Catalytic Properties Of Platinum Nanozyme Based On Microfluidic Droplets

Nanozyme with low-budget,high stability,and wide range of p H and temperature adaptability,is a hot research field in recent years.Nanozyme can be regulated by size,shape and surface modification,and they can be used in disease treatment,medical imaging,biological detection and immunoassay.Platinum nanoparticles(Pt NPs)and their alloys show excellent catalytic performance due to their relatively lower activation energy.At present,they have been confirmed to have a variety of enzyme activities,such as oxidase(OXD)-like,peroxidase(POD)-like,catalase(CAT)-like and superoxide dismutase(SOD)-like activities.However,compared with natural enzyme,the catalytic ability of Pt nanozyme is still insufficient.It is necessary to optimize the activity of nanozyme to make it a substitute for natural enzyme.The activity of nanozyme can be affected by adjusting the size,morphology,surface modification,valence,composition and structure of active site and so on.In this paper,we systematically studied the effects of size,surface modification,substrate and other factors on the activity of Pt nanozyme.For the first time,we studied the activity of Pt nanozyme based on the fluorescent substrate Amplex Red(AR)reaction system,and further realized the detection of nanozyme activity in the micro droplet reaction system on the microfluidic chip.It lays a foundation for nanozyme to replace natural enzyme to realize biomolecular detection based on microfluidic droplets.Firstly,sodium citrate(Cit),polyethyleneimine(PEI)and polyethylene glycol(PEG)were used as stabilizers respectively,and H₂Pt Cl₆was reduced by Na BH₄to obtain Pt NPs(Pt@Cit,Pt@PEI,Pt@PEG).TEM,XPS,SEM and Zeta potential were used to characterize the Pt NPs products.The results showed that the average size of Pt@cit was 2.9nm,the average sizes of Pt@PEI were 2.01 nm,1.87 nm,1.19 nm and 1.16 nm respectively,and the average sizes of Pt@PEG were 2.61 nm,1.89 nm,1.38 nm and 0.95 nm respectively.More over,each kind of Pt NPs were uniform,well dispersed,and stable-charged.In addition,Pt composite polystyrene microspheres(PS@Pt)were prepared by electrostatic adsorption and in-situ reduction,which were characterized by SEM as well.Secondly,the CAT-like activity of Pt NPs(using H₂O₂as a substrate),and OXD-like activity(using AR as a substrate),POD-like activity(using AR,H₂O₂as substrates)were systematically studied.The results showed that the OXD-like and POD-like activities of Pt@PEG of 2.6nm size were better than other Pt NPs,but the POD-like activity was weaker.However,Pt@PEI of 1.1nm size showed the highest POD-like activity.In addition,the enzyme-like activities of Pt NPs,Prussian blue and Fe₃O₄nanozymes were compared.The results showed that the OXD-like and POD-like activities of Pt NPs were significantly better than those of the other two nanozymes.Further more,the differences of OXD-like and POD-like catalytic activities of Pt NPs for TMB,ABTS and AR substrates were also compared.Finally,a microfluidic droplet chip was designed and fabricated,in which the mixed aqueous solution of substrate and nano enzyme Pt NPs were used as the dispersion phase,and the flow rate of water phase and oil phase was adjusted to realize the formation of size controllable droplets.The fluorescence microscope images of the droplet array showed that the generated droplets were uniform,stable and high-red-fluorescent.Based on the fluorescence images of microfluidic droplets,the OXD-like and POD-like activities of Pt NPs in the droplets were studied,and the linear relationship between the fluorescence intensity and the concentration of Pt NPs was obtained,which laid a foundation for the application of Pt nanozyme instead of natural enzyme in the biological detection based on microfluidic droplets.