Reporter Selection for Nanotags in Multiplexed Surface Enhanced Raman Spectroscopy Assays

Abstract

We report a quantitative evaluation of the choice of reporters for multiplexed surface-enhanced Raman spectroscopy (SERS). An initial library consisted of 15 reporter molecules that included commonly used Raman dyes, thiolated reporters, and other small molecules. We used a correlation matrix to downselect Raman reporters from the library to choose five candidates: 1,2-bis(4-pyridyl)ethylene, 4-mercaptobenzoic acid, 3,5-dichlorobenzenthiol, pentachlorothiophenol, and 5,5′-dithiobis(2-nitrobenzoic acid). We evaluated the ability to distinguish the five SERS reporters in a dipstick immunoassay for the biomarker human IgG. Raman nanotags, or gold nanostars conjugated to the five reporters and anti-human IgG polyclonal antibodies were constructed. A linear discriminant analysis approach was used to evaluate the separation of the nanotag spectra in mixtures of fixed ratios.

Designing Paper-Based Immunoassays for Biomedical Applications

“…A wide range of molecules can be bound to NPs to form the probes used in paper-based assays, to enable numerous possible targets and applications. Furthermore, multiple probes functionalized with different molecules can be used to simultaneously test for a number of targets [20,21,22]. Device design geometry can be augmented to suit use-specific requirements [19,23].…”

Recent Advancement in the Surface-Enhanced Raman Spectroscopy-Based Biosensors for Infectious Disease Diagnosis

“…It will help eliminating non-specific biological interactions hence reducing SERS spectral interference and ensuring colloidal stability for stable SERS signal acquisition. An appreciable amount of effort is currently being invested by the research community to investigate these fundamental aspects of the nanoparticle-based SERS diagnostic assays [7,17,70,73,125,126]. Combining the current exponential improvement in both fundamental chemistry and device engineering, the widespread use of SERS-based diagnostics for infectious diseases at the POC and clinical setting is only a very few years away from us.…”

“…In comparison to the enzyme-linked immunosorbent assay (ELISA)-based method, the SERS-based LFA provides approximately three orders of magnitude higher detection sensitivity. Hamad-Schifferli's group also developed LFAs using SERS nanotags for dengue and Zika detection [70,125]. The importance of their study is that they systematically examined the importance of gold nanostar morphological features and Raman reporter selection in order optimize the SERS enhancement and multiplexed detection in LFAs, respectively.…”

“…In addition, they also observed that the SERS intensity distribution within the test zone is greater in the area where the fluid first encounters the immobilized antibody, and such factors can be used to strategically design LFA to decrease the overall assay cost. In 2018, the same group focused on selecting Raman reporters for quantitative multiplexed SERS detection of protein (IgG) biomarkers in a dipstick immunoassay [125]. In this study, gold nanostars conjugated to polyclonal anti-human IgG antibodies were tagged with 15 different Raman reporters.…”

“…quantitative multiplexed SERS detection of protein (IgG) biomarkers in a dipstick immunoassay. [125] In this study, gold nanostars conjugated to polyclonal anti-human IgG antibodies were tagged with 15 different Raman reporters. They used machine learning to evaluate the spectral overlap and quantitative contribution of Raman reporters.…”