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The native vaginal microbiome plays a crucial role in maintaining vaginal health and disruption can have significant consequences for women during their lifetime. While the composition of the vaginal microbiome is important, current methods for monitoring this community are lacking. Clinically used techniques routinely rely on subjective analysis of vaginal fluid characteristics or time-consuming microorganism culturing. Surface-enhanced Raman spectroscopy (SERS) can aid in filling this gap in timely detection of alterations in the vaginal microbiome as it can discriminate between bacterial species in complex solutions including bacterial mixtures and biofluids. SERS has not previously been applied to study variations in vaginal Lactobacillus, the most common species found in the vaginal microbiome, in complex solutions. Herein, the SERS spectra of Lactobacillus crispatus (L. crispatus) and Lactobacillus iners (L. iners), two of the most common vaginal bacteria, was characterized at physiologically relevant concentrations. Subsequently, the ability of SERS to detect L. crispatus and L. iners in both pure mixtures and when mixed with a synthetic vaginal fluid mimicking solution was determined. In both pure and complex solutions, SERS coupled with partial least squares regression predicted the ratiometric bacterial content with less than 10% error and strong goodness of prediction (Q2 > 0.9). This developed method highlights the applicability of SERS to predict the dominant Lactobacillus in the vaginal micro-environment toward the monitoring of this community.
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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.
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INTRODUCTION: Biochemical alterations in the esophagus of patients with eosinophilic esophagitis (EoE) are poorly understood. We used Raman spectroscopy through a pediatric endoscope to identify key Raman features reflective of the esophageal biochemical composition to differentiate between children with EoE from non-EoE controls and between children with active (aEoE) and inactive EoE (iEoE). METHODS: Spectral measurements were obtained using a customized pediatric endoscope-compatible fiber-optic Raman probe in real time during an esophagogastroduodenoscopy. Chemometric analysis was performed to identify key Raman features associated with EoE. Pearson correlation analysis was used to assess relationship between the key Raman features and EoE activity indices. Their diagnostic utility was ascertained using the receiver operator characteristic curve analysis. RESULTS: Forty-three children were included in the study (EoE = 32 [74%] and non-EoE control = 11 [26%]; aEoE = 20 [63%] and iEoE = 12 [37%]). Raman intensities assigned to lipids, proteins, and glycogen:protein ratio accurately distinguished children with EoE from those without EoE and aEoE from iEoE. They significantly correlated with EoE activity indices. The Raman peak ratio for lipids had 90.6% sensitivity, 100% specificity, and an area under the curve of 0.95 to differentiate children with EoE from non-EoE controls. The glycogen:protein ratio had 70% sensitivity, 91.7% specificity, and an area under the curve of 0.75 to distinguish children with aEoE from iEoE. DISCUSSION: Real-time intraendoscopy Raman spectroscopy is an effective method for identifying spectral markers reflective of the esophageal biochemical composition in children with EoE. This technique may aid in the diagnosis and monitoring of EoE and help to elucidate EoE pathogenesis.
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Enterite , Eosinofilia , Esofagite Eosinofílica , Gastrite , Humanos , Criança , Esofagite Eosinofílica/diagnóstico , Esofagite Eosinofílica/patologia , Análise Espectral Raman , Glicogênio , LipídeosRESUMO
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.
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Otitis media (OM) is a common disease of the middle ear, affecting 80% of children before the age of three. The otoscope, a simple illuminated magnifier, is the standard clinical diagnostic tool to observe the middle ear. However, it has limited contrast to detect signs of infection, such as clearly identifying and characterizing middle ear fluid or biofilms that accumulate within the middle ear. Likewise, invasive sampling of every subject is not clinically indicated nor practical. Thus, collecting accurate noninvasive diagnostic factors is vital for clinicians to deliver a precise diagnosis and effective treatment regimen. To address this need, a combined benchtop Raman spectroscopy (RS) and optical coherence tomography (OCT) system was developed. Together, RS-OCT can non-invasively interrogate the structural and biochemical signatures of the middle ear under normal and infected conditions.In this paper, in vivo RS scans from pediatric clinical human subjects presenting with OM were evaluated in parallel with RS-OCT data of physiologically relevant in vitro ear models. Component-level characterization of a healthy tympanic membrane and malleus bone, as well as OM-related middle ear fluid, identified the optimal position within the ear for RS-OCT data collection. To address the design challenges in developing a system specific to clinical use, a prototype non-contact multimodal handheld probe was built and successfully tested in vitro. Design criteria have been developed to successfully address imaging constraints imposed by physiological characteristics of the ear and optical safety limits. Here, we present the pathway for translation of RS-OCT for non-invasive detection of OM.
