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This work explores the use of Surface-Enhanced Raman Spectroscopy (SERS) combined with artificial neural network (ANN) models to detect and quantify growth hormone (GH) and testosterone (TE) in the blood of Sprague Dawley (SD) rats. SERS spectra were recorded from blood samples of SD rats injected with GH, TE, both hormones, and non-injected controls using 785 nm laser excitation. The samples were mixed with silver nanoparticles (AgNPs) synthesized in distilled water, applied onto a microscope slide, and air-dried. The resulting SERS spectra displayed similar profiles with intensity variations depending on the hormone, revealing specific bands at 658, 798, 878, 914, 932, 1064, 1190, 1354, 1410, and 1658 cm-1. PCA analysis indicated time-dependent intensity changes in bands centered around 1378 (all groups), 658 and 1614 cm-1 (GH-injected rats), and others for different hormone combinations. These variations reflect subtle biochemical changes induced by hormone injections. The ANN models, trained with six PCA scores of blood spiked with various hormone concentrations, showed high accuracy, with coefficients of determination greater than 87.71% and low root mean square error (RMSE) values below 0.6436. The hormone levels in injected rats increased initially and later declined, a trend confirmed by ELISA kits. Although ELISA and SERS produced similar results, SERS offered advantages such as rapid analysis (about two minutes), simple sample preparation, small sample volumes, and non-specificity to hormones. This suggests that SERS, combined with ANN models, could be used to detect exogenous sports dopants. These findings expand the potential applications of SERS in sports science, clinical diagnostics, and biomedical research.

This work reports the potential use of surface enhanced Raman spectroscopy (SERS) in rapid, label-free assaying of testosterone (TE) and growth hormone (GH) in whole blood. Biomarker SERS spectral bands from the two hormones (TE and GH) in intentionally spiked water for injection and in male Sprague-Dawley (SD) rat blood are reported. Abuse of the two hormones (TE and GH) singly or simultaneously is widespread and not only has prolonged side effects such as hypertension and liver failure, but their illegal use by athletes is against clean competition. Currently used highly label-dependent doping detection methods involve complex and time-consuming procedures, rendering them unsuitable for rapid analysis. In blood, the most concentration-sensitive bands (in both TE and GH), as deduced through Principal Component Analysis (PCA) and Analysis of Variance (ANOVA), were around 684 cm-1 (assigned to C-C stretching) and 1614 cm-1 (assigned to C-C stretching) in GH; and 786 cm-1 (assigned to N-H wagging), 856 cm-1 (assigned to C-C stretching), and 1490 cm-1 (assigned to CH2 bending) in TE. In addition, a characteristic variance was noted in the bands around 1510 cm-1 (attributable to CH2 stretching) in GH and 1636 cm-1 (C-C stretching) in TE, which could be used as biomarker bands for the respective hormones in the blood. This work has shown the capability of SERS for potential hormone concentration level determination when concentration-sensitive or biomarker bands are employed. This discovery opens new possibilities for the use of SERS in fields such as sports science, clinical diagnostics, and biomedical research.