A novel portable Raman scattering platform for antibiotic screening in pig urine.

Background and Aim: Public health and food safety are gaining attention globally. Consumer health can be protected from chemical residues in meat by early detection or screening for antibiotic residues before selling the meat commercially. However, conventional practices are normally applied after slaughtering, which leads to massive business losses. This study aimed to use portable surface-enhanced Raman spectroscopy (SERS) equipped with multivariate curve resolution-alternation least squares (MCR-ALS) to determine the concentrations of enrofloxacin, oxytetracycline, and neomycin concentrations. This approach can overcome the problems of business loss, costs, and time-consumption, and limit of detection (LOD). Materials and Methods: Aqueous solutions of three standard antibiotics (enrofloxacin, oxytetracycline, and neomycin) with different concentrations were prepared, and the LOD for each antibiotic solution was determined using SERS. Extracted pig urine was spiked with enrofloxacin at concentrations of 10, 20, 50, 100, and 10,000 ppm. These solutions were investigated using SERS and MCR-ALS analysis. Urine samples from pigs at 1 and 7 days after enrofloxacin administration were collected and investigated using SERS and MCR-ALS to differentiate the urinary enrofloxacin concentrations. Results: The LOD of enrofloxacin, oxytetracycline, and neomycin in aqueous solutions were 0.5, 2.0, and 100 ppm, respectively. Analysis of enrofloxacin spiking in pig urine samples demonstrated the different concentrations of enrofloxacin at 10, 20, 50, 100, and 10,000 ppm. The LOD of spiking enrofloxacin was 10 ppm, which was 10 times lower than the regulated value. This technique was validated for the first time using urine collected on days 1 and 7 after enrofloxacin administration. The results revealed a higher concentration of enrofloxacin on day 7 than on day 1 due to consecutive administrations. The observed concentration of enrofloxacin was closely correlated with its circulation time and metabolism in pigs. Conclusion: A combination of SERS sensing platform and MCR-ALS is a promising technique for on-farming screening. This platform can increase the efficiency of antibiotic detection in pig urine at lower costs and time. Expansion and fine adjustments of the Raman dataset may be required for individual farms to achieve higher sensitivity.

Microbial Poly(hydroxybutyrate-co-hydroxyvalerate) Scaffold for Periodontal Tissue Engineering.

In this study, we fabricated three dimensional (3D) porous scaffolds of poly(hydroxybutyrate-co-hydroxyvalerate) with 50% HV content. P(HB-50HV) was biosynthesized from bacteria Cupriavidus necator H16 and the in vitro proliferation of dental cells for tissue engineering application was evaluated. Comparisons were made with scaffolds prepared by poly(hydroxybutyrate) (PHB), poly(hydroxybutyrate-co-12%hydroxyvalerate) (P(HB-12HV)), and polycaprolactone (PCL). The water contact angle results indicated a hydrophobic character for all polymeric films. All fabricated scaffolds exhibited a high porosity of 90% with a sponge-like appearance. The P(HB-50HV) scaffolds were distinctively different in compressive modulus and was the material with the lowest stiffness among all scaffolds tested between the dry and wet conditions. The human gingival fibroblasts (HGFs) and periodontal ligament stem cells (PDLSCs) cultured onto the P(HB-50HV) scaffold adhered to the scaffold and exhibited the highest proliferation with a healthy morphology, demonstrating excellent cell compatibility with P(HB-50HV) scaffolds. These results indicate that the P(HB-50HV) scaffold could be applied as a biomaterial for periodontal tissue engineering and stem cell applications.

Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies.

We demonstrate a novel bio-spectroscopic technique, "simultaneous Raman/GFP microspectroscopy". It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti-Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two-component multivariate curve resolution with alternate least squares (MCR-ALS) analysis in the anti-Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR-ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm-1 , previously reported as "Raman spectroscopic signature of life of yeast."

Detection of Metal-Molecule-Metal Junction Formation by Surface Enhanced Raman Spectroscopy.

Vibrational modes play a key role in characterizing metal-molecule-metal junctions, but their detection currently either requires single-molecule sensitivity or the generation of defect-free large-scale junctions. Here we demonstrate that surface-enhanced Raman scattering (SERS) on nonideal surfaces can provide a significant amount of information despite many defects in the layer. We determine the vibrational signature of the molecular electronic junction for palladium ions complexed and reduced on 4-mercaptopyridine adsorbed on rough gold and gold nanoparticles using SERS and density functional theory. We show that these nonideal surfaces can be used to probe kinetics of metal ion complexation and establish the success of electrochemical metallization. SERS on nonideal surfaces is thus revealed as a useful tool to rapidly establish the key process parameters in making molecular electronic junctions before embarking on more detailed studies on single molecules or single crystal surfaces.

Discrimination between hydrogen bonding and protonation in the spectra of a surface-enhanced Raman sensor.

We investigate the surface-enhanced Raman spectra of 4-mercaptopyridine on gold in a variety of acids. 4-Mercaptopyridine is a known pH sensor which exhibits characteristic spectral changes when the pH is changed. Here we show with the help of experiment and density functional calculations that the ring breathing mode is also highly sensitive to hydrogen bonding. Its spectral signature is a broad band with up to three contributions from free, protonated and hydrogen-bonded 4-mercaptopyridine. Unlike pyridine in solution, where protonation leads to a higher ring breathing frequency than hydrogen-bonding, we find that protonated adsorbed 4-mercaptopyridine possesses a frequency which is lower than the corresponding hydrogen-bonded species. The Raman spectra indicate an orientation change of the aromatic ring in acidic solutions, which could be caused by a cation/π interaction between protonated and deprotonated 4-mercaptopyridine. As the frequencies of the three species are well separated, adsorbed 4-mercaptopyridine can probe more complex changes in the solution environment than just pH.