Construction Of Structure-stabilized Electrochemiluminescence Nanoparticle And Its Signal Amplification For Immunoassay Reaction

During long-term preservation,nanoparticles often exhibit structural instability,prone to particle swelling,and even complete structural destruction.In general,the shelf life of clinical test kits should not be less than 6 months,thus the structural instability limits the practical application of nanoparticles as markers.Based on this,this article constructed doped nanoparticles with macromolecular polymers as the electrochemiluminescence（ECL）signal molecule ruthenium complex carrier and small molecules doped to realize the enhancement of the structural stability of the particles and signal amplification for immunodetection reactions.First,synthesized the epoxy derivative（Ru-epo）of the ECL signal molecule terpyridine ruthenium.An intermediate product diligand was obtained through the coordination reaction of ruthenium trichloride（Ru Cl₃）and 2,2’-bipyridine（bpy）,and then the diligand was further coordinated with 5,6-dioxo-[1,10]phenanthroline（phen-epoxide）to obtain Ru（bpy）₂（phen-epoxide）（PF₆）₂.The complex had an active group epoxy group,and the epoxy group easily undergone a ring-opening reaction with an amino group,a carboxyl group,and the like.Based on this,this paper used two macromolecular polymers,namely polyethyleneimine（PEI）and polyacrylic acid（PAA）as the carrier of Ru-epo,to construct nanoparticles with macromolecular polymer as the carrier.A strong hydrophobic macromolecule PEI-Ru was prepared by the ring-opening reaction of the amino group of the polyethylene imine PEI with the epoxy group of Ru-epo.Nanoparticle o PEI-Ru was prepared based on hydrophobic interactions,and further doped with small molecular polyacrylic acid,hydrophobic interaction and electrostatic interaction were combined to prepare doped nanoparticle d PEI-Ru.Good structural stability was maintained for the doped nanoparticle d PEI-Ru during 298 days.There are a large number of residual amino groups on the surface of doped nanoparticle d PEI-Ru,and amino and carboxyl groups are commonly used labeling groups.In order to obtain ECL nanoparticles with surface residual groups of carboxyl groups,this paper also used macromolecular polyacrylic acid PAA as the carrier and difunctional reagent ethylenediamine as the connection bridge to prepare another strongly hydrophobic macromolecule PAA-Ru.Similarly,the nanoparticle o PEI-Ru was prepared based on hydrophobic interaction,and the small molecule PEI was further doped to prepare the doped nanoparticle d PAA-Ru.The doped nanoparticle d PAA-Ru was still uniformly distributed and maintained good structural stability on the 31st day.The properties and structures of the two kinds of doped nanoparticles were characterized,and the feasibility of constructing stable nanoparticles with macromolecules as carriers was proved.In the immunoassay reaction system,the concentration of ECL luminescent molecules was often low,and the sensitivity of conventional detection methods was difficult to meet the requirements.To solve this problem,this article studied from the perspective of improving the co-reaction reagent system and the detection cell.On the one hand,Triton X-100,a non-ionic surfactant,was added to a conventional tripropylamine（TPA）solution to form a new co-reactive reagent solution system.The results showed that Triton X-100enhanced the luminous intensity up to 22.1 times compared to that of the TPA solution alone.On the other hand,a new type of electrochemical detection cell was constructed in this paper.Compared with the purchased electrochemical detection cell,the signal strength of the self-made new detection cell was about 19 times for the same concentration Ru-COOH.Also,the self-made detection cell had better reproducibility,high sensitivity.For carboxylated ruthenium terpyridine derivative Ru-COOH,the lowest detection limit was 0.035 f M（S/N=3）.On the strength of the new type of co-reaction reagent system and detection cell,the prepared doped nanoparticles used Ru-COOH as a control,and compared the ECL signals at their optimal antibody labeling concentrations,respectively.The results showed that the doped nanoparticle d PAA-Ru had a signal amplification of 2.3times compared to Ru-COOH,doped nanoparticle d PEI-Ru had a signal amplification of5.6 times compared to Ru-COOH,d PEI-Ru NP had better signal amplification ability.In summary,the doped nanoparticle d PEI-Ru prepared in this article had excellent nanoparticle structure stability,which is far longer than the 6-month shelf life of the commonly used small molecule labeled immunoassay kits in clinical practice;At the same time,it had obvious immunodetection reaction signal amplification,which indicates that the ECL nanoparticles can replace small molecule ruthenium complexes as signal markers for immunodetection,showing broad application prospects.