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

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 <i>filtfilt</i> filter from <i>scipy.signals</i>, baseline corrected using the Improved Asymmetric Least Squares (<i>isals</i>) function from the <i>pybaselines.Whittaker</i> library. The results showed the common peaks at 867, 1312, 2894, 3026, and 3258 cm^-1 corresponding to glycine, alanine <i>ν</i> (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.