Open Hardware for Microfluidics: Exploiting Raspberry Pi Singleboard Computer and Camera Systems for Customisable Laboratory Instrumentation.

The integration of Raspberry Pi miniature computer systems with microfluidics has revolutionised the development of low-cost and customizable analytical systems in life science laboratories. This review explores the applications of Raspberry Pi in microfluidics, with a focus on imaging, including microscopy and automated image capture. By leveraging the low cost, flexibility and accessibility of Raspberry Pi components, high-resolution imaging and analysis have been achieved in direct mammalian and bacterial cellular imaging and a plethora of image-based biochemical and molecular assays, from immunoassays, through microbial growth, to nucleic acid methods such as real-time-qPCR. The control of image capture permitted by Raspberry Pi hardware can also be combined with onboard image analysis. Open-source hardware offers an opportunity to develop complex laboratory instrumentation systems at a fraction of the cost of commercial equipment and, importantly, offers an opportunity for complete customisation to meet the users' needs. However, these benefits come with a trade-off: challenges remain for those wishing to incorporate open-source hardware equipment in their own work, including requirements for construction and operator skill, the need for good documentation and the availability of rapid prototyping such as 3D printing plus other components. These advances in open-source hardware have the potential to improve the efficiency, accessibility, and cost-effectiveness of microfluidic-based experiments and applications.

Dilution Reduces Sample Matrix Effects for Rapid, Direct, and Miniaturised Phenotypic Antibiotic Susceptibility Tests for Bovine Mastitis.

The time-consuming nature of current methods for detecting antimicrobial resistance (AMR) to guide mastitis treatment and for surveillance, drives innovation towards faster, easier, and more portable technology. Rapid on-farm testing could guide antibiotic selection, reducing misuse that contributes to resistance. We identify challenges that arise when developing miniaturized antibiotic susceptibility tests (AST) for rapid on-farm use directly in milk. We experimentally studied three factors: sample matrix (specifically milk or spoiled milk); the commensal bacteria found in fresh bovine milk; and result time on the performance of miniaturised AST. Microfluidic "dip-and-test" devices made from microcapillary film (MCF) were able to monitor Gram-negative bacterial growth colourimetrically even in the presence of milk and yoghurt (used to simulate spoiled milk samples), as long as this sample matrix was diluted 1:5 or more in growth medium. Growth detection kinetics using resazurin was not changed by milk at final concentrations of 20% or lower, but a significant delay was seen with yoghurt above 10%. The minimum inhibitory concentration (MIC) for ciprofloxacin and gentamicin was increased in the presence of higher concentrations of milk and yoghurt. When diluted to 1% all observed MIC were within range, indicating dilution may be sufficient to avoid milk matrix interfering with microfluidic AST. We found a median commensal cell count of 6 × 105 CFU/mL across 40 healthy milk samples and tested if these bacteria could alter microfluidic AST. We found that false susceptibility may be observed at early endpoint times if testing some pathogen and commensal mixtures. However, such errors are only expected to occur when a susceptible commensal organism is present at higher cell density relative to the resistant pathogen, and this can be avoided by reading at later endpoints, leading to a trade-off between accuracy and time-to-result. We conclude that with further optimisation, and additional studies of Gram-positive organisms, it should be possible to obtain rapid results for microfluidic AST, but a trade-off is needed between time-to-result, sample dilution, and accuracy.

Insights into implementation planning for point-of-care testing to guide treatment of chronic obstructive pulmonary disease exacerbation: a mixed methods feasibility study.

The purpose of this mixed methods feasibility study was to gain insights into unmet clinical needs, stakeholder preferences and potential barriers and enablers to adoption for planning the implementation of point-of-care testing for earlier detection and guided treatment of chronic obstructive pulmonary disease (COPD) acute exacerbation in the NHS in England. Exacerbations of COPD cause considerable mortality and morbidity. Earlier identification of exacerbations and guided treatment would lead to reduced exacerbation duration, reduced hospitalizations and mortality, improve health-related quality of life, reduce unnecessary treatments (including inappropriate antibiotic prescribing) which could save the NHS over £400 per patient. During the early stages of product design, we took a multi-disciplinary approach to evidence generation, gaining insights from key stakeholders to test the product concept and inform evidence-based implementation planning. Primary data was collected from 11 health care and service professionals involved in the management of acute COPD exacerbations. Overall, participants agreed that by earlier differentiation of acute exacerbation from stable COPD, patients could be started on appropriate treatment. To implement point-of-care testing into clinical practice, evidence is required to demonstrate the accuracy of differentiating between exacerbation etiologies and to provide information on the beneficial impact to the system in terms of optimized management, reduced long-term side effects, admission avoidance, and cost-effectiveness. This research provides an evidence base for future implementation planning of point-of-care testing for earlier detection and guided treatment of COPD acute exacerbation. Moreover, the technology developers can decide whether to refine the product design and value proposition thereby de-risking product development.

PiRamid: A compact Raspberry Pi imaging box to automate small-scale time-lapse digital analysis, suitable for laboratory and field use.

