Brian M. Cummins

Design of a self-cleaning thermoresponsive nanocomposite hydrogel membrane for implantable biosensors.

Following implantation of a biosensor, adhesion of proteins and cells and eventual fibrous encapsulation will limit analyte diffusion and impair sensor performance. A thermoresponsive nanocomposite hydrogel was developed as a self-cleaning biosensor membrane to minimize the effect of the host response and its utility for an optical glucose sensor, demonstrated here. It was previously reported that thermoresponsive nanocomposite hydrogels prepared from photopolymerization of an aqueous solution of N-isopropylacrylamide (NIPAAm) and polysiloxane colloidal nanoparticles released adhered cells with thermal cycling. However, poly(N-isopropylacrylamide) hydrogels exhibit a volume phase transition temperature (VPTT) of approximately 33-34 degrees C, which is below body temperature. Thus, the hydrogel would be in a collapsed state in vivo, which would ultimately limit diffusion of the target analyte (e.g., glucose) to the encapsulated sensor. In this study, the VPTT of the nanocomposite hydrogel was increased by introducing N-vinylpyrrolidone (NVP) as a comonomer, so that the hydrogel was in the swollen state in vivo. This thermoresponsive nanocomposite hydrogel was prepared by the photopolymerization of an aqueous solution of NIPAAm, NVP, and polysiloxane colloidal nanoparticles. In addition to a VPTT a few degrees above body temperature, the hydrogel also exhibited good mechanical strength, glucose diffusion, and in vitro cell release upon thermal cycling. Thus, this nanocomposite hydrogel may be useful as a biosensor membrane to minimize biofouling and extend the lifetime and efficiency of implantable glucose sensors and other biosensors.

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 × 106 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.

Modular pumps as programmable hydraulic batteries for microfluidic devices

Simple fluid pumps have been developed to improve microfluidic device portability, but they cannot be easily programmed, produce repeatable pumping performance, or generate complex flow profiles — key requirements for increasing the functionality of portable microdevices. We present a detachable, paper-based, “hydraulic battery” that can be connected to the outlet of a microfluidic channel to pump fluid at varying flow rates over time, including step changes, ramping flows, and oscillating flows.

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 ConAs full binding site and a fluorophore that can effectively track the ligands 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 ConAs 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.

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.

Time-Dependent Model for Fluid Flow in Porous Materials with Multiple Pore Sizes

An understanding of fluid transport through porous materials is critical for the development of lateral flow assays and analytical devices based on paper microfluidics. Models of fluid transport within porous materials often assume a single capillary pressure and permeability value for the material, implying that the material comprises a single pore size and that the porous material is fully saturated behind the visible wetted front. As a result, current models can lead to inaccuracies when modeling transport over long distances and/or times. A new transport model is presented that incorporates a range of pore sizes to more accurately predict the capillary transport of fluid in porous materials. The model effectively predicts the time-dependent saturation of rectangular strips of Whatman filter no. 1 paper using the manufacturers data, published pore-size distribution measurements, and the fluids properties.

Limitations of current fluorescent glucose sensing assays based on competitive binding

Competitive binding assays based on the protein Concanavalin A (ConA) have been proposed as potential sensors for continuous glucose monitoring applications. However, ConA-based assays in the literature have primarily displayed a lack of sensitivity or a lack of repeatability in their glucose response. This work explores this apparent trade-off by separating the measured glucose response into the recognition and fluorescence transduction mechanisms. The recognition responses are modeled for typical competing ligands/assays used in the literature, and they are combined with an optimized fluorescence approach to yield expected fluorescent glucose responses. Because aggregation is known to increase the apparent affinity between multivalent ligands and multivalent receptors, preliminary models are generated for assays that were initially optimized with multivalent ligands but increase in affinity over time. These models accurately predict the low sensitivity for monovalent ligands and the lack of repeatability in the responses with multivalent ligands as seen in the literature. This subsequently explains the aforementioned trade-off no matter the optical approach.

ConA-based glucose sensing using the long-lifetime azadioxatriangulenium fluorophore

Fluorescent glucose sensing technologies have been identified as possible alternatives to current continuous glucose monitoring approaches. We have recently introduced a new, smart fluorescent ligand to overcome the traditional problems of ConA-based glucose sensors. For this assay to be translated into a continuous glucose monitoring device where both components are free in solution, the molecular weight of the smart fluorescent ligand must be increased. We have identified ovalbumin as a naturally-occurring glycoprotein that could serve as the core-component of a 2nd generation smart fluorescent ligand. It has a single asparagine residue that is capable of displaying an N-linked glycan and a similar isoelectric point to ConA. Thus, binding between ConA and ovalbumin can potentially be monovalent and sugar specific. This work is the preliminary implementation of fluorescently-labeled ovalbumin in the ConA-based assay. We conjugate the red-emitting, long-lifetime azadioxatriangulenium (ADOTA+) dye to ovalbumin, as ADOTA have many advantageous properties to track the equilibrium binding of the assay. The ADOTA-labeled ovalbumin is paired with Alexa Fluor 647-labeled ConA to create a Förster Resonance Energy Transfer (FRET) assay that is glucose dependent. The assay responds across the physiologically relevant glucose range (0-500 mg/dL) with increasing intensity from the ADOTA-ovalbumin, showing that the strategy may allow for the translation of the smart fluorescent ligand concept into a continuous glucose monitoring device.

A fluorescence polarization based assay for glucose sensing

A fluorescence polarization (FP) assay was developed to determine concentrations of glucose using concanavalin A (ConA) and fluorescently-labeled dextran. Predictive FP responses to glucose were elicited for different assay configurations using mathematical modeling and displayed herein. Using 4 kDa FITC-dextran, we predicted a change of 0.120 P units from 0 mg/dL glucose to 500 mg/dL. This shows the potential that a homogenous, reproducible FP assay can be engineered to measure glucose concentrations using tetrameric ConA and 4k kDa FITC-dextran.

Long term response of a Concanavalin-A based fluorescence glucose sensing assay

Competitive binding assays comprised of the protein Concanavalin A (ConA) have shown potential for use in continuous glucose monitoring devices. However, its time-dependent, thermal instability can impact the lifetime of these ConA based assays. In an attempt to design sensors with longer in vivo lifetimes, different groups have immobilized the protein to various surfaces. For example, Ballerstadt et al. have shown that immobilizing ConA onto the interior of a micro-dialysis membrane and allowing dextran to be freely suspended within solution allowed for successful in vivo glucose sensing up to 16 days. This work explores the glucose response of an assay comprised of modified ConA and a single fluorescently labeled competing ligand in free solution to increase the in vivo sensing lifetime without immobilization,. The behavior of this assay in the presence of varying glucose concentrations is monitored via fluorescence anisotropy over a 30 day period.