The developed nanoprobe has been tested for the detection of immunologically significant C-reactive protein (CRP) in ELISA format. developed nanoprobe was performed with ELISA developed on conventional guidelines, the proposed immunoassay showed an increase of 12-fold sensitivity for detecting CRP due to the high loading of 6Xhis peptide and binding of multiple Ni2+-HRP on Imipenem a platinum nanoparticle. Additionally, the proposed assay provides a simple, fast, and cost-efficient (not requiring multiple antibodies) detection of CRP with easy nanoprobe synthesis. Moreover, the developed Histag-HRP functionalized nanoconjugate immunoassay is usually flexible and can be applied to other biomarkers efficiently by using disease specific antibody. is the absorption (AU) at the peak SPR, is the amount of gold used in the synthesis (moles/L), and and are constants defined by values ?4.75, 0.314, respectively. For determining the concentration of AuNPs, is the number density of AuNPs per mL, is the absorption (AU) at 450 nm, and is the common diameter of the AuNPs. The concentration of bare AuNPs and size were estimated to be 2.72 nM and 20 nm (Supplementary Materials Physique S1). Thereafter, titration study was performed using BSA to confer the maximum amount of protein that can be adsorbed on the surface of 100 L of Imipenem 20 nm AuNPs, which was inferred from your minimum amount of protein required to stabilize the AuNPs . Physique 1a represented the color change from grey to pink as the BSA concentration increased causing redshift in the absorbance spectra due to subsequent conjugation of BSA around the AuNPs surface. No significant aggregation and color switch were observed above 2 g/mL of BSA. After the estimation of the nanoparticle size, concentration, and maximum protein binding capacity, Imipenem the antibody, 6Xhis, and Ni2+-HRP were sequentially conjugated around the AuNPs. The conjugation was confirmed through quantifying the spectrophotometric changes of AuNPs. As the protein or peptide was conjugated around the AuNPs surface, local refractive index, and absorbance of AuNPs changes. This switch was characterized by the redshift in the wavelength of peak absorbance in the visible range . Thus, the developed nanoprobe was simultaneously evaluated by UV-vis-spectrophotometry (400C700 nm) for the subsequent conjugation of CRP antibody and 6Xhis peptide-Ni2+-HRP complex. The average diameter of AuNPs before conjugation was estimated to be 20 nm (Supplementary Materials Physique S1) while after conjugation of the abCRP antibody and 6Xhis-Ni-HRP, the size gradually shifted to 24 nm ITM2A and 28 nm as calculated from the peak obtained at 524 nm and 527 nm, respectively (Equations (1) and (2)). As expected, the spectral shift confirmed the covering of abCRP/6Xhis-Ni2+HRP on the surface of AuNPs (Physique 1b). Open in a separate window Physique 1 Plot depicting (a) the titration of platinum nanoparticle with BSA for the binding saturation study, each data point (black dots) represent the error bar, obtained from three units of experiments. (b) UV-Visible characterization of a developed peptide nanoprobe. After the successful conjugation, the total quantity of Imipenem HRP attached to the AuNPs was decided because the HRP is usually directly responsible for generating the colorimetric signals through TMB oxidation. In standard ELISA, a 1:1 antibody to HRP ratio is usually achieved by using a main or secondary antibody conjugated HRP. This ratio can be increased to 1:3 through streptavidin-biotin chemistry [20,36]. Streptavidin, a 60 kDa tetrameric protein, has a high binding affinity to biotin and four biotin binding sites . Generally, biotinylated antibody and streptavidin-conjugated HRP are used in ELISA to enhance the detection in ELISA . However, the option of further transmission enhancement is with streptavidin-biotin chemistry is limited. Thus, instead of using biotinylated antibody and streptavidin-HRP binding chemistry, we have exploited 6Xhis peptide and AuNPs binding chemistry. Multiple 6Xhis can be conjugated on the surface of AuNPs, moreover, one molecule.