Enzyme-Free Nucleic Acid Amplification Assay Using a Cellphone-Based Well Plate Fluorescence Reader

Abstract

Nucleic acids, DNA and RNA, provide important fingerprint information for various pathogens and have significant diagnostic value; however, improved approaches are urgently needed to enable rapid detection of nucleic acids in simple point-of-care formats with high sensitivity and specificity. Here, we present a system that utilizes a series of toehold-triggered hybridization/displacement reactions that are designed to convert a given amount of RNA molecules (i.e., the analyte) into an amplified amount of signaling molecules without any washing steps or thermocycling. Fluorescent probes for signal generation were designed to consume products of the catalytic reaction in order to push the equilibrium and enhance the assay fold amplification for improved sensitivity and reaction speed. The system of toehold-assisted reactions is also modeled to better understand its performance and capabilities, and we empirically demonstrate the success of this approach with two analytes of diagnostic importance, i.e., influenza viral RNA and a micro RNA (miR-31). We also show that the amplified signal permits using a compact and cost-effective smartphone-based fluorescence reader, an important requirement toward a nucleic-acid-based point-of-care diagnostic system.

Portable Spectroscopy

“…534 Ozcan 535 has described imaging and diagnostics on mobile phones, and the use of a mobile phone as a well-plate fluorescence ready for RNA, moving towards nucleic-acid-based point-of-care medicine. 536 Soh reported a low-cost polymerase chain reaction (PCR) system using a smartphone as the fluorescence reader, 537 and Askim and Suslick described 538 a handheld reader for colorimetric sensor arrays. Sia et al described a smartphone dongle, deriving power from the headphone jack, which was the reader for a microfluidic-based diagnostic test for HIV and syphilis based on the standard immunoassay.…”

An enzymatic assay to measure long-term adherence to pre-exposure prophylaxis and antiretroviral therapy

“…The assay could be performed using fingerprick samples since < 5 µL of blood is required per assay and previous studies have established correlations between HIV drug metabolites in venous and fingerprick blood draws. 62 The RESTRICT assay has the potential to be further integrated into a near-patient test for use in a doctor's office or a patient's home by replacing the microplate reader with a portable and inexpensive fluorescence reader or mobile phone 63,64 and thus be a critical tool for preventing HIV transmission and drug resistance. We envision a test where assay reagents are lyophilized and stored in a tube or cartridge and the assay is initiated by adding blood and dilution buffer, incubating, and reading out results in a simple instrument.…”

Mobile Microfluidics

“…While these tests provide cheap ad-hoc diagnosis, they fail to explore the programmability aspect of microfluidics. To overcome that, recent developments combine paper microfluidics with the computational power of smartphones, e.g., using their cameras to read the results for HIV tests [51,52] and pathogen tests in urine [53,54].…”