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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session B

Poster F4 - Design of a paper-based surface-enhanced Raman spectroscopy substrate for bacteria detection in biofluid

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Bacteria detection is an important area of research for clinical settings. Current methods of testing for bacterial infections lack efficiency. Enzyme-linked immunosorbent assays and polymerase chain reactions are the gold standard methods of testing. However, these processes are costly in labor and materials, and other methods require a high concentration of analytes.

Surface-enhanced Raman spectroscopy (SERS) presents a solution to these problems. Raman spectroscopy measures the molecular “fingerprint” of a sample, depending on the inelastic scattering of incident light. SERS enhances this via electromagnetic and chemical enhancement occurring in the presence of colloidal metal nanoparticles or roughened metallic surfaces, often silver or gold, increasing the Raman scatter typically in the range of 108 - 1011 orders of magnitude.

SERS is emerging as a non-destructive technique that can detect low analyte concentrations, making SERS attractive for bacteria detection in biofluids. By combining SERS with a filtration mechanism to mitigate spectral interference from complex media, a robust SERS platform for bacteria detection can be developed. Cellulose and nitrocellulose-based papers are beneficial materials for this platform, as the pores of the paper can trap bacteria-sized molecules while allowing other components of the biofluid to pass through.

This study proposes a method of in-situ growth of gold nanoparticles on nitrocellulose paper to create a SERS platform for rapid bacteria detection in biofluid. We hypothesize that by optimizing gold nanoparticle synthesis for size and deposition, this platform can yield high SERS enhancement, enabling rapid, sensitive detection of bacterial pathogens.

Materials and

Methods:  Gold nanoparticles (AuNPs) embedded in 450 nm pore nitrocellulose paper (NC) were synthesized using modified published protocols. The NC papers (2-x-2-cm) were suspended in a round bottom flask containing 110 mM sodium citrate (TSC). A low temperature (LT) and high temperature (HT) procedure was explored to study the effect of temperature on the incorporation and size of the particles. The TSC solution was set to 210 ℃ (LT) or 250 ℃ (HT) to synthesize the initial gold seeds. Then, 25 mM chloroauric acid (HAuCl4) was added and allowed to react for 15 mins. After the initial color change, the temperature was reduced to 145 ℃ (LT) or 215 ℃ (HT) to begin the growth process. Two different growth cycles were explored to investigate the effect of the nanoparticle size on the SERS signal. For each cycle, 60 mM TSC and 25 mM HAuCl4 were injected with 30-minute wait times between each cycle. The HT and LT papers were removed for analysis after 1, 3, 5, and 16 hours of the last injection to investigate the growth period.

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were used to analyze AuNP distribution and size within the papers. SERS spectra were collected with 4-mercaptobenzoic acid (MBA), a Raman-active molecule, to determine which procedure yields optimal SERS enhancement. Spectra were collected under 20X magnification using a Renishaw inViaTM Raman microscope and a 785 nm excitation laser. Finally, human vaginal fluid (IRB#222199) was applied to the platform to analyze the bacterial microbiome.

Results, Conclusions, and Discussions: The SEM images of the AuNP papers made from the LT procedure showed larger particles relative to the HT procedure, which had a more significant amount of smaller particles distributed throughout the paper. Additionally, EDS images of the different numbers of growth cycles show that the LT papers with 2X cycles have less gold and significant aggregates throughout the surface. However, the NC papers with 3X cycles have a more even distribution of comparatively smaller particles. Inversely, the HT papers with 2X cycles show a more even dispersion of gold than those with 3X cycles, which have much larger aggregates across their surfaces. Thus, the effect of extra growth steps had opposite effects on the nanoparticle distribution for the different temperatures, indicating that both factors influence the formation of AuNP aggregations.

Furthermore, the SEM images reveal that LT AuNP papers incubated for 1, 3, and 5 hours have relatively similar amounts of smaller gold nanoparticles on their surfaces. However, the 16-hour incubation period papers have approximately two times larger nanoparticles. This indicates that a longer incubation time is needed to grow larger nanoparticles.

To determine which temperature yields higher SERS enhancement, the SERS spectra of the LT and HT papers incubated for 16 hours were tested using MBA. The results show that the LT papers with MBA display stronger signal intensities when analyzing the well-known MBA vibrational bonds at 1075 cm-1 and 1589 cm-1 compared to the HT papers. This difference is because the LT papers have larger nanoparticles, leading to improved enhancement. Thus, the 16-hour LT paper prepared with 2X growth cycles shows promise for its use as a sensitive detection platform.

To test this hypothesis, human vaginal fluid was tested on the 2X 16-hour LT papers. The resultant spectra show enhanced bacterial Raman peaks, including the characteristic 730 cm-1 peak representing cell wall components, with minimal signal seen from the other vaginal fluid components. This highlights the potential of the developed platform for separating and detecting bacteria from biofluids using SERS with minimal sample preparation.

Acknowledgements (Optional):  Vanderbilt Biophotonics Center, Vanderbilt University. Department of Biomedical Engineering, Vanderbilt University. Department of Chemistry, Vanderbilt University. The SyBBURE Searle Undergraduate Research Program.