The SERS Immunoassay for Detecting Protein A by Using Molecular Probes-Modified Gold Nanoparticles

• Main Authors: Yan-Fu Chen, 陳彥夫
• Other Authors: Hsien-Chang Chang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/63088529240577303978

碩士 === 國立成功大學 === 醫學工程研究所碩博士班 === 95 === According to the statistics of National Nosocomial Infection Surveillance System in Centers for Disease Control and Prevention and Surveillance and Control of Pathogens of Epidemiologic Importance, Staphylococcus aureus was the most common infectant of nosocomial bloodstream microbial infection detected. It accounts for 13 percentages at the high rate. Sepsis induced by bacteremia is one of the main causes of death of critically ill patients in intensive care unit. In clinical tests, the most traditional medical examination technique is enzyme-linked immunosorbent assay (ELISA). However, this method in the sample pretreament and measurement steps is rather cumbersome, and would entail a lot of manpower and time.For this reason, this study is modified from traditional sandwich ELISA model as prototype. We mixed gold colloidal solution (20 nm, 5.4×1011 particles/mL) and Immunoglobulin G (IgG) (80 μg/ml) in phosphate buffer solution (pH=6.5) at pH below isoelectric point (pI=7.4) of antibodies isovolumically. After centrifugation, 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) can be employed as Raman-active molecular probes to modify on IgG-coated gold nanoparticles. This molecule has strong Raman signals at 1340 cm-1, and the gold nanoparticle can amplify the Raman scattering of molecular probe. The enzyme-linked secondary antibodies in traditional immunoassay are replaced with the immunogold molecular probes we fabricated. Protein A is the specific antigen of Staphylococcus aureus, will be used as a model system. The antigen concentrations were adjusted to be 10-6~10-13 g/ml, and detected by surface-enhanced Raman scattering (SERS). As a result, it showed that the detection of limit could reach to 10-11 g/ml. Comparing with traditional immunoassay only to 10-8~10-9 g/ml, the sensitivity could promote 2~3 order of magnitude higher. In addition to improve the sensitivity at low concentration, this method may also provide us Raman spectra and information of biological samples. We may (1) choose better molecular probes, and (2) change type of nanoparticles with higher SERS efficiency (3) increase roughness of substrate, and (4) switch to shorter LASER excitation wavelength (e.g. 532 nm Nd : YAG LASER), to lower the detection limit.