Characterization of lamina propria remodeling in pediatric eosinophilic esophagitis using second harmonic generation microscopy.

Eosinophilic esophagitis (EoE) is a chronic inflammatory condition characterized by an intense infiltration of eosinophils into the esophageal epithelium. When not adequately controlled, eosinophilic inflammation can lead to changes in components of the extracellular matrix (ECM) of the lamina propria. Particularly, alterations to the collagen fiber matrix can lead to lamina propria fibrosis (LPF), which plays an important role in the fibrostenotic complications of EoE. Current approaches to assess LPF in EoE are prone to inter-observer inconsistencies and provide limited insight into the structural remodeling of the ECM. An objective approach to quantify LPF can eliminate inter-observer inconsistencies and provide novel insights into the fibrotic transformation of the lamina propria in EoE. Second harmonic generation (SHG) microscopy is a powerful modality for objectively quantifying disease associated alterations in ECM collagen structure that is finding increasing use for clinical research. We used SHG with morphometric analysis (SHG-MA) to characterize lamina propria collagen fibers and ECM porosity in esophageal biopsies collected from children with active EoE (n = 11), inactive EoE (n = 11), and non-EoE (n = 11). The collagen fiber width quantified by SHG-MA correlated positively with peak eosinophil count (r = 0.65, p < 0.005) and histopathologist scoring of LPF (r = 0.52, p < 0.005) in the esophageal biopsies. Patients with active EoE had a significant enlargement of ECM pores compared to inactive EoE and non-EoE (p < 0.005), with the mean pore area correlating positively with EoE activity (r = 0.76, p < 0.005) and LPF severity (r = 0.65, p < 0.005). These results indicate that SHG-MA can be utilized to objectively characterize and provide novel insights into lamina propria ECM structural remodeling in children with EoE, which could aid in monitoring disease progression.

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.

Differentiation of otitis media-causing bacteria and biofilms via Raman spectroscopy and optical coherence tomography.

In the management of otitis media (OM), identification of causative bacterial pathogens and knowledge of their biofilm formation can provide more targeted treatment approaches. Current clinical diagnostic methods rely on the visualization of the tympanic membrane and lack real-time assessment of the causative pathogen(s) and the nature of any biofilm that may reside behind the membrane and within the middle ear cavity. In recent years, optical coherence tomography (OCT) has been demonstrated as an improved in vivo diagnostic tool for visualization and morphological characterization of OM biofilms and middle ear effusions; but lacks specificity about the causative bacterial species. This study proposes the combination of OCT and Raman spectroscopy (RS) to examine differences in the refractive index, optical attenuation, and biochemical composition of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Pseudomonas aeruginosa; four of the leading otopathogens in OM. This combination provides a dual optical approach for identifying and differentiating OM-causing bacterial species under three different in vitro growth environments (i.e., agar-grown colonies, planktonic cells from liquid cultures, and biofilms). This study showed that RS was able to identify key biochemical variations to differentiate all four OM-causing bacteria. Additionally, biochemical spectral changes (RS) and differences in the mean attenuation coefficient (OCT) were able to distinguish the growth environment for each bacterial species.

Optimization of electron beam-deposited silver nanoparticles on zinc oxide for maximally surface enhanced Raman spectroscopy.

Surface enhanced Raman spectroscopy enables robust, rapid analysis on highly dilute samples. To be useful, the technique needs sensing substrates that will enhance intrinsically weak Raman signals of trace analytes. In particular, three-dimensional substrates such as zinc oxide nanowires decorated with electron-beam deposited silver nanoparticles are easily fabricated and serve the dual need of structural stability and detection sensitivity. However, little has been done to optimize electron beam-deposited silver nanoparticles for maximal surface enhancement in the unique dielectric environment of the zinc oxide substrate. Herein, fabrication and anneal parameters of electron beam-deposited silver nanoparticles were examined for the purpose of maximizing surface enhancement. Specifically, this work explored the effect of changing film thickness, deposition rate, anneal temperature, and anneal time on the surface plasmon resonance of Ag nanoparticles. In this study, multiple sets of fabrication and annealing parameters were discovered that optimized surface plasmon resonance for maximal enhancement to Raman signals acquired with a 532 nm laser. This work represents the first characterization of the fabrication and annealing parameters for electron beam-deposited silver nanoparticles on zinc oxide.

Nanoparticle-based assay for detection of S100P mRNA using surface-enhanced Raman spectroscopy.

