Surface Enhanced Raman Spectroscopy for Single Molecule Protein Detection

Lamyaa M Almehmadi,Scott A Tenenbaum,Natalya A Tokranova,Igor K Lednev,Stephanie M Curley

doi:10.1038/s41598-019-48650-y

Lamyaa M Almehmadi, Scott A Tenenbaum + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-48650-y

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Abstract

A two-step process of protein detection at a single molecule level using SERS was developed as a proof-of-concept platform for medical diagnostics. First, a protein molecule was bound to a linker in the bulk solution and then this adduct was chemically reacted with the SERS substrate. Traut’s Reagent (TR) was used to thiolate Bovine serum albumin (BSA) in solution followed by chemical cross linking to a gold surface through a sulfhydryl group. A Glycine-TR adduct was used as a control sample to identify the protein contribution to the SER spectra. Gold SERS substrates were manufactured by electrochemical deposition. Solutions at an ultralow concentration were used for attaching the TR adducts to the SERS substrate. Samples showed the typical behavior of a single molecule SERS including spectral fluctuations, blinking and Raman signal being generated from only selected points on the substrate. The fluctuating SER spectra were examined using Principle Component Analysis. This unsupervised statistics allowed for the selecting of spectral contribution from protein moiety indicating that the method was capable of detecting a single protein molecule. Thus we have demonstrated, that the developed two-step methodology has the potential as a new platform for medical diagnostics.

Highlights

Tenenbaum and co-workers[10] have recently developed a novel approach for biomedical diagnostics based on the binding of an engineered RNA molecule to a specific targeted microRNA molecule that ‘switches’ the structural conformation of the engineered RNA strand upon binding[10]
In this study we examine the potential of protein detection at a single molecule level when small linker molecules react with a protein in bulk solution followed by attachment to the SERS substrate
SER spectra obtained from substrates with high and low loadings of Bovine serum albumin (BSA)-Traut’s reagent (TR) could be differentiated by visual inspection

Summary

Introduction

Tenenbaum and co-workers[10] have recently developed a novel approach for biomedical diagnostics based on the binding of an engineered RNA molecule to a specific targeted microRNA molecule that ‘switches’ the structural conformation of the engineered RNA strand upon binding[10]. We speculated that surface enhanced Raman spectroscopy (SERS) might be an ideal technique for detecting the sxRNA bound protein with a high degree of sensitivity and specificity With this long-term goal in mind, we first developed the two-step process described here for the simple detection of a protein at the single molecule level using SERS. In this study we examine the potential of protein detection at a single molecule level when small linker molecules react with a protein in bulk solution followed by attachment to the SERS substrate. This two-step process is followed by acquiring Raman spectra from multiple points on the substrate via automatic mapping. Two typical approaches are used for generating a SER spectral dataset, (i) acquiring spectra from multiple spots on a SERS substrate using automatic mapping for example and (ii) recording multiple consecutive spectra from individual substrate spots[18,19]

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