Our lab group is currently developing a fluorescent competitive binding assay between the Alexa fluor 647 labeled
lectin, Concanavalin A, and highly structured glycosylated dendrimers to be sensitive to varying levels of glucose.
Previously, this chemistry has elicited a high sensitivity to additions of physiological concentrations of glucose.
However, the exact mechanism behind the sensing has not yet been well understood. This work presents a conceptual
model of the response in which competitive binding results in different distributions of aggregates size to varying
amounts of glucose. Preliminary experiments were performed by using Numerical Tracking Analysis (NTA) which
correlates the movement of particles, positioned by light scattering, to the equivalent Brownian motion associated with
particles of a certain spherical diameter. Using this method, the sensing chemistry was exposed to two different glucose
concentrations and histograms of the size distribution for glucose concentrations were obtained. Herein the aggregation
profile, mean aggregate size, and the number of aggregates (aggregates per mL) for two glucose concentrations are
displayed, showing a correlation between the aggregation and glucose concentration.
Fluorescent microarrays have the ability to detect and monitor multiple analytes simultaneously and noninvasively,
following initial placement. This versatility is advantageous for several biological applications including drug
discovery, biohazard detection, transplant organ preservation and cell culture monitoring. In this work, poly(ethylene
glycol) hydrogel microarrays are described that can be used to measure multiple analytes, including H+ and dissolved
oxygen. The array elements are created by filling micro-channels with a hydrogel precursor solution containing analyte
specific fluorescent sensors. A photomask is used to create the microarray through UV polymerization of the PEG
precursor solution. A compact imaging system composed of a CCD camera, high powered LED, and two optical filters
is used to measure the change in fluorescence emission corresponding to analyte concentration. The proposed system
was tested in aqueous solution by altering relevant analyte concentrations across their biological ranges.
A minimally invasive biosensor is undergoing development to detect physiological concentrations of glucose within
interstitial fluid. The sensor is based on a chemical assay consisting of Alexa Fluor 647 labeled concanavalin A lectin
and dendrimer macromolecules functionalized to contain peripheral glucose moieties. The two components form large
cross-linked particles that result in loss of fluorescent emission through shielding of interior fluorophores. As glucose is
introduced into the assay, it competes with the glycodendrimers for binding to concanavalin A to disrupt the cross-linked
complex and produce a reversible change in fluorescence intensity that is dependent on glucose concentration.
Chemical analogs of the original glycodendrimer have been created and analyzed with the purpose of creating more
stable and consistent dendrimers in order to maximize the response of the assay so that its signal can be better detected
through dermal tissue and provide a better understanding of the sensor binding mechanics.
Poly(ethylene glycol) (PEG) microspheres have been used to sense a variety of analytes by encapsulating fluorescently labeled molecules into a PEG hydrogel matrix. This matrix is designed to retain the sensing molecules while simultaneously allowing nearly unhindered analyte diffusion. Some sensing assays, however, depend on the
conformational rearrangement or binding of large macromolecular compounds which may be sterically prohibited in a dense polymer matrix. A new microporation process has been developed in order to create small cavities in the spheres containing aqueous solution and the assay components. This configuration insures a small mesh size for the supporting polymer, which limits leaching, while allowing the large assay components space to react within the aqueous cavities.
Three hydrogel compositions (100% PEG, 50% PEG hydrogels, and microporated 100% PEG) were studied by embedding traditional pH (FITC) and oxygen sensitive fluorophores (Ru(Phen)). These hydrogels were analyzed for leaching and dynamic response to evaluate the functionality of the new microporated hydrogel.
A fluorescent assay based on the competitive binding between glycosylated PAMAM dendrimer and glucose with the sugar-binding lectin Concanavalin A has been developed. This assay, composed of the glycodendrimer and Alexa Fluor 647 labeled Concanavalin A, has shown a large dynamic response to physiological concentrations of glucose. The larger dynamic range is believed to be due to the spheriodal shape of the dendrimer molecule, which eliminates the multiple
binding of the same dextran chain to the Concanavalin A tetramer that plagued previous approaches. However, in order to further understand the operation of the assay and optimize the dynamic response, the dendrimer construction must be modified to determine the optimum degree of glycosylation. In this paper, a description of the assay function and the change in fluorescence response with various formulations of glycodendrimers are shown. Theories are also presented as means of understanding the various assay responses with different degrees of dendrimer functionalization.
A combined dissolved oxygen and pH sensitive poly(ethylene glycol) hydrogel microarray was fabricated for use in monitoring cell culture media. The sensor was prepared by filling micro-channels with a hydrogel precursor solution containing pH or oxygen sensitive fluorophores. This solution was then cured by passing UV light through a mask placed to the channels, creating array with 100 μm elements. An imaging system with a monochrome CCD camera and
appropriate interference filters was used to capture the fluorescence image induced by excitation of microstructure in transmission mode. The sensor performance was characterized in buffer solution (PBS) and cell culture media (MEM) across the biological range of pH (6-8) and dissolved oxygen (3-21%).
