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The team chose to use saliva rather than nasopharyngeal swab samples as their collection method, because it’s
easier for users to collect saliva and studies have shown that SARS-CoV-2 is detectable in saliva for a greater
number of days post-infection. But unprocessed saliva presents challenges of its own: It contains enzymes
that degrade various molecules, producing a high rate of false positives.

The researchers developed a novel technique to solve that problem. First, they added two chemicals called DTT
and EGTA to saliva and heated the sample to 95°C for 3 minutes, which eliminated the false-positive signal
from the untreated saliva and sliced open any viral particles. They then incorporated a porous membrane that
was engineered to trap RNA on its surface, which could finally be added directly to the SHERLOCK reaction to

generate a result.

To integrate the saliva sample preparation and the SHERLOCK reaction into one diagnostic test, the team
designed a simple battery-powered device with two chambers: a heated sample prep chamber and an unheated
reaction chamber.

A user spits into the sample prep chamber, turns on the heat, and waits three to six minutes for the saliva to be
wicked into the filter. The user removes the filter and transfers it to the reaction chamber column, then pushes
a plunger that deposits the filter into the chamber and punctures a water reservoir to activate the SHERLOCK
reaction. Fifty-five minutes later, the user looks through the tinted transilluminator window into the reaction
chamber and confirms the presence of a fluorescent signal. They can also use a smartphone app that analyzes
the pixels being registered by the smartphone’s camera to provide a clear positive or negative diagnosis.

The researchers tested their diagnostic device using clinical saliva samples from 27 COVID-19 patients and 21
healthy patients, and found that miSHERLOCK correctly identified COVID-19-positive patients 96% of the
time and patients without the disease 95% of the time. They also tested its performance against the Alpha,
Beta, and Gamma SARS-CoV-2 variants by spiking healthy human saliva with full-length synthetic viral RNA
containing mutations representing each variant, and found that the device was effective across a range of viral
RNA concentrations.

One of the great things about miSHERLOCK is that it’s entirely modular. The device itself is separate from the

assays, so you can plug in different assays for the specific sequence of RNA or DNA you’re trying to detect.
Assays for new targets can be created in about two weeks, enabling the rapid development of tests for new
variants of COVID-19 and other diseases.

The miSHERLOCK team created their device with low-resource settings in mind. By solving the sample
preparation problem, the team ensured that this device is virtually ready for consumers to use as-is, and they
are excited to work with industrial partners to make it commercially available.

This research was supported by the Wyss Institute for Biologically Inspired Engineering at Harvard
University, the Paul G. Allen Frontiers Group, the Harvard University Center for AIDS Research (an NIH-
funded program that is supported by the following NIH co-funding and participating institutes and centers:
NIAID, NCI, NICHD, NIDCR, NHLBI, NIDA, NIMH, NIA, NIDDK, NINR, NIMHD, FIC, OAR), The
Burroughs-Wellcome American Society of Tropical Medicine and Hygiene, an American Gastroenterological
Association Takeda Pharmaceutical Research Scholar Award, and an MIT-TATA Center fellowship.

~Ms. Alaika Anford D'Souza
(T.Y.BSc 2020-21)
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