Development of a Low-Cost Paper-Based Platform for Coffee Ring-Assisted SERS.

The need for highly sensitive, low-cost, and timely diagnostic technologies at the point of care is increasing. Surface-enhanced Raman spectroscopy (SERS) is a vibrational spectroscopic technique that is an advantageous technique to address this need, as it can rapidly detect analytes in small or dilute samples with improved sensitivity compared to conventional Raman spectroscopy. Despite the many advantages of SERS, one drawback of the technique is poor reproducibility due to variable interactions between nanoparticles and target analytes. To overcome this limitation, coupling SERS with the coffee ring effect has been implemented to concentrate and localize analyte-nanoparticle conjugates for improved signal reproducibility. However, current coffee ring platforms require laborious fabrication steps. Herein, we present a low-cost, two-step fabrication process for coffee ring-assisted SERS, utilizing wax-printed nitrocellulose paper. The platform was designed to produce a highly hydrophobic paper substrate that supports the coffee ring effect and tested using gold nanoparticles for SERS sensing. The nanoparticle concentration and solvent were varied to determine the effect of solution composition on ring formation and center clearance. The SERS signal was validated using 4-mercaptobenzoic acid (MBA) and tested with Moraxella catarrhalis bacteria to ensure functionality for chemical and biological applications. The limit of detection using MBA is 41.56 nM, and the biochemical components of the bacterial cell wall were enhanced with low spectral variability. The developed platform is advantageous due to ease of fabrication and use, representing the next step toward implementing low-cost coffee ring-assisted SERS for point-of-care sensing.

Measurement of rat and human tissue optical properties for improving the optical detection and visualization of peripheral nerves.

Peripheral nerve damage frequently occurs in challenging surgical cases resulting in high costs and morbidity. Various optical techniques have proven effective in detecting and visually enhancing nerves, demonstrating their translational potential for assisting in nerve-sparing medical procedures. However, there is limited data characterizing the optical properties of nerves in comparison to surrounding tissues, thus limiting the optimization of optical nerve detection systems. To address this gap, the absorption and scattering properties of rat and human nerve, muscle, fat, and tendon were determined from 352-2500 nm. The optical properties highlighted an ideal region in the shortwave infrared for detecting embedded nerves, which remains a significant challenge for optical approaches. A 1000-1700 nm hyperspectral diffuse reflectance imaging system was used to confirm these results and identify optimal wavelengths for nerve imaging contrast in an in vivo rat model. Optimal nerve visualization contrast was achieved using 1190/1100 nm ratiometric imaging and was sustained for nerves embedded under ≥600 µm of fat and muscle. Overall, the results provide valuable insights for optimizing the optical contrast of nerves, including those embedded in tissue, which could lead to improved surgical guidance and nerve-sparing outcomes.

Development of a platform for broadband, spectra-fitted, tissue optical phantoms.

Significance: Current methods of producing optical phantoms are incapable of accurately capturing the wavelength-dependent properties of tissue critical for many optical modalities. Aim: We aim to introduce a method of producing solid, inorganic phantoms whose wavelength-dependent optical properties can be matched to those of tissue over the wavelength range of 370 to 950 nm. Approach: The concentration-dependent optical properties of 20 pigments were characterized and used to determine combinations that result in optimal fits compared to the target properties over the full spectrum. Phantoms matching the optical properties of muscle and nerve, the diffuse reflectance of pale and melanistic skin, and the chromophore concentrations of a computational skin model with varying oxygen saturation ( StO 2 ) were made with this method. Results: Both optical property phantoms were found to accurately mimic their respective tissues' absorption and scattering properties across the entire spectrum. The diffuse reflectance phantoms were able to closely approximate skin reflectance regardless of skin type. All three computational skin phantoms were found to have emulated chromophore concentrations close to the model, with an average percent error for the StO 2 of 4.31%. Conclusions: This multipigment phantom platform represents a powerful tool for creating spectrally accurate tissue phantoms, which should increase the availability of standards for many optical techniques.

Simultaneous Exposure to Intracellular and Extracellular Photosensitizers for the Treatment of Staphylococcus aureus Infections.

Staphylococcus aureus is a serious threat to public health due to the rise of antibiotic resistance in this organism, which can prolong or exacerbate skin and soft tissue infections (SSTIs). Methicillin-resistant S. aureus is a Gram-positive bacterium and a leading cause of SSTIs. As such, many efforts are under way to develop therapies that target essential biological processes in S. aureus. Antimicrobial photodynamic therapy is an effective alternative to antibiotics; therefore we developed an approach to simultaneously expose S. aureus to intracellular and extracellular photosensitizers. A near infrared photosensitizer was conjugated to human monoclonal antibodies (MAbs) that target the S. aureus iron-regulated surface determinant (Isd) heme acquisition proteins. In addition, the compound VU0038882 was developed to increase photoactivatable porphyrins within the cell. Combinatorial photodynamic treatment of drug-resistant S. aureus exposed to VU0038882 and conjugated anti-Isd MAbs proved to be an effective antibacterial strategy in vitro and in a murine model of SSTIs.

