An Enzyme Cascade-Triggered Fluorogenic and Chromogenic Reaction Applied in Enzyme Activity Assay and Immunoassay

Abstract

An enzyme cascade-triggered reaction with novel signal generation mechanism is beneficial for the development and insight of the enzyme cascade, which is extensively used for signal transduction in potential applications. Inspired by the fluorogenic and chromogenic reaction between dopamine and resorcinol, and the specific catalytic properties of alkaline phosphatase (ALP) and tyrosinase, we designed and synthesized an unconventional substrate of ALP, named p-aminoethyl-phenyl phosphate disodium salt (PAPP). As expected, the ALP and tyrosinase-incubated PAPP solution exhibited pale yellow with intense blue fluorescence upon addition of resorcinol, owing to the ALP-catalyzed transformation of PAPP into an intermediate tyramine, and the tyrosinase-catalyzed hydroxylation of tyramine to dopamine, as well as the specific reaction between dopamine and resorcinol. Therefore, an enzyme cascade system has been developed herein based on the ALP and tyrosinase coupled enzymes-triggered fluorogenic and chromogenic reaction. According to the direct relationship between the activity of ALP/tyrosinase and absorbance/fluorescence intensity of the resultant solution, the proposed enzyme cascade-triggered reaction was utilized for assaying ALP and tyrosinase activity with fluorometric and colorimetric dual-readout signals. Furthermore, such enzyme cascade catalysis process was integrated into the ALP-based cascade enzyme-linked immunosorbent assay with dual-readout signals, resulting in the sensitive detection of cardiac troponin I in diluted serum.

Alkaline Phosphatase-Triggered in Situ Formation of Silicon-Containing Nanoparticles for a Fluorometric and Colorimetric Dual-Channel Immunoassay

“…Typical sigmoidal curves can be achieved when plotting the fluorescence intensity and absorbance versus the logarithm of the Na 3 VO 4 concentrations. The IC 50 (the inhibitor concentration required for 50% inhibition of the enzyme activity) values for fluorometric and colorimetric detections are calculated to be approximately 20.6 and 17.3 μM, respectively, which are in good accord with the reported values in other works. , The above results clearly manifested that our method is prospective for the screening of ALP inhibitors.…”

“…The IC 50 (the inhibitor concentration required for 50% inhibition of the enzyme activity) values for fluorometric and colorimetric detections are calculated to be approximately 20.6 and 17.3 μM, respectively, which are in good accord with the reported values in other works. 9,42 The above results clearly manifested that our method is prospective for the screening of ALP inhibitors. Dual-Readout ELISA Strategy for PSA Detection.…”

Detection of tyrosine and monitoring tyrosinase activity using an enzyme cascade-triggered colorimetric reaction

“…19 We therefore developed a sequential cascade reaction involving multiple enzymes to serve as a signal lter and amplier. 20,21 Tyrosinase converts Tyr to L-DOPA; i.e., the substrate of DOPA-dioxygenase (DOD). Note that DOD is an oxidase that opens the cyclic ring between carbon 4 and 5 on L-DOPA, resulting in the synthesis of yellow betalamic acid, [22][23][24] which is a precursor of betalains/betaxanthins used as readouts of optical biosensors (Scheme 1).…”

Chemiluminescence “turn-on” detection of tyrosinase activity via in situ generation of dopamine based on a lucigenin and riboflavin system

“…23 Yang et al applied the specific catalysis of alkaline phosphatase (ALP) and tyrosinase in the fluorescence chromogenic reaction of dopamine and resorcinol, and in the fluorescence detection of ALP and tyrosinase activity. 25 Wu et al developed a luminescent assay for tyrosinase activity using AMP-Tb/Ag + lanthanide coordination polymer nanoparticles and an in situ reaction between tyrosinase and tyramine to generate dopamine. 26 Since tyrosinase can catalyze the oxidation of tyramine hydrochloride to dopamine, and then to dopamine- o -quinone, Zhao et al developed a fluorescent assay of tyrosinase based on the acceleration of the oxidation of dopamine to dopamine- o -quinone by ˙OH radical.…”