Picoliter Droplet Generation and Dense Bead-in-Droplet Encapsulation via Microfluidic Devices Fabricated via 3D Printed Molds.

Picoliter-scale droplets have many applications in chemistry and biology, such as biomolecule synthesis, drug discovery, nucleic acid quantification, and single cell analysis. However, due to the complicated processes used to fabricate microfluidic channels, most picoliter (pL) droplet generation methods are limited to research in laboratories with cleanroom facilities and complex instrumentation. The purpose of this work is to investigate a method that uses 3D printing to fabricate microfluidic devices that can generate droplets with sizes <100 pL and encapsulate single dense beads mechanistically. Our device generated monodisperse droplets as small as ~48 pL and we demonstrated the usefulness of this droplet generation technique in biomolecule analysis by detecting Lactobacillus acidophillus 16s rRNA via digital loop-mediated isothermal amplification (dLAMP). We also designed a mixer that can be integrated into a syringe to overcome dense bead sedimentation and found that the bead-in-droplet (BiD) emulsions created from our device had <2% of the droplets populated with more than 1 bead. This study will enable researchers to create devices that generate pL-scale droplets and encapsulate dense beads with inexpensive and simple instrumentation (3D printer and syringe pump). The rapid prototyping and integration ability of this module with other components or processes can accelerate the development of point-of-care microfluidic devices that use droplet-bead emulsions to analyze biological or chemical samples with high throughput and precision.

REASSURED Multiplex Diagnostics: A Critical Review and Forecast.

The diagnosis of infectious diseases is ineffective when the diagnostic test does not meet one or more of the necessary standards of affordability, accessibility, and accuracy. The World Health Organization further clarifies these standards with a set of criteria that has the acronym ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users). The advancement of the digital age has led to a revision of the ASSURED criteria to REASSURED: Real-time connectivity, Ease of specimen collection, Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free or simple, and Deliverable to end-users. Many diagnostic tests have been developed that aim to satisfy the REASSURED criteria; however, most of them only detect a single target. With the progression of syndromic infections, coinfections and the current antimicrobial resistance challenges, the need for multiplexed diagnostics is now more important than ever. This review summarizes current diagnostic technologies for multiplexed detection and forecasts which methods have promise for detecting multiple targets and meeting all REASSURED criteria.

Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples.

Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response of Escherichia coli present within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility of E. coli directly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.

Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma.

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.

Flow-through Capture and in Situ Amplification Can Enable Rapid Detection of a Few Single Molecules of Nucleic Acids from Several Milliliters of Solution.

Detecting nucleic acids (NAs) at zeptomolar concentrations (few molecules per milliliter) currently requires expensive equipment and lengthy processing times to isolate and concentrate the NAs into a volume that is amenable to amplification processes, such as PCR or LAMP. Shortening the time required to concentrate NAs and integrating this procedure with amplification on-device would be invaluable to a number of analytical fields, including environmental monitoring and clinical diagnostics. Microfluidic point-of-care (POC) devices have been designed to address these needs, but they are not able to detect NAs present in zeptomolar concentrations in short time frames because they require slow flow rates and/or they are unable to handle milliliter-scale volumes. In this paper, we theoretically and experimentally investigate a flow-through capture membrane that solves this problem by capturing NAs with high sensitivity in a short time period, followed by direct detection via amplification. Theoretical predictions guided the choice of physical parameters for a chitosan-coated nylon membrane; these predictions can also be applied generally to other capture situations with different requirements. The membrane is also compatible with in situ amplification, which, by eliminating an elution step enables high sensitivity and will facilitate integration of this method into sample-to-answer detection devices. We tested a wide range of combinations of sample volumes and concentrations of DNA molecules using a capture membrane with a 2 mm radius. We show that for nucleic acid detection, this approach can concentrate and detect as few as ∼10 molecules of DNA with flow rates as high as 1 mL/min, handling samples as large as 50 mL. In a specific example, this method reliably concentrated and detected ∼25 molecules of DNA from 50 mL of sample.

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15†Minutes of Antibiotic Exposure.

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The "holy grail" of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.