Avidin Conjugates Research Articles

Glucose oxidase‐mediated polymerization as a platform for dual‐mode signal amplification and biodetection

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc.

Fabrication and Characterization of Planar Plasmonic Substrates with High Fluorescence Enhancement

The use of plasmonic nanostructures for fluorescence signal amplification is currently a very active research field. The detection of submonolayers of proteins labeled with organic dyes is a widely used technique in surface-based immunoassays and DNA hybridization. There is a strong interest in the development of new optical and chemical methods to increase the signal from ultralow concentrations of dyes on the surface of sensor substrates. Herein, we have explored the possibility of using vacuum-deposited silver nanostructures on dielectric layers and silver mirrors as potential plasmonic substrates that effectively amplify fluorescence over a broad spectral range. By optimizing deposition parameters for dielectric layers and silver nanostructures and applying thermal annealing processes, we observed large fluorescence amplifications from three different dye-strept(avidin) conjugates: about 7-fold for a UV/blue dye AF350-Av, 49-fold for a blue-green dye AF488-SA, and up to 208-fold for red-emitting AF647-SA dye. The observed amplification factors for the ensemble of fluorophores are very promising for development of surface-based bioassays. These substrates can be prepared using simple vacuum deposition in which we circumvent using the expensive nanofabrication methods. In addition, unlike most nanofabrication methods, the present approach is appropriate for large scale fabrication of substrates with microscope slide surface area suitable for sensing applications.

Iminobiotin Binding Induces Large Fluorescent Enhancements in Avidin and Streptavidin Fluorescent Conjugates and Exhibits Diverging pH-Dependent Binding Affinities

The pH-dependent binding affinity of either avidin or streptavidin for iminobiotin has been utilized in studies ranging from affinity binding chromatography to dynamic force spectroscopy. Regardless of which protein is used, the logarithmic dependence of the equilibrium dissociation constant (K(d)) on pH is assumed conserved. However a discrepancy has emerged from a number of studies which have shown the binding affinity of streptavidin for iminobiotin in solution to be unexpectedly low, with the K(d) at values usually associated with non-specific binding even at strongly basic pH levels. In this work we have utilized a Bodipy fluorescent conjugate of avidin and an Oregon Green fluorescent conjugate of streptavidin to determine the K(d) of the complexes in solution in the pH range of 7.0 to 10.7. The study was made possible by the remarkable fluorescent enhancement of the two fluorescent conjugates (greater than 10 fold) upon saturation with iminobiotin. The streptavidin-iminobiotin interaction exhibited almost no pH dependence over the range studied, with K(d) consistently on the order of 10(-5) M. In contrast, under identical experimental conditions the avidin-iminobiotin interaction exhibited the expected logarithmic dependence on pH. We discuss the possible origins for why these two closely related proteins would diverge in their binding affinities for iminobiotin as a function of pH.

Abstract LB-139: Laser scanning cytometry for the imaging and quantitation of membrane lipid rafts in cancer cells

Abstract The role of lipid trafficking in the physiological and patho-physiological processes is a subject of intense investigation. The enrichment of a large array of signal transduction components in caveolae and lipid rafts provide testimony to their role as signal portals. Similarly, the formation of intracellular lipid droplets, which were originally thought to be lipid storage organelles, now are known to play an important role in signaling and lipid trafficking. While these cellular components can be assessed using TPLC, mass spectrometry or western blotting, they can also be identified based on the staining of specific lip ids and proteins and analyzed by flow cytometry or using a microscope. We have employed fluorescent based techniques with the laser scanning cytometry (LSC), which allows quantification and localization of fluorescence signals as well as generating digital based images, to investigate lipid rafts and lipid droplets in 3 different cell culture models: 1) B cell lymphomas(Raji and DG75 cell lines) in which we previously reported mislocalization of the apoptosis adaptor protein APAF-1 in the lipid rafts. Nystatin, methyl-beta cyclodextrin and cytocholasin B were used to validate the labeling technique, 2) macrolipophagy, induced during autophagy, in the lung cancer cell line A549 and the cervical cancer cell line HeLa (ECACC) to evaluate the changes in lipid droplets accompanying the induction of autophagic vesicles, and 3) the formation of lipid droplets and raft structures in A549 cells following exposure to a panel of anti-cancer agents. To monitor these events, a sequential multi-colored labeling strategy was employed that allowed for the labeling of the plasma membrane using an antibody to pan-cadherin or by staining with Alexa Fluor 647 conjugated wheat germ agglutinin, prior to fixation, in combination with the labeling of lipid rafts using biotin cholera toxin B coupled with avidin conjugate Q dot 655 and antibodies to caveolin and the pro-apoptotic protein APAF-1. This labeling protocol was used in both cell cultures and in tissues. To detect the formation of lipid droplets, nile red and oil red were used to characterize the lipid content and where possible the number and localization of droplets. A threshold based segmentation strategy was employed that allows for the detection of changes to the total amount of lipid rafts, droplets, or vesicles, together with the digital maxpixel which permits monitoring of clustering or diffusion of events, and the correlation with the cell cycle. Using the profile and segmentation tools, the distance from the plasma membrane or nucleus was calculated and the localization of the events in the cytoplasm, nucleus and plasma membrane were determined using peripheral contouring and masking filters. Furthermore the digital generated images were processed using a set of image filters to generate a “3D” image of the cell or tissue giving a better resolution of events. Results provide a novel imaging strategy for the detection of lipid raft-like structures in cells and tissues, and given their importance in diverse biological systems and pathways could have potential clinical implications Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-139.

