Precipitates produced via horseradish peroxidase (HRP-) catalyzed reaction was exploited to enhance the sensitivity of a surface acoustic wave (SAW) immunosensor for monitoring airborne house dust mite (HDM) allergens. Three candidate substrates, 4-chloro-1-naphthol (4CN), 3,3′-diaminobenzidine (DAB) and 3,3′,5,5′-tetramethylbenzidine (TMB), were systematically compared in terms of signal amplification and suitability to rapid and repeated measurement. Each substrate solution was applied to the sensor surface where group 2 HDM allergens of Dermatophagoides farinae (Der f 2) were sandwiched by capture and HRP-conjugated detection antibodies. The signal amplification and sensor regeneration rate were evaluated from the sensor output induced by precipitated product of HRP-catalyzed reaction and the sensor signal recovery after the regeneration via various pH reagents, respectively. As a result, it was revealed that 4CN showed the highest surface regeneration rate among the three substrates with use of NaOH adjusted to pH 13 as well as the second largest enhancement of the sensor output. Use of 4CN enabled to enhance the sensor output by a factor of 13 for 1 ng/mL Der f 2 with respect to that without the HRP-catalyzed reaction. The limit of detection of the precipitate-enhanced SAW immunosensor was 35 pg/mL (equivalent to 2.3 pM), which was 1.4-fold and 206-fold better than those of ELISA and the SAW immunosensor without a precipitate, respectively. These indicate that exploiting 4CN under the optimal conditions in the SAW immunosensor realizes ultra-sensitive monitoring of HDM allergens.
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