Fluorescent glucose assays based on the affinity reaction between Concanavalin A and dextran have been extensively studied. However, advancements in polymer science have allowed for new macromolecules capable of replacing dextran which may improve the performance of this well-known assay. Dendrimer macromolecules, being highly
ordered and spherical, allow for the binding of specific residues to the terminal (peripheral) binding sites, enabling researchers to customize the molecule. In this research, glycosylated dendrimers have been engineered to replace dextran to allow for more controlled chemical and fluorescent responses (eliminate multivalent binding and improve reversibility). This new assay has been shown to form small aggregate particles containing many Con A and glycosylated dendrimers resulting in a substantial loss in fluorescent intensity. Overall, this assay shows promise for use as part of an implantable glucose monitoring device, but more research needs to be done to increase sensor stability and optimize the sensor response to glucose.
An implantable sensor is being created that allows measurement of blood glucose through fluorescent detection of an embedded chemical assay. The sensor is based on the competitive binding reaction between the protein Concanavalin A and various saccharide molecules, specifically a glycodendrimer and glucose. Previous studies have shown the ability of an embedded chemical assay using Con A and dextran with shorter wavelength dyes to both sense changes in glucose and generate sufficient fluorescent emission to pass through the dermal tissue. However, due to the chemical constituents of the assay, multivalent binding was evident resulting in poor spectral change due to glucose within the biological range. Use of a glycodendrimer and longer wavelength dyes has improved the sensor’s spectral change due to glucose and the overall signal to noise ratio of the sensor. In this work, a description of this sensor and the results obtained from it will be presented showing a large dynamic range of fluorescence with glucose.
The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.
A preliminary in vivo study using photopolymerized poly(ethylene glycol) (PEG) microspheres containing tetramethylrhodamine isothiocyanate labeld concanavalin A (TRITC-Con A) fluroescein isothiocyanate labeld dextran (FITX-dextran) as an implantable glucose sensor was performed using hairless rats. The glucose sensor works by affinity reaction between the two fluorescent labeled molecules binding together to form a fluorescent energy transfer system in which the FITC peak is quenched by the TRITC peak. The addition of glucose to the sensors local environment displces the dextran disrupting the FRET pair and the quenching. The change in fluroescent peak ratio (TRITC/FITC) therefore can be related to glucose. The microspheres in this study were implanted below the dermal skin layer of the lower abdomen by injection. A bolus injection of glucose was given through the tail vein to simulate glucose consumption. Spectra were obtained by shining and collecting light through the skin using an optical fiber delivery system via a 488nm argon laser and a spectrophometer. The preliminary results showed quantifiable changes in the ratio between the two peaks in response to the changae in glucose levels in the interstitial fluid of the rat.
Low molecular weight molecules are typically very difficult to detect directly in solution using commercially available SPR (surface plasmon resonance) instruments. This is because the mass change on binding is not sufficient to cause a detectable change in refractive index on binding to surface- bound receptors (e.g., antibodies). Some receptors, however, undergo extensive changes in tertiary structure upon binding ligands. Here we present data suggesting conformational changes in surface-bound receptors such as periplasmic binding proteins and calcium-binding proteins can be detected by SPR. This SPR response can be used to monitor specific binding of carbohydrates and calcium even though the molecular weight of these analytes would be difficult to detect using traditional SPR methods. Therefore this approach has potential applications for developing optical biosensors for such small molecules.
Glucose monitoring is of critical importance in the life of Type I and many Type II diabetics. This research furthers work toward a minimally invasive implantable glucose sensor based on fluorescence detection. Current experimental models use heterogeneous fluorescence resonance energy transfer (FRET) systems for sensing; ideally, the response of one fluorophore bound to a large polysaccharide is enhanced greatly in the presence of glucose while the other fluorophore bound to a glucose sensitive protein is diminished or unaffected. Many fluorophores are affected by environmental factors such as pH and temperature. FRET experiments using two fluorophores, tetramethylrodamine isothiocyanate (TRITC) and fluoroscein isothiocyanate (FITC), are performed evaluating the effects of fluctuations over the range of pH 4-8 and temperature 25-45 degree(s)C for various concentrations of glucose in a flow cell. TRITC is bound to the lectin Concanavalin A (Con A), and FITC is bound to dextran molecules of varying sizes.
Progress towards a painless and hygienic glucose monitoring procedure for diabetics continues as the growth of diabetes mellitus reaches epidemic proportions in the American population. Utilizing an implantable fluorescence based glucose assay, the minimally invasive approach presented here has previously shown promise towards this goal in terms of glucose specificity and quantification for in vitro environments. However, in realistic physiological circumstances the depth of the implant can vary and optical properties of skin can change due to normal physiological conditions. Additionally, naturally occurring auto-fluorescence can obscure the sensor signal. An important concern under these conditions is that variations of fluorescent intensity due to these or other causes might be mistaken for glucose concentration fluctuations. New data shows that fluorescence-based glucose assays can be probed and interpreted in terms of glucose concentrations through pig skin at depths of up to 700 mm when immobilized in a bio-compatible polymer. When a combination of two fluorophores are employed as demonstrated here, reasonable changes in skin thickness and the confounding effects of the variations inherent in skin can be overcome for this glucose sensing application.
A painless monitoring procedure for diabetics has proven to be an elusive goal. While completely noninvasive measurements are the desired technique, minimally invasive procedures using implanted fluorescence sensor chemistry offer significant advantages in specificity over current noninvasive approaches. The goal was to evaluate the potential for transdermal glucose sensing using intensity measurements from implanted microspheres. A fiber-optic probe and spectrometer were custom-built for collection of in vivo data. Comparisons with commercial fluorometers show the constructed device is adequate for this project.