Increased Robustness of Single-Molecule Counting with Microfluidics, Digital Isothermal Amplification, and a Mobile Phone versus Real-Time Kinetic Measurements

Abstract

Quantitative bioanalytical measurements are commonly performed in a kinetic format, and are known to not be robust to perturbation that affects the kinetics itself, or the measurement of kinetics. We hypothesized that the same measurements performed in a “digital” (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a two-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7×10−7), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20-minute change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R2 = 0.9997 vs. true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries, and more generally to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular measurements under LRS. Furthermore, it may provide more general principles describing robustness of chemical and biological networks in digital formats.

Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering

“…Robustness, that is, consistent performance despite variation in external conditions, is a crucial requirement in low-resource settings and one of the key advantages of digital single-molecule counting approaches 67 . Because of its reliance on binary outputs, the digital format gives isothermal amplification chemistries an unexpected high degree of robustness with respect to perturbations in temperature 64 , reaction time and imaging quality.…”

“…Because of its reliance on binary outputs, the digital format gives isothermal amplification chemistries an unexpected high degree of robustness with respect to perturbations in temperature 64 , reaction time and imaging quality. Digital isothermal assays have been read using a mobile phone camera that captures an image of a chip and transmits the pattern of the positive and negative compartments wirelessly to a cloud server for analysis 67 .…”

Measuring Fate and Rate of Single‐Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA

“…Single-molecule, or “digital” [5] LAMP [6] is attractive for quantification under limited-resource settings due to its high intensity fluorescent output with calcein chemistry. [7] Digital RT-LAMP for the quantification of human immunodeficiency virus RNA was shown to be robust to perturbations in reaction conditions, imaging, temperature, and automatic cloud-based analysis, enabling robust cell phone–based quantification. [7b] In this work, we used RT-LAMP primers (see Table S1 in Supporting Information (SI)) modified from previous work targeting the conserved 5′-untranslated region (5′UTR) of HCV.…”

“…[7] Digital RT-LAMP for the quantification of human immunodeficiency virus RNA was shown to be robust to perturbations in reaction conditions, imaging, temperature, and automatic cloud-based analysis, enabling robust cell phone–based quantification. [7b] In this work, we used RT-LAMP primers (see Table S1 in Supporting Information (SI)) modified from previous work targeting the conserved 5′-untranslated region (5′UTR) of HCV. [8] RE-based digestion is a reliable method to recognize specific nucleic acid sequences of multiple letters in length and cleave at specific sites.…”

Deployment and exploitation of nanotechnology nanomaterials and nanomedicine

“…With optical reporters, the results may be read using a mobile phone camera and relayed wirelessly to a cloud server for remote analysis [102]. By parallel analysis of aliquots of varying size, digital quantitation may be adapted to a wide dynamic range, and has proven very robust due to its relying on binary outputs, hence may be particularly suitable for home/field testing.…”