More with Less for Point-of-Care Testing

(From left) Andres Martinez observes Miguel Vazquez-Alvarado and Alyssa Pama injecting reagent solutions with micropipettes onto test strips. Photos by Alexis Kovacevic

CAL POLY BIOTECH RESEARCHERS WORK TO CREATE MORE RELIABLE AND ACCESSIBLE DIAGNOSTIC DEVICES WITH LESS COST

APRIL 2024
by nick wilson

Since the outbreak of the COVID-19 pandemic, a familiar household lab procedure has become routine: swabbing the nose; mixing the sample in a chemical solution; and squeezing droplets on paper test strips to await a color change indicating the test results. Because these paper-based tests are inexpensive, portable and easy to use, a team of researchers in Cal Poly’s Center for Applications in Biotechnology wants to expand their range.

"Working on this project is so important and rewarding knowing that it could be applied to the prevention of
infectious diseases and help provide healthcare accessibility to so many areas of the world.

Alyssa Pama

Cal Poly biochemistry undergraduate researcher

Since the outbreak of the COVID-19 pandemic, a familiar household lab procedure has become routine: swabbing the nose; mixing the sample in a chemical solution; and squeezing droplets on paper test strips to await a color change indicating the test results. Because these paper-based tests are inexpensive, portable and easy to use, a team of researchers in Cal Poly’s Center for Applications in Biotechnology wants to expand their range.

“What we’re trying to do is cut down the costs of testing devices by using something as cheap, accessible and easy to get rid of as paper,” said Miguel Vazquez-Alvarado (Biochemistry, ’21), a student researcher who coauthored a journal article on the team’s research findings. “Paper is better than microchips, better than a lab. Let’s say you run a blood test in a lab. That’s not cheap, and the supplies are not as simple to dispose of as paper.”

In addition to detecting disease, paper test strips can track health indicators and monitor the environment. Two limitations of current strips are that the results are only qualitative (yes/no) and the limits of detection are not sufficient to reveal biomarkers, such as the presence of a virus at low concentrations.

(From left) Martinez, Pama and Vazquez-Alvarado wait for a Formulatrix Mantis Liquid Dispenser to complete its operation and provide data on substrate analysis.

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The research team is led by Professors Andres W. Martinez, chemistry and biochemistry, and Nathaniel W. Martinez, biological sciences. The brothers and their student researchers are working to address these limitations by developing the next generation of paper test strips called microfluidic paper-based analytical devices, or microPADs.

A microPAD is a strip of paper that is coated with a substrate, a chemical compound that changes colors upon contact with specific enzymes. Enzymes accelerate chemical reactions and are used routinely in diagnostic tests, or assays, to produce color changes.

In a recent study, the team analyzed six different substrates. The goal was to evaluate and optimize the performance of the substrates on microPADs when reacting with horseradish peroxidase (HRP), a commonly used testing enzyme. HRP catalyzes the conversion of the substrates from colorless to a brightly colored final state. This color change can be harnessed to detect a wide variety of biomarkers, including potentially deadly respiratory viruses such as SARS-CoV-2.

“Certain substrates can become highly colored, fluorescent, or chemiluminescent products upon reacting with HRP, making the combination of HRP and a suitable substrate a powerful analytical tool,” the team wrote in their journal article.
The research compared 33 substrate formulations tested with 20 distinct concentrations of enzyme.

“The novelty of the work is in the fact that we took the time to directly compare all these different substrate formulations in a head-to-head matchup,” Andres Martinez said. “Often researchers look at one or maybe two of these substrates independently, but it’s impossible to compare two different studies to each other because the experimental conditions are different.”
Martinez said their work could lead to improved paper test strips capable of detecting disease or contamination before proliferation.

“Our results showed that some of these substrates have orders of magnitude better sensitivity than others,” he said. “Commercial COVID tests don’t necessarily catch the early stages of the illness when you may be contagious but don’t yet have symptoms. Tests will usually start coming back positive once your symptoms are fully developed, and by then it’s too late to prevent the spread of the disease.”

Surprisingly, the overall results did not identify a single most-effective substrate. For example, some substrates are more suited for detecting high concentrations of analytes — the chemical substance that is the subject of analysis — while others are better for detecting low concentrations of analytes. Some substrates produce insoluble colored spots, while other substrates produce colored products that remain soluble in water. Finally, each substrate produces a unique color, which could be helpful for determining test results if the sample has a strong color of its own.

These results provide foundational knowledge that could help other researchers develop paper-based tests.

“The value of this work is that researchers around the globe can now refer to these results and select the substrate that’s going to work best for their specific application without having to go through extensive optimization experiments,” Martinez concluded.

Close-up view of the Formulatrix Mantis Liquid Dispenser in operation.

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The findings of Cal Poly’s research could help accelerate the development of new paper-based tests that are portable, cost-effective and more broadly accessible, expanding testing that can be done at or near the point of care, both at home in the U.S. and in developing countries.

The impact of this work is already being felt in a chemistry classroom at Lompoc High School where former student researcher Vazquez-Alvarado is now a teacher. He recalled the hours of lab work, analyzing large sets of data and collaborating with student peers. “The skills I learned were priceless. Presenting to faculty and peers, and public speaking, although nerve-wracking, is something that I practice every day now,” he said. “Everything that I learned in that research setting I’m now using.”

Alyssa Pama, a fourth-year biochemistry major, also contributed to the project. She summed up her experience: “I think working on this project is so important and rewarding knowing that it could be applied to the prevention of infectious diseases and help provide healthcare accessibility to so many areas of the world.”

Research contacts:  
Andres Martinez, awmartin@calpoly.edu
Nathaniel Martinez, nmarti32@calpoly.edu

Funding contributors to this research:

1. National Science Foundation Grant
2. National Institutes of Health: Bridges to the Baccalaureate Grant
3. The Society for Analytical Chemists of Pittsburgh Undergraduate Analytical Research Program Grant
4. Cal Poly College-Based Fees
5. William and Linda Frost Fund