Unusual switchable peroxidase-mimicking nanozyme for the determination of proteolytic biomarker

Abstract

Detection of enzyme biomarkers originating from either bio-fluids or contaminating microorganisms is of utmost importance in clinical diagnostics and food safety. Herein, we present a simple, low-cost and easy-to-use sensing approach based on the switchable peroxidase-mimicking activity of plasmonic gold nanoparticles (AuNPs) that can catalyse for the oxidation of 3,3',5'5-tetramethylbenzidine (TMB) for the determination of protease enzyme. The AuNP surface is modified with casein, showing dual functionalities. The first function of the coating molecule is to suppress the intrinsic peroxidase-mimicking activity of AuNPs by up to 77.1%, due to surface shielding effects. Secondly, casein also functions as recognition sites for the enzyme biomarker. In the presence of protease, the enzyme binds to and catalyses the degradation of the coating layer on the AuNP surface, resulting in the recovery of peroxidase-mimicking activity. This is shown visually in the development of a blue colored product (oxidised TMB) or spectroscopically as an increase in absorbance at 370 and 650 nm. This mechanism allows for the detection of protease at 44 ng•mL −1 in 90 min. The nanosensor circumvents issues associated with current methods of detection in terms of ease of use, compatibility with point-of-care testing, low-cost production and short analysis time. The sensing approach has also been applied for the detection of protease spiked in ultra-heat treated (UHT) milk and synthetic human urine samples at a limit of detection of 490 and 176 ng•mL −1 , respectively, showing great potential in clinical diagnostics, food safety and quality control.

Gold Nanozymes: From Concept to Biomedical Applications

“…Biswas et al found gold nanorods (AuNRs) of 2.8 aspect ratio to possess a catalytic efficiency for 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation slightly higher than cysteamine-capped gold nanospheres (AuNSs) of 34 nm. However, McVey et al [102] observed that smaller AuNSs (14 nm diameter) showed a much higher catalytic efficiency, in agreement with the catalytic model studies previously mentioned. Moreover, these smaller AuNSs have overcome the efficiency of biological enzymes (Fig.…”

“…Most applications encompassed in this category are related to diagnostic tools to detect toxic contaminants in environmental waters. This mainly includes heavy metals [136,[176][177][178][179][180][181][182][183], pesticides [100,184,185] or micro-organisms [102,[186][187][188][189], for being the most common contaminants, but it could potentially include any others. Nonetheless, these are not the only examples of gold nanozyme applications in the environmental sector, especially since their catalytic activities started to be exploited for wastewater treatment to degrade or convert toxic molecules into innocuous products [190,191].…”

Amalgamated gold-nanoalloys with enhanced catalytic activity for the detection of mercury ions (Hg2+) in seawater samples

“…1(c), blue triangles). Due to the overlapping of plasmonic peak from AuNP aggregation (~ 650 nm) with the second peak from oxidised TMB (oxTMB), the most prominent peak at 370 nm was selected for analysis [17]. From these results, it is evident that the presence of AuNPs notably enhances the rate of TMB oxidation therefore, confirming the strong peroxidase-mimicking activity of bare-AuNPs.…”

“…Previously, plasmonic AuNPs have been widely exploited for the colorimetric detection of Hg 2+ through T-Hg 2+ -T co-ordination chemistry [10][11][12] and interactions with molecules such as cysteamine [13], tween-20 [14], papain [15] and thioctic acid [16]. Additionally, AuNPs can mimic the activities of natural enzymes (termed nanozymes), for example horseradish peroxidase (HRP) [17], glucose oxidase [18][19][20], superoxide dismutase [21] and catalase [22], which have been well documented for detecting metal ions, including Pb, Co, K and Hg [23][24][25]. However, some of these mimicking behaviours can exhibit relatively low catalytic activity [26].…”

The Steadfast Au@Pt Soldier: Peroxide-Tolerant Nanozyme for Signal Enhancement in Lateral Flow Immunoassay of Peroxidase-Containing Samples

“…We developed the single-step approach for the detection of nanozymes with peroxidase- peroxidase-mimicking activity by shielding the catalytic surface. 31 For lateral flow immunoassay, additional reduction occurs because of the trapping of nanozymes inside membrane pores. 41,42 To overcome the blocking, the surface of nanozymes can be recuperated by overgrowth with a bare catalytic layer over initial particles.…”