Controllable SERS Substrates For Proteins Detection

Proteins are essential for living organisms, because they are main components of the physiological metabolic pathways of cells. As an ultrasensitive technique, surface-enhanced Raman scattering (SERS) has been proven to have great potential in high throughput biomolecules detection, especially in proteins detection. Proteins are biomacromolecules whose adsorption behavior on SERS-active substrates differs significantly from that of small organic molecules. Therefore, it is very important to prepare homogeneous, stable and biocompatible SERS-active substrates for highly sensitive and reliable protein detection. The major contributions of this work are as follows:(1) Detection of Proteins on Silica-Silver Core-Shell Substrates by Surface-Enhanced Raman Spectroscopy.We have employed the proposed Silica-Silver Core-Shell (SSCS) SERS-active substrates combine self-assembly and a seeding method for silver plating on silica-decorated glass slide. This proposed method enables to precise control and reproducibility of the structure of silver cluster coating on the silica bead, which induces a large homogeneity in their local structure and large enhancement of Raman scattering. We can produce closely packed silver nanoparticles on the surface of the silica beads when plating for 90 seconds, which adapt to protein adsorption and produce strong SERS enhancement with high reproducibility. This method allows direct and rapid detection of label-free proteins (cytochrome c and lysozyme) on the SSCS substrates, as well as labeled protein (FITC-anti human IgG) and atto610-labeled biotin/avidin recognition. SERS spectra of these proteins and Raman labels on the SSCS substrates show both high sensitivity and reproducibility, which are due to electromagnetic SERS enhancement with additional localization field within closely packed Ag nanoparticles decorated on the SiO2 nanoparticles. We have found that the SERS intensities of atto610-biotin/avidin adsorbed on the SSCS substrates are about 20 times stronger than those from Ag plating on Au-decorated substrate. Moreover, the broad surface plasmon resonance (SPR) of the proposed substrates will extend SERS applications to more biological molecules with different laser excitations.(2) Tunable Plasmon Properties of Fe2O3@,Ag Substrate for protein-small molecule recognition.We introduce a new approach that combines the self-assembly and seeding methods for silver plating of the proposed Fe2O3@Au-seed substrate. The proposed method could effectively control surface morphology, which is one of the pivotal factors in the fabrication of excellent SERS substrates. By adjusting the silver plating time between 0 and 360 s, the optical resonance of the substrates clearly varied from visible to near-infrared. To investigate the potential of these substrates for use in surface-enhanced Raman scattering (SERS) applications, SERS spectra of thiophenol (TP),4-aminothiophenol (PATP) and rhodamine 6G (R6G) were compared with spectra for an internal standard of silicon at three excitation wavelengths (532,633 and 785 nm). Analysis of the SERS spectra clearly demonstrated that the SERS effect depend strongly on the nature of the substrate surface and the nature and electronic resonance of the probe molecules. The calculated enhancement factor (EF) of TP adsorbed onto a Fe2O3@Ag substrate was～10'following three laser excitations. More importantly. these SERS substrates can successfully be utilized for detection of small molecules at very low concentrations. (3) Magnetic Assistance Highly Sensitive Protein Assay Based on Surface-Enhanced Resonance Raman Scattering.The simple and effective SERS-based protocol for detection of protein-small molecule interaction has been developed. We employed silver coated magnetic particles (AgMNPs) which can provide high SERS-active as protein carrier to capture small molecule. Combining magnet separation and SERS method for protein detection, highly reproducible SERS spectra of protein-small molecule complex with sensitivity can be obtained and a single assay can be finished in only one minute (contains magnet separation and SERS assay). This time saving method just employed the external magnetic field induce the AgMNPs aggregate which increase the amount of avidin-biotin complex in a unit area. Furthermore, by decreasing the amount of magnetic nanoparticles and increasing the time of protein capture small molecule, their SERS intensity of atto610 increased. Because of the contribution from the AgMNPs aggregation to the SERS. it has great potential in practical high-throughput protein-small molecule complex and antigen-antibody immunocomplex.(4) Biomagnetic Glass Beads for Protein Separation and Detection Based on Surface-Enhanced Raman Scattering.A novel surface-enhanced Raman scattering (SERS)-based detection method for proteins attached to magnetic glass beads have been developed in this study. Silica coated magnetic nanoparticles (Fe3O4U) are prepared and aldehyde-functionalized for capturing proteins in solutions by Schiff reaction, which is more biocompatible than previous magnetic nanoparticle and SERS-based detection methods. Target proteins are captured and separated by the magnetic nanoparticles. followed by colloid silver staining for SERS detection. Agglomerating the magnetic nanobeads by an external magnetic force resulted in the increase of the amount of conjugated proteins per unit area, which enhanced the sensitivity of the proposed method combined with SERS technique. Moreover, this biomagnetic glass beads and detection protocol are found to be useful in protein-ligand recognitions even in mixture system (two protein ligands). The proposed magnetic nanoparticles are biocompatible. manipulable by an external magnetic force and highly sensitive due to silver nanoparticles staining, which make the protocol quite promising in high throughput protein assays.