Improved lateral-flow immunoassays for chlamydia and immunoglobulin M by sequential rehydration of two-phase system components within a paper-based diagnostic

Frequent screening of at-risk individuals using paper-based point-of-care diagnostics is a promising method for attenuating the spread of chlamydia and HIV. However, such diagnostics typically suffer from limited sensitivity. The authors describe a method to improve the sensitivity of the lateral-flow immunoassay (LFA), in which the target molecules are thermodynamically concentrated using aqueous two-phase systems (ATPSs) to improve the detection limit. The novel concept of sequential ATPS component resolubilization within paper for both a PEG-potassium phosphate and a UCON-50-HB-5100-potassium phosphate system was established to provide a diagnostic tool which only requires the sample to be added without any sample preparation steps. The automated ATPS phase separation and concentration of the target into the leading phase within the paper matrix can be visually demonstrated due to the use of gold nanoparticles. The importance of resolubilization order of the dehydrated polymers and salts is discussed. The designs presented improve the detection limit of a conventional LFA setup for Chlamydia trachomatis bacteria (158 μg·mL−1) and human immunoglobulin M (IgM) antibodies (0.31 μg·mL−1) to 15.8 μg·mL−1 and 0.031 μg·mL−1, respectively. This is a 10-fold detection limit improvement and results are available in 15 min. Compared to other methods that improve LFA sensitivity, this particular method does not require additional equipment, reagents, or sample preparation steps as all necessary components are embedded into the initial setup. Therefore, this advancement enables a more sensitive LFA that can be operated by untrained or minimally trained personnel, significantly expanding its applicability as a point-of-care test.