Dual detection of spinal cord injury biomarkers in rat model using gold nanorod array substrate based on surface-enhanced Raman scattering

Tumor necrosis factor alpha (TNF-a) and glial fibrillary acidic protein (GFAP) are candidate biomarkers for early diagnosis and evaluation of spinal cord injury (SCI). In fact, their concentrations in serum, plasma, or cerebrospinal fluid are highly correlated with the SCI severity and neurological prognosis. Therefore, development of a rapid, ultrasensitive, and accurate technique for dual-detection of these important biomarkers is critical for SCI diagnosis, monitoring, and recovery assessment. We synthesized gold nanorods (AuNRs) with stable aspect ratio and fabricated an AuNR array parallelly aligned on a substrate using evaporation-induced self-assembly. The uniform morphology and neat arrangement of the AuNRs produce many hotspots that increase the sensitivity and reproducibility of the array. For dual detection of TNF-alpha and GFAP, the self-assembled AuNR array substrate was modified with anti-TNF-alpha and anti-GFAP. Then, special Raman probe molecules, specific antibodies, and AuNRs were combined to fabricate surface-enhanced Raman scattering (SERS) tags of AuNR@4-MPBA and AuNR@NBA modified with anti-TNF-alpha and anti-GFAP, respectively. Sandwich immunoassay using standard solutions of TNF alpha and GFAP provided limits of detection of 0.023 and 0.018 pg/mL, respectively. The detector was evaluated on concentrations of TNF- a and GFAP in plasma of SCI rat models. Cross-validation against ELISA (enzyme-linked immunosorbent assay) indicated that the SERS sandwich immunoassay detector based on AuNR array substrate has a high dual-detection accuracy. Overall, the proposed detector provides high sensitivity, reproducibility, stability, and accuracy, and it correctly performs dual detection. The fabricated detector platform seems promising for detection of multiple molecules to achieve early and accurate disease diagnosis in clinical practice.