Cytokines secreted by stromal cells in TNBC microenvironment as potential targets for cancer therapy.

In triple-negative breast cancer (TNBC), the lack of therapeutic markers and effective targeted therapies result in an incurable metastatic disease associated with a poor prognosis. Crosstalks within the tumor microenvironment (TME), including those between cancer and stromal cells, affect the tumor heterogeneity, growth, and metastasis. Previously, we have demonstrated that IL-6, IL-8, and CCL5 play a significant role in TNBC growth and metastasis. In this study, we performed a systematic analysis of cytokine factors secreted from four stromal components (fibroblasts, macrophages, lymphatic endothelial cells, and blood microvascular endothelial cells) induced by four TNBC cell types. Through bioinformatic analysis, we selected putative candidates of secreted factors from stromal cells, which are involved in EMT activity, cell proliferation, metabolism, and matrisome pathways. Among the candidates, LCN2, GM-CSF, CST3, IL-6, IL-8, and CHI3L1 are ranked highly. Significantly, Lipocalin-2 (LCN2) is upregulated in the crosstalk of stromal cells and four different TNBC cells. We validated the increase of LCN2 secreted from four stromal cells induced by TNBC cells. Using a specific LCN2 antibody, we observed the inhibition of TNBC cell growth and migration. Taken together, these results propose secreted factors as molecular targets to treat TNBC progression via crosstalk with stromal components.

Optimization of optical parameters for improved photodynamic therapy of Staphylococcus aureus using endogenous coproporphyrin III.

BACKGROUND: It has recently been shown that endogenous photosensitization of Gram-positive bacteria is achieved through the accumulation of the heme precursor coproporphyrin III and not protoporphyrin IX, as was previously assumed. As previous studies have operated under this assumption, the efficacy of optimal targeting of the absorption peaks of coproporphyrin III has not been explored. METHODS: Staphylococcus aureus was endogenously photosensitized through the addition of either the small molecule VU0038882, aminolevulinic acid, or both. The efficacy of five different LEDs whose wavelengths target different coproporphyrin III absorption peaks were determined in vitro. Based on these in vitro measurements, the effectiveness of utilizing these LEDs to treat a skin infection was predicted using a Monte Carlo simulation to estimate the fluence rates and resulting bacterial reductions as a function of depth. RESULTS: Optimal targeting of the Soret band provided a 4.7-log improvement as compared to previously utilized wavelengths. Activation of the Q-bands was found to provide similar cytotoxic effects but required significantly larger doses of light. Despite near sterilization in vitro, it was predicted that Soret band targeted light would only provide at least a 2 log-reduction up to 430 µm into the skin while Q-band targeted light could remain effective up to 1 mm in depth. Multiplexing these different wavelengths was found to provide a further 0.5-1.0 log-reduction in bacterial viability. CONCLUSIONS: Accurate targeting of coproporphyrin III has shown that endogenous photodynamic therapy has the potential to be further developed into an effective treatment of skin and soft tissue infections caused by Gram-positive bacteria.

A genomic investigation of ecological differentiation between free-living and Drosophila-associated bacteria.

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome-scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.

A Phantom Study of Terahertz Spectroscopy and Imaging of Micro- and Nano-diamonds and Nano-onions as Contrast Agents for Breast Cancer.

THz imaging is effective in distinguishing between cancerous, healthy, and fatty tissues in breast tumors, but a challenge remains in the contrast between cancerous and fibroglandular (healthy) tissues. This work investigates carbon-based nanoparticles as potential contrast agents for terahertz imaging of breast cancer. Microdiamonds, nanodiamonds, and nanometer-scale onion-like carbon are characterized with terahertz transmission spectroscopy in low-absorption backgrounds of polydimethylsiloxane or polyethylene. The refractive index and absorption coefficients are calculated based on the measured electric fields. Nanodiamonds show little effect on the terahertz signal, microdiamonds express resonance-like, size-dependent absorption peaks, and onion-like carbon provides a uniform increase in the optical properties even at low concentration. Due to its strong interaction with terahertz frequencies and ability to be activated for selective binding to cancer cells, onion-like carbon is implemented into engineered three-dimensional breast tumor models composed of phantom tissue mimicking infiltrating ductal carcinoma surrounded by a phantom mimicking healthy fibroglandular tissue. This model is imaged using the terahertz reflection mode to examine the effectiveness of contrast agents for differentiation between the two tissue types. In both spectroscopy and imaging, a 10% concentration of onion-like carbon shows the strongest impact on the terahertz signal and holds promise as a terahertz contrast agent.