Surface-Enhanced Raman Scattering Based Detection Methods For Proteins

As an ultrasensitive analytical technique, suface-enhanced Raman scattering has been attracted increasing attention in biological science. SERS-based methods have salient advantages of high sensitivity, photostability and spectral multiplexing over conventional methods, based on which we successfully developed several SERS-based detection methods for protein identification, detection of protein-protein and protein-small molecule interactions. The major contributions of this work are as follows:(1) Silver colloid staining and protein-mediated SERS-active substratesWe developed a new staining method by using silver colloid, which is an excellent SERS-active substrate. The proposed method is facile and sensitive, and the most important thing is after staining, we can determine different proteins or protein-ligand interactions by SERS, wich is unaccessible for the conventional silver staining.In most SERS-based studies, SERS-active substrates are first prepared and then analytes are assembled on these substrates for further SERS detection. In our studies we use a reverse way to produce a SERS-active substrate which is mediated by target proteins and used for detection of these proteins. Silver nanoparticles can adsorb on immobilized target proteins because of strong interactions (i.e., hydrophobic, electrostatic, and covalent interactions) between metal nanoparticles and the proteins, which result in aggregation of silver nanoparticles and a consequent SERS effect. In this way, we combine protein staining with SERS by silver colloid and take advantages of both to detect proteins. (2) Label-free multi-protein detection on a NC membraneBased on Western blot and SERS, we developed a new analytical procedure for label-free protein detection designated"Western-SERS", consisting of protein electrophoresis, protein blotting, colloidal silver staining, and SERS detection. The most important feature of this method is that it enables multi-protein detection on one nitrocellulose (NC) membrane, which is inaccessible for other SERS-based detection methods.This"Western-SERS"method offers the dual advantages of simplicity and high sensitivity. Compared with Western blot and mass spectrometry, we can detect label-free proteins directly on an NC membrane without time and reagent-consuming procedures. Moreover, the detection limit of Western-SERS is almost consistent with the detection limit of colloidal silver staining (2 ng/band), and SERS signals do not self-quench, unlike fluorescence. Thus, the new method has great potential for identifying proteomic components or proteins of differential expression in some proteomes.(3) Label-free highly sensitive protein detection in aqueous solutionsRoughened metal surfaces and dried colloids are difficult to obtain reproducible SERS spectra especially for those proteins with no chromophores because of irreproducible SERS substrates and different orientation of analytes on a metal surface. Otherwise, no previous SERS-based study for proteins allows routine detection of label-free proteins with high sensitivity in an aqueous solution because halide ions, which are commonly used aggregation reagents, can form a strongly bonded surface layer which repels adsorption of proteins.According to weak binding of SO42- on silver surfaces that can induce much stronger SERS, we use acidified sulphate as an aggregation agent and obtained strong SERS of targert proteins with high sensitivity. Moreover, when silver nanoparticles are aggregated by the proposed protein involved aggregation agent, target proteins with net positive charges are sandwiched among silver nanoparticles in aqueous solutions, and then vibration information of whole proteins would be probably displayed in their SERS spectra with remarkable enhanced reproducibility. It can be used as a practical method to directly detect label-free proteins with the advantages of rapidness, reproducibility and appropriately high sensitivity.(4) Fluorescence-linked immunoabsorbent assayBy combining enzyme-linked immunoabsorbent assay (ELISA) with SERS, we developed a SERRS-based immunoassay on bottom of microtiter plates. In this method, SERRS spectra of FITC are measured after several continuous steps of antigen coating, blocking, antibody adding, and colloidal silver staining. Protein-mediated formation of silver aggregates results in electromagnetic enhancement of resonance Raman scattering.The proposed method has several advantages over ELISA and other immunoassays. 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 highly sensitive. Therefore, it may have great potential as a high-sensitivity and highthroughput immunoassay.(5) Detection of protein-ligand interactionsProtein chips with versatile applications play an important role in high-throughput proteomic studies. The purpose of protein arrays is to quickly probe the activity of a given protein against many targets simultaneously, and the currently preferred detection method for protein chips is fluorescence. In our study, we used SERRS instead of fluorescence as detection method for protein chips.Silver nanoparticles were assembled to the protein-ligand complexes via covalent and noncovalent binding, resulting in the formation of SERS-active silver aggregates. We observed both SERRS and Surface-enhanced fluorescence of the target labels, and they are more photostable and sensitive than their corresponding fluorescence. Therefore, the proposed method for detection of protein-ligand interactions has great potential in high-sensitivity and high-throughput chip-based protein function determination.(6) Metal sandwich substrate for versatile protein detectionAs a more advanced system, we develop a novel type of SERRS-active sandwich substrates bridged by the target proteins for the detection of these proteins. The most significant advantage of the present method over other SERS-based methods for protein detections is that a self-assembling gold nanoparticle monolayer is used for both capturing proteins and forming the SERS-active substrate with the second metal layer.Highly reproducible SERRS and SERS spectra can be obtained by the present gold-protein-gold (Au/Au) and gold-protein-silver (Au/Ag) sandwiches, and we find that the latter yields about seven times stronger SERRS than the former. Moreover, besides labeled immunoassays, the present method can identify label-free proteins and protein-drug interactions without Raman dyes. Because of contributions from the two metal layers to the SERS, this sandwich strategy holds great potential in highly sensitive and reproducible protein detections.