Extended Data Fig. 9: Comparison of LODs of model ssDNA with real RPA amplicons and gold nanoparticles.

a, The dilution series of the real RPA amplicons and the model ssDNA ‘amplicons’ were plotted against concentration for 600 nm FNDs (dots showing means with error bars showing s.d., n = 3–9 technical replicates, n = 3 measurement replicates) with their respective linear fits (solid lines with 95% confidence intervals of the fit shown shaded). The curves are similar, with fitted K_D values of 29 and 22 fM for model and real amplicons, respectively, and similar dynamic ranges. The real amplicons showed increased variation in the blanks, leading to a higher blank cutoff giving a higher LOD, and slightly reduced signal-to-blank ratio. b, The dilution series of model ssDNA ‘amplicons’ were plotted against concentration for 120, 200 and 600 nm FNDs (dots showing means with error bars showing s.d., n = 3 technical replicates, n = 3 measurement replicates) with their respective linear fits (solid lines with 95% confidence intervals of the fit shown shaded). The LODs are 3.7, 3.6 and 0.8 fM respectively. c, Comparison of 600 nm FNDs with 40 nm gold nanoparticles on LFAs, often used in LFAs owing to a good compromise between stability (and therefore ease of functionalization), and sensitivity⁵⁸. Serial dilutions are plotted (dots showing means with error bars showing s.d., n = 3 technical replicates, n = 3 measurement replicates for the FNDs; and dots with error bars showing the s.d. across the test line, n = 1 technical replicate, n = 1 measurement replicate for the gold nanoparticles). LODs are calculated as previously, giving 800 aM and 6.0 pM, respectively. d, e, A Monte Carlo simulation of the signal variation that can be explained by the FND size distribution (from DLS measurements in Extended Data Fig. 3b) was performed (n = 200,000). The violin plots (d) show the normalized simulated random variation in lock-in amplitudes due to the 600-nm FND size distribution in the clinical sample assays in Fig. 4d (negative plasma control and clinical standard). The experimental data are overlaid, showing that FND size distribution explains approximately 8–9% of the total experimental signal variance. Full details of the simulation are given in Supplementary Information section 3. A further approximately 0.1–2% of the variance is explained by periodic drift in modulation amplitude, shown over 45 min in e, normalized to the mean. f, A plot of the variation in lock-in amplitude due to small changes in the modulation frequency, F_m. The variance of the frequency is 3 × 10⁻⁸% over the same period, giving negligible differences in lock-in amplitude.