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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.
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Otite Média , Análise Espectral Raman , Bactérias , Biofilmes , Haemophilus influenzae , Humanos , Tomografia de Coerência ÓpticaRESUMO
Primary healthcare centers (PHC) are the first point of contact for people in low-resource settings, and laboratory services play a critical role in early diagnosis of any disease. In recent years, several smartphone-based spectroscopic systems have been demonstrated to translate lab-confined healthcare applications into point-of-care environments to improve their accessibility. Due to constraints, such as the low availability of skilled personnel and consumables in a PHC, batch processing would be ideal for a large number of samples. Therefore, high-throughput and multi-channel detection is equally critical as affordability and portability. To date, most point-of-care systems are designed to perform a single type of analysis at a time. Herein, we introduce a smartphone-based spectroscopic system based on the use of line-beam illumination to achieve high-throughput sensing (15 channels simultaneously) within a 3d-printed microfluidic device. We also developed a smartphone application to process the spectral data and provide the results in real-time. Bland-Altman analysis revealed that the proposed device performs similarly to a laboratory spectrophotometer. The availability of the developed system will enable detection of multiple samples rapidly in low-resource settings with the existing limited manpower and infrastructures. The fast turnaround time may eventually help in timely diagnosis of patients during situations of high sample load, such as during disease outbreaks.
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Técnicas Biossensoriais , Aplicativos Móveis , Técnicas Biossensoriais/métodos , Humanos , Dispositivos Lab-On-A-Chip , Sistemas Automatizados de Assistência Junto ao Leito , Smartphone , EspectrofotometriaRESUMO
Infrared neural stimulation (INS) uses pulsed infrared light to yield label-free neural stimulation with broad experimental and translational utility. Despite its robust demonstration, INS's mechanistic and biophysical underpinnings have been the subject of debate for more than a decade. The role of lipid membrane thermodynamics appears to play an important role in how fast IR-mediated heating nonspecifically drives action potential generation. Direct observation of lipid membrane dynamics during INS remains to be shown in a live neural model system. We used hyperspectral stimulated Raman scattering microscopy to study biochemical signatures of high-speed vibrational dynamics underlying INS in a live neural cell culture model. The findings suggest that lipid bilayer structural changes occur during INS in vitro in NG108-15 neuroglioma cells. Lipid-specific signatures of cell stimulated Raman scattering spectra varied with stimulation energy and radiation exposure. The spectroscopic observations agree with high-speed ratiometric fluorescence imaging of a conventional lipophilic membrane structure reporter, 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)pyridinium hydroxide. The findings support the hypothesis that INS causes changes in the lipid membrane of neural cells by changing the lipid membrane packing order. This work highlights the potential of hyperspectral stimulated Raman scattering as a method to safely study biophysical and biochemical dynamics in live cells.
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Microscopia Óptica não Linear , Análise Espectral Raman , Bicamadas Lipídicas , Imagem Óptica , Análise Espectral Raman/métodos , VibraçãoRESUMO
Spatial and temporal trends of marine harmful algal events in Canada over the last three decades were examined using data from the Harmful Algal Event Database (HAEDAT). This database contains the most complete record of algal blooms, phycotoxins and shellfish harvesting area closures in Canada since 1987. This 30-year review of 593 Canadian HAEDAT records from 1988 to 2017, together with other Canadian data and publications, shows that recurring harmful algal events have been widespread throughout both the Atlantic and Pacific coastal regions. The 367 paralytic shellfish toxin (PST) reports revealed annual and frequent recurrence throughout both the Atlantic and Pacific regions, including multi-year PST events in the Bay of Fundy, the Estuary and Gulf of St. Lawrence and the Strait of Georgia. The 70 amnesic shellfish toxin (AST) records revealed no recognizable trend, as these events were usually area specific and did not recur annually. The increasing frequency of diarrhetic shellfish toxin (DST) events over the period of this review, in total 59 records, can be at least partially explained by increased sampling effort. Marine species mortalities caused by harmful algae (including diatoms, dictyochophytes, dinoflagellates, and raphidophytes), were a common occurrence in the Pacific region (87 reports), but have been reported much less frequently in the Atlantic region (10 reports). Notable Canadian records contained in HAEDAT include the first detection worldwide of amnesic shellfish poisoning (ASP), attributed to the production of domoic acid (an AST) by a diatom (Pseudo-nitzschia multiseries) in Prince Edward Island in 1987. The first proven case of diarrhetic shellfish poisoning (DSP) in Canada and North America was recorded in 1990, and the first closures of shellfish harvesting due to DST (associated with the presence of Dinophysis norvegica) occurred in Nova Scotia in 1992, followed by closures in Newfoundland and Labrador in 1993. In 2008, mass mortalities of fishes, birds and mammals in the St. Lawrence Estuary were caused by Alexandrium catenella and high levels of PST. During 2015, the Pacific coast experienced a large algal bloom that extended from California to Alaska. It resulted in the closure of several shellfish harvesting areas in British Columbia due to AST, produced by Pseudo-nitzschia australis. Data from the Canadian Arctic coast is not included in HAEDAT. However, because of the emerging importance of climate change and increased vessel traffic in the Arctic, information on the occurrence of harmful algal species (pelagic and sympagicâ¯=â¯sea ice-associated) in that region was compiled from relevant literature and data. The results suggest that these taxa may be more widespread than previously thought in the Canadian Arctic. Information in HAEDAT was not always robust or complete enough to provide conclusions about temporal trends. Compilation of spatial and temporal information from HAEDAT and other records is nevertheless important for evaluating the potential role of harmful algae as a stressor on Canadian marine ecosystems, and will support the next step: developing a knowledge gap analysis that will establish research priorities for determining their consequences on human and ecosystem health.
