobacteriaceae family, and P. mirablis. To confirm that the advantage translated to other assay types we compared the PNA beacon and dsPNA in a prototype droplet microfluidic device. Beyond the bulk fluid assay and droplet devices, use of dsPNA probes may be advantageous in a wide variety of assays that employ homogenous nucleic acid hybridization.

Simple and Precise Counting of Viable Bacteria by Resazurin-Amplified Picoarray Detection.

Simple, fast, and precise counting of viable bacteria is fundamental to a variety of microbiological applications such as food quality monitoring and clinical diagnosis. To this end, agar plating, microscopy, and emerging microfluidic devices for single bacteria detection have provided useful means for counting viable bacteria, but they also have their limitations ranging from complexity, time, and inaccuracy. We present herein our new method RAPiD (Resazurin-Amplified Picoarray Detection) for addressing this important problem. In RAPiD, we employ vacuum-assisted sample loading and oil-driven sample digitization to stochastically confine single bacteria in Picoarray, a microfluidic device with picoliter-sized isolation chambers (picochambers), in <30 s with only a few minutes of hands-on time. We add AlamarBlue, a resazurin-based fluorescent dye for bacterial growth, in our assay to accelerate the detection of "microcolonies" proliferated from single bacteria within picochambers. Detecting fluorescence in picochambers as an amplified surrogate for bacterial cells allows us to count hundreds of microcolonies with a single image taken via wide-field fluorescence microscopy. We have also expanded our method to practically test multiple titrations from a single bacterial sample in parallel. Using this expanded "multi-RAPiD" strategy, we can quantify viable cells in E. coli and S. aureus samples with precision in ∼3 h, illustrating RAPiD as a promising new method for counting viable bacteria for microbiological applications.

Complements Spurned: Our Experience with Atypical Hemolytic Uremic Syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare disorder resulting from a dysregulated activation of the alternative pathway of the complement system. It results in significant morbidity and mortality if not diagnosed and treated promptly. It lends itself to myriad renal and extrarenal manifestations, all potentially disabling. Eculizumab, a monoclonal antibody to complement C5 is now the widely accepted norm for treatment. However, in resource-limited settings, plasma exchange if instituted early may be as beneficial. We report a case of aHUS treated with extended plasma exchange with excellent results. Critical care monitoring is essential for the management of the disease in view of a tendency to develop multiple complications. Long-term immunosuppression may be successful in maintaining remission.

Clinically HIV but negative serology: Think of idiopathic CD4(+) lymphocytopenia.

idiopathic CD4(+) lymphocytopenia (ICL) is a rare disorder characterized by the presence of depleted CD4 cell line without the presence of HIV infection. Slight male preponderance is noticed and is usually seen in the middle age group. Opportunistic infections are the reason for their discovery and here we describe a case where a man was diagnosed as having Pneumocystis jiroveci pneumonia and oral candidiasis.

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. To fill the gap, we introduce herein a droplet device that features a new Tubing Eliminated Sample Loading Interface (TESLI). TESLI integrates a network of programmable pneumatic microvalves that regulate vacuum and pressure sources so that successive sub-microliter samples can be directly spotted onto the open-to-atmosphere TESLI inlet, vacuumed into the device, and pressurized into nanoliter droplets within the device with minimal wastage. The same vacuum and pressure regulation also endows TESLI with cleaning and sample switching capabilities, thus enabling scalable processing of many samples in succession. Moreover, we implement a pair of TESLIs in our device to parallelize and alternate their operation as means to minimizing idle time. For demonstration, we use our device to successively process 44 samples into droplets-a number that can further scale. Our results demonstrate the feasibility of tubing-free sample loading and a promising approach for advancing droplet microfluidics.

A Cascaded Droplet Microfluidic Platform Enables High-Throughput Single Cell Antibiotic Susceptibility Testing at Scale.

The global threat of antibiotic resistance underscores critical but unmet needs for rapid antibiotic susceptibility testing (AST) technologies. To this end, droplet microfluidic-based single-cell AST offers promise by achieving unprecedented rapidity, but its potential for clinical use is marred by the capacity of testing one to few antibiotic conditions per device, which falls short from the required scale in clinically relevant scenarios. To lift the scalability constraint in rapid single-cell AST technologies, a new cascaded droplet microfluidic platform that can streamline bacteria/antibiotic mixing, single-cell encapsulation within picoliter droplets, incubation, and detection in a continuous, assembly-line-like workflow is developed. The scalability of the platform is demonstrated by generating 32 groups of ≈10 000 droplets with custom antibiotic conditions within a single device, from which a new statistics-based method is used to analyze the single cell data and produce clinically useful antibiograms with minimum inhibitory concentrations in ≈90 min for the first antibiotic, plus 2 min for each subsequent antibiotic condition. Potential clinical utility of this platform is demonstrated by testing three clinical isolates and eight urine specimens against four frequently used antibiotics, and 100% and 93.8% categorical agreements are achieved compared to laboratory-based results that became available after 48 h.

Digital CRISPR/Cas-Assisted Assay for Rapid and Sensitive Detection of SARS-CoV-2.

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.

Droplet-Based Single-Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min.

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 transcriptomic reveals molecular diversity and developmental heterogeneity of human stem cell-derived oligodendrocyte lineage cells.

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.

ddRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform.

