Researchers develop a COVID-19 testing method that uses a smartphone microscope to analyze saliva samples and deliver results in about 10 minutes


Researchers at the University of Arizona are developing a COVID-19 testing method that uses a smartphone microscope to analyze saliva samples and deliver results in about 10 minutes.

The UArizona research team, led by biomedical engineering professor Jeong-Yeol Yoon, aims to combine the speed of existing nasal swab antigen tests with the high accuracy of nasal swab PCR, or polymerase chain reaction, tests.

The researchers are adapting an inexpensive method that they originally created to detect norovirus – the microbe famous for spreading on cruise ships – using a smartphone microscope.

They plan to use the method in conjunction with a saline swish-gargle test developed by Michael Worobey, head of the UArizona Department of Ecology and Evolutionary Biology and associate director of the University of Arizona BIO5 Institute.

The team’s latest research using water samples—done in collaboration with Kelly A. Reynolds, chair of the Department of Community, Environment and Policy in the UArizona Mel and Enid Zuckerman College of Public Health – is published today in Nature Protocols.

“We’ve outlined it so that other scientists can basically repeat what we did and create a norovirus-detecting device,” said Lane Breshears, a biomedical engineering doctoral student in Yoon’s lab. “Our goal is that if you want to adapt it for something else, like we’ve adapted it for COVID-19, that you have all the ingredients you need to basically make your own device.”

Yoon – a BIO5 Institute member who is also a professor of biosystems engineering, animal and comparative biomedical sciences, and chemistry and biochemistry – is working with a large group of undergraduate and graduate students to develop the smartphone-based COVID-19 detection method.

“I have a couple of friends who had COVID-19 that were super frustrated, because their PCR results were taking six or seven days or they were getting false negatives from rapid antigen tests.

But when they got the final PCR tests, they found out they had been sick, like they’d suspected,” said Katie Sosnowski, a biomedical engineering doctoral student who works in Yoon’s lab. “It’s really cool to be working on a detection platform that can get fast results that are also accurate.”

Cheaper, Simpler Detection

Traditional methods for detection of norovirus or other pathogens are often expensive, involve a large suite of laboratory equipment or require scientific expertise. The smartphone-based norovirus test developed at UArizona consists of a smartphone, a simple microscope and a piece of microfluidic paper – a wax-coated paper that guides the liquid sample to flow through specific channels. It is smaller and cheaper than other tests, with the components costing about $45.

The basis of the technology, described in a 2019 paper published in the journal ACS Omega, is relatively simple. Users introduce antibodies with fluorescent beads to a potentially contaminated water sample. If enough particles of the pathogen are present in the sample, several antibodies attach to each pathogen particle.

Under a microscope, the pathogen particles show up as little clumps of fluorescent beads, which the user can then count. The process – adding beads to the sample, soaking a piece of paper in the sample, then taking a smartphone photograph of it under a microscope and counting the beads – takes about 10 to 15 minutes.

It’s so simple that Yoon says a nonscientist could learn how to do it by watching a brief video.

The version of the technology described in the Nature Protocols paper makes further improvements, such as creating a 3-D-printed housing for the microscope attachment and microfluidic paper chip. The paper also introduces a method called adaptive thresholding.

Previously, researchers set a fixed value for what quantity of pathogen constituted a danger, which limited precision levels. The new version uses artificial intelligence to set the danger threshold and account for environmental differences, such as the type of smartphone and the quality of the paper.

On-Campus Impact

The researchers plan to partner with testing facilities at the University of Arizona to fine-tune their method as they adapt it for COVID-19 detection.

Pending approval of the university’s institutional review board, students who are already being tested on campus through other methods will have the option to provide written consent for their sample to be run through the smartphone-based testing device as well.

Figure 1

Ultimately, the researchers envision distributing the device to campus hubs so that the average person – such as a resident assistant in a dorm – could test saliva samples from groups of people.

“Adapting a method designed to detect the norovirus – another highly contagious pathogen – is an outstanding example of our researchers pivoting in the face of the pandemic,” said University of Arizona President Robert C. Robbins. “This promising technology could allow us to provide fast, accurate, affordable tests to the campus community frequently and easily. We hope to make it a regular part of our ‘Test, Trace, Treat’ strategy, and that it will have a broader impact in mitigating the spread of the disease.”

Yoon and his team are also working on another idea, based on a 2018 paper they published in Chemistry – A European Journal, which is even simpler but leaves slightly more room for error.

It involves the same technology, but instead of a smartphone microscope and specially designed enclosure, users would only need to download a smartphone app and use a microfluidic chip stamped with a QR code.

“Unlike the fluorescent microscope technique, where you get the chip into just the right position, you just take a snapshot of the chip,” said biomedical engineering master’s student Pat Akarapipad. “No matter the angle or distance the photo is taken from, the smartphone app can use AI and the QR code to account for variances and run calculations accordingly.”

The method requires no training, so, if perfected, it could potentially allow students to pick up microfluidic chips from a campus location and test their own samples.

The team is also working with other members of the university’s COVID-19 testing group, including Deepta Bhattacharya, an associate professor in the Department of Immunobiology.

