SUMMARY The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.

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This paper reports the development of a method to control the flow rate of fluids within paper-based microfluidic analytical devices. We demonstrate that by simply sandwiching paper channels between two flexible films, it is possible to accelerate the flow of water through paper by over 10-fold. The dynamics of this process are such that the height of the liquid is dependent on time to the power of 1/3. This dependence was validated using three different flexible films (with markedly different contact angles) and three different fluids (water and two silicon oils with different viscosities). These covered channels provide a low-cost method for controlling the flow rate of fluid in paper channels, and can be added following printing of reagents to control fluid flow in selected fluidic channels. Using this method, we redesigned a previously published bidirectional lateral flow pesticide sensor to allow more rapid detection of pesticides while eliminating the need to run the assay in two stages. The sensor is fabricated with sol-gel entrapped reagents (indoxyl acetate in a substrate zone and acetylcholinesterase, AChE, in a sensing zone) present in an uncovered "slow" flow channel, with a second, covered "fast" channel used to transport pesticide samples to the sensing region through a simple paper-flap valve. In this manner, pesticides reach the sensing region first to allow preincubation, followed by delivery of the substrate to generate a colorimetric signal. This format results in a uni-directional device that detects the presence of pesticides two times faster than the original bidirectional sensors.

Water soluble pullulan films were formatted into paper-based microfluidic devices, serving as a controlled time shutoff valve. The utility of the valve was demonstrated by a one-step, fully automatic implementation of a complex pesticide assay requiring timed, sequential exposure of an immobilized enzyme layer to separate liquid streams. Pullulan film dissolution and the capillary wicking of aqueous solutions through the device were measured and modeled providing valve design criteria. The films dissolve mainly by surface erosion, meaning the film thickness mainly controls the shutoff time. This method can also provide time-dependent sequential release of reagents without compromising the simplicity and low cost of paper-based devices.

We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.