Direct comparison of colorimetric signal amplification techniques in lateral flow immunoassays

The lateral flow immunoassay (LFIA) is widely adopted for point-of-care testing, but its limit of detection (LoD) falls short of that of laboratory-based immunoassays. Several techniques have been proposed to enhance the LoD of LFIAs using visual colorimetric readouts, yet a direct comparison of the LoDs achieved by these techniques has not been performed. In this work, we measure the LoDs of LFIAs designed for the detection of a malaria protein, PfHRP2, using four different colorimetric signal generation techniques: (i) AuNP(40 nm)-tagged detection antibodies (base case), (ii) AuNP-based enhancement of AuNP(40 nm) signal, (iii) oxidation of chloronaphthol/diaminobenzidine (CN/DAB) using HRP-tagged detection antibodies, and (iv) oxidation of CN/DAB using polyHRP(400)-tagged detection antibodies. The LoDs and the 95% confidence intervals of the LoDs achieved by the 4 techniques were 19.34 (13.37-27.62) ng mL