miRNA probe integrated biosensor platform using bimetallic nanostars for amplification-free multiplexed detection of circulating colorectal cancer biomarkers in clinical samples

Abstract

There is a critical need for sensitive and rapid detection technologies utilizing molecular biotargets such as microRNAs (miRNAs), which regulate gene expression and are a promising class of diagnostic biomarkers for disease detection. Here, we present the development and fabrication of a highly reproducible and robust plasmonic bimetallic nanostar biosensing platform to detect miRNA targets using surfaced-enhanced Raman scattering (SERS)-based gene probes called the inverse Molecular Sentinel (iMS). We investigated and optimized the integration of iMS gene probes onto this SERS substrate, achieving ultra-sensitive detection with limits of detection of 6.8 and 16.7 zmol within the sensing region for two miRNA sequences of interest. Finally, we demonstrated the biomedical usefulness of this nanobiosensor platform with the multiplexed detection of upregulated miRNA targets, miR21 and miR221, from colorectal cancer patient plasma. The resulting SERS data are in excellent agreement with PCR data obtained from patient samples and can distinguish between healthy and cancerous patient samples. These results underline the potential of the iMS-integrated substrate nanobiosensing platform for rapid and sensitive diagnostics of cancer biomarkers for point-of-care applications.

Keywords: Biosensor; Early detection; Plasmonic nanoparticle; SERS Substrate; miRNA detection.

Fig. 1.

A) Illustration of the iMS probe assay. B) Example of multiplexed iMS SERS spectra going from OFF to ON. C) Illustration showing the substrate and well system. D) Schematic of the miR21 iMS probe, placeholder, and synthetic target designs. E) Schematic of the miR221 iMS probe, placeholder, and synthetic target designs. Created with BioRender.com.

Fig. 2.

A-C) EDS STEM images of BNS, A) gold only, B) silver only, C) overlay. D) HAADF of BNS. E) SEM of BNS monolayered substrate at two different magnifications. F) Absorbance spectra of BNS. G) Percent surface covering of several SERS substrates with BNS. A,B,C,D N = 10. All N = 40. H) Stability of SERS signal across 4 substrates using Raman reporter p-mercaptobenzoic acid. N = 3.

Fig. 3.

A) Normalized SERS Intensity, Fluorescent background intensity, and SERS: Fluorescence ratios of substrate regions loaded with iMS solutions of varying ionic strength, N = 3. B) Normalized SERS Intensity, Fluorescent background intensity, and SERS: Fluorescence ratios of substrate regions exposed to iMS loading solutions with varying ratios of MCH to iMS probe, N = 3. C) SERS ratios for before: after placeholder and after target: after placeholder for difference MCH concentrations are graphed on the left axis, and SERS after target divided by SERS before placeholder is plotted on the right axis, N = 3. D) SERS intensity of substrate regions before and after the addition of placeholder are plotted on the left y-axis, and the ON to OFF ratios of those regions are plotted on the right y-axis, N = 10. E) The SERS intensity of three substrate regions before and after adding 500 fmol target solution, N = 3. F) the normalized standard curves of miR21 and miR221 iMS probes, N = 3. G) Illustration of steric hindrance and MCH co-loading effects on iMS probe functionality. Created with BioRender.com.

Fig. 4.

A) SERS spectra of the miR221 iMS probe, the miR21 iMS probe, a multiplexed spectra of a substrate region containing both probes after adding placeholder. B) Top: PCR results showing the relative expression levels or miR21, N = 3. Bottom: The signal increase factor of miR21 iMS probe from the multiplexed spectra taken of patient sample analysis, N = 3. C) Top: PCR results showing the relative expression levels of miR221, N = 3. Bottom: The signal increase factor of miR221 iMS probe from the multiplexed spectra taken of patient sample analysis, N = 3.

Fig. 5.

A) Scatter plot of miR21 and miR221 probes signal increase factors from each multiplexed spectra recorded from patient samples. B) Combined SERS data for miR21 probes of all patients, grouped by patient status, N = 9,6, Welsh’s t-test, P value = 0.0004. D) Combined SERS data for miR221 probes of all patients, grouped by patient status, N = 9,6, Welsh’s t-test, P value = 0.0005. D) Principal component analysis of multiplexed spectra recorded from patient samples, N = 9,6.