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Ecossistema , Fitoplâncton , Alaska , Regiões Árticas , Colúmbia Britânica , Humanos , América do Norte , Nova EscóciaRESUMO
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.
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Bacterial infection is a global burden that results in numerous hospital visits and deaths annually. The rise of multi-drug resistant bacteria has dramatically increased this burden. Therefore, there is a clinical need to detect and identify bacteria rapidly and accurately in their native state or a culture-free environment. Current diagnostic techniques lack speed and effectiveness in detecting bacteria that are culture-negative, as well as options for in vivo detection. The optical detection of bacteria offers the potential to overcome these obstacles by providing various platforms that can detect bacteria rapidly, with minimum sample preparation, and, in some cases, culture-free directly from patient fluids or even in vivo. These modalities include infrared, Raman, and fluorescence spectroscopy, along with optical coherence tomography, interference, polarization, and laser speckle. However, these techniques are not without their own set of limitations. This review summarizes the strengths and weaknesses of utilizing each of these optical tools for rapid bacteria detection and identification.
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Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico por imagem , Infecções Bacterianas/fisiopatologia , Óptica e Fotônica/tendências , Biofilmes , Meios de Cultura , Humanos , Hibridização in Situ Fluorescente , Lactobacillus acidophilus , Lasers , Microscopia de Interferência , Testes Imediatos , Espectrometria de Fluorescência , Espectrofotometria Infravermelho , Análise Espectral Raman , Streptomyces , Tomografia de Coerência Óptica , Raios Ultravioleta , VibraçãoRESUMO
INTRODUCTION: Elucidating esophageal biochemical composition in eosinophilic esophagitis (EoE) can offer novel insights into its pathogenesis, which remains unclear. Using Raman spectroscopy, we profiled and compared the biochemical composition of esophageal samples obtained from children with active (aEoE) and inactive EoE (iEoE) with non-EoE controls, examined the relationship between spectral markers and validated EoE activity indices. METHODS: In vitro Raman spectra from children with aEoE (n = 8; spectra = 51) and iEoE (n = 6; spectra = 48) and from non-EoE controls (n = 10; spectra = 75) were acquired. Mann-Whitney test was used to assess the differences in their Raman intensities (median [interquartile range]) and identify spectral markers. Spearman correlation was used to evaluate the relationship between spectral markers and endoscopic and histologic activity indices. RESULTS: Raman peaks attributable to glycogen content (936/1,449 cm) was lower in children with aEoE (0.20 [0.18-0.21]) compared with that in non-EoE controls (0.24 [0.23-0.29]). Raman intensity of proteins (1,660/1,209 cm) was higher in children with aEoE compared with that in non-EoE controls (3.20 [3.07-3.50] vs 2.91 [2.59-3.05]; P = 0.01), whereas that of lipids (1,301/1,260 cm) was higher in children with iEoE (1.56 [1.49-1.63]) compared with children with aEoE (1.40 [1.30-1.48]; P = 0.02). Raman peaks attributable to glycogen and lipid inversely correlated with eosinophilic inflammation and basal zone hyperplasia. Raman mapping substantiated our findings. DISCUSSION: This is the first study to identify spectral traits of the esophageal samples related to EoE activity and tissue pathology and to profile tissue-level biochemical composition associated with pediatric EoE. Future research to determine the role of these biochemical alterations in development and clinical course of EoE can advance our understanding of EoE pathobiology.