The focus of this work is toward the development of a point-of-care (POC) handheld technology for the noninvasive early detection of salivary biomarkers. The initial of focus was the detection and quantification of S100 calcium-binding protein P (S100P) mRNA found in whole saliva for use as a potential biomarker for oral cancer. Specifically, a surface-enhanced Raman spectroscopy (SERS)-based approach and assay were designed, developed, and tested for sensitive and rapid detection of S100P mRNA. Gold nanoparticles (AuNPs) were conjugated with oligonucleotides and malachite green isothiocyanate was then used as a Raman reporter molecule. The hybridization of S100P target to DNA-conjugated AuNPs in sandwich assay format in both free solution and a vertical flow chip (VFC) was confirmed using a handheld SERS system. The detection limit of the SERS-based assay in free solution was determined to be 1.1 nM, whereas on the VFC the detection limit was observed to be 10 nM. SERS-based VFCs were also used to quantify the S100P mRNA from saliva samples of oral cancer patients and a healthy group. The result indicated that the amount of S100P mRNA detected for the oral cancer patients is three times higher than that of a healthy group.

A Layer-by-Layer Approach To Retain a Fluorescent Glucose Sensing Assay within the Cavity of a Hydrogel Membrane.

A continuous glucose monitoring device that resides fully in the subcutaneous tissue has the potential to greatly improve the management of diabetes. Toward this goal, we have developed a competitive binding glucose sensing assay based on fluorescently labeled PEGylated concanavalin-A (PEGylated-TRITC-ConA) and mannotetraose (APTS-MT). In the present work, we sought to contain this assay within the hollow central cavity of a cylindrical hydrogel membrane, permitting eventual subcutaneous implantation and optical probing through the skin. A "self-cleaning" hydrogel was utilized because of its ability to cyclically deswell/reswell in vivo, which is expected to reduce biofouling and therefore extend the sensor lifetime. Thus, we prepared a hollow, cylindrical hydrogel based on a thermoresponsive electrostatic double network design composed of N-isopropylacrylamide and 2-acrylamido-2-methylpropanesulfonic acid. Next, a layer-by-layer (LbL) coating was applied to the inner wall of the central cavity of the cylindrical membrane. It consisted of 5, 10, 15, 30, or 40 alternating bilayers of positively charged poly(diallyldimethylammonium chloride) and negatively charged poly(sodium 4-styrenesulfonate). With 30 bilayers, the leaching of the smaller-sized component of the assay (APTS-MT) from the membrane cavity was substantially reduced. Moreover, this LbL coating maintained glucose diffusion across the hydrogel membrane. In terms of sensor functionality, the assay housed in the hydrogel membrane cavity tracked changes in glucose concentration (0 to 600 mg/dL) with a mean absolute relative difference of ∼11%.

Foreign Body Reaction to a Subcutaneously Implanted Self-Cleaning, Thermoresponsive Hydrogel Membrane for Glucose Biosensors.

Towards achieveing a subcutaneously implanted glucose biosensor with long-term functionality, a thermoresponsive membrane previously shown to have potential to house a glucose sensing assay was evaluated herein for its ability to minimize the foriegn body reaction (FBR) and the resulting fibrous capsule. The severity of the FBR proportionally reduces diffusion of glucose to the sensor and hence sensor lifetime. However, efforts to reduce the FBR have largedly focused on anti-fouling materials that passively inhibit cellular attachment, particularly poly(ethylene glycol) (PEG). Herein, the extent of the FBR of a subcutaneously implanted "self-cleaning" cylindrical membrane was analyzed in rodents. This membrane represents an "actively anti-fouling" approach to reduce cellular adhesion. It is a thermoresponsive double network nanocomposite hydrogel (DNNC) comprised of poly(N-isopropylacrylamide) (PNIPAAm) and embedded polysiloxane nanoparticles. The membrane's cyclical deswelling/reswelling response to local body temperature fluctuations was anticipated to limit cellular accumulation. Indeed, after 30 days, the self-cleaning membrane exhibited a notably thin fibrous capsule (~30 µm) and increased microvascular density within 1 mm of the implant surface in comparison to a non-thermoresponsive, benchmark biocompatible control (PEG diacrylate, PEG-DA).

Self-Cleaning, Thermoresponsive P (NIPAAm-co-AMPS) Double Network Membranes for Implanted Glucose Biosensors.

A self-cleaning membrane that periodically rids itself of attached cells to maintain glucose diffusion could extend the lifetime of implanted glucose biosensors. Herein, we evaluate the functionality of thermoresponsive double network (DN) hydrogel membranes based on poly(N-isopropylacrylamide) (PNIPAAm) and an electrostatic co-monomer, 2-acrylamido-2-methylpropane sulfonic acid (AMPS). DN hydrogels are comprised of a tightly crosslinked, ionized first network [P(NIPAAm-co-AMPS)] containing variable levels of AMPS (100:0-25:75 wt% ratio of NIPAAm:AMPS) and a loosely crosslinked, interpenetrating second network [PNIPAAm]. To meet the specific requirements of a subcutaneously implanted glucose biosensor, the volume phase transition temperature is tuned and essential properties, such as glucose diffusion kinetics, thermosensitivity, and cytocompatibility are evaluated. In addition, the self-cleaning functionality is demonstrated through thermally driven cell detachment from the membranes in vitro.

