ABSTRACT
The unprecedented global pandemic known as SARS-CoV-2 has exercised to its limits nearly all aspects of modern viral diagnostics. In doing so, it has illuminated both the advantages and limitations of current technologies. Tremendous effort has been put forth to expand our capacity to diagnose this deadly virus. In this work, we put forth key observations in the functionality of current methods for SARS-CoV-2 diagnostic testing. These methods include nucleic acid amplification-, CRISPR-, sequencing-, antigen-, and antibody-based detection methods. Additionally, we include analysis of equally critical aspects of COVID-19 diagnostics, including sample collection and preparation, testing models, and commercial response. We emphasize the integrated nature of assays, wherein issues in sample collection and preparation could impact the overall performance in a clinical setting.
Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Pandemics , SARS-CoV-2/isolation & purification , COVID-19/virology , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , RNA, Viral/analysis , SARS-CoV-2/genetics , Specimen Handling/methodsABSTRACT
A microfluidic PCR device was developed that enables DNA amplification at speeds as fast as 2 s/cycle, with concurrent detection and amplification. Two targets were amplified from human genomic DNA. By observing the fluorescence emitted by a DNA dye while the sample is amplified, it is possible to obtain both qPCR and spatial melting information about the amplified product. The speed and integration of the device make it conducive to while-you-wait diagnostic tests that do not require post-PCR analysis.
Subject(s)
DNA/genetics , Microfluidic Analytical Techniques , Nucleic Acid Amplification Techniques , Real-Time Polymerase Chain Reaction , Temperature , DNA/analysis , Fluorescence , Humans , Optical ImagingABSTRACT
Transparent surfaces within microfluidic devices are essential for accurate quantification of chemical, biological, and mechanical interactions. Here, we report how to create low-cost, rapid 3D-printed microfluidic devices that are optically free from artifacts and have transparent surfaces suitable for visualizing a variety of fluid phenomenon. The methodology described here can be used for creating high-pressure microfluidic systems (significantly higher than PDMS-glass bonding). We develop methods for annealing Poly-Lactic Acid (PLA) microfluidic devices demonstrating heat resistance typically not achievable with other plastic materials. We show DNA melting and subsequent fluorescent imaging analysis, opening the door to other high-temperature applications. The FDM techniques demonstrated here allow for fabrication of microfluidic devices for precise visualization of interfacial dynamics, whether mixing between two laminar streams or droplet tracking. In addition to these characterizations, we include a printer troubleshooting guide and printing recipes for device fabrication to facilitate FDM printing for microfluidic device development.
Subject(s)
Hot Temperature , Lab-On-A-Chip Devices , Pressure , Printing, Three-Dimensional , Diffusion of Innovation , Optical Imaging , Polyesters/chemistryABSTRACT
As one type of non-Newtonian fluid, viscoelastic fluids exhibit unique properties that contribute to particle lateral migration in confined microfluidic channels, leading to opportunities for particle manipulation and separation. In this paper, particle focusing in viscoelastic flow is studied in a wide range of polyethylene glycol (PEO) concentrations in aqueous solutions. Polystyrene beads with diameters from 3 to 20 µm are tested, and the variation of particle focusing position is explained by the coeffects of inertial flow, viscoelastic flow, and Dean flow. We showed that particle focusing position can be predicted by analyzing the force balance in the microchannel, and that particle separation resolution can be improved in viscoelastic flows.
ABSTRACT
OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.
Subject(s)
Azoospermia , Microdissection , Microfluidics , Reproductive Techniques, Assisted , Sperm Retrieval/instrumentation , Testis/pathology , Adult , Azoospermia/complications , Azoospermia/diagnosis , Biopsy/methods , Clinical Laboratory Techniques , Embryology/methods , Equipment Design , Humans , Infertility, Male/diagnosis , Infertility, Male/etiology , Male , Microdissection/instrumentation , Microdissection/methods , Microfluidics/instrumentation , Microfluidics/methods , Specimen Handling/instrumentation , Specimen Handling/methods , Sperm Count , Sperm MotilityABSTRACT
Non-obstructive azoospermia (NOA) is the most severe form of male factor infertility. It is characterized by a lack of spermatogenesis in the seminiferous tubules. Microdissection testicular sperm extraction (microTESE) has significantly improved testicular sperm retrieval rates compared to conventional techniques for NOA. Following testicular biopsy, the sperm is usually non-motile and contained within seminiferous tubules requiring extensive laboratory processing to find individual sperm sufficient for artificial reproductive technologies (ART). Current techniques include mechanical and enzymatic processing which is time-consuming and often damaging to sperm. We review novel techniques that may help improve sperm retrieval rates after microTESE including microfluidics (dielectrophoretic cell sorting, spiral channel sorting, and pinched flow fractionation), fluorescence-activated cell sorting (FACS), and magnetic-activated cell sorting (MACS).