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 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.
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.
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.