High Affinity Mannotetraose as an Alternative to Dextran in ConA Based Fluorescent Affinity Glucose Assay Due to Improved FRET Efficiency.

Diabetes mellitus affects millions of people worldwide and requires that individuals tightly self-regulate their blood glucose levels to minimize the associated secondary complications. Continuous monitoring devices potentially offer patients a long-term means to tightly monitor their glucose levels. In recent years, fluorescent affinity sensors based on lectins (e.g., Concanavalin A (ConA)) have been implemented in such devices. Traditionally, these sensors pair the lectin with a multivalent ligand, like dextran, in order to develop a competitive binding assay that changes its fluorescent properties in response to the surrounding glucose concentrations. This work introduces a new type of fluorescent ligand for FRET-based assays in an attempt to improve the sensitivity of such assays. This ligand is rationally designed to present a core trimannose structure and a donor fluorophore in close proximity to one another. This design decreases the distance between the FRET donor and the FRET acceptors on ConA to maximize the FRET efficiency upon binding of the ligand to ConA. This work specifically compares the FRET efficiency and sensitivity of this new competing ligand with a traditional dextran ligand, showing that the new ligand has improved characteristics. This work also tested the long-term thermal stability of the assay based on this new competing ligand and displayed a MARD of less than 10% across the physiological range of glucose after 30 days incubation at 37 °C. Ultimately, this new type of fluorescent ligand has the potential to significantly improve the accuracy of continuous glucose monitoring devices based on the competitive binding sensing approach.

Overcoming the aggregation problem: a new type of fluorescent ligand for ConA-based glucose sensing.

Competitive binding assays based on the lectin Concanavalin A (ConA) have displayed significant potential to serve in continuous glucose monitoring applications. However, to date, this type of fluorescent, affinity-based assay has yet to show the stable, glucose predictive capabilities that are required for such an application. This instability has been associated with the extensive crosslinking between traditionally-used fluorescent ligands (presenting multiple low-affinity moieties) and ConA (presenting multiple binding sites) in free solution. The work herein introduces the design and synthesis of a new type of fluorescent ligand that can avoid this aggregation and allow the assay to be sensitive across the physiologically relevant glucose concentration range. This fluorescent ligand (APTS-MT) presents a single high-affinity trimannose moiety that is recognized by ConA's full binding site and a fluorophore that can effectively track the ligand's equilibrium binding via fluorescent anisotropy. This is confirmed by comparing its measured fluorescent lifetime to experimentally-determined rotational correlation lifetimes of the free and bound populations. Using an assay comprised of 200 nM APTS-MT and 1 µM ConA, the fluorescence anisotropy capably tracks the concentration of monosaccharides that are known to bind to ConA's primary binding site, and the assay displays a MARD of 6.5% across physiologically relevant glucose concentrations. Ultimately, this rationally-designed fluorescent ligand can facilitate the realization of the full potential of ConA-based glucose sensing assays and provide the basis for a new set of competing ligands to be paired with ConA.

PEGylation of concanavalin A to improve its stability for an in vivo glucose sensing assay.

Competitive binding assays utilizing concanavalin A (ConA) have the potential to be the basis of improved continuous glucose monitoring devices. However, the efficacy and lifetime of these assays have been limited, in part, by ConA's instability due to its thermal denaturation in the physiological environment (37 °C, pH 7.4, 0.15 M NaCl) and its electrostatic interaction with charged molecules or surfaces. These undesirable interactions change the constitution of the assay and the kinetics of its behavior over time, resulting in an unstable glucose response. In this work, poly(ethylene glycol) (PEG) chains are covalently attached to lysine groups on the surface of ConA (i.e., PEGylation) in an attempt to improve its stability in these environments. Dynamic light scattering measurements indicate that PEGylation significantly improved ConA's thermal stability at 37 °C, remaining stable for at least 30 days. Furthermore, after PEGylation, ConA's binding affinity to the fluorescent competing ligand previously designed for the assay was not significantly affected and remained at ~5.4 × 10(6) M(-1) even after incubation at 37 °C for 30 days. Moreover, PEGylated ConA maintained the ability to track glucose concentrations when implemented within a competitive binding assay system. Finally, PEGylation showed a reduction in electrostatic-induced aggregation of ConA with poly(allylamine), a positively charged polymer, by shielding ConA's charges. These results indicate that PEGylated ConA can overcome the instability issues from thermal denaturation and nonspecific electrostatic binding while maintaining the required sugar-binding characteristics. Therefore, the PEGylation of ConA can overcome major hurdles for ConA-based glucose sensing assays to be used for long-term continuous monitoring applications in vivo.