Studies On The Interactions Between Colloidal Gold And Proteins Or Drugs By Spectral Analysis

Immunoassay, which is based on the specific immunoreaction between antibodyand corresponding antigen, has received a great deal of attention. In recent years,immunoassay has been widely used in clinical diagnoses, food safety, environmentcontrol and biochemical studies because of its high sensitivity, specificity andselectivity.Colloidal gold has been widely used in immunoassay because of its uniqueoptical and chemical properties. Au nanoparticles can be easily prepared in a widerange of sizes from 2 to 100 nm. They are more suitable for conjugation withbiological systems because of their good biocompatibility. The detection methods ofcolloidal gold labels include transmission electron microscopy, inductively coupledplasma mass spectrometry, electrochemical analysis and light scatting analysis.In this work, the determination of human immunoglobulin G ( IgG ) based on acolloidal gold label by fluorospectrophotometry was developed. The sandwichimmunoreaction among goat anti human IgG, human IgG and goat anti human IgGlabeled with colloidal gold nanoparticles was applied in this experiment. First, thesandwich immunocomplex was formed on the surface of 96 well clear polystyrene microplate. After the formation of sandwich immunocomplex, a solution was added todissociate the immunocomplex at room temperature. Then the solution of each wellwas transferred into the corresponding test tube. Thirdly, the rhodamine B solutionwas injected into each test tube. The rhodamine B chloraurate was extracted into etherand measured with spectrofluorometer. The experimental results indicated that thefluorescence intensity increased with the increase of human IgG concentration. Thefluorescence intensity of rhodamine B chloraurate at 570 nm was proportional to thelogarithm of human IgG concentration in the range from 10 to 5×105 ng mL 1. It wasshowed that the determination of human IgG was easily made with the proposedfluorospectrophotometry.From above studies, it can be seen that most determination methods of antigen orantibody using the colloidal gold label technique are based on the specialimmunoreaction between antigen and antibody. By taking advantage of using a highsensitivity detector to detect colloidal gold signal or other reagents to enhance thecolloidal gold signal, the analyte can be easily detected. NH2OH HCl isthermodynamically capable of reducing Au3+ to bulk metal, and the reaction isdramatically accelerated by Au surface.In this paper, to enhance the signal resultingfrom gold nanoparticles coated on the microplate surface, the signal amplificationmethod in which the reduction of Au3+ by NH2OH HCl was accelerated on the Ausurface was applied. By taking advantage of sandwich immunoreaction and signalamplification, a simple spectrophotometric immunoassay for human IgG wasdeveloped. The good linear relationship between the signal of colloidal gold andlogarithm of human IgG concentration was obtained.Resonance light scattering (RLS), a Rayleigh scattering phenomenon at awavelength near or at the absorption bands of the scattering molecules, can provide ahigh signal level and good selectivity. Over the last decade, RLS technique has beendeveloped to study nucleic acids, proteins, pharmaceutical drugs and sugars. In theseexperiments, RLS not only meets sensitivity and selectivity criteria but also offers theadditional benefits of simplicity and versatility.In this paper, RLS spectrometry was applied to studying the interaction between drugs containing thiol and gold colloid. The RLS intensity is enhanced in the presenceof trace amount of drugs containing thiol due to the interaction between drugscontaining thiol and gold colloid. The binding of colloidal gold to drugs containingthiol results in ligand induced aggregation of colloidal gold, which was characterizedby RLS and ultraviolet visible (UV vis) spectrometry. The effect of pH, the amount ofbuffer or gold colloid, the temperature or the incubated time on the system wereinvestigated. The RLS intensities at 605.0 nm were found to be proportional to theconcentration of penicillamine over the range of 0.089 2.984 mg L 1 with thedetection limit of 29.8 g L 1. The RLS intensities were found to be proportional tothe concentration of 6 MP over the range of 0.085 2.55 mg L 1 with the detection limitof 8.5 g L 1. The RLS intensities were found to be proportional to the concentrationof 6 TG over the range of 0.017 1.67 mg L 1 with the detection limit of 1.67 g L 1.The RLS intensities were found to be proportional to the concentration ofDimercaprol over the range of 0.089 2.984 mg L 1 with the detection limit of 62g L 1. The RLS intensities were found to be proportional to the concentration ofCysteamine over the range of 0.057 1.136 mg L 1 with the detection limit of 11 g L 1.With the aids of absorption spectrometry, the combination of RLS technique andcolloidal gold may be an effective method to detect thiol functioned substances, suchas amino acids or proteins.