range close to pathophysiological glucose have been created. ggbp contains five tryptophan residues, two of which, w183 in the binding site and w284 in the n-terminal domain (which can bind calcium), affect the autofluorescence spectra upon glucose binding. some studies with ggbp and solvatochromic dyes work not to create a sensor, but to elucidate the chemistry behind the conformational change of ggbp. examples of this include a study utilizing l255c with acrylodan and ruthenium at the n-terminus revealing three conformational states closed and twisted, the fluorescence and phosphorescence of the tryptophan w183 under normal conditions , under high pressure and with or without calcium. 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). the response of an environment sensitive dye attached to a specific residue of ggbp depends not only on the labelling site, which has a certain environment, but also on the nature of the dye, which interacts differently depending on its geometry. the interaction between a given dye and its environment is hard to predict in silico. as a result, in order to obtain a working sensor, several independent studies have screened a set of environmentally sensitive dyes attached to several possible sites in the binding pocket (endosteric site), near it (peristeric site) or away from it (allosteric site). one advantage of ggbp is that in the wild-type there are no cysteine residues, making the introduction of this residue in a specific location ideal for labelling. a team led by homme hellinga conducted two large screens. in the first (2002) they made of a series (320 constructs) of labelled mutants of 11 bacterial periplasmic binding protein including ggbp for which they made nine mutants introducing a cysteine in a specific spot (y10c, n15c, e93c, e149c, h152c, w183c, l255c, d257c, v296c) and tested the response when labelled with one out of eight dyes (pyrene (340, 390); acrylodan (390, 500); fluorescein (485, 520); nbd (490, 540); nbde (490, 530); jpw4039 (485, 590); jpw4042 (470, 640); and jpw4045 (470, 640)). out of the 72 combinations made, ggbp labelled with acrylodan in position w183c had a fivefold change and kd=5mm. 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. 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. a very different approach was taken by pitner in bd, who used a single dye (ianbd) attached to e149c as a starting point for a directed evolution screen, in which a mutant library is created and selected for "winners", namely mutants that meet the selection criteria. with this approach they identified e149c/a213r/l238s with kd of 10mm and an eightfold increase in fluorescence; this mutant was later used for spr. independently another group (j pickup) tested two mutants (h152c and m182c) labelled with badan (6-bromoacetyl-2-dimethylaminonaphtalene), bound to the thiol group of the cisteine introduced at site 152 (h152c mutant). 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), 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). tissue autofluorescence another pair of papers suggest that the natural fluorescence (autofluorescence) of tissues may be harnessed to track glucose concentrations. these studies took advantage of the fact that nad(p)h, in its reduced form, is autofluorescent, and that metabolites such as glucose cause a predictable increase in nad(p)h reduction. sensing in vivo an alternative way of measuring the change in environment of environmental dyes is their change in lifetime, which may give better results in some sensors, using lanthanides or e.g. the aforementioned ruthenium (ru) metal-ligand complex, either with gox or as an fret acceptor of an environment sensitive dye as in the case of ans26-ggbp in a ruthenium-coated cuvette that shows little increase in intensity but a substantial change in lifetime. the construction of the fluorescent protein is only one subsystem of a clinically viable monitoring device: the sensing protein has to be immobilized and its fluorescence has to be read by a detecting subsystem that, in turn, informs the user. 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. the general approach for fluorescent sensors is to attach the protein to one extremity of an optic fibre implanted under the skin whilst the other extremity is connected to the detection subsystem, which includes a path splitter (sliced fibre or dichroic mirror) to allow the fibre to transmit both the excitation and the emitted light, a filtered light source (in general, a laser), and a filtered photodetector (a ccd or a pmt). the information thus collected is then analysed with a computer. smart tattoo the skin is permeable to near-infrared light (nir). as a consequence, near-infrared dyes can be measured across the skin without the need of an optic fibre; this has been termed a "smart tattoo" by mcshane who created a near-infrared oxygen quenching assay contained in microspheres. however, there is a limited amount of commercially available fluorescent dyes, and a limited amount of environmentally sensitive dyes, such as cyanine cy7. as a consequence, pitner made a reactive nile red dye, but to date no study with a nile red-ggbp sensor has been conducted. nevertheless, several studies with nir dyes have been done. pickup and birch made a nir fret sensor measuring both the time-resolved counts or by nanotomography of allophycocyanin-cona/malachite green-dextran, where allophycocyanin is a nir fluorescent protein. in another study, the autofluorescence of naph, an energy carrier in cells, was assessed as an indirect indicator. a group at biotex inc, led by mcnichols and ballerstadt, created a nir fret sensor based on cona, with nir dyes alexa 647 and alexa 750 (originally alexa 647 & cy7) enclosed in a dialysis fibre attached to the end of an optic fibre, which they have dubbed "fas" (fluorescent). to improve the stability they attached the protein to a sephadex, a macroporous hydrogel. 