Digital nucleic acid amplification tests (digital NAATs) have emerged as a popular tool for nucleic acid detection due to their high sensitivity and specificity. Most current digital NAAT platforms, however, are limited to a "one-color-one-target" approach wherein each target is encoded with a specific fluorescently-labeled probe for single-plex fluorometric detection. This approach is difficult to multiplex due to spectral overlap between any additional fluorophores, and multiplexability of digital NAATs has therefore been limited. As a means to scale multiplexability, we have developed a multiplexed digital NAAT platform, termed Droplet Digital Ratiometric Fluorescence Coding (ddRFC), via a padlock probe-based nucleic acid detection assay which encodes each nucleic acid target with a unique combination of 2 fluorophores. We detect this encoded two-color fluorescence signature of each target by performing digital amplification in microfluidic droplets. To demonstrate the utility of our platform, we have synthesized 6 distinct padlock probes, each rendering a unique two-color fluorescence signature to a nucleic acid target representing a clinically important sexually transmitted infection (STI). We proceed to demonstrate broad-based, two-plex, four-plex, and six-plex detection of the STI targets with single-molecule resolution. Our design offers a cost-effective approach to scale up multiplexability by simply tuning the number of molecular beacon binding sites on the padlock probe without redesigning amplification primers or fluorescent molecular beacons. With further development, our platform has the potential to enable highly multiplexed detection of nucleic acid targets, with potentially unrestricted multiplexability, and serve as a diagnostic tool for many more diseases in the future.

Investigating cone photoreceptor development using patient-derived NRL null retinal organoids.

Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal development and disease. We utilized iPSC organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop S-opsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development.

Customizing droplet contents and dynamic ranges via integrated programmable picodroplet assembler.

Droplet microfluidic technology is becoming increasingly useful for high-throughput and high-sensitivity detection of biological and biochemical reactions. Most current droplet devices function by passively discretizing a single sample subject to a homogeneous or random reagent/reaction condition into tens of thousands of picoliter-volume droplets for analysis. Despite their apparent advantages in speed and throughput, these droplet devices inherently lack the capability to customize the contents of droplets in order to test a single sample against multiple reagent conditions or multiple samples against multiple reagents. In order to incorporate such combinatorial capability into droplet platforms, we have developed the fully Integrated Programmable Picodroplet Assembler. Our platform is capable of generating customized picoliter-volume droplet groups from nanoliter-volume plugs which are assembled in situ on demand. By employing a combination of microvalves and flow-focusing-based discretization, our platform can be used to precisely control the content and volume of generated nanoliter-volume plugs, and thereafter the content and the effective dynamic range of picoliter-volume droplets. Furthermore, we can use a single integrated device for continuously generating, incubating, and detecting multiple distinct droplet groups. The device successfully marries the precise control and on-demand capability of microvalve-based platforms with the sensitivity and throughput of picoliter droplet platforms in a fully automated monolithic device. The device ultimately will find important applications in single-cell and single-molecule analyses.

Droplet microfluidics for high-sensitivity and high-throughput detection and screening of disease biomarkers.

Biomarkers are nucleic acids, proteins, single cells, or small molecules in human tissues or biological fluids whose reliable detection can be used to confirm or predict disease and disease states. Sensitive detection of biomarkers is therefore critical in a variety of applications including disease diagnostics, therapeutics, and drug screening. Unfortunately for many diseases, low abundance of biomarkers in human samples and low sample volumes render standard benchtop platforms like 96-well plates ineffective for reliable detection and screening. Discretization of bulk samples into a large number of small volumes (fL-nL) via droplet microfluidic technology offers a promising solution for high-sensitivity and high-throughput detection and screening of biomarkers. Several microfluidic strategies exist for high-throughput biomarker digitization into droplets, and these strategies have been utilized by numerous droplet platforms for nucleic acid, protein, and single-cell detection and screening. While the potential of droplet-based platforms has led to burgeoning interest in droplets, seamless integration of sample preparation technologies and automation of platforms from biological sample to answer remain critical components that can render these platforms useful in the clinical setting in the near future. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.

Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform.

There remains an urgent need for rapid diagnostic methods that can evaluate antibiotic resistance for pathogenic bacteria in order to deliver targeted antibiotic treatments. Toward this end, we present a rapid and integrated single-cell biosensing platform, termed dropFAST, for bacterial growth detection and antimicrobial susceptibility assessment. DropFAST utilizes a rapid resazurin-based fluorescent growth assay coupled with stochastic confinement of bacteria in 20 pL droplets to detect signal from growing bacteria after 1h incubation, equivalent to 2-3 bacterial replications. Full integration of droplet generation, incubation, and detection into a single, uninterrupted stream also renders this platform uniquely suitable for in-line bacterial phenotypic growth assessment. To illustrate the concept of rapid digital antimicrobial susceptibility assessment, we employ the dropFAST platform to evaluate the antibacterial effect of gentamicin on E. coli growth.

Non-contact method for directing electrotaxis.

We present a method to induce electric fields and drive electrotaxis (galvanotaxis) without the need for electrodes to be in contact with the media containing the cell cultures. We report experimental results using a modification of the transmembrane assay, demonstrating the hindrance of migration of breast cancer cells (SCP2) when an induced a.c. electric field is present in the appropriate direction (i.e. in the direction of migration). Of significance is that migration of these cells is hindered at electric field strengths many orders of magnitude (5 to 6) below those previously reported for d.c. electrotaxis, and even in the presence of a chemokine (SDF-1α) or a growth factor (EGF). Induced a.c. electric fields applied in the direction of migration are also shown to hinder motility of non-transformed human mammary epithelial cells (MCF10A) in the presence of the growth factor EGF. In addition, we also show how our method can be applied to other cell migration assays (scratch assay), and by changing the coil design and holder, that it is also compatible with commercially available multi-well culture plates.