Nitrocellulose-based SERS immunosensor for detection of biological molecules

We have developed a simple and potentially a low-cost method for the sensitive detection of target proteins via surface-enhanced Raman scattering (SERS). The immunosensor constructed by the conjugation of monoclonal antibodies to 20 nm diameter gold nanoparticles via the bifunctional Raman reporter molecule, 5, 5'dithiobis (succinimidyl-2-nitrobenzoate) (DSNB) is the basis of a membrane-bound detection system. Traditionally, a common laboratory technique called a dot blot, which is a colorimetric method where detection of proteins is accomplished through the application of assorted dyes followed by their measurement via a densitometer. Dot blotting is a convenient and time saving method that involves the spotting of a protein onto an immobilizing matrix, such as nitrocellulose (NC) or polyvinylidene fluoride (PVDF) membrane. We found that for detection via SERS spectroscopy NC is the matrix of choice because it offers low background, minimal preparation prior to protein application, and optimal position of Raman bands. Furthermore, SERS detection of protein on NC requires only minimal sample preparation and demonstrates increased sensitivity when compared to other dot blot detection methods. Depending on the dye used for visualization, dot blots analyzed by commonly used optical methods have limits of detection in the nanogram range, some as low as 20 pg/ml. Here we demonstrate the use of the dot blot method for detecting target proteins (e.g., protein A and prostate specific protein (PSA)) by SERS spectroscopy down to a concentration of 100 fg/ml.