Study On The Effect Of Surface Probe Density Of Upconversion Fluorescent Probes On Their Binding Affinity

C-reactive protein(CRP)is a protein with inflammatory markers that is typically produced by the body in response to bacterial or viral infections.Early detection of CRP contributes to improved diagnosis and treatment of infectious diseases,while ultra-sensitive CRP detection serves as one of the risk factors for cardiovascular diseases such as coronary heart disease,cardiac conditions,and stroke.Therefore,in daily life,early and highly sensitive CRP detection is of significant importance for preventing chronic disease infections.However,conventional clinical detection methods are time-consuming,costly,and reliant on laboratory conditions,greatly limiting the motivation for early patient testing.The development of instant detection techniques based on highly specific and visually interpretable fluorescence probes holds the potential to overcome the limitations of traditional detection methods by offering advantages such as fast detection speed,simple operation,portability,and low cost.Nevertheless,the conventional fluorescence probes suffer from low detection signal and background fluorescence interference,which reduces the signal-to-noise ratio and hampers their application in the detection of ultra-sensitive C-reactive protein(hs CRP).This study utilizes upconversion nanoparticles(UCNPs)with a unique upconversion luminescence mechanism as fluorescent signals.By surface modification and biofunctionalization,a specific upconversion fluorescence probe for capturing CRP is constructed.The upconversion fluorescence probe is integrated onto a lateral flow assay(LFA)platform,which is user-friendly,portable,fast,and cost-effective.By optimizing the fluorescence intensity of UCNPs and the probe density on the surface of UCNPs,the amplification of the fluorescence signal in response to the biological signal is achieved,enabling the detection of hs CRP.The main research objectives and findings are outlined as follows:(1)Four types of UCNPs with different fluorescence intensities were successfully prepared using a high-temperature decomposition method.The process parameters of doping ion concentration and insulation duration were investigated.The hydrophobic UCNPs were rendered hydrophilic by encapsulating them with-COOH groups.Subsequently,the-NH2 groups of anti-CRP Ab 8# probe reacted with the-COOH on the surface of UCNPs,forming the upconversion fluorescent bio-probe.Ultimately,four types of upconversion fluorescent bio-probes were prepared,exhibiting a fluorescence gradient,high stability,and long lifespan,capable of specifically capturing CRP.(2)An upconversion fluorescent bio-probe-based lateral flow assay platform(UCNPsLFA)was developed for instant detection.Firstly,the upconversion fluorescent bio-probe was sprayed onto the conjugate pad.Secondly,the capture probe and control probe were immobilized on the NC membrane.Finally,the sample pad and absorbent pad were attached.By optimizing the system parameters,a UCNPs-LFA detection platform capable of specifically detecting CPR concentration within 15 minutes was successfully constructed.(3)The impact of controlling the fluorescence intensity of UCNPs and the probe density on the enhancement of sensitivity in the UCNP-LFA platform was systematically investigated.By adjusting the fluorescence probe intensity and varying the size of UCNPs from 50 nm to500 nm,the sensitivity of the UCNPs-LFA was improved by nearly fourfold(LOD for 50 nm UCNPs: 0.852 ng/m L;LOD for 500 nm UCNPs: 0.247 ng/m L),confirming the theoretical relationship between increased fluorescence probe intensity and enhanced detection sensitivity.Although increasing the particle size of UCNPs enhances fluorescence intensity,it also leads to an increase in the number of available surface modification sites.However,excessive probes can decrease the response between fluorescence signals and sample concentration,thereby reducing the detection sensitivity.Therefore,the control of probe density on the surface of UCNPs was achieved by adjusting the proportions of anti-CRP Ab 8# and blank probe(BSA)to 90%,70%,50%,30%,and 10%.Experimental results demonstrated that as the surface probe density decreased,the UCNPs-LFA was capable of detecting lower concentrations of CRP,achieving amplification of the fluorescence signal in response to the biological signal.It was confirmed that a more dispersed distribution of anti-CRP Ab 8# on the surface of UCNPs resulted in stronger binding ability with CRP.(4)By optimizing the UCNPs probe concentration and the reading device,the optimized UCNPs-LFA detection platform was integrated with a domestically produced desktop fluorescence analyzer,enabling high-performance instant detection capabilities.The sensitivity of the optimized UCNPs-LFA detection platform for CRP was increased by nearly20 times,with a detection limit of 0.046 ng/m L and a detection range of 0.2-300 ng/m L.The application of this platform in the detection of CRP in clinical serum samples yielded results consistent with clinical trials,validating its clinical utility.Furthermore,this optimization method holds promise for enhancing other nanoparticle-based bio-probes,thus enabling a broader range of instant detection applications.This research provides healthcare professionals and patients with a fast,convenient,cost-effective,and versatile means of detection.