Establishment And Application Of The Nucleic Acid Code Assay For Ultrasensitive Detection Of Proteins

Objective:Combining nanotechnology and immuno-detection assay,the nucleic acid code assay was established basic on preparation of immunomagnetic microspheres and double labeled gold nanoparticle probes.The nucleic acid code assay could had the promising prospect for ultrasensitive detection of proteins in the clinical diagnosis.Methods:Colloidal gold nanoparticles were prepared through chlorauric acid reduction.To establish a preparative method of double labeled gold nanoparticle probes by labelling with antibody and thiol-terminated oligonucleotides at the same time via electrostatic force and covalent binding forming.By using Transmission electron microscopy(TEM),UV-vis spectra to observe the morphology and spectroscic properties of probes.The antibody activity was detected by dot-immunogold filtration assay and immunogold-silver staining.The reduction of thiol-modified oligonucleotides by dithiothreitol(DTT) was carried out by time evolution of UV-vis spectra.The fluorescent molecules labeled to the oligonucleotides was used to calculate the coverage of the oligonucleotides onto the surface of the probes.The oligonucleotides onto the gold nanoparticles probes were also characterized by PCR-PAGE techniques.The immunomagnetic microspheres were designed as Fe₃O₄/alginate microspheres,prepared by heptane as organic dispersed medium and AOT as surfactant,then cross-linked by calcium ions and antibodies(human monoclonal antibodies).By using Transmission electron microscopy(TEM),iverson Fluorescence microscope,Flow cytomentryto to observe the morphology andntibody activity of immuomagnetic microspheres.After building ways of gold nanoparticle probes and immuomagnetic microspheres,To establish the nucleic acid code assay together with immunomagnetic microspheres modifying PSA monoclonal antibofy and 30nm gold nanoparticle probes labeling rabbit anti-human PSA polyclonal antibody and thiol-terminated oligonucleotides.According double-antibody sandwich immuno-detection,the magnetic microspheres probes reacted with the clinical samples first,then added gold nanoparticle probes by mixing,placed on magnet and remove supernant. Bio-ssDNA-SH was released in DTT.Through tracing the the signal of code DNA(Bio-ssDNA-SH) binding to streptoacidin and alkaline phosphatase on nylon memvrane,PSA(Prostate specific antigen,PSA)was detected and the nucleic acid code assay was established.Evaluation of this method for detection range,sensitivity,specificity,reproducibility, accuracy,and so on.15 cases of Prostate cancer patients,45 benign prostate hyperplasia,15 cases of patients with various other tumors and 30 cases of normal serum were chosen for analysis in the use of the nucleic acid code assay,and the result was compared with the enzyme-linked immunosorbent assay(ELISA) and radioactive immunization method(RIA),in the assessment of clinical examination rate and the clinical relevance to the traditional method.Results:By using Transmission electron microscopy(TEM),the average size of gold nanoparticle probes was(10±3)nm with uniform sizes.Antibody(IgG) and thiol-terminated oligonucleotides were showed pretty well activity when gold nanoparticles were functionalized with antibody(IgG) and thiol-terminated oligonucleotides.The surface coverage of gold nanoparticles probes is(8±3) IgG and(92±20) oligonucleotides per gold nanoparticles. The result also showed that the immunomagnetic microspheres was of generally spherical appearance,and most had well distributions and magnetic response,it's about 118μg antibodies per milligram of magnetic microspheres with Coomassie Brilliant Blue G-250 test.And antibodies on the surface of magnetic microspheres were active.With the nucleic acid code assay for ultrasensitive detection of proteins,it's best to test PSA when 5 mg / ml immunomagnetic microspheres and 10 nM of gold nanoparticle probes with volume ratio of 1:2.Through tracing the the Marker signal of nucleic acid code,the minimum value of PSA testing can be 1 fg / ml and had a good repeatability and stability.The sensitivity of clinical examination for positive serum of PSA was 92%and specificity was 68%.In comparison with ELISA and RIA,the result showed that r correlation coefficient of 0.950 and 0.967,respectively,the correlation between good.And the K coefficient of 0.918 to 0.960,anastomosis of the strong,similar with the standard method in the current clinical application.Coneluetion:The study mentioned above proved that the nucleic acid code has significant advantages in amplifying the signals.The nucleic acid code detection result of PSA showed high sensitivity,excellent specificity,no need for special device.Therefore,This method can be widely applied with ultrasensitive detection of proteins and had a favorable prospect.