Fluorescent glucose biosensors are devices that measure the concentration of glucose in diabetic patients by means of sensitive protein that relays the concentration by means of fluorescence, an alternative to amperometric sension of glucose. Due to the prevalence of diabetes, it is the prime drive in the construction of fluorescent biosensors. A recent development has been approved by the FDA allowing a new continuous glucose monitoring system called EverSense, which is a 90-day glucose monitor using fluorescent biosensors.
Application
Keeping glucose levels in check is crucial to minimize the onset of the damage caused by diabetes.The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med, 1993. 329(14): p. 977-86. As a consequence, in conjunction with insulin administrations, the prime requirement for diabetic patients is to regularly monitor their blood glucose levels. 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. Some continuous glucose monitors are commercially available, but suffer from the severe drawback of a short working life of the probe. The majority of these work amperometrically. 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.Pickup, J.C., et al., Fluorescence-based glucose sensors. Biosens Bioelectron, 2005. 20(12): p. 2555-65.
Various strategies exist to detect glucose levels using fluorescence, the firstMeadows, D.L. and J.S. Schultz, Design, manufacture and characterization of an optical fiber glucose affinity sensor-based on an homogeneous fluorescence energy transfer assay system. Analytica Chimica Acta, 1993. 280(1): p. 21-30. and most common being a competition assay between glucose and a labelled glucose polymer for the binding site of Concanavalin A.Ballerstadt, R. and J.S. Schultz, A fluorescence affinity hollow fiber sensor for continuous transdermal glucose monitoring. Anal Chem, 2000. 72(17): p. 4185-92.Sato, K. and J. Anzai, Fluorometric determination of sugars using fluorescein-labeled concanavalin A-glycogen conjugates. Anal Bioanal Chem, 2006. 384(6): p. 1297-301.Liao, K.C., et al., Percutaneous fiber-optic sensor for chronic glucose monitoring in vivo. Biosens Bioelectron, 2008. 23(10): p. 1458-65.Ballerstadt, R., et al., In vivo performance evaluation of a transdermal near-infrared fluorescence resonance energy transfer affinity sensor for continuous glucose monitoring. Diabetes Technol Ther, 2006. 8(3): p. 296-311. Over the years, using a combination of rational design and screening approaches, many possible combinations of fluorescent sensor for glucose have been studied with varying degrees of success: In most approaches, the glucose concentration is translated into a change in fluorescence either by using a pairChinnayelka, S. and M.J. McShane, RET nanobiosensors using affinity of an apo-enzyme toward its substrate. Conf Proc IEEE Eng Med Biol Soc, 2004. 4: p. 2599-602.McCartney, L.J., et al., Near-infrared fluorescence lifetime assay for serum glucose-based on allophycocyanin-labeled concanavalin A. Anal Biochem, 2001. 292(2): p. 216-21.Hanashi, T., et al., BioCapacitor-A novel category of biosensor. Biosens Bioelectron, 2009.Sakaguchi-Mikami, A., et al., Engineering of ligand specificity of periplasmic binding protein for glucose sensing. Biotechnol Lett, 2008. 30(8): p. 1453-60. or by using environment sensitive (solvatochromic) dyesKhan, F., L. Gnudi, and J.C. Pickup, Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: a comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies. Biochem Biophys Res Commun, 2008. 365(1): p. 102-6.Brown, J.Q., et al., Enzymatic fluorescent microsphere glucose sensors:evaluation of response under dynamic conditions. Diabetes Technol Ther, 2006. 8(3): p. 288-95.Salins, L.L., et al., A novel reagentless sensing system for measuring glucose-based on the galactose/glucose-binding protein. Anal Biochem, 2001. 294(1): p. 19-26. in a variety of combinations, the fluorescent small molecule, proteinTeasley Hamorsky, K., et al., A bioluminescent molecular switch for glucose. Angew Chem Int Ed Engl, 2008. 47(20): p. 3718-21.Ye, K. and J.S. Schultz, Genetic engineering of an allosterically based glucose indicator protein for continuous glucose monitoring by fluorescence resonance energy transfer. Anal Chem, 2003. 75(14): p. 3451-9. or quantum dotTang, B., et al., A new nanobiosensor for glucose with high sensitivity and selectivity in serum-based on fluorescence resonance Energy transfer () between CdTe quantum dots and Au nanoparticles. Chemistry, 2008. 14(12): p. 3637-44. have been used in conjunction with a glucose binding moiety either a boronic acid functionalized fluorophoreUwira, N., N. Opitz, and D.W. Lubbers, Influence of Enzyme Concentration and Thickness of the Enzyme Layer on the Calibration Curve of the Continuously Measuring Glucose Optode. Advances in Experimental Medicine and Biology, 1984. 169: p. 913-921. or a protein, such as glucose oxidase,Schultz, J.S., S. Mansouri, and I.J. Goldstein, Affinity sensor: a new technique for developing implantable sensors for glucose and other metabolites. Diabetes Care, 1982. 5(3): p. 245-53. concanavalin A, glucose/galactose-binding protein, glucose dehydrogenase and glucokinase.Schaffar, B.P.H. and O.S. Wolfbeis, A Fast Responding Fiber Optic Glucose Biosensor Based on an Oxygen Optrode. Biosensors & Bioelectronics, 1990. 5(2): p. 137-148 In general, the change seen with competition assays is small (see below).
