Smartphone-read phage lateral flow assay for point-of-care detection of infection

Abstract

The COVID-19 pandemic has highlighted the urgent need for sensitive, affordable, and widely accessible testing at the point of care. Here we demonstrate a new, universal LFA platform technology using M13 phage conjugated with antibodies and HRP enzymes that offers high analytical sensitivity and excellent performance in a complex clinical matrix. We also report its complete integration into a sensitive chemiluminescence-based smartphone-readable lateral flow assay for the detection of SARS-CoV-2 nucleoprotein. We screened 84 anti-nucleoprotein monoclonal antibody pairs in phage LFA and identified an antibody pair that gave an LoD of 25 pg mL^-1 nucleoprotein in nasal swab extract using a FluorChem gel documentation system and 100 pg mL^-1 when the test was imaged and analyzed by an in-house-developed smartphone reader. The smartphone-read LFA signals for positive clinical samples tested (N = 15, with known Ct) were statistically different (p < 0.001) from signals for negative clinical samples (N = 11). The phage LFA technology combined with smartphone chemiluminescence imaging can enable the timely development of ultrasensitive, affordable point-of-care testing platforms for SARS-CoV-2 and beyond.

Fig. 1

Schematic illustration of the chemiluminescent phage lateral flow assay for the detection of SARS-CoV-2 nucleoprotein. The sample flows along a nitrocellulose membrane on which immobilized (rabbit) antibodies capture the nucleoprotein molecules on the LFA test line (TL). Murine detection antibodies bind to the captured nucleoprotein molecules and are then detected by anti-mouse antibody-HRP M13 phage reporters. Light signal from the HRP-mediated chemiluminescent reaction is captured and analyzed by a smartphone and associated app. Created with BioRender.com.

Fig. 2

Phage LFA-based antibody screening. The performance of the 84 antibody pairs was initially evaluated with a no-target and 5 ng mL⁻¹ nucleoprotein sample in LFA extraction buffer. Values shown are the differences between the TL/CL ratios of positive and negative strips; darker blues correspond to higher differences. The top 6 antibody pairs that were available in sufficient stock (shown in bold borders) were chosen for the next round of screening using 1 ng mL⁻¹ nucleoprotein.

Fig. 3

Sub-picomolar (25 pg mL⁻¹=0.53 pM) detection of nucleoprotein in nasal swab extract using the phage LFA. Nucleoprotein was spiked in presumed-negative fresh nasal swab extracts; capture: rabbit monoclonal antibody 12F1 (#12) ExonBio and detection: mouse monoclonal antibody 1G1-F2 (#3) Ray Biotech. LFA strips were imaged 6 minutes after adding the ECL substrate, on a FluorChem gel documentation system. Data are mean ± s.d; the experiments were repeated at least three times. The dashed red line denotes the estimated background as the average plus three times the standard deviation (μ + 3σ) of the negative tests.

Fig. 4

Individual results of clinical specimens (nasal swab extracts from Labcorp) tested using the phage LFA and read with lab instrumentation. The box plots display the phage LFA TL/CL ratios of negative (n = 12) and positive samples (n = 15). Horizontal lines on each box plot, from bottom to top beginning with the bottom whisker are: 10th percentile, 25th percentile, median, 75th percentile, and 90th percentile. + symbols indicate outliers (low Ct value samples, 21.1, 19.5 and 18.7). *** symbol indicates that the two groups are statistically different by the Mann-Whitney U test, p < 0.001. TL/CL as a function of Ct value is shown in Figure S6 in the ESI.

Fig. 5

Smartphone-based picomolar detection of nucleoprotein using the phage LFA. Nucleoprotein was spiked in presumed-negative nasal swab extracts; capture: rabbit monoclonal antibody 12F1 (#12); ExonBio and detection: mouse monoclonal antibody 1G1-F2 (#3); Ray Biotech. Data are mean ± s.d.; n = 3. The dashed blue line denotes the estimated background as the average plus three times the standard deviation (μ + 3σ) of the negative tests.

Fig. 6

Individual results of clinical specimens (nasal swab extracts from Labcorp) tested using the phage LFA read with a smartphone. The box plots display the LFA signals (TL/CL) of negative (n = 11) and positive samples (n = 15). One negative sample used in the experiments presented in Fig. 4 was exhausted before testing with smartphone imaging and analysis. Horizontal lines on each box plot, from bottom to top beginning with the bottom whisker are: 10th percentile, 25th percentile, median, 75th percentile, and 90th percentile. + symbols indicate outliers (low Ct value samples, 21.1, 19.5 and 18.7). *** symbol indicates that the two groups are statistically different by the Mann-Whitney U test, p < 0.001. TL/CL as a function of Ct value is shown in Figure S8 in the ESI.