# Ultrasensitive Enzyme-Free Detection of Proteins on Magnetic Beads

## TL;DR

A new enzyme-free method for detecting proteins with high sensitivity could improve diagnostics in resource-limited settings.

## Contribution

An enzyme-free platform (efMOSAIC) is developed for ultrasensitive protein detection with simplified workflows and stable reagents.

## Key findings

- efMOSAIC achieves detection limits comparable to Simoa for most proteins.
- Signal amplifier reagents remain effective when stored at 21 °C for six months.
- The platform successfully detects cytokines from human plasma and enables multiplexed biomarker detection.

## Abstract

Ultrasensitive measurements
of low abundance protein biomarkers
from minimally invasive biofluids (e.g., plasma or
urine) can enable detection of diseases and transform clinical diagnostics
and treatment. Digital protein detection assays, such as Single Molecule
Arrays (Simoa) and Molecular On-bead Signal Amplification for Individual
Counting (MOSAIC), enable ultrasensitive protein quantification, achieving
detection limits that are at least 1000-fold lower than those of conventional
ELISA assays. Currently, these ultrasensitive assays are performed
in centralized laboratories that have access to specialized instrumentation
and stable supply chains, which are rarely available in low- and middle-income
countries (LMIC) or at the point-of-care (POC) in resource-limited
settings. To address the need for ultrasensitive, accessible diagnostic
assays, we have developed an enzyme-free platform for the detection
of protein biomarkers with simple assay workflows. This approach uses
enzyme-free signal amplifiers generated using hybridization chain
reaction to achieve detection limits comparable to Simoa for most
proteins. The signal amplifier reagent retains full performance when
stored in the dark at 21 °C for up to six months. To investigate
the utility of efMOSAIC for clinical workflows, we validated the platform
for the sensitive detection of cytokines from human plasma and demonstrated
multiplexed biomarker detection. With simple assay workflows and temperature
stable reagents, efMOSAIC is poised to transform ultrasensitive protein
assays making these technologies more accessible for use in LMICs.