Fluorescent glucose biosensor

[2] As a consequence, in conjunction with insulin administrations, the prime requirement for diabetic patients is to regularly monitor their blood glucose levels.

[2] The monitoring systems currently in general use have the drawback of below optimal number of readings, due to their reliance on a drop of fresh blood.

As a result, there is an effort to create a sensor that relies on a different mechanism, such as via external infrared spectroscopy or via fluorescent biosensors.

Fluorescence is a property present in certain molecules, called fluorophores, in which they emit a photon shortly after absorbing one with a higher energy wavelength.

[23] An alternative approach is to use solvatochromic dyes,[13][14][15] which change their properties (intensity, half-life, and excitation, and emission spectra), depending on the polarity and charge of their environments.

[23] An additional property of fluorescence that has found a large usage is Förster resonance energy transfer (Fret) in which the energy of the excited electron of one fluorophore, called the donor, is passed on to a nearby acceptor dye, either a dark-quencher (non-emitting chromophore) or another fluorophore, which has an excitation spectrum that overlaps with the emission spectrum of the donor dye, resulting in a reduced fluorescence.

Over the years, using a combination of rational design and screening procedures, many possible typologies of fluorescent sensors for glucose have been created with varying degrees of success.

In general, these sensors rely either on Fret[4][5][6][7][9][10][11][12] or on sensitivity to polarity changes[13][14][15] to translate the glucose concentration into fluorescent intensity.

In Biotex Inc. (Houston), McNichols and Ballastardt created a dialysis fibre-enclosed ConA Fret sensor, which has undergone testing in animal models for several years.

[27][28][29][30][31][32] To be specific, Endo[31] and Pasic[32] have used this GOx-based oxygen-quenching assay to make a fibre-based sensor, whilst McShane uses GOx-based oxygen-quenching assay in microspheres made with the aim of subcutaneous injection in order to create what the group has coined a "smart tattoo", a sensor operating non-invasively by reporting across the skin, taking advantage of the fact the skin is permeable to near-infrared light.

In addition, this group has created several Fret completion assays, first using ConA (TRITC-Con A /FITC-dextran (500kDa)),[24] but then switching to GOx apoenzyme in 2004 (TRITC-apo-GOx /FITC-dextran (500kDa)),[9] and in 2009 testing sensors (QSY-21-apo-GOx /Alexa647- dextran) in microspheres.

[34][35] One particular GOx oxygen ruthenium-quenching assay was used in a study in Ingo Klimant's group, in a fully functional sensor to measure glucose levels in a healthy volunteer.

Sensors have been made using QD as Fret donors and a small molecule or gold nanoparticle (dark quencher) as acceptors.

The binding affinity of Ggbp changes when it is labelled endosterically or peristerically, so several mutants that work at range close to pathophysiological glucose have been created.

Examples of this include a study utilizing L255C with acrylodan and ruthenium at the N-terminus revealing three conformational states closed and twisted,[56] the fluorescence and phosphorescence of the tryptophan W183 under normal conditions [52], under high pressure[61] and with or without calcium.

[62] Sode et al. made a series of mutants of Ggbp to increase the Kd in the unlabelled form near physiological range (Phe16Ala) and remove galactose specificity (Asp14Glu).

[64] In a subsequent study (2007), using the heat-stable Ggbp from Thermotoga maritima they screened five mutants (Y13C, W14C, Y189C, S131C and M239C) with four dyes (Ianbd, Acrylodan, Cy5 and Cy3) identifying Y13C-Cy5 conjugate, which gave a maximal increase of 50% and affinity at 15mM.

[63] A group led by Daunert used three endosteric mutants (G148C, H152C and M182C) in combination with four dyes (acrylodan, 1,5-IAEDANS, MDCC and Ianbd ester) identifying M182C–MDCC, which gave a 30% change in fluorescence.

This showed a threefold increase (200% change) upon saturating glucose binding, making it an ideal candidate for a sensor.

Later work, adopting the mutations identified by Pitner (above),[38] generated a Badan-labelled Ggbp mutant (H152C/A213R/L238S), with a dissociation constant in the human physiological glucose range (Km=11mM) and a twofold increase in fluorescence (100% change).

In the ideal situation, the detector could be implanted with the immobilized protein and queried by radio frequency, however this has currently been achieved only with amperormetric sensors.

Despite the change in Fret of only 35% across the pathophysiological range (possibly 40% maximum change form no glucose to saturation), the sensor has been shown to decrease in functionality by only 20% after 450 days incubation at 37 °C (99 °F) and to monitor glucose as well as the Medtronic/Minimed CGMS sensor in animal models (mouse, pig, and dog); however their stated aim is to create a smart tattoo.

In fact, the Menarini Diagnostics' GlucoDay sensor has an improved lifetime because the injected probe uses a dialysis membrane, although to drastically increase the diffusion rate it is coupled with a pump.

[77] Regarding its application in fluorescent sensing of glucose, the first glucose biosensor by fluorescence, which, as mentioned, was made in 1982 by means of a Fret competition assay for the binding site of ConA, was entrapped in a sealed microdialysis tube,[21] in the same lab, namely of J Schultz, in 2001 another study was published using microdialysis fibres using a Fret ConA sensor but with different labels and using sephadex instead of dextran (the former being several orders of magnitude larger).

Hydrogels can be classified according to their polymers constituents, which can be natural (Hyaluronan, alginic acid, pectin, carrageenan, chondroitin sulfate, dextran and dextran sulphate, chitosan, polylysine, collagen, carboxymethyl chitin, fibrin, agarose, pullulan), or synthetic (PEG, PLA, PLGA, PCL, PHB, PVA, PNVP, P(HEMA),[clarification needed] p(biscarboxy-phenoxy-phosphazene), p(GEMA-sulfate), and others), or a hybrid of the two.

[89] All the various hydrogels have different advantages and disadvantage, such as biocompatibility, protein stability, toxicity, or lifetime; for example, the gelling conditions for sol-gels may damage the protein, and, as a result, several copolymers, such as chitosan, may be added (making hybrid gels)[90] or alternative monomers, such as glycol-modified tetraethoxysilane as it is more biocompatible than the commonly used methoxy- or ethoxy-modified tetraethoxysilane.

this sensor was composed of a photocrosslinked diacrylate-modified PEG hydrogel containing the tetra-rhodamine (TRITC), labelled Fret competitor betacyclodextrin and the quantum dot-labelled apoenzyme Concanavalin A.

This used a 2-hydroxyethyl methacrylate hydrogel as a scaffold onto which two dyes were attached one a fluorescent anionic dye and a cationic quencher (to be specific, a viologen) functionalized with boronic acid, which assumes a negative charge when bound to glucose, making the net charge of the molecule neutral and less attracted to the fluorophore, hence modulating its intensity based on glucose concentration.

To be more specific, it relied on a GOx oxygen-ruthenium quenching assay where the protein was mixed with AWP (azide-functionalized polyvinyl alcohol, a photocrosslinkable polymer) and cross-linked to a dialysis membrane that was rolled around a premade ruthenium oxygen probe (ocean optics) and inserted into an 18-gauge needle with eight holes on the side (akin to a recorder).