In situ amplified colorimetric immunoassay coupled with a dual-enzyme-functionalized UiO-66(Ce) framework for quantitative detection of the dengue virus†

Signal amplification and noise reduction are very crucial for the sensitive determination of disease-related biomarkers. The development of rapid and laborious testing is urgently required due to extended incubation time and high mortality. Herein, we developed a Ce-based mesoporous metal–organic framework-based multi-enzyme encapsulation device for the realization of enzyme-linked immunosorbent assay (ELISA) for the dengue virus (DENV). Briefly, a UiO-66(Ce) framework structure with uniform mesopores was synthesized by a one-pot method, which allowed efficient encapsulation of natural enzymes with an encapsulation efficiency of 700%. The developed multi-enzyme reaction probes were able to maintain more than 90% of catalytic activity stable at room temperature for 60 days while ensuring efficient enzyme immobilization. Sensitive evaluation of DENV was achieved by encapsulating glucose oxidase and horseradish peroxidase, combined with the formation of an immune sandwich in the presence of DENV. The developed sensor enabled flexible detection of recombinant dengue virus serotype 2 NS1 protein (DENV2-NS1) from 0.05 to 100 ng mL⁻¹, with a low limit of detection of 39.7 pg mL⁻¹. In addition, there were no significant differences in the test results of the samples obtained through the developed multi-enzyme probes when compared to commercially available ELISA kits. This work provides new horizons for the development of efficient enzyme encapsulation systems.