Microfluidic System for the Label-Free Multiplexed Electrical Detection of Viruses using Hydrogel Beads

Introduction:: Delivering rapid and accurate diagnostics are crucial to providing better patient outcomes and improving in disease management. Point of care technologies that do not require expensive and bulky laboratory equipment and highly trained operators allow for these diagnostics to be more readily accessible in low resource areas and lead to a faster response time for patient care. Additionally, because many conditions have overlapping symptoms, multiplexed testing can further improve diagnostic capacities by increasing the amount of information you can get from a single test. Current diagnostics for viruses tend to center around either PCR tests or antibody tests. However, PCR tests require bulky equipment and temperature cycling and aren’t suitable for point of care, and multiplexing with nucleic acid tests is currently difficult. Antibody tests on the other hand are limited by the fact that antibodies are not present during early stages of infection when the body has not had time to produce them yet. Here we introduce our work that takes our preexisting electrical detection platform the multiplexed detection of circulating protein biomarkers and apply it to capturing and detecting whole viruses. We show the ability of our platform to use micro-sized hydrogel beads to allow label-free detection of Parvovirus B19 (B19V) and Hepatitis A Virus (HAV), without the need to disrupt the viral particle, down to the FDA required limits individually in buffer, as well as demonstrate multiplexed detection of the two viruses in plasma.


Materials and Methods:: We utilized an electrical differential counting system in order to detect the presence of viruses. Two sets of polyacrylamide hydrogel beads were synthesized which each have distinct impedance signals when flowed through our on-chip coulter counter. Micro-sized beads (13 µm diameter) with 55 and 80% gel content were functionalized with antibodies against HAV or B19V respectively using EDC-NHS chemistry. These beads can be distinguished from each other electrically due to the difference in gel content. To assess our ability to detect the viruses, we spiked known concentrations of inactivated HAV or B19V VLPS into PBS buffer and mixed the sample (75 µL) with a reaction mixture (224 µL) containing the a biotinylated detection antibody and pre-incubated (10 minutes) before adding the beads with the capture antibody and incubating (40 minutes). The resulting solution was diluted to half concentration and run through our electrical system. First it was flown past a counter electrode to obtain an entrance count, then it is flown through a PDMS capture chamber functionalized with Streptavadin which will specifically capture beads bound to viruses via Streptavadin-biotin affinity with the detection antibody. Finally, the beads flow through an exit electrode and an exit count is obtained. This data is processed in MATLAB and used to obtain a percentage of capture by considering the difference between entrance and exit counts. After validating the assay in buffer, we performed multiplexed tests of both viruses in plasma samples.


Results, Conclusions, and Discussions:: Results and Discussion

We were able to successfully distinguish positive virus samples from negative samples in spiked buffer for all concentrations down to or below the FDA required levels (2x10³ IU/mL for HAV; 3x10⁶ for B19V), achieving detection at 2x10³ IU/mL of inactivated virus for HAV and 1 x10⁶ particles/mL of B19V VLPs. Additionally, we demonstrated our ability to perform multiplexed detection of the two viruses spiked in plasma samples. Our system was able to not only to detect both viruses when present in the sample, but also distinguish HAV from B19V.

Conclusions

We have demonstrated the capacity of our system to perform multiplexed detection of whole viruses in plasma. This should expand the diagnostic capabilities of our platform and lead to more robust and versatile diagnostics in the future. Next steps for this technology include demonstrating its effectiveness with live viruses as opposed to VLPs or inactivated viruses, as well as demonstrating the ability of our platform to capture viruses with more complex structures such as envelope viruses, as opposed to B19V and HAV which have relatively simple structures composed of only a small number of capsid proteins. In addition, as this platform has already demonstrated it’s capacity to capture circulating protein biomarkers in past work, it should be possible to combine virus and protein detection to allow for even more versatile diagnostic capabilities. In addition, by adding more beads with distinct electrical signals we can increase the multiplexing capabilities even further allowing for even more versatile diagnostics.


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