Understandably, over 13 billion in IVD assays are performed annually. To drive healthcare forward and facilitate more predictive, personalized medical care, the testing of biological fluid using point-of-care (POC) technologies will become increasingly paramount. Many optical approaches have been utilized for POC applications including Raman spectroscopic approaches. Advancements in nanotechnology, biochemical sensing methods and the miniaturization of optics have greatly improved spectroscopic platforms for biomarker detection. The use of reporter molecules with Raman spectroscopy becomes popular in other spectroscopic platforms beyond fluorescence. The spectra of the same dye reporter observed using Raman produce spectral peaks with a full width half maximum 10-100 times narrower than the peaks typically obtained using fluorescence. These thin spectral bands provide the ability for surface enhanced Raman spectroscopy (SERS) to be used for multiplexed detection of several biomarkers from a single measurement.
Raman spectroscopy relies on the loss or gain in energy of an inelastically scattered photon due to a molecular vibrational event. It provides a “chemical fingerprint” and can enhance the Raman signal by several orders of magnitude by amplifying the electron cloud density around metallic nanostructures. There are two distinct mechanisms, electromagnetic enhancement and chemical enhancement, that make the SERS signal enhanced. For both mechanisms to occur simultaneously, an analyte must be adsorbed onto or reside very close to a dielectric surface. Typical SERS signal enhancement factors (EF) are observed between 106 and 108 with some reporting EFs as high as 1014, thereby indicating that single-molecule detection is possible. The dramatic signal enhancement makes it useful to detect extremely low analyte concentrations. Many scientists have documented the success of SERS for detecting nano-grams per milliliter analytes concentrations. Moreover, successful analyte detection at pico-gram per milliliter concentrations and even claim single-molecule detection have also been observed.
SERS nanoprobes have recently been used to improve the detection capabilities of immunoassays and have the potential to rival the popular ELISA techniques. Combining SERS and ELISA, also known as “SLISA”, has proven to be an effective approach for improving the limits of detection (LODs) due to the intrinsic enhancement capabilities of SERS. Besides, the ability to speed up the assay reaction times can be obtained due to the 3D architecture of functionalized colloidal nanoparticles, and the ability to capitalize on the narrow spectra bands can be achieved with Raman. Researches have found that SLISA has similar accuracy as ELISA but improves upon the indirect enzyme-based method by being reusable, faster, more direct, and easier to use. SLISA was also more sensitive (five times) than ELISA while providing qualitative information on the immunosensor’s chemical characterization and antigen-antibody binding. This allows direct detection with less uncertainty, which is a stringent limitation of all label-based biosensor technologies, including ELISA.
The scattered light intensity can be measured by using the corresponding optical techniques, such as Raman spectroscopy and Rayleigh scattering spectroscopy, etc. Studies have shown that gold nanoparticles (AuNPs) have extremely strong light scattering ability at the plasmon-resonance wavelength. This scattering ability is several orders of magnitude higher than that of nonmetallic materials of the same size, and over a million times greater than fluorescent molecules. This ultra-strong light scattering property makes AuNPs one of the most widely used optical labels in light scattering-based sensing technologies. To date, a majority of biosensors have been developed for chemical and biological analysis using the phenomenon of elastic light scattering of AuNPs, such as resonance light scattering correlation spectroscopy (RLSCS)-based sensors and dynamic light scattering (DLS)-based assays. Moreover, for inelastic scattering, Raman scattering-based biosensing methods have also been extensively proposed for target analyte detection by directly measuring the scattered light from specific nanoassemblies or the enhanced scattered light from the Raman active dye molecules located on the surface of nanomaterials, as for metal nanoparticles.
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