Unconventional Application of Conventional Enzymatic Substrate: First Fluorogenic Immunoassay Based on Enzymatic Formation of Quantum Dots

Abstract

In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) leads to the formation of p-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd(2+) with S(2-) ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL(-1) of analyte antibody with a linear range up to 10 ng mL(-1). The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric p-nitrophenyl phosphate assay.

Optical Nanoprobes for Ultrasensitive Immunoassay

“…52 In this work, the substrate of alkaline phosphatase (ALP) was the conventional chromogenic substrate pNitrophenyl phosphate (pNPP), which could be hydrolyzed to produce p-nitrophenol (pNP) and phosphate ions. With the addition of Cd 2+ and S 2− ions, phosphate-stabilized fluorescent CdS QDs could be formed in situ.…”

Enzymatic synthesis of a DNA-templated alloy nanocluster and its application in a fluorescence immunoassay

“…16,17 Nevertheless, the high sensitivity of these plasmonic enzymelinked immunosorbent assay was dependent on the complex plasmonic structure or tedious modication process. 22 However, the toxicity of quantum dots hampered the clinical application of this technology. [18][19][20][21] As ALP was the most common enzyme in labelling antibodies, this strategy was also applied to the detection of bovine serum albumin.…”

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

“…simplicity, practicality, low cost, and rapid/direct readout with the naked eye614. Compared with commercialized ELISA kits and CL/FL-based immunoassays, unfortunately, the majority of the currently developed colorimetric immunoassays usually involve poor sensitivity because of using the vulnerable/limited signal amplification strategies1415. In contrast, the increasing demand for screening diseases at the early stage of development calls for ultrasensitive detection of biologically relevant species at an extremely low level of expression16.…”

“…horseradish peroxidase (HRP), alkaline phosphatase (ALP) and β -galactosidase, due to their high catalytic activity, high specificity, mild reaction conditions, and easy conjugation to antibodies171819. Moreover, a single enzyme molecule can also catalytically transform many molecules of substrate into a product15. In the conventional enzyme-based colorimetric immunoassays, unfavorably, there is for steric reasons usually a ratio of 1:1 for enzyme and detection antibody used.…”