Real-time Colorimetric Quantitative Molecular Detection of Infectious Diseases on Smartphone-based Diagnostic Platform

Abstract

Rapid diagnostics of infectious diseases and accurate identification of their causative pathogens play a crucial role in disease prevention, monitoring, and treatment. Conventional molecular detection of infectious pathogens requires expensive equipment and well-trained personnel, thus limiting its use in centralized clinical laboratories. To address this challenge, a portable smartphone-based quantitative molecular detection platform, termed "smart connected pathogen tracer" (SCPT), has been developed for pathogen monitoring and disease surveillance. The platform takes advantage of synergistically enhanced colorimetric loop-mediated isothermal amplification (LAMP) assay and smartphone-based color analysis, enabling simple, rapid and reliable nucleic acid quantification without need for expensive fluorescence detection equipment. The SCPT platform has been successfully applied to quantitatively detect: i) HPV DNA in saliva and clinical vaginal swab samples, and ii) HIV RNA in plasma samples with comparable sensitivity to state-of-art machine. It has also been demonstrated for disease spatiotemporal mapping and pathogen tracking by wireless connection and web-based surveillance. Such simple, cost-affordable, portable molecular detection platform has great potential for on-site early disease detection, remote healthcare monitoring, and epidemic surveillance.

Figure 1

Optimization of real-time colorimetric LAMP assay. (A) Detection mechanism of real-time colorimetric LAMP assay based on synergistic enhancement effect of H⁺ and PPi^- ions, two byproducts of LAMP reaction. (B) Comparison of color change between positive control (PC) (1 × 10⁴ copies HPV 16 DNA template) and negative control (NC) (no DNA template) in colorimetric LAMP assay with: i) non-buffered LAMP reaction solution, and ii) commercially available NEB LAMP reaction buffer. Inner images are the corresponding photos of PC and NC samples. (C) Effect of different metal ion indicators on the colorimetric LAMP assay. (D) Effect of different Mg²⁺ ion concentration on the colorimetric LAMP assay.

Figure 2

Hue-based colorimetric analysis. (A) Comparison of signal-to-noise (S/N) of RGB model and hue value analysis (HSI model) in colorimetric LAMP assay. (B) 3D printed device containing four cylindrical chambers with various depths (5, 10, 15 and 20 mm) for hue value measurement of the EBT indicator: i) schematic illustration of the 3D printed device; ii) a photograph of the device filled with the EBT indictor; iii) the interface of our smartphone app for hue value quantitative measurement and iv) Hue value (in degree), as a function of the depths of the chambers (in mm). (C) Comparison of hue value detection and absorbance measurement of the EBT indicator solution with various volumes in 96-well plate. (n = 5).

Figure 3

Hue-based quantitative molecular detection by our SCPT platform. (A) A photograph of our SCPT platform. (B) A photograph of our microfluidic chip. (C) A sequence of optical images obtained by smartphone camera during colorimetric LAMP assay of HPV 16 DNA ranging from 0 to 10⁴ copies per reaction. The images were taken at 0, 20, 30, 40 and 60 min after the start of LAMP incubation. (D) Real-time hue value change (∆Hue) monitoring of LAMP amplification. (E) Threshold time Tt (in minutes), as a function of the HPV 16 DNA concentration (expressed in terms of HPV 16 DNA copies per reaction).

Figure 4

Clinical validation and smart, connected health monitoring on the SCPT platform. (A) A process of on-chip nucleic acid extraction from different clinical samples. (B) Comparison of HPV 16 DNA quantitative detection in spiked saliva samples with: i) the SCPT platform and ii) qPCR method. (C) Quantitative detection of clinical vaginal swab samples by the SCPT platform and qPCR method. (D) Representative images of Pap smear results for negative clinical sample (clinical sample 1 in Fig. 4C) and positive clinical sample (clinical sample 9 in Fig. 4C). NILM and HSIL stand for High-Grade Squamous Intraepithelial Lesion and Negative for Intraepithelial Lesion or Malignancy, respectively. (E) Smart, connected disease monitoring and pathogen tracking by the SCPT platform.