Enzyme-Based Paper Tests for Target Detection in Low-Resource Settings

Paper analytic devices (PADs) are a cheap and effective way to detect a variety of analytes, such as antibiotics, illicit drugs, and contaminants in water. PADs consist of a number of “micro-reactor” regions. These regions are separated by hydrophobic wax barriers, which are deposited onto the card using wax printers or crayons. Liquid reagents are dropped into each region and then dried onto the cards. Solid samples are swiped across the regions (or dropped onto the card for liquid samples), and the card is placed in water to re-wet the reagents via capillary action. Samples react in each region to produce detailed color changes, which mark the concentration of various analytes. Each PAD costs around $1 to manufacture and does not require any laboratory equipment to run. This makes PADs particularly useful in low-resource areas. For example, many countries in the developing world do not have the technological infrastructure or regulatory resources to keep low quality and counterfeit medicines off market shelves. PADs can quickly determine whether a drug tablet contains the correct medicines, and they can quickly be deployed at scale anywhere a problem with pharmaceutical quality is suspected.

My research focused on inventing novel, enzyme-based PADs to detect targets that have proven difficult to analyze with traditional chemical reagents. I designed a colorimetric enzyme-on-paper test to detect lactose in illicit drug samples. Lactose is commonly used as a cutting agent in illicit drugs in order to increase the sellable product volume. Lactose is difficult to detect in drug samples without expensive methods such as LC-MS or HPLC. My PAD utilized a 3-enzyme system and was able to detect lactose in concentrations as low as 5% by mass in drug samples. I used principal component analysis and color analysis to validate that this test was semi-quantitative for lactose concentration based on the intensity of blue-green color formation. The lactose test was then incorporated into a 12-lane paper test card (idPAD) that can detect various illicit drugs and cutting agents. I am currently working on extending this work into more enzyme-on-paper tests for other common cutting agents in illicit drug samples, such as mannitol.

I created a stand-alone card, sugarPAD, based on my work with lactose. The sugarPAD can detect 4 different sugars (lactose, galactose, glucose, fructose) via 4 enzyme-based spot tests at concentrations as low as 5% by mass. This card was designed for at-home instruction of undergraduate biochemistry courses during the COVID-19 pandemic. The sugarPAD does not contain any hazardous or highly reactive compounds. I wrote a take-home experiment on enzyme kinetics and administered it to undergraduate students. The experiment used the sugarPAD to evaluate the efficacy of a consumer enzyme supplement, which claimed to convert fructose into glucose.

I am working towards creating a new enzyme-based color test to detect oxytocin. Oxytocin is commonly used to prevent postpartum hemorrhaging. It is difficult to detect on paper due to its extremely low therapeutic dosage. I developed a HPLC assay that detects the cleavage product formed by exposing oxytocin to a serine protease enzyme. I also developed a preliminary color test that detects the cleavage product on paper. I am currently working on the formulation for leveraging these results in a PAD.

The tests that I invented have a real-world impact in low-resource and field settings, as well as in educational curriculum development. The PADs are inexpensive to produce, field- and user-friendly, and require no electricity or specialized instrumentation. Novice users with little to no background training can easily use these devices. The tests allow for accurate analyses in areas where it would normally not be possible, such as roadside drug testing, low-resource healthcare settings, and at home experiments for remote learning during a global pandemic.