fluorescent glucose biosensor fluorescent glucose biosensor 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 main article: diabetes management keeping glucose levels in check is crucial to minimize the onset of the damage caused by diabetes. 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. various strategies exist to detect glucose levels using fluorescence, the first and most common being a fret competition assay between glucose and a labelled glucose polymer for the binding site of concanavalin a. 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 fret pair or by using environment sensitive (solvatochromic) dyes in a variety of combinations, the fluorescent small molecule, protein or quantum dot have been used in conjunction with a glucose binding moiety either a boronic acid functionalized fluorophore or a protein, such as glucose oxidase, concanavalin a, glucose/galactose-binding protein, glucose dehydrogenase and glucokinase. in general, the change seen with fret competition assays is small (see below). theory of fluorescence absorption and emission spectra of fluorescein 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. 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. 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 fret 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 cartoon of fret between two protein interacting protein, labelled with fluorescein and tetramethylrhodamine 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 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 fret 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, 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 fret sensor, which has undergone testing in animal models for several years. 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. the activity of glucose oxidase has also been used to make lifetime-based fluorescent/phosphorescent sensors, taking advantage of the fact that the protein oxidises glucose, utilizing molecular oxygen, and that oxygen quenches the fluorescence of ruthenium. this was done by uwira and colleagues in 1984 and followed by several groups. to be specific, endo and pasic 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)), but then switching to gox apoenzyme in 2004 (tritc-apo-gox /fitc-dextran (500kda)), and in 2009 testing sensors (qsy-21-apo-gox /alexa647- dextran) in microspheres. several other groups have constructed smart tattoos and are reviewed below. 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. the sensor was constructed by functionalizing an oxygen sensor with glucose oxidase and inserting it into the external part of a catheter used for monitoring. apoenzymes can still bind glucose but, due to the lack of cofactors (in vitro), cannot catalyse their reaction so are less likely to get damaged. other proteins that have been used are glucokinase from a thermophile in d'auria group and glucose-galactose-binding protein (ggbp), which is not an enzyme but a periplasmic protein involved in chemotaxis that undergoes a large conformational change. the majority of the fluorophores used for the sensors are small molecules, although some sensors have been made using quantum dots (qd) or fluorescent protein. sensors have been made using qd as fret donors and a small molecule or gold nanoparticle (dark quencher) as acceptors. an example of the former is loeb's sensil, an optic fibre system in which the quantum dot is attached to cona whilst tetramethylrhodamine is attached to cyclodextran, which in turn is attached to the peg diacrylate scaffold. an example of the latter is tang with qds-cona-beta-cds-aunps. fluorescent protein can be made into a fusion protein with a desired protein, circumventing the labelling steps. shultz made a ggbp molecule with two gfp at each end. it has not been reported in the literature, but in theory it is possible to improve this by doing a directed in vitro evolution using facs. this is not easily done by labelling, although a screening has been attempted by pitner. fluorescence is not the only type of luminescence achievable in biological systems: chemiluminescence, the generation of light by means of chemical reactions, is produced by some proteins, such as aqueorin from symbiont in jellyfish and luciferase from symbiont in fireflies. these have been used to make glucose sensors: daunert makes a ggbp-split aqueorin sensor and in 2009 koji sode made ggbp-luciferase with asp459asn (glc not gal). in addition to small-molecule dyes, fluorescent proteins have been used: one group made a near-infrared (nir) fret sensor detected by means of time-resolved/nanotomography allophycocyanin-cona/malachite green-dextran, regarding fret with allophycocyanin, which maccoll has reviewed. in addition to protein as the glucose-binding moiety, boronic acid functionalized molecules have been used. boronic acid binds to vicinal groups, preferably hydroxyl; therefore, it has a high affinity for carbohydrates. the use of the boronic acid group for the recognition of saccharide has been widely studied by shinkai, james and their collaborators. to take advantage of this several approaches have been taken. one approach is by fret quenching, in which the system can work through the modulation of the quenching of a dye by a boronic acid functionalized viologen. an alternative approach is by photo-induced electron transfer (pet), a mechanism of fluorescence-quenching due to the electron-rich tertiary amino group near the fluorophore, which is affected by the change in charge of the nearby boronate group when glucose is bound. this has been used in combination with lifetime by one group., not only in fluorescence but as nmr agent for imaging with a europium (3+) boronic acid dye. environment sensing dyes example of an environmentally sensitive dye, badan, which having a tertiary amine and a ketone at opposite ends displays a large change in dipole momentum when excited (including an internal charge transfer), which results in turn in a significant lowering of the energy when solvent relaxation occurs the majority of the sensors adopting environmentally sensitive dyes have utilized ggbp, a transport protein that binds to d-glucose and d-galactose and transports them to the membrane-bound trg receptor triggering the chemotaxis of the bacterium towards that glucose source. it belongs to the malg family in escherichia coli, which includes the maltose-binding protein, which, depending on the presence of glucose, can adopt two distinct conformations or possibly three generating a 31° hinge movement between the two globular domains connected by a hinge, which is the glucose-binding pocket. its affinity for glucose is k= 0.2 μm, which is much lower than the pathophysiological range of glucose found in diabetes (1.7-33 mm). as a consequence, several studies have been done to lower the affinity of ggbp, which otherwise would result in near-saturation of ggbp throughout the pathophysiological glucose concentrations. the binding affinity of ggbp changes when it is labelled endosterically or peristerically, so several mutants that work at