Glucose Probes

Immunoassays constitute a large and diverse family of assays, some of which are based on the principles described above, whereas others are based on unique princi- 

ELISA assays exist in a number of formats. In some cases the reaction product absorbs light, and in other cases the product is strongly fluorescent. The second antdxidy is not always labeled with enzyme but rather is sometimes detected with yet another antibody that contains the bound enzyme. This procedure eliinioates the need to attach probe or enzyme to a specific antibody which may be in short supply. 

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G. Palleschi, M.A. Nabi Rahni, G.J. Lubrano, J.N. Nwainbi and G.G. Guilbault, A study of interferences in glucose measurements in blood by hydrogen peroxide based glucose probes, Anal. Biochem., 159 (1986) 114-121. 

Optodes provided with non-fluorescent esters of fluorophores have been used for the determination of external enzyme activities. The fluorophores are liberated by the enzymes and then seen by the optical Ober [214], As ecamples of p(02)-modulated optical biosensors, a glucose probe [213] and an ethanol probe [216] can be mentioned sensors based on glucose, alcohol, and other oxidases were reviewed by Opitz and Lttbbers [217]. The advantages of these 02-dependent optical biosensors are that, unlike corresponding amperometric sensors, they do not consume O2 and that they are strictly diffusion limited in their response. Fiber-optical devices are also available for the determination of substrates of dehydrogenases the NADH fluorescence produced by the immobilized enzyme is measured as a function of time [218, 219]. 

How can the princiides of analyte recognition be used to develop a fluorophore which is sensitive to glucose An itUrinsic glucose probe would be simpler and more stable than a sensor based [Pg.559]

Amine A, Cremisini C, Palleschi G (1995) Determination ofmercury(ll), methyl-mercury and ethyhnercury in the ng/ml range with an electrochcanical enzyme glucose probe. Microchim Acta 121 183-190... 

Biosensors (qv) and DNA probes ate relatively new to the field of diagnostic reagents. Additionally, a neat-infrared (nit) monitoring method (see Infrared TECHNOLOGY AND RAMAN SPECTROSCOPY), a teagenfless, noninvasive system, is under investigation. However, prospects for a nit detection method for glucose and other analytes ate uncertain. 

FIGURE 6-4 Schematic of a first-generation glucose biosensor (based on a probe manufactured by YSI Inc.). 

Several simple deoxyfluoro sugars and glycosyl fluorides were utilized " as probes for hydrogen bonding in the glycogen phosphorylase-o-glucose complex. 

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

Fig. 2. Northern analysis of pgaX expression using a 2.0 kb pgaX Pst fragment as a probe. A. tubingensis was pregrown for 20 h on 1% sucrose and transferred to medium with different carbon sources. Lane 1 1% glucose lane 2 2% glucose lane 3 1% galacturonic acid lane 4 1% galacturonic acid, 1% glucose.

Figure 5. 1% agarose gel showing PCR amplification products for PG cDNA obtained from mRNA isolate. Lanes 1, 2, 4 and 6 apple pectin culture medium, lanes 3, 5 and 7 glucose culture medium, lanes 1, 2 and 3 to isolate r,3 and lanes 4, 5, 6 and 7 to isolate rz. Lane 8 corresponds to the amplification of the 742 pb probe cloned in the vector PCR TM (In-vitrogen) digested with Eco R1 and lane 9 A digested with Pstl. 

Petersons pH probe also was modified in order to give a miniature fiber optic sensor potentially suitable for glucose measurements90. Kopelman et al.91 developed a fiber-optic pH nanosensor for physiological measurements using a dual-emission sensitive dye. The performance of a pH sensor was reported92. An unclad fiber was dip-coated with a thin layer of porous cladding within which a pH-sensitive dye was entrapped. The fundamental... 

Narayanaswamy R., Sevilla F., An optical fiber probe for the determination of glucose based on immobilized glucose dehydrogenase, Anal. Lett. 1988 21 1165. 

Nicolas, J.-C., Balaguer, R, Terouanne, B., Villebrun, M.A., and Boussioux, A.-M. (1992) Detection of glucose 6-phosphate dehydrogenase by bioluminescence. In Nonisotopic DNA Probe Techniques (L.J. Kricka, ed.), p. 207. Academic Press, New York. 

P. Audebert, C. Demaille, and C. Sanchez, Electrochemical probing of die activity of glucose oxidase embedded sol-gel matrixes. Chem. Mater. 5, 911—913 (1993). 

The decrease of the reduction current has been employed to determine the glucose concentration and probe the enzyme activity of GOD. The peak current responds linearly to the glucose concentration ranging from 0.5 to ll.lmM (r = 0.998), with a sensitivity of 7.0 pA mM"1 and the estimated detection limit of 0.05mM at a signal-to-noise ratio of 3. The Zmapp is equal to 5.1 mM. 

The application of near-IR spectroscopy for real-time monitoring of glucose, lactic acid, acetic acid and biomass in liquid cultures of microorganisms of the genera Lactobacillus and Staphylococcus has been recently published [76]. The NIR spectrum acquired by the optical-fibre probe immersed in the culture is exploited using a partial least squares (PLS) calibration step, a classical method for IR techniques. 

The design of fluorescent sensors is of major importance because of the high demand in analytical chemistry, clinical biochemistry, medicine, the environment, etc. Numerous chemical and biochemical analytes can be detected by fluorescence methods cations (H+, Li+, Na+, K+, Ca2+, Mg2+, Zn2+, Pb2+, Al3+, Cd2+, etc.), anions (halide ions, citrates, carboxylates, phosphates, ATP, etc.), neutral molecules (sugars, e.g. glucose, etc.) and gases (O2, CO2, NO, etc.). There is already a wide choice of fluorescent molecular sensors for particular applications and many of them are commercially available. However, there is still a need for sensors with improved selectivity and minimum perturbation of the microenvironment to be probed. Moreover, there is the potential for progress in the development of fluorescent sensors for biochemical analytes (amino acids, coenzymes, carbohydrates, nucleosides, nucleotides, etc.). 

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