Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme.

The alkaline phosphatase (ALP) biocatalysis followed by the in situ enzymatic generation of a visible light responsive nanozyme is coupled to elucidate a novel amplification strategy by enzymatic cascade reaction for versatile biosensing. The enzymatic hydrolysis of o-phosphonoxyphenol (OPP) to catechol (CA) by ALP is allowed to coordinate on the surface of TiO2 nanoparticles (NPs) due to the specificity and high affinity of enediol ligands to Ti(IV). Upon the stimuli by CA generated from ALP, the inert TiO2 NPs is activated, which demonstrates highly efficient oxidase mimicking activity for catalyzing the oxidation of the typical substrate of 3,3',5,5'-tetramethylbenzidine (TMB) under visible light (λ ≥ 400 nm) irradiation utilizing dissolved oxygen as an electron acceptor. On the basis of the cascade reaction of ALP and the nanozyme of CA coordinated TiO2 (TiO2-CA) NPs, we design exquisitely colorimetric biosensors for probing ALP activity and its inhibitor of 2, 4-dichlorophenoxyacetic acid (2,4-DA). Quantitative probing of ALP activity in a wide linear range from 0.01 to 150 U/L with the detection limit of 0.002 U/L is realized, which endows the methodology with sufficiently high sensitivity for potentially practical applications in real samples of human serum (ALP level of 40-190 U/L for adults). In addition, a novel immunoassay protocol by taking mouse IgG as an example is validated using the ALP/nanozyme cascade amplification reaction as the signal transducer. A low detection limit of 2.0 pg/mL is attained for mouse IgG, which is 4500-fold lower than that of the standard enzyme-linked immuno-sorbent assay (ELISA) kit. Although only mouse IgG is used as a proof-of-concept in our experiment, we believe that this approach is generalizable to be readily extended to other ELISA systems. This methodology opens a new horizon for amplified and versatile biosensing including probing ALP activity and following ALP-based ELISA immunoassays.

Label-free colorimetric sensor for mercury(II) and DNA on the basis of mercury(II) switched-on the oxidase-mimicking activity of silver nanoclusters.

In this paper, a novel colorimetric biosensor for Hg(2+) and DNA molecules is presented based on Hg(2+) stimulated oxidase-like activity of bovine serum albumin protected silver clusters (BSA-Ag NCs). Under mild conditions, Hg(2+) activated BSA-Ag NCs to show high catalytic activity toward the oxidation of 3,3',5, 5'-tetramethylbenzidine (TMB) using ambient dissolved oxygen as an oxidant. The oxidase-like activity of BSA-Ag NCs was "switched-on" selectively in the presence of Hg(2+), which permitted a novel and facile colorimetric sensor for Hg(2+). As low as 25 nmol L(-1)Hg(2+) could be detected with a linear range from 80 nmol L(-1) to 50 mmol L(-1). In addition, the sensing strategy was also employed to detect DNA molecules. Hg(2+) is known to bind very strongly and specifically with two DNA thymine bases (T) to form thymine-Hg(2+)-thymine (T-Hg(2+)-T) base pairs. The hairpin-structure was disrupted and Hg(2+) ions were released after hybridization with the DNA target. By coupling the Hg(2+) switched-on the oxidase-mimicking activity of BSA-Ag NCs, we developed a novel label-free strategy for facile and fast colorimetric detection of DNA molecules. More important, target DNA can be detected as low as 10 nmol L(-1) with a linear range from 30 to 225 nmol L(-1). Compared with other methods, this method presents several advantages such as the independence of hydrogen peroxide, high sensitivity and good selectivity, avoiding any modification or immobilization of DNA, which holds a great potential of metal NCs for clinical application in biosensing and biotechnology.

Intrinsic enzyme mimicking activity of gold nanoclusters upon visible light triggering and its application for colorimetric trypsin detection.

In this research, a novel enzyme mimetics based on the photochemical property of gold nanoclusters was demonstrated. It was found that the bovine serum albumin (BSA) stabilized red or blue emitting gold nanoclusters (Au NCs) exhibited enzyme-like activity under visible light irradiation. The BSA-Au NCs had better stability against stringent conditions compared to natural enzyme. In addition, the photostimulated enzyme mimetics of BSA-Au NCs showed several unprecedented advantages over natural peroxidase or other existing alternatives based on nanomaterials, such as the independence of hydrogen peroxide on activity and the easily regulated activity by light irradiation. The mechanism of the photoresponsive enzyme-like activity of BSA-Au NCs was investigated. The photoactivated BSA-Au NCs was designed to develop a facile, cheap, and rapid colorimetric assay to detect trypsin through trypsin digestion of the protein template of BSA-stabilized Au NCs. The limit of detection for trypsin was 0.6 µg/mL, which was much lower than the average level of trypsin in patient's urine or serum.