Nanozymes in Point-of-Care Diagnosis:An Emerging Futuristic Approach for Biosensing

Nanomaterial-based artificial enzymes(or nanozymes) have attracted great attention in the past few years owing to their capability not only to mimic functionality but also to overcome the inherent drawbacks of the natural enzymes.Numerous advantages of nanozymes such as diverse enzyme-mimicking activities,low cost,high stability,robustness,unique surface chemistry,and ease of surface tunability and biocompatibility have allowed their integration in a wide range of biosensing applications. Several metal,metal oxide,metal–organic framework-based nanozymes have been exploited for the development of biosensing systems,which present the potential for point-of-care analysis. To highlight recent progress in the field,in this review,more than 260 research articles are discussed systematically with suitable recent examples,elucidating the role of nanozymes to reinforce,miniaturize,and improve the performance of point-of-care diagnostics addressing the ASSURED(a ordable,sensitive,specific,user-friendly,rapid and robust,equipment-free and deliverable to the end user) criteria formulated by World Health Organization. The review reveals that many biosensing strategies such as electrochemical,colorimetric,fluorescent,and immunological sensors required to achieve the ASSURED standards can be implemented by using enzyme-mimicking activities of nanomaterials as signal producing components. However,basic system functionality is still lacking. Since the enzyme-mimicking properties of the nanomaterials are dictated by their size,shape,composition,surface charge,surface chemistry as well as external parameters such as pH or temperature,these factors play a crucial role in the design and function of nanozyme-based point-of-care diagnostics. Therefore,it requires a deliberate exertion to integrate various parameters for truly ASSURED solutions to be realized. This review also discusses possible limitations and research gaps to provide readers a brief scenario of the emerging role of nanozymes in state-of-the-art POC diagnosis system development for futuristic biosensing applications.

Gold Nanozymes:From Concept to Biomedical Applications

In recent years,gold nanoparticles have demonstrated excellent enzyme-mimicking activities which resemble those of peroxidase,oxidase,catalase,superoxide dismutase or reductase.This,merged with their ease of synthesis,tunability,biocompatibility and low cost,makes them excellent candidates when compared with biological enzymes for applications in biomedicine or biochemical analyses.Herein,over 200 research papers have been systematically reviewed to present the recent progress on the fundamentals of gold nanozymes and their potential applications.The review reveals that the morphology and surface chemistry of the nanoparticles play an important role in their catalytic properties,as well as external parameters such as pH or temperature.Yet,real applications often require specific biorecognition elements to be immobilized onto the nanozymes,leading to unexpected positive or negative effects on their activity.Thus,rational design of efficient nanozymes remains a challenge of paramount importance.Different implementation paths have already been explored,including the application of peroxidase-like nanozymes for the development of clinical diagnostics or the regulation of oxidative stress within cells via their catalase and superoxide dismutase activities.The review also indicates that it is essential to understand how external parameters may boost or inhibit each of these activities,as more than one of them could coexist.Likewise,further toxicity studies are required to ensure the applicability of gold nanozymes in vivo.Current challenges and future prospects of gold nanozymes are discussed in this review,whose significance can be anticipated in a diverse range of fields beyond biomedicine,such as food safety,environmental analyses or the chemical industry.

Fabrication of plasmonic nanopyramidal array as flexible SERS substrate for biosensing application

The proposed work aims to develop a sensitive surface-enhanced Raman spectroscopy(SERS)nano-biosensor.The inverted nano pyramid array on silicon substrate fabricated using electron beam lithography(EBL)was utilised as a master template and the mold was later replicated via nanoimprinting process to prepare gold-coated polymer nanopyramid three-dimensional(3D)SERS substrate.The fast and versatile replication process using nanoimprinting lithography(NIL)can produce polymer nanopyramids in a low-cost and reproducible fashion.Also,the proposed fabrication protocol can be easily upscale for large scale fabrication.The intense electric field confinement at nanotips and four edges of gold-coated polymer nanopyramid enhanced the Raman signal of probe molecules,i.e.,Rhodamine 6G with a limit of detection down to 3.277×10−9 M was achieved.This work also underlines the efficiency of gold-coated polymer nanopyramid arrays in the spectral detection of hemoglobin proteins at low concentrations.The Raman signal enhancement mechanism was further studied through the electromagnetic simulation using COMSOL Multiphysics.In addition,bending test experiments were performed to understand the effect of flexibility on SERS signal response.The fabricated gold-coated polymer nanopyramids arrays could pave the way for the development of low-cost SERS platforms for the detection of hazardous biological and chemical compounds at ultra-low concentrations in practical applications.

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

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.

Amalgamated gold-nanoalloys with enhanced catalytic activity for the detection of mercury ions(Hg^2+)in seawater samples

Mercury(Hg)is extremely toxic,and continues to cause major threats to aquatic life,human health and the environment.Hg^2+mainly derives from seawater as a product of atmospheric deposition,therefore there is great demand for sensing approaches that can detect Hg^2+in seawater samples.Herein,we demonstrate that the peroxidase-mimicking activity of gold nanoparticles(AuNPs)or so-called nanozymes,can be exploited for the detection of Hg^2+ions in various water samples.In a high electrolyte environment,the catalytic activity for the oxidation of 3,3’,5,5’-tetramethylbenzidine(TMB)was significantly diminished due to poor stability of the bare-AuNPs.This activity was reduced by-73.7%when the NaCl concentration was higher than 1.168%,which is much lower than that of seawater(-3.5%),thus presenting its unsuitability for detecting Hg^2+in harsh water matrices.To overcome this limitation,AuNPs were first functionalized with oligo-ethylene glycol(OEG),of which their colloidal form presented high stability in NaCl concentrations up to 20%and across a wide range of pHs from 1-14.Interestingly,the catalytic activity of OEG-AuNPs for the oxidation of TMB was strongly suppressed by the coating,but enhanced upon formation of Au-Hg amalgamation.This novel finding underlies a straightforward,sensitive,and highly selective detection platform for Hg^2+in water samples.The approach could detect the exposure limit level for Hg^2+in drinking water(i.e.,2 ppb for tap and bottled water)as set by the United States Environmental Protection Agency(EPA)and the World Health Organization(WHO).When Hg^2+was spiked into a 3.5%saline solution and a coastal seawater certified reference material(CRM),the detection limits were found to be 10 ancM 3 ppb,respectively,which exceed the Hg^2+concentrations commonly found within seawater(-60-80 ppb).The whole procedure takes less than 45 min to conduct,providing a highly innovative,rapid and low-cost approach for detecting Hg^2+in complex water matrices.