Sensitive and quantitative detection of cardiac troponin I with upconverting nanoparticle lateral flow test with minimized interference

Figure 1. The effect of sample pre-treatment on plasma interference. All samples were analysed as unspiked (blank) and spiked with 2000 ng/L of cTnI before the pre-treatment. Pooled EDTA plasma samples were used in this experiment. IgM and RF commercial clean up solution was compared to 250 mM EDTA, IgM scrub line (prior to test line) and to different combinations. The error bars represent standard deviations of three replicate LFIA strips.

Figure 2. Analytical performance of the developed proof of concept cTnI LF test, showing the standard curve, precision profile and parallelism. (A) Calibration curve for the UCNP-LFIA. The error bars represent the standard deviation of three replicate strips. Equation of the curve was y = 0.76x + 2.52, R2 = 0.996. (B) Precision profile determined from the calibration curve measurements. CV% describes the variation between the calculated cTnI concentrations. (C) Parallelism of the UCNP-LFIA. Three clinical LiH-plasma samples containing variable amounts of endogenous cTnI were serially diluted to 1/81-fold with blank LiH-plasma pool.

Figure 3. Method comparison between the UCNP-LFIA and the in-house well-based reference assay (n = 188). (A) Correlation of the assays and (B) Bland–Altman analysis of agreement. The relative difference is calculated as UCNP-LFIA concentration subtracted by in-house well-based assay concentration divided by mean concentration. The mean difference (39.4%) is presented with a solid line and the 95% limits of agreement (from − 55.1 to 134%) are shown with dashed lines.

Figure 4. Method comparison between the UCNP-LFIA and Siemens Advia Centaur TnI-Ultra reference assay (n = 191). (A) Correlation of the assays and (B) Bland–Altman analysis of agreement. The relative difference is calculated as UCNP-LFIA concentration subtracted by Siemens Advia Centaur TnI-Ultra concentration divided by mean concentration. The mean difference (− 107.7%) is presented with a solid line and the 95% limits of agreement (from − 25.1 to − 190.4%) are shown with dashed lines.

Figure 5. A schematic illustration of the cTnI epitopes and the binding sites of the antibodies in the developed LFIA. The developed UCNP-LFIA utilized the (3 + 1) assay design, with three capture antibodies against the N-terminus, midfragment and C-terminus of cTnI and one tracer antibody against the C-terminus of cTnI molecule. Created with www.BioRender.com.

Figure 6. Schematic illustration of the protocol of the developed lateral flow assay and the strip design. Created with www.BioRender.com.