Multiplexed reverse-transcriptase quantitative PCR using plasmonic nanoparticles for point-of-care COVID-19 diagnosis

The COVID-19 pandemic exposed limitations in bringing molecular diagnostic testing to the point-of-care. To close that gap, we developed an RT-qPCR platform utilizing heating via plasmonic AuNRs that gives results in 23 minutes, bringing us a step closer toward making point-of-care PCR a reality.
Multiplexed reverse-transcriptase quantitative PCR using plasmonic nanoparticles for point-of-care COVID-19 diagnosis

This study started with a lockdown in New York City, amidst the country’s first wave of COVID-19 in March 2020.  While the university hospital, located adjacent to our lab, was experiencing a crushing wave of COVID patients, research labs, like the City itself, were shutting down.  

 

But there was an exception made for research labs working on COVID.  Up to that point, our lab was embarking on a project to miniaturize PCR, the gold-standard laboratory technique for detecting nucleic acids.  We had previous experience in miniaturizing a multiplexed ELISA, which detects proteins, and had tested the device in resource-limited field settings (Chin et al, 2010; Laksanasopin et al, 2015).  For PCR, we held a similar ambition.  When the first COVID test from the U.S. CDC came out, we pivoted our approach to detect SARS-CoV-2.

 

The campus was hauntingly quiet, but the project presented an opportunity to use our technical skills towards the public good, addressing an extraordinary public health challenge in real time.  We had to do the research safely, and abide by all health regulations, but ultimately recruited to our pre-existing small team a wide set of researchers consisting of Master's students (8 of whom are co-authors on this paper), additional doctoral students, and research technicians with backgrounds in biomedical, mechanical, and electrical engineering.  The team worked on CAD designs and electronics, sprawled out in a new Makerspace at Columbia Engineering, which was normally buzzing with student activity but for now acted as an extended space for this project, in addition to our research lab which focused on biochemistry and systems integration and validation.

 

The project was propelled forward by partnering with Rover Diagnostics, a company co-founded by one of us (Sia) and a serial entrepreneur, Dr. Mark Fasciano.  This collaboration brought to the table additional experts who had industry experience, to help develop scientific concepts into testable prototypes. 

 

In Summer 2020, we were accepted into the NIH RADx-Tech program.  It provided a huge boost and sharp focus to our effort.  From the beginning of our work on plasmonic thermocycling, nanoparticles were thought to quench the fluorescence of common qPCR probes and preclude the real-time monitoring and quantitative measurement of cycle threshold values that are central to qPCR.  But we noticed the quenching did not preclude meaningful fluorescence measurements.  Working with our Rover Diagnostics colleague Dr. Martin Jaspan, the team found that a spectrometer with a deconvolution algorithm can measure multiple fluorescence emission signals emanating from a single excitation wavelength.  In short, the advantages of plasmonic thermocycling – where the solutions can be rapidly heated from the inside with compact optics – could co-exist with the real-time fluorescence monitoring central to qPCR.

 

The entire team was galvanized with an “all hands-on-deck” mentality, working tirelessly and pulling all-nighters to prepare for our pilot test with partners in Emory University’s ACME Point-of-Care Testing Center in Fall 2020.  Afterwards, we built off this RADx pilot work.  We tested the extraction-free approach first with saliva and expanded the approach to nasal specimens (the pace of research was constrained by access to specimens, which we needed to be available as dry swabs or at least not in an extraction buffer).  As the pandemic evolved, we performed validation on variants.

 

The value of point-of-care diagnostics has become widely known and experienced.  For the first time, the words “PCR” has seeped into public consciousness.  The public is now proficient with rapid testing, but the relatively low sensitivity of antigen tests, while still serving an important public health function, also leaves open important scenarios where a sensitive and rapid test will be needed.  Is it possible to have the best of both worlds, a lab-quality qPCR test that is fast and accessible everywhere?  Our effort here is to move closer to that goal.

 

For more information, please check out our recent publication in Nature Nanotechnology:

Blumenfeld, N.R., Bolene, M.A.E., Jaspan, M. et al. Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis. Nat. Nanotechnol. (2022). https://doi.org/10.1038/s41565-022-01175-4

(full-text access here)

References:
Chin, C. D., Laksanasopin, T., Cheung, Y. K., Steinmiller, D., Linder, V., Parsa, H., Wang, J., Moore, H., Rouse, R., Umviligihozo, G., Karita, E., Mwambarangwe, L., Braunstein, S. L., van de Wijgert, J., Sahabo, R., Justman, J. E., El-Sadr, W. & Sia, S. K. Microfluidics-based diagnostics of infectious diseases in the developing world. Nat Med 17, 1015–1019 (2011). https://doi.org/10.1038/nm.2408


Laksanasopin, T., Guo, T. W., Nayak, S., Sridhara, A. A., Xie, S., Olowookere, O. O., Cadinu, P., Meng, F., Chee, N. H., Kim, J., Chin, C. D., Munyazesa, E., Mugwaneza, P., Rai, A. J., Mugisha, V., Castro, A. R., Steinmiller, D., Linder, V., Justman, J. E., Nsanzimana, S. & Sia, S. K. A smartphone dongle for diagnosis of infectious diseases at the point of care. Sci. Transl. Med. 7, (2015). https://doi.org/10.1126/scitranslmed.aaa0056