Simplified protocol for detection of protein-ligand interactions via surface-enhanced resonance Raman scattering and surface-enhanced fluorescence.

A simple and effective protocol for detections of protein-protein and protein-small molecule interactions has been developed. After interactions between proteins and their corresponding ligands, we employed colloidal silver staining for producing active substrates for surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF). Tetramethylrhodamine isothiocyanate (TRITC) and Atto610 were used for both Raman and fluorescent probes. We detected interactions between human IgG and TRITC-anti-human IgG, and those between avidin and Atto610-biotin by surface-enhanced resonance Raman scattering (SERRS) and SEF. The detection limits of the proposed SERRS-based method are comparable to those of the proposed SEF-based one, 0.9 pg/mL for anti-human IgG and 0.1 pg/mL for biotin. This protocol exploits several advantages of simplicity over other SERS and SEF-based related methods because of the protein staining-based strategy for silver nanoparticle assembling, high sensitivity from SERRS and SEF, and high stability in photostability comparing to fluorescence-based protein detections. Therefore, the proposed method for detection of protein-ligand interactions has great potential in high-sensitivity and high-throughput chip-based protein function determination.

Fluorescein isothiocyanate linked immunoabsorbent assay based on surface-enhanced resonance Raman scattering.

By using fluorescein isothiocyanate (FITC) as a Raman probe, we have developed a simple and sensitive method for an immunoassay based on surface-enhanced resonance Raman scattering (SERRS). For the first time, a SERRS-based immunoassay on the bottom of a microtiter plate is reported. We have applied the main pretreatment method of enzyme-linked immunoabsorbent assay (ELISA) to the present study. In this method, SERRS spectra of FITC are measured after several continuous steps of antigen coating, blocking, antibody adding, and colloidal silver staining. Human immunoglobulin G (IgG) and FITC-antihuman IgG are used for the immunoreaction. The proposed method has several advantages for immunoassay. First, we can determine the concentration of antigens via the intensity of a SERRS signal of FITC molecules that are attached to antibodies without an enzyme reaction, and thus the process is simple and reagent saving. Second, one can obtain SERRS spectra of FITC directly from silver aggregates on the bottom of a microtiter plate without displacement. Third, by using SERRS of FITC, the present method is sensitive enough to detect antigens at the concentration of 0.2 ng/mL, which is comparable to ELISA. Results are presented to demonstrate that the proposed SERRS-based immunoassay may have great potential as a high-sensitivity and high-throughout immunoassay.

Analytical technique for label-free multi-protein detection based on Western blot and surface-enhanced Raman scattering.

We have developed a new analytical procedure for label-free protein detection designated "Western SERS", consisting of protein electrophoresis, Western blot, colloidal silver staining, and surface-enhanced Raman scattering (SERS) detection. A novel method of silver staining for Western blot that uses a silver colloid, an excellent SERS-active substrate, is first proposed in the present study. During the process of silver staining, interactions between proteins and silver nanoparticles result in the emergence of SERS of proteins. In the present study, we use myoglobin (Mb) and bovine serum albumin (BSA) as model proteins. From different protein bands on a nitrocellulose (NC) membrane, we have observed surface-enhanced resonance Raman scattering (SERRS) spectra of Mb and SERS spectra of BSA. The proposed technique offers dual advantages of simplicity and high sensitivity. On one hand, after the colloidal silver staining, we can detect label-free multi-proteins directly on a NC membrane without digestion, extraction, and other pretreatments. On the other hand, the detection limit of the Western SERS is almost consistent with the detection limit of colloidal silver staining, and the SERRS detection limit of Mb is found to be 4 ng/band. This analytical method, which combines the technique of protein separation with SERS, may be a powerful protocol for label-free protein detection in proteomic research.