Reimagining Existing Technologies for Faster Time-to-Detection of Biomarkers of Interest
PublisherThe University of Arizona.
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AbstractWith the goal of reducing the overall assay times of key diagnostic technologies, four platforms have been developed over the course of several years. Each platform seeks to take a gold standard diagnostic test for a given target of interest and reduce the time-to-results. The primary gold standard techniques being challenged are cell subpopulation analysis via flow cytometry, bacteria/virus detection via PCR/RT-PCR, and microRNA detection via RT-PCR. The first diagnostic platform utilizes a smartphone-based device to both characterize natural killer cell subpopulation ratio and quantify circulating natural killer cell and IL-2 cytokine concentrations in blood. This machine learning-based analysis used the cell chromatography profile of samples that had flowed through the paper chip and had an overall accuracy of 89% at correctly classifying the CD56bright: CD56dim ratio without the need for complex instrumentation beyond a paper microfluidic chip, a smartphone microscope attachment, and a simple 3D-printed enclosure within a 10-minute assay. Meanwhile, the flow rate-based cell/cytokine quantification aspect of the platform allowed for the detection of just 98 IU/mL IL-2 and 68 natural killer cells/mL, allowing for sensitive and accurate quantification of circulating biomarkers tied to patient immune response. The next three platforms all contain the use of emulsified loop-mediated isothermal amplification (eLAMP) reactions to compartmentalize the reaction process to increase specificity while also reducing assay time due to requiring smaller reaction volume changes over the course of the assay. These platforms monitor these reactions using light scatter technology, which allows for real-time monitoring without the use of a target-specific bioreceptor or fluorophore, thus reducing the overall cost of the reaction. The first of these three platforms was developed to detect whole bacteria (E. Coli O157:H7 in this case) with a limit of detection of 103 CFU/µL using either an OceanOptics spectrophotometer or a smartphone camera to monitor the light scatter change within as little as 3 minutes. This platform was also able to provide a higher assay specificity when compared to conventional LAMP reactions. The second eLAMP platform was developed with the goal of rapid yet sensitive SARS-CoV-2 detection in mind. By using the eLAMP assay mechanism, we could combine the rapid time-to-results of conventional antigen tests with the sensitivity/specificity of RT-PCR tests. The platform was able to detect as little as 10 viral copies/µL in saliva in just 5 minutes. Finally, the last eLAMP platform was built by making a minor modification to an existing real-time fluorometer, thus removing the need for an engineering-heavy skillset to build the technology. Within this fluorometer, microRNAs could be used as a primer in an emulsified LAMP reaction, thus controlling the rate of reaction based on the presence of the microRNA of interest. With this platform, the successful detection of 1 fM miR-21 could be made in just 66% of the time of a conventional LAMP reaction, and this assay also proved to be specific to single base-pair differences in the microRNA target, rendering it highly specific.
Degree ProgramGraduate College