Restoring the Oxidase-Like Activity of His@AuNCs for the Determination of Alkaline Phosphatase

Abstract

In this paper, we propose a simple colorimetric method for the sensitive and selective detection of alkaline phosphatase (ALP) activity based on the turn off/turn on oxidase mimic activity of His@AuNCs. His@AuNCs/graphene oxide hybrids (His@AuNCs/GO) were easily obtained using the self-assembly method with poly (diallyldimethylammonium chloride) (PDDA)-coated GO and showed high oxidase-like activity compared with His@AuNCs. We found that the pyrophosphate ion (P₂O₇^4-, PPi) could effectively inhibit the oxidase mimic activity of His@AuNCs/GO, and the hydrolysis of PPi by ALP restored the inhibited activity of His@AuNCs/GO, enabling them to efficiently catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate the blue oxidized product oxTMB. The intensity of the color showed a linear dependency with the ALP activity. ALP was detected in the linear range of 0-40 mU/mL with a low detection limit (LOD) of 0.26 mU/mL (S/N = 3). The proposed method is fast, easy, and can be applied to monitor the ALP activity in serum samples accurately and effectively, which suggests its practicability and reliability in the detection of ALP activity in clinical practice.

Keywords: ALP; His@AuNCs/GO; PPi; colorimetric detection; oxidase-like activity.

Scheme 1

Illustration of the colorimetric sensing strategy for ALP activity based on PPi-mediated catalytic activity of His@AuNCs/GO.

Figure 1

Characterization of His@AuNCs, GO, andHis@AuNCs/GO, respectively. (A) TEM of His@AuNCs, (B) TEM of GO, (C) TEM of His@AuNCs/GO, (D) FT-IR spectra of GO (black), His@AuNCs (red) and His@AuNCs/GO (blue). Insert in (A), (C) show HR-TEM of His@AuNCs and His@AuNCs/GO, respectively.

Figure 2

(A) The UV–vis absorption spectra of His@AuNCs, PDDA/GO, and His@AuNCs/GO in the colorimetric system with TMB as a substrate. The inset shows the corresponding photographs. Inset: photograph of corresponding solution (from left to right: His@AuNCs, PDDA/GO, and His@AuNCs/GO). (B) Michaelis–Menten curve of nanomaterials (His@AuNCs, His@AuNCs/GO) with different concentrations of TMB (inset: the Lineweaver–Burk plot of nanomaterials with different concentrations of TMB).

Figure 3

(A) The UV–vis absorption spectra of the His@AuNCs/GO-TMB system in the presence of various concentrations of PPi. Inset shows the photos of the corresponding solutions. (B) Linear relationship for PPi detection. Absorbance changes with the increase of PPi concentration from 4 to 60 μM (inset). Error bars represent the standard deviations (n = 3).

Figure 4

(A) The UV–vis absorption spectra of the His@AuNCs/GO-TMB system in the absence and presence of various concentrations of ALP; the inset shows the photos of corresponding solutions. (B) Linear relationship between the absorbance at 652 nm and the ALP concentration. Absorbance changes with the increase of ALP concentration from 0.5 to 60 mU/mL (inset). Error bars represent the standard deviations (n = 3) (C) Selectivity of the assay for PPi compared to other potential interferences. Experimental conditions: PPi (40 μM), other anions (200 μM), and amino acids (500 μM). Error bars represent the standard deviations (n = 3).