Rapid, accurate, and minimally-invasive biosensors for glucose measurement have the potential to enhance management
of diabetes mellitus and improve patient outcome in intensive care settings. Recent studies have indicated that
implantable biosensors based on Förster Resonance Energy Transfer (FRET) can provide high sensitivity in quantifying
glucose concentrations. However, standard approaches for determining the potential for interference from other
biological constituents have not been established. The aim of this work was to design and optimize a FRET-based
glucose sensor and assess its specificity to glucose. A sensor based on competitive binding between concanavalin A and
dextran, labeled with long-wavelength acceptor and donor fluorophores, was developed. This process included
optimization of dextran molecular weight and donor concentration, acceptor to donor ratio, and hydrogel concentration,
as well as the number of polymer layers for encapsulation. The biosensor performance was characterized in terms of its
response to clinically relevant glucose concentrations. The potential for interference and the development of test methods
to evaluate this effect were studied using a potential clinical interferent, maltose. Results indicated that our biosensor had
a prediction accuracy of better than 11% and that the robustness to maltose was highly dependent on glucose level.
We present a biosensing platform that uses spatial electroluminescent (EL) illumination combined with charge-coupled
device (CCD)-based detection for fluorescence measurements. The resulting EL-CCD detector platform was used to
monitor different protease activities with substrates labeled for fluorescence resonance energy transfer (FRET)-based
assays. The first uses a commercial FITC/DABCYL-SNAP-25 peptide substrate to monitor the activity of the light
chain derivative (LcA) of botulinum neurotoxin A, achieving a limit of detection (LOD) of 1.25 nM (62 ng/ml). The
second protease activity assay measured trypsin proteolysis using peptide substrates immobilized onto semiconductor
quantum dot (QD) nanoparticles with a LOD of 6.2 nM trypsin (140 ng/ml). The specific ovomucoid inhibition of
trypsin activity was also monitored. The highlighted studies clearly demonstrate the utility of the EL-CCD detector
platform for monitoring fluorescent-based protease activity assays with potential healthcare applications, including
point-of-care diagnostics.
We present a characterization of the metal-affinity driven self-assembly between luminescent CdSe-ZnS core-shell
semiconductor quantum dots (QDs) and either peptides or proteins appended with various length terminal
polyhistidine tags. We first monitor the kinetics of self-assembly between surface-immobilized QDs and
proteins/peptides under flow conditions (immobilized). To accomplish this, the QDs were immobilized onto
functionalized substrates and then exposed to dye-labeled peptides/proteins. By using evanescent wave excitation of
the substrate, self-assembly was assessed by monitoring the time-dependent changes in the dye fluorescence. This
configuration was complemented with experiments using freely diffusing QDs and proteins/peptides (solution-phase)
via energy transfer between QDs and dye-labeled proteins/peptides. Cumulatively, these measurements
allowed determination of kinetic parameters, including association and dissociation rates (kon and koff) and the
binding constant (Kd). We find that self-assembly is rapid with an equilibrium constant Kd-1 in the low nM. We next
demonstrate the importance of understanding this self-assembly by creating QD-peptide bioconjugates which we
employ as substrates to monitor the cleavage activity of proteolytic enzymes. This confirms that metal-affinity
interactions can provide QD-bioconjugates that are functional and stable.
An array biosensor developed for performing simultaneous analysis of multiple samples for multiple analytes has been miniaturized and fully automated. The biochemical component of the multi-analyte biosensor consists of a patterned array of biological recognition elements ("capture" antibodies) immobilized on the surface of a planar waveguide. A fluorescence assay is performed on the patterned surface, yielding an array of fluorescent spots, the locations of which are used to identify what analyte is present. Signal transduction is accomplished by means of a diode laser for fluorescence excitation, optical filters and a CCD camera for image capture. A laptop computer controls the miniaturized fluidics system and image capture. Data analysis software has been developed to locate each spot and quantify the fluorescent signal in each spot. The array biosensor is capable of detecting a variety of analytes including toxins, bacteria and viruses and shows minimal interference from complex physiological sample matrices such whole blood and blood components, fecal matter, saliva, nasal secretions, and urine. Some results from recent field trials are presented.
Array biosensors provide the capability of immobilizing multiple capture biomolecules onto a single surface and therefore offer the exciting prospect of multi-analyte detection. A miniaturized, fully automated, stand-alone biosensor is reported which can simultaneously test multiple samples for multiple analytes. This portable system (< 10 lbs) is particularly appropriate for on-site monitoring for food safety, infectious disease detection, and biological warfare defense. The surface-selective nature of this technology allows determination of binding constants and tracking of both specific and non-specific binding events as they occur. Thus, it provides an exciting new research tool for characterizing the interactions of biomolecules with surfaces or immobilized receptors in real time. This capability has important implications for development of new materials and sensors.
The array biosensor is capable of detecting and identifying multiple analytes in multiple samples simultaneously. Using fluorescence immunoassays on a planar waveguide and miniaturized fluidics, the sensor is automated and portable. Assays are sensitive and require 12 minutes to perform. Environmental contaminants in the sample fail to generate false positive or false negative results in tests performed to date. Measurements can be conducted in real time using spots as small as 80 micrometers . The waveguide can be coated with indium tin oxide (ITO) to create a charged field at the surface to further regulate the interaction of sample components with the surface.
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