Human enteric viruses are small infectious agents that can cause gastrointestinal disease upon ingestion of very low doses. Detection of these viruses requires an extremely low limit of detection (LOD), especially when assessing viruses in reclaimed wastewater or unconfined aquifers used as sources of drinking water. Norovirus is one of such well-known examples and is the most common cause of epidemic and sporadic gastroenteritis worldwide.(1)

Studies have indicated that norovirus infection can occur upon exposure to as few as 18 virions.(2,3) Highly sensitive detection methods are needed for assessing exposure to norovirus, especially considering that the methods for virus recovery and concentration from environmental matrices are rather inefficient.

In addition, the infectivity of human noroviruses by in vitro cell culture has proven to be quite complex (only possible in stem cell-derived human enteroids),(4) which prevents the use of traditional culture-based assays for evaluating virus infectivity in environmental matrices. Because of this limitation, norovirus has been assayed by either reverse transcription polymerase chain reaction (RT-PCR)(5) or sandwich immunoassay(6) techniques.

While RT-PCR-based techniques do provide necessary specificity for detection and identification of norovirus, these molecular methods are susceptible to inhibition by multiple components associated with environmental matrices and fail to provide sufficient rapidity and field-applicability.(7)

Immunoassay techniques are simpler than RT-PCR and have the potential to be incorporated on a microfluidic platform. Specifically, microfluidic paper analytic devices (μPADs) have shown numerous advantages over silicone-based microfluidic devices, as they are lightweight, easy to fabricate via wax printing (no lithography), use spontaneous flow by capillary action, and have potential on-chip filtration capability.(8,9)

However, optical detection of low concentrations of pathogens has rarely been demonstrated on paper substrates because paper is optically opaque and non-homogeneous (porous), generating substantial background scatter and reflection. So far, single virus copy level detection of norovirus has rarely been demonstrated on paper substrates (including lateral flow assays and μPADs).

While single copy level detection of other virus targets has indeed been demonstrated on paper substrates (20 copies of Ebola, 20 copies/μL of pseudorabies, and 1 copy/μL of HIV), all of them required nucleic acid amplifications, most notably isothermal methods such as loop-mediated isothermal amplification (LAMP).(10−12) Such methods are not sufficiently simple for field-based applications (requiring a heater and thermostat system plus an expensive isothermal amplification kit) and cannot be considered near-real-time (just the amplification part can take from 15 min to 2.5 h).

As described previously, immunoassay on μPAD without sample concentration and/or nucleic acid amplification is the ideal method for field-based norovirus detection, which has unfortunately not been demonstrated at the single virus copy level. The LODs of paper-based norovirus immunoassays ranged from 104 to 106 copies/μL (=10 fg/μL to 1 pg/μL, as the weight of a single norovirus particle is approximately 10 ag considering its diameter of 35–40 nm)(13) without concentration or amplification(14,15) and 102 copies/μL with 1 h reaction of signal amplification.(16)

In this work, we attempted to “visualize” the norovirus-induced particle immunoagglutination down to the single virus copy level directly on a μPAD toward field-based applications. Antibody-conjugated, submicron, fluorescent polystyrene particles were used on μPAD to quantify norovirus. The μPAD allows the antibody-conjugated particles and norovirus to “flow” through paper pores spontaneously via capillary action, which is much faster and more effective than passive, diffusional mixing. As the submicron particles move much slower than norovirus, unbound noroviruses can also be washed from the antibody-conjugated particles, potentially eliminating a separate washing step.(17)

The extent of particle aggregation caused by antibody–antigen binding was correlated to the norovirus concentration in the samples. A smartphone-based fluorescence microscope was used to identify and quantify these aggregated particles to provide additional field applicability.

Only the aggregated particles could be isolated through image processing, enabling extremely sensitive detection down to the single virus copy level. Neither sample concentration nor nucleic acid amplification steps are necessary due to such an extremely low LOD.

This novel method is wholly different from other optical biosensing methods where their signals are ensemble-averaged, that is, specific, nonspecific, and background signals are not fully isolated. By securing direct evidence of particle aggregation, credibility and accuracy of the assay could be improved. In addition, it is also entirely different from other imaging-based virus counting methods, where host cells are infected with target viruses.(18) Such methods require in vitro cultivation of noroviruses, which is costly and time-intensive,(19) and most importantly, are complex and difficult for norovirus.

To accomplish our goal, we designed and tested a smartphone-based fluorescence microscope to image aggregated particles directly on a wax-printed μPAD (Figure 1a). In this novel method, norovirus target solutions (5 μL each) were first loaded on μPADs, followed by the addition of antibody-conjugated, yellow-green fluorescent polystyrene particle suspension that resulted in particle aggregation (i.e., immunoagglutination).

This alternative approach enabled the antibody-conjugated particles to spread and flow through the entire channel, allowing them to be imaged separately and minimizing nonspecific aggregation. In addition, much lower concentration of the antibody-conjugated particles (0.001–0.002%) was used for the particle suspension than those used in other particle immunoassays, which also contributed to minimizing nonspecific aggregation.

A smartphone-based fluorescence microscope (Figure 1b) was constructed to fluorescently image the several different areas of a μPAD channel. Through a novel image processing algorithm, only the aggregated particles were isolated to relate them to the norovirus concentration. Field water samples—tap water and reclaimed wastewater—were also evaluated.

reference link :

More information: Nature Protocols (2021). DOI: 10.1038/s41596-020-00460-7


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