<p>Pathogen detection via viscosity quantification in biological systems has long been an essential aspect of biomedical research. The importance of persistent testing of pathogens such as <em>V. cholerae</em> and HIV has consistently been recognized but limited in regions where systematic and financial resources are unavailable. Current methods require the samples be transported to research labs primarily in large cities or different countries. For consistent pathogen testing to be performed in remote areas, detection methods must be designed for portability with laboratory standards and simplicity for use without much technical background in place. </p> <p>Particle Diffusometry is a visualization method on the result of the amplification of pathogen by quantifying the Brownian motion of suspended particles in a solution. The amplification usually occurs in the specialized machine; then, the fluid sample gets inserted into the microfluidic chip for optical observation for Brownian motion. The technique has been used in particle sizing and measuring viscosity change in the biomolecular solution. In use with limitations, I present the improvements on the existing Particle Diffusometry technique to expand its use in broader biomedical applications.</p> <p>We address the portability of the technique. In the emerging and fast-growing mobile technology market, we have developed a smartphone-based portable platform capable of performing par quality tasks compared to traditional lab-based microscopy. We successfully measure the presence of <em>V. cholerae</em> as few as 6 cells/reaction, a waterborne pathogen, where its DNA is spiked into environmental water sources in just under 35 minutes. To further make the overall technology portable, we developed an on-chip amplification method accompanied by the portable heating unit. A mobile heating unit removes the need for the qPCR machine to amplify the biomolecular structure. Also, it opens the capability of on-chip amplification, further simplifying the steps needed to identify the pathogen in the source. We confirm the validity of the developed setup by measuring the presence of as low as 50 SARS-CoV-2 virus particles within 10% saliva. </p> <p>Addressing two main limitations of the existing Particle Diffusometry technique, improvements in the algorithm occur. First, we improve the algorithm to calculate diffusion coefficients even when the particles suspended in the sample are experiencing unified patterns, hence the flow, when recording is taking place. The improved algorithm correctly identified the diffusion coefficient within  margin of error using simulation and experimental verification for the sample under simple shear flow types, uniform, Couette, and Poiseuille. Second, we address the mismatch between the frame rate of the camera and the Brownian motion of particles at elevated temperatures. By configuring the correction equation for the frame mismatch behavior, we corrected the deviation of the diffusion coefficient in the range of 3E-13 to 3E-12 m²/s. Ultimately, we applied the improved flow algorithm to the elevated temperature simulation, showing the error propagation does not differ by the temperature; the percentage of error in computing the diffusion coefficient for the sample exhibiting flow only depends on the flow velocity. </p> <p>Applying these two improvements, we perform measurements on over-time viscosity change using the hydrogel formation. We characterize the hydrogel formation time using the diffusion gradient plane and variation of the initiator. By applying the addressed improvements on the real-time detection of HIV amplification on-chip, we further validate the applicative nature of the extended Particle Diffusometry technique. </p> <p>Real-time flow-adjusted Particle Diffusometry is, therefore, a feasible method for detecting viscosity changes in both chemical and biomolecular solutions in real-time. This approach opens up an alternative method for measuring biological amplification in real-time. The improvements further open the existing Particle Diffusometry technique to be widely used in the field involving rheology and pathogen detection not only in the traditional lab-based setting but also out in the field. </p>

<div>Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.</div><div><br></div><div>Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use.</div>

<p dir="ltr">Since the beginning of the HIV/AIDS epidemic, 85.6 million people worldwide have become infected with HIV; more than half of whom have died from AIDS-related complications.[1] Sustained viral suppression below the clinically relevant threshold (1000 copies per mL) with highly active antiretroviral therapy (HAART) has proven effective at managing and prolonging the life expectancy of people living with HIV (PLHIV). However, in 2022, 11.3 million PLHIV had still not achieved viral suppression and may become susceptible to both HIV transmission and a variety of opportunistic infections. Of particular importance is the complex issue of patient non-compliance in global HIV management due to social, economic, behavioral, and healthcare access barriers, potentially disconnecting many PLHIV from the HIV care continuum. Therefore, to boost patient engagement in clinical care and to improve overall patient outcomes, new approaches to viral load monitoring practices need to be developed to increase access, particularly in regions of high HIV prevalence.</p><p dir="ltr">Nucleic acid amplification tests (NAATs) have emerged as potent tools for monitoring viral load, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) being recognized as the benchmark due to its sensitivity and ability for real-time quantification enabled by fluorescence signal emission. Nevertheless, RT-qPCR is burdened by drawbacks including extended processing times, high operational costs, and the requirement for specialized laboratory facilities. In this study, we propose a novel method for HIV-1 viral load monitoring by integrating reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) with real-time particle diffusometry (PD). This approach allows for the continuous monitoring of changes in the diffusion of 400 nm fluorescent particles during RT-LAMP amplification, targeting the <i>p24</i> gene region of HIV-1 RNA. This enables the real-time detection of amplification curves, achieving a detection sensitivity in water samples as low as 25 virus particles per μL within a short duration of 30 minutes. Additionally, to address challenges related to amplification inhibition in complex human specimens, we developed a power-free sample processing system specifically designed for extracting HIV-1 RNA from both whole blood and plasma.Top of FormBottom of FormThis system modifies a commercially available spin-column protocol by integrating a syringe device and handheld bulb dryer, thus eliminating the requirement for a centrifuge. The adaptation allows for the completion of the entire extraction procedure, encompassing viral lysis, RNA capture, washing, and elution of purified HIV-1 RNA, within a timeframe of less than 16 minutes. Subsequent analyses, including RT-LAMP and RT-qPCR, demonstrate a limit of detection of 100 copies per μL and an average RNA recovery of 32% (for blood) and 70% (for plasma) in the elution fraction. Further investigations emphasize the significant presence of purified RNA in the spin column volume (termed as dead volume), and the cumulative recovered RNA copies align with those obtained using the gold standard centrifugation extraction method. Ultimately, we incorporated the real-time quantitative PD-RT-LAMP assay onto a field-compatible handheld portable platform suitable for field use, featuring built-in quality control measures. This platform enables sample-to-answer viral load testing near the point of care (POC). Subsequently, we undertook essential preparatory steps, such as reagent drying to obviate the need for cold storage, initial device calibration, and hands-on training of laboratory personnel regarding device operation, to validate device performance within a cohort of individuals living with HIV (PLHIV). These innovations facilitate quick and comprehensive viral load determination, offering promise for enhanced HIV management and patient care</p>