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.

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.

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.

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.

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.

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.