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Esofagite Eosinofílica/diagnóstico , Eosinófilos/imunologia , Mucosa Esofágica/patologia , Esofagoscopia/métodos , Análise Espectral Raman , Adolescente , Biópsia , Criança , Esofagite Eosinofílica/imunologia , Esofagite Eosinofílica/patologia , Mucosa Esofágica/citologia , Mucosa Esofágica/diagnóstico por imagem , Mucosa Esofágica/imunologia , Feminino , Humanos , Masculino , Estudos ProspectivosRESUMO
Researchers have found that decreased levels of circulating citrulline could be an indicator of intestinal failure. Typically, this amino acid, which is produced by the intestinal mucosa cells, circulates in the blood at a physiological level of â¼40 µM. The current methodology for measuring this level involves the use of bulky equipment, such as mass spectroscopy and analysis at a central laboratory, which can delay diagnosis. Therefore, the current detection method is unsuited for routine monitoring at a doctor's office. Our research group proposes the development of a point-of-care (POC) device to overcome this issue. The proposed device utilizes surface-enhanced Raman spectroscopy (SERS) coupled with a specifically designed aptamer, capable of binding to citrulline, conjugated to colloidal gold nanoparticles. The assay is then embedded within a vertical flow paper-fluidic platform as a deliverable at the POC, and a handheld Raman spectrometer (638-nm excitation) was used to interrogate the sample. Results showed good dynamic range and specificity with an average 73% decrease in SERS signal intensity with increasing concentrations of citrulline (0 to 50 µM) in phosphate-buffered saline compared to its controls: glycine, glutamine, histidine, and valine, which showed less than 10% average decrease in the presence of 200 µM of each analyte. Further, the limit of detection (LOD) within a chip was determined to be 0.56 µM, whereas the LOD across chips was below 10 µM.
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Citrulina/sangue , Dispositivos Lab-On-A-Chip , Sistemas Automatizados de Assistência Junto ao Leito , Análise Espectral Raman/métodos , Aptâmeros de Nucleotídeos , Bioensaio/instrumentação , Desenho de Equipamento , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Papel , Análise Espectral Raman/instrumentaçãoRESUMO
Esophageal diseases result in significant mortality, morbidity, and health care costs worldwide. Current approaches to detect and monitor esophageal diseases have severe limitations. Advanced imaging technologies are being developed to complement current approaches to improve diagnostic, therapeutic and surveillance protocols in order to advance the field. Raman spectroscopy-based technologies hold promise to increase the sensitivity for detection of diseased and high-risk lesions in vitro and in vivo in real time. This technique allows for the investigation of microstructural changes and also facilitates the discovery of disease-specific biochemical alterations with the potential to provide novel insights into the pathobiology of these conditions. Raman spectroscopy has been increasingly applied in precancerous and cancerous esophageal conditions. However, its application in benign esophageal diseases is still in the early stages. Continuing its application in cancerous and precancerous conditions and expanding its use to benign esophageal disorders could lay a foundation for integration of this technology in clinical practice and diagnostic paradigms and development of an accurate and cost-effective tool for use in a clinical setting. Furthermore, Raman spectroscopy can also be used as an innovative technique to advance our understanding of the biochemical transformations associated with esophageal diseases and answer a myriad of fundamental questions in the field. In this review, we described the principles of Raman spectroscopy and instrumentation while providing an overview of current applications, challenges, and future directions in the context of esophageal diseases with an emphasis on its clinical translational application.
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Doenças do Esôfago/diagnóstico , Análise Espectral Raman/métodos , Humanos , Análise Espectral Raman/instrumentaçãoRESUMO
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.
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Proteínas de Ligação ao Cálcio/análise , Carcinoma de Células Escamosas/diagnóstico , Nanopartículas Metálicas/química , Neoplasias Bucais/diagnóstico , Proteínas de Neoplasias/análise , Sistemas Automatizados de Assistência Junto ao Leito , RNA Mensageiro/análise , Saliva/química , Biomarcadores/análise , Carcinoma de Células Escamosas/metabolismo , Ouro/química , Humanos , Limite de Detecção , Microscopia Eletrônica de Transmissão , Neoplasias Bucais/metabolismo , Nanotecnologia , Ligação Proteica , Corantes de Rosanilina , Análise Espectral RamanRESUMO
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 â¼11%.
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DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies.
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MicroRNAs/análise , Nanopartículas/química , Análise Espectral Raman/métodos , Corantes , DNA/química , Isotiocianatos , MicroRNAs/genética , Testes Imediatos , Corantes de RosanilinaRESUMO
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).
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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.
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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.