Theory of fluorescence
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.
To be more specific, in order for an electron in the outer orbital of a molecule to jump from a ground-state orbital to an excited state orbital, it requires a fixed amount of energy, which, in the case of chromophores (molecules that absorb light), can be acquired by absorbing a photon with an energy equal or slightly higher. This state is short-lived, and the electron returns to the ground-level orbital, losing the energy either as heat or in the case of fluorophores by emitting a photon, which, due to the loss of the difference between the energy of the absorbed photon and the excitation energy required, will have a lower energy than the absorbed photon, or, expressed in terms of wavelength, the emitted photon will have a longer wavelength. The difference between the two wavelengths is called Stokes' shift.
This property can be found in quantum dots, certain lanthanides and certain organic molecules with delocalized electrons.
These excited molecules have an increase in dipole momentum and in some cases can undergo internal charge rearrangement. When they possess an electron withdrawing group and an electron donating group at opposite ends of the resonance structure, they have a large shift in charge distribution across the molecule, which causes the solvent molecules to reorient to a less energetic arrangement, called solvent relaxation. By doing so, the energy of the excited state decreases, and the extent of the difference in energy depends on the polarity of the solvent surrounding the molecule.
An alternative approach is to use solvatochromic dyes, which change their properties (intensity, half-life, and excitation, and emission spectra), depending on the polarity and charge of their environments. Hence, they are sometimes loosely referred to as environmentally sensitive dyes. These can be positioned on specific residues that either change their spatial arrangement due to a conformational change induced by glucose or reside in the glucose-binding pocket whereby the displacement of the water present by glucose decreases the polarity.Lakowicz, J.R., Principles of Fluorescence Spectroscopy. 3rd ed. 2006, New York: Springer. xxvi, 954 p.
An additional property of fluorescence that has found a large usage is Förster resonance energy transfer () 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.
For sensing purposes, this property is, in general, used either in combination with a biomolecule, such as a protein, which undergoes a conformational change upon ligand binding, changing the distance between the two labels on this protein, or in a competition assay, in which the analyte has to compete with a known concentration of a fixed labelled ligand for the labelled binding site of protein. Therefore, the between the binding site and the competing ligand decreases when the analyte concentration is increased. In general, the competing ligand in the case of glucose is dextran, a long glucose polymer attached to the scaffolding or to the enzyme.
Förster resonance energy transfer
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 or on sensitivity to polarity changes to translate the glucose concentration into fluorescent intensity.
In addition to the fluorophores, these sensors contain a molecule that confers glucose specificity, usually a protein. A variety of proteins have been used for this purpose, often with different labs concentrating on one particular protein.
The first glucose biosensor reported in the literature was made in 1982 by Schultz's group using a competition assay between glucose and a labelled glucose polymer for the binding site of Concanavalin A entrapped in a hollow dialysis fibre. As a result, Con A was widely used in subsequent sensors in several labs,Russell, R.J., et al., A fluorescence-based glucose biosensor using concanavalin A and dextran encapsulated in a poly(ethylene glycol) hydrogel. Anal Chem, 1999. 71(15): p. 3126-32. however Con A suffers from the downside of high toxicity and low reversibility. As a result, other glucose-binding proteins were and are being explored by several labs.
In Biotex Inc. (Houston), McNichols and Ballastardt created a dialysis fibre-enclosed ConA sensor, which has undergone testing in animal models for several years.Dutt-Ballerstadt, R., et al., Preclinical in vivo study of a fluorescence affinity sensor for short-term continuous glucose monitoring in a small and large animal model. Diabetes Technol Ther, 2008. 10(6): p. 453-60.
Amperometric biosensors, in contrast, can utilise only glucose oxidase as a protein, as it is a redox enzyme. This protein has also been used in fluorescent sensing either simply as an apoenzyme or as a holoenzyme. An exception to this group of sensors is the Biocapacitor A Sode's group, which relies on glucose dehydrogenase instead.