SERS-based immunoassay using gold-patterned array chips for rapid and sensitive detection of dual cardiac biomarkers

Ziyi Cheng【程子译】,†^a Rui Wang【王锐】,†^a Yanlong Xing【邢艳珑】,^a Linlu Zhao【赵琳璐】,^a Jaebum Choo *^b and Fabiao Yu【于法标】 *^a

^a. Institute of Functional Materials and Molecular Imaging, Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue, College of Clinical Medicine, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China.

^b. Department of Chemistry, Chung-Ang University, Seoul 06974, South Korea

† These authors contributed equally.

From the themed collection: Bioanalytical tools for enabling precision medicine

The article was first published on 20 Sep 2019
Analyst, 2019, Advance Article
https://doi.org/10.1039/C9AN01260E

Cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) are important diagnostic biomarkers for acute myocardial infarction (AMI). Many efforts have been undertaken to develop highly sensitive detection methods for the quantitative analysis of these dual targets. However, current immunoassay methods are inadequate for accurate measurement of cTnI and CK-MB, due to their limited detection sensitivity. Thus, there is still an urgent demand for a new technique that will enable ultrahigh sensitive detection of these biomarkers. In this study, we developed a surface-enhanced Raman scattering (SERS)-based sandwich immunoassay platform for the ultrasensitive detection of cTnI and CK-MB. In this study, a monoclonal-antibody-immobilized gold-patterned chip was used as a SERS active template. Target samples and polyclonal-antibody-conjugated Au@Ag core-shell nanoparticles were then added. Using this SERS platform, the concentration of biomarkers could be quantified by monitoring the characteristic Raman peak intensity of Raman reporter molecules. Under optimized conditions, the limits of detection (LODs) were estimated to be 8.9 pg/mL and 9.7 pg/mL for cTnI and CK-MB, respectively. Thus, the proposed SERS-based immunoassay has great potential to be an effective diagnostic tool for the rapid and accurate detection of cTnI and CK-MB.

Scheme 1 Schematic illustration of SERS-based sandwich immunoassays for quantitative analysis of cTnI and CK-MB. (A) Gold-patterned chip and SERS probes for the detection of dual biomarkers. (B) Monoclonal antibodies conjugated (cTnI and CK-MB) onto the gold-patterned chip for the capture of target antigens. SERS probe addition for the formation of sandwich immunocomplexes. Raman detection of immunocomplexes using 632.8 nm laser.

Fig. 1 Fabrication and characterization of Au@Ag core-shell nanoparticles. (A) Fabrication process of MGITC-labeled Au@Ag core-shell nanoparticles. (B) Photographic images for different thickness of Ag shell. (C) Different volumes of AA and AgNO₃ silver-staining solutions for the control of silver thickness. (D) Corresponding UV-vis spectra. (E) Variation of SERS signal intensity at 1615 cm^-1 for different silver staining solutions.

Fig. 2 Comparison of Raman spectra for different types of SERS nanoprobes.

Fig.3  (A) TEM image of Au@Ag core-shell nanoparticles. (B) Two-dimensional model of two Au@Ag core-shell nanoparticles in FDTD simulation. The red arrow indicates the polarization direction of the incident light. (C) Maps for the enhancement of local electric fields under the illumination of a linearly polarized wave. Computational simulations were performed for Au@Ag nanoparticles with four different thicknesses of sliver shell (i: 2 nm, ii: 3 nm, iii: 4 nm and iv: 5nm). The scale bar shows a colour decoding bar for different Raman intensities.

Fig. 4 Preparation and characterization of antibody-conjugated Au@Ag core-shell SERS nanoprobes. (A) Antibody conjugation on the surface of nanoparticles. (B) TEM image of SERS nanoprobes. (C) DLS distributions, (D) UV-vis spectra and (E) SERS spectra of nanoparticles before (black) and after (red) bioconjugation.

Fig. 5 (A) Fabrication process of gold-patterned chip: (1) silicon wafer washing using piranha solution; (2) Functionalization of amino groups with APTES; (3) gold nanoparticles coating on the functionalized silicon wafer; (4) carboxyl group modification on the gold surface. Self-assembly of 11-MUA on the surface of the nanoparticles-embedded gold substrate. (B) Raman spectra of MGITC measured for 9 spots randomly chosen from 2D substrate.

Fig. 6 SERS spectra for increasing concentrations of (A) cTnI and (C) CK-MB. Corresponding calibration curves of the SERS signal intensity at 1615 cm^-1 as a function of the logarithm of the concentrations of (B) cTnI and (D) CK-MB.