Colorimetric Polymerase Chain Reaction Enabled by a Fast Light-Activated Substrate Chromogenic Detection Platform

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Miniaturization of nucleic acid tests (NATs) is crucial for point-of-care (POC) diagnostics.
• Development of simple, quantitative, and versatile colorimetric readouts for portable NATs is highly desired.
• Existing fluorescent PCR assays require specialized equipment, limiting their POC applicability.

Purpose of the Study:

• To develop a novel colorimetric nucleic acid detection method for POC settings.
• To establish a fast light-activated substrate chromogenic polymerase chain reaction (FLASH PCR) system.
• To demonstrate the utility of FLASH PCR with portable readout platforms.

Main Methods:

• Developed FLASH PCR utilizing DNA intercalating dyes (DIDs) and chromogenic substrates.
• Leveraged light-induced singlet oxygen production to drive colorimetric signal generation.
• Fabricated a portable electronic FLASH reader and a paper-based FLASH strip for readout.

Main Results:

• Successfully converted fluorescent PCR assays to colorimetric FLASH PCR.
• Achieved a limit of detection (LOD) of 60 DNA copies using the FLASH reader, comparable to commercial quantitative PCR.
• Demonstrated reader-free detection using the FLASH strip by converting colorimetric signals to visual distance measurements.
• Validated FLASH PCR for detecting nucleic acid markers in clinical and biological samples.

Conclusions:

• FLASH PCR provides a versatile, sensitive, and portable colorimetric method for nucleic acid detection and quantification.
• The developed readout platforms enhance the applicability of FLASH PCR for POC diagnostics.
• FLASH PCR holds significant potential for disease diagnosis in resource-limited settings.