Two-Fluorophore Mobile Phone Imaging of Biplexed Real-Time NAATs Overcomes Optical Artifacts in Highly Scattering Porous Media

Abstract

Nucleic acid amplification tests (NAATs) are common in laboratory and clinical settings because of their low time to result and exquisite sensitivity and specificity. Laboratory NAATs include onboard positive controls to reduce false negatives and specialized hardware to enable real-time fluorescence detection. Recent efforts to translate NAATs into at-home tests sacrifice one or more of the benefits of laboratory NAATs, such as sensitivity, internal amplification controls (IACs), or time to result. In this manuscript, we describe a mobile-phone-based strategy for real-time imaging of biplexed NAATs in paper. The strategy consisted of: (1) using mobile phones with multipass excitation and emission filters on the flash and camera to image the signal from distinct fluorophore-labeled probe types in a biplexed NAAT in a glass fiber membrane; and (2) analyzing the differential fluorescence signal between the red and green color channels of phone images to overcome a strong evaporation-induced optical artifact in heated glass fiber pads due to changes in the refractive index. We demonstrated that differential fluorescence imaging enabled low limits of detection (316 copies of methicillin-resistant Staphylococcus aureus DNA) in our lab's "MD NAAT" platform, even in biplexed isothermal strand displacement amplification reactions containing 100k copies of coamplifying IAC DNA templates. These results suggest that two-fluorophore mobile phone imaging may enable translating the benefits of extant laboratory-based, real-time NAATs to the point of care.

Near-digital amplification in paper improves sensitivity and speed in biplexed reactions

“…30 Optical segmentation increased signal-to-noise ratios by ~10× (without leading to false positives or false negatives by following the principles outlined by Rolando et al 18 ), improved limits of detection (from above 1000 copies of MRSA genomic DNA to between 100 and 316 copies in biplexed reactions), and reduced time to result (from 45 minutes of ampli cation to 15-20 minutes for positive samples and 25 minutes for negative samples). These results were generalizable to multiple assays (e.g., mecA and ldh1 genes for MRSA), 31 phone models with vastly different camera spectra (e.g. Nexus 5X and Pixel 2), and more complex samples (e.g.…”

“…The near-digital assay with optically segmented mobile phone imaging described in this manuscript has several advantages over standard assays that lack segmentation. This was enabled in part by integrating the recent algorithmic 31 , assay, and hardware innovations (that can be found in the PhD thesis of K. G. Shah, available from the University of Washington). 30 Optical segmentation increased signal-to-noise ratios by ~10× (without leading to false positives or false negatives by following the principles outlined by Rolando et al 18 ), improved limits of detection (from above 1000 copies of MRSA genomic DNA to between 100 and 316 copies in biplexed reactions), and reduced time to result (from 45 minutes of ampli cation to 15-20 minutes for positive samples and 25 minutes for negative samples).…”

“…A mobile phone uorescence reader (Nexus 5X or Pixel 2 equipped with multipass excitation and emission lters) detailed in another manuscript under review was placed about 15 cm above the MD NAAT and imaged the pads every 30 seconds using the Camera FV-5 application. 31…”

Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis

“…However, it is well known that differences in phone components (i.e. camera quality and/or flash spectrum) can result in differing signals, 61,62 potentially confounding quantification if not properly calibrated. Amplification nucleation site analysis does not rely on relative fluorescence or precise pixel intensity values and may prove to be a more robust and consistent method of quantification across inexpensive optics as well as mobile phone types and models.…”

Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis

“…However, it is well known that differences in phone components (i.e. camera quality and/or flash spectrum) can result in differing signals, 49,50 potentially confounding quantification if not properly calibrated. Amplification nucleation site analysis does not rely on relative fluorescence or precise pixel intensity values and may prove to be a more robust and consistent method of quantification across inexpensive optics as well as mobile phone types and models.…”