<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>