Digital imaging permits the quantitation of many experiments, such as microbiological growth assays, but laboratory digital imaging systems can be expensive and too specialised. The Raspberry Pi camera platform makes automated, controlled imaging affordable with accessible customisation. When combined with open source software and open-source 3D printed hardware, the control over image quality and capture of this platform permits the rapid development of novel instrumentation. Here we present "PiRamid", a compact, portable, and inexpensive enclosure for autonomous imaging both in the laboratory and in the field. The modular three-piece 3D printed design makes it easy to incorporate different camera systems or lighting configurations (e.g., single wavelength LED for fluorescence). The enclosed design allows complete control of illumination unlike a conventional digital camera or smartphone, on a tripod or handheld, under ambient lighting. The stackable design permits rapid sample addition or camera focus adjustment, with a corresponding change in magnification and resolution. The entire unit is small enough to fit within a microbiological incubator, and cheap enough (∼£100) to scale out for larger parallel experiments. Simply, Python scripts fully automate illumination and image capture for small-scale experiments with an ∼110×85 mm area at 70-90 µm resolution. We demonstrate the versatility of PiRamid by capturing time-resolved, quantitative image data for a wide range of assays. Bacterial growth kinetics was captured for conventional microbiology (agar Petri dishes), 3D printed custom microbiology labware and microfluidic microbiology. To illustrate application beyond microbiology, we demonstrate time-lapse imaging of crystal growth and degradation of salad leaves. Minor modifications permit epi-illumination by addition of a LED ring to the camera module. We conclude that PiRamid permits inexpensive digital capture and quantitation of a wide range of experiments by time-lapse imaging to simplify both laboratory and field imaging.

3D-Printed Dip Slides Miniaturize Bacterial Identification and Antibiotic Susceptibility Tests Allowing Direct Mastitis Sample Analysis.

The early detection of antimicrobial resistance remains an essential step in the selection and optimization of antibiotic treatments. Phenotypic antibiotic susceptibility testing including the measurement of minimum inhibitory concentration (MIC) remains critical for surveillance and diagnostic testing. Limitations to current testing methods include bulky labware and laborious methods. Furthermore, the requirement of a single strain of bacteria to be isolated from samples prior to antibiotic susceptibility testing delays results. The mixture of bacteria present in a sample may also have an altered resistance profile to the individual strains, and so measuring the susceptibility of the mixtures of organisms found in some samples may be desirable. To enable simultaneous MIC and bacterial species detection in a simple and rapid miniaturized format, a 3D-printed frame was designed for a multi-sample millifluidic dip-slide device that combines panels of identification culture media with a range of antibiotics (Ampicillin, Amoxicillin, Amikacin, Ceftazidime, Cefotaxime, Ofloxacin, Oxytetracycline, Streptomycin, Gentamycin and Imipenem) diluted in Muëller-Hinton Agar. Our proof-of-concept evaluation confirmed that the direct detection of more than one bacterium parallel to measuring MIC in samples is possible, which is validated using reference strains E. coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 10145, and Staphylococcus aureus ATCC 12600 and with mastitis milk samples collected from Reading University Farm. When mixtures were tested, a MIC value was obtained that reflected the most resistant organism present (i.e., highest MIC), suggesting it may be possible to estimate a minimum effective antibiotic concentration for mixtures directly from samples containing multiple pathogens. We conclude that this simple miniaturized approach to the rapid simultaneous identification and antibiotic susceptibility testing may be suitable for directly testing agricultural samples, which is achieved through shrinking conventional tests into a simple "dip-and-incubate" device that can be 3D printed anywhere.

Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples.

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device-termed Cygnus-with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-µl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58-0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63-0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.

Direct microfluidic antibiotic resistance testing in urine with smartphone capture: significant variation in sample matrix interference between individual human urine samples.

Rapid and portable direct tests for antibiotic resistance in human clinical samples such as urine could reduce misuse of precious antimicrobials, by allowing treatment decisions to be informed by microfluidic diagnostic tests. We demonstrate that the variable composition of human urine can significantly affect the antibiotic minimum inhibitory concentration (MIC) measured using microfluidic devices. The urine sample matrix interference was not observed in pooled normal urine, emphasising the critical importance of assessing matrix interference with a wide range of individual urine samples, rather than a few standardised or pooled controls. Both dilution into assay medium and inclusion of buffer could reduce the matrix interference, but dilution may affect analytical sensitivity by increasing the minimum bacterial cell density needed in a sample for growth to be detected, especially for miniaturised devices that test small sample volumes. We conclude it is vital to fully assess and optimise novel analytical microbiology tools using multiple individual urine samples, otherwise the high variation in matrix interference will compromise the clinical performance of these rapid diagnostics that are urgently needed to tackle the global threat of antimicrobial resistance.

MicroMI: A portable microbiological mobile incubator that uses inexpensive lithium power banks for field microbiology.

Incubation at controlled temperature is a key step in culture based microbiological tests. Access to culture-based microbiological testing requires access to conventional incubators in a laboratory. Portable incubators allow microbiological testing in the field and in resource-limited settings, and can eliminate the challenge of sample transportation, minimising the chance of sample degradation. Recent studies have reported low-cost portable incubator designs suitable for field or off-grid use, but these either need an external power supply (e.g. mains AC or 12 V DC), or rely on passive heating without thermostatic control. Here we report that small inexpensive uninterruptable power supply (UPS) products manufactured for consumer electronics and powered by lithium-ion battery packs allowing thermostatic temperature control in small portable incubators that can maintain precise temperatures with or without external power. We present an open-source design for a Microbiological Mobile Incubator (MicroMI) in two sizes for field use. The MicroMI is built from simple and widely available components and is easy to set up. The open source design can be customised for different numbers of samples. The smallest and most efficient design uses a vacuum insulated food flask that allows longer operation with smaller, lower capacity UPS. The larger flight case design has space for more samples, but depletes the battery faster. The UPS maintains a typical microbiology incubation temperature for up to 24 h without external power- ideal for typical incubation needed for culture methods. The battery capacity, incubator design, and external ambient temperature all affected duration of operation without requiring external power. We validated the MicroMI by conducting classical microbiological tests using agar petri dishes, slant cultures and dip slides, and biochemical tests. We conclude the MicroMI design allows inexpensive lithium battery products to be used to simplify field microbiology and increase access to vital analytical microbiology testing.