Targeted FT-NIR and SERS Detection of Breast Cancer HER-II Biomarkers in Blood Serum Using PCB-Based Plasmonic Active Nanostructured Thin Film Label-Free Immunosensor Immobilized with Directional GNU-Conjugated Antibody

Abstract

This work describes our recent PCB-based plasmonic nanostructured platform patent (US 11,828,747B2) for the detection of biomarkers in breast cancer serum (BCS). A 50 nm thin gold film (TGF) was immersion-coated on PCB (i.e., PCB-TGF) and immobilized covalently with gold nanourchin (GNU) via a 1,6-Hexanedithiol (HDT) linkage to produce a plasmonic activated nanostructured thin film (PANTF) platform. A label-free SERS immunosensor was fabricated by conjugating the platform with monoclonal HER-II antibodies (mAb) in a directional orientation via adipic acid dihydrazide (ADH) to provide higher accessibility to overexpressed HER-II biomarkers (i.e., 2+ (early), 3+ (locally advanced), and positive (meta) in BCS. An enhancement factor (EF) of 0.3 × 105 was achieved for PANTF using Rhodamine (R6G), and the morphology was studied by scanning electron microscopy (SEM) and atomic force microscope (AFM). UV-vis spectroscopy showed the peaks at 222, 231, and 213 nm corresponding to ADH, mAb, and HER-II biomarkers, respectively. The functionalization and conjugation were investigated by Fourier Transform Near Infrared (FT-NIR) where the most dominant overlapped spectra of 2+, 3+, and Pos correspond to OH-combination of carbohydrate, RNH2 1st overtone, and aromatic CH 1st overtone of mAb, respectively. SERS data were filtered using the filtfilt filter from scipy.signals, baseline corrected using the Improved Asymmetric Least Squares (isals) function from the pybaselines.Whittaker library. The results showed the common peaks at 867, 1312, 2894, 3026, and 3258 cm−1 corresponding to glycine, alanine ν (C-N-C) assigned to the symmetric C-N-C stretch mode; tryptophan and α helix; C-H antisymmetric and symmetric stretching; NH3+ in amino acids; and N-H stretch primary amide, respectively, with the intensity of Pos > 3+ > 2+. This trend is justifiable considering the stage of each sample. Principal Component Analysis (PCA) and Linear Discrimination Analysis (LDA) were employed for the statistical analysis of data.