Localized surface plasmon resonance and vibrational spectroscopic study of trenbolone acetate dopant towards its rapid and label-free detection

Abstract

Biomolecular sensing and interaction monitoring have formed the basis for label-free analytical methodologies. The methodology involves the immobilization of the molecular receptor or biomolecule binding on the substrate without interference from modified analytes. The advantages of label-free methods explain their usage in biomedical and analytical techniques. In this work, localized surface plasmon resonance (LSPR) and vibrational-based optical techniques; Raman, FTIR, and surface-enhanced Raman spectroscopy (SERS) have been demonstrated as feasible methods applied in the detection of trenbolone acetate, anabolic androgenic steroid prohibited by the World Anti-doping Agency (WADA) due to its potential to enhance muscle mass and positively alter an athlete’s endurance ability, resulting in unfair competition. Laser ablation in Liquid (LAL) was employed to synthesize silver nanoparticles from pristine silver granules. The nanoparticles were then characterized using SEM-EDS, TEM, LIBS, XRD, and EDXRF. SEM and TEM showed that the average particle size was 22nm. The silver nanoparticles were then used as the surface on which trenbolone acetate was adsorbed, leveraging their large surface area relative to their volume, giving ample sites for the molecule to attach. After adsorption, the plasmon band of silver nanoparticles was monitored with the help of UV-VIS spectroscopy. The results showed that after binding with the molecule, an average wavelength redshift of + 18nm of the plasmon band was noted. The value of the shift in the localized surface plasmon band exponentially reduces at lower concentrations before increasing with the increase in the concentration of trenbolone acetate until a saturation point is reached. Besides the shift in the LSPR band position, the band-broadening behavior of the Tren/Ac complex was monitored, showing an inverse exponential relation with analyte concentration. The results demonstrated that mixing silver nanoparticles with trenbolone acetate changes the refractive index of the dielectric environment and the impact can be monitored using UV-VIS spectroscopy. Raman, FTIR, and surface-enhanced Raman spectroscopy were also employed to characterize the molecule. The Raman and FTIR spectra were obtained experimentally and also simulated with the help of density functional theory (DFT) implemented in Gaussian 09W software package. The results showed that the simulated spectra had a 99% similarity with what was obtained experimentally. Vibrational Energy Distribution Analysis (VEDA) helped to properly assign the vibrational bands to their functional groups through potential energy distribution (PED). Proper assigning of the molecule marks the hallmark of understanding unique vibrational bands that act as a fingerprint for the molecule. Such knowledge is particularly key in the anti-doping campaign since it allows declassifying trenbolone acetate from other steroids that have similar structural traits. The spectral characterization of the molecule was followed by surface-enhanced Raman spectroscopic studies with silver nanoparticles being used as a substrate. The electromagnetic field potential of silver nanoparticles allowed signal amplification of the trenbolone signal by X11. The signal was even enhanced when SERS was coupled to dry coating deposition Raman. The SERS mixture was drop-casted on an Aluminium foil hydrophobic surface resulting in X60 signal enhancement. The SERS amplification was achieved due to the effective tunability of trenbolone Acetate on silver nanoparticles. The orientation of trenbolone Acetate on silver nanoparticles was monitored by observing shifts in active SERS bands and through HOMO-LUMO studies of the molecule. SERS showed that some bands such as 1618cm-1, 1748cm-1, and 2058cm-1 reported an average of 8nm blue shift in the wavenumbers. This region is assigned to the C=O ketone group which is attached to the acetate part of the steroid. The frontier molecular orbital analysis showed that the energy gap of trenbolone acetate was 3.798eV while the chemical potential of the molecule was 2.094 eV. Consequently, trenbolone acetate can easily participate in a chemical reaction since its potential is lower than the energy gap. The HOMO-LUMO also showed that O2- is the most electronegative and hence the most active adsorbing site in the molecule. The chemical reaction between Ag+ and O2- created an Ag2O complex that influences both the plasmon behavior and the enhancement tendency in SERS. Based on the results of the synthesis and characterization of the silver nanoparticles, their use in surface plasmon resonance and as substrates in SERS, and finally the adsorption studies of Tren Act/ AgNPs complex using DFT, it is possible to develop a cost-effective analytical technique for androgen anabolic steroids. The work suggests using the results as a framework for developing a biosensor prototype for on-site analytical detection of performance-enhancement drugs prohibited in sports competitions.