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. draper laboratory are also developing a smart tattoo, and are currently testing on animals. the performance and the identity of the sensor have not been revealed. encapsulation in dialysis membranes despite the higher benefit of smart tattoos compared to a transdermal optic fibre, no in vivo smart tattoo has yet been demonstrated, whereas fibre-based systems have been shown to be potential sensors. the majority of the sensors mentioned in the previous sections consisted of labelled proteins in solution. the only sensors to progress towards an implantable sensor have been either gox–ruthenium oxygen-quenching assay sensors or fret competition assay sensors; to date, no environment sensitive dye-based sensors attached at the end of a fibre has been published. for fibre-based biosensors to work, the protein must be immobilized to the fibre that can either entrapped in a hollow tube made of dialysis membrane or entrapped in a hydrogel. a hollow dialysis tube is a tube with sub-millimetre diameter whose walls are composed of porous crosslinked cellulose designed to allow small solutes through but not large biomolecules, such as protein with a cut-off ranging from 0.5 to 20 kda. as a consequence, they are well suited for sensory applications, where the analyte is free to diffuse across whilst proteins cannot, both the sensor protein inside and the blood/interstitial tissue proteases. 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. encapsulation in a hydrogel 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, 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). after, dr. ballastardt joined biotex as a senior scientist under dr roger mcnichols, the chief scientist, where for the past seven years they have been testing the previously mentioned fas sensor, which used the same fret system in a dialysis tube. to be specific, the labelled protein was loaded with a p10 tip into a dialysis tube 200 μm wide that had been sealed with cyanoacrylate (superglue) at one end with or without an optic fibre end inserted inside. in the field of sensors of analytes, glucose sensors have been at the forefront due to the large amount of research into glucose sensors as a result of the prevalence of diabetes, nevertheless, a wide breadth of optic fibre-based biosensors, mainly using enzymes, immunoassays, nucleic acids, whole cells or biomimetic materials and relying on different detection methods (fluorescence, absorbance, chemiluminescence, and scattering), and attachment methods (coating, hydrogels, or membranes). the majority of these sensors, however, rely on entrapping the protein in hydrogels, as these are more sturdy and protect the protein more than a simple coating or membrane. a hydrogel is a porous crosslinked polymer matrix filled with water. several types of hydrogel exist and have been used to entrap small molecules such as dyes, biomolecules, such as enzymes or whole cells. in the case of protein, they can work either by physically entrapping the protein having pores smaller than the proteins or by chemical linkage of the protein to the matrix. in physically entrapping gels, the protein has to be added when the gel is crosslinked, so the conditions used must not damage the protein, excluding the hydrogel, which requires non-aqueous solvents or harsh chemicals, an example being temed-persulphate-catalysed (peroxide radical initiation) acrylamide or acrylate, which is used for sds page but for not protein encapsulation. hydrogels have been extensively studied, mainly in the entrapment of small molecules for drug delivery, including cases where the hydrogel nanoparticles slowly release the drug to a targeted site. 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), p(biscarboxy-phenoxy-phosphazene), p(gema-sulfate), and others), or a hybrid of the two. in addition to organic hydrogels there are sol-gels, which are oxygen-bridged silicates (or titanium oxide), that polymerise in water. an additional classification can be by method of polymerization, which can be physical (freezing or heating) or chemical (γ-ray, oxygen or photo-induced radical polymerization in the case of acrylates, vinyls and acrylamides). 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) or alternative monomers, such as glycol-modified tetraethoxysilane as it is more biocompatible than the commonly used methoxy- or ethoxy-modified tetraethoxysilane. fibers with hydrogel regarding fibre-optic-based biosensors, several hydrogels have been used but mainly acrylate-based polymers and sol-gels, either by chemical or physical entrapment. in the case of acetylcholinesterase, the target of many pesticides, a sensors has been made chemically linking the enzyme to an acrylate hydrogel or physically entrapping the enzyme in solgel. an optic-fibre-based hydrogel-entrapped biosensor for glucose was made in the lab of loeb (liao and colleagues) and was named sencil. this sensor was composed