Delivery of NADPH-Cytochrome P450 Reductase Antisense Oligos Using Avidin−Biotin Approach

Although avidin-mediated intracellular delivery of oligonucleotides or proteins has been shown before, the efficacy studies are lacking. Here, we tested the effectiveness of avidin for delivery of a cytochrome P450 reductase (CPR) antisense oligo in rat liver epithelial cells. A phosphorodiamidate morpholino oligo (PMO) against CPR was biotinylated using four reagents with short, cleavable, or long linkers, followed by conjugation with avidin. The dose-inhibitory response of the unmodified PMO in the presence of a transfection reagent (Endoporter, EP) and the effectiveness of the EP-assisted and avidin-assisted delivery of biotinylated PMOs were tested by Western blot analysis. Additionally, in a preliminary study, the avidin-biotin PMO with a long linker was also tested in vivo in rats. The biotinylated oligos were at least as effective as the unmodified oligo. Whereas the avidin conjugate of biotinylated PMO with the short linker was ineffective, those with the long linkers showed significant reductions in CPR protein expression. Finally, the in vivo study showed modest, but significant, reductions in CPR activity. In conclusion, these studies show for the first time that avidin-mediated intracellular delivery of biotinylated oligos can effectively knock down target genes in vitro, depending on the length of the linker. Additionally, the avidin-biotin approach may be of potential value for in vivo gene knockdown.

Detection of the Ribosomal RNA Gene in Pear (Pyrus spp.) using Fluorescence in situ Hybridization

The locations of the 18S-5.8S-25S ribosomal RNA gene (rDNA) in the seedlings of Japanese pear [Pyrus pyrifolia (Burm. F.) Nakai] ‘Osa Gold’ and European pear (P. communis L.) ‘La France’ and the 18S ribosomal rDNA in ‘Osa Gold’ were determined using fluorescence in situ hybridization (FISH). 18S-5.8S-25S and 18S rDNA probes were labeled with biotin-16-dUTP. The probes were detected using a fluorescein isothiocyanate (FITC)-avidin conjugate with chromosomes counterstained with propidium iodide (PI). The 18S-5.8S-25S and 18S rDNA sites of ‘Osa Gold’ and the former of ‘La France’ were detected in telomeric positions of six chromosomes. The number and positions of rDNA sites were stable.

Enzyme‐Amplified Amperometric Detection of DNA Using Redox Mediating Films on Gold Microelectrodes

AbstractHere we describe an amperometric assay for enzyme‐labeled detection of DNA hybridization based on a redox polymer film, cross‐linked and co‐immobilized, with a 20‐mer oligonucleotide, to pre‐adsorbed cysteamine on gold microelectrode surfaces. Hybridization between the immobilized probe DNA and a biotin‐modified target DNA (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)‐avidin conjugate and glucose, results in bioelectrocatalytic oxidation of glucose mediated by the redox polymer, generating significant amplification. The use of gold microelectrodes (25 μm, 40 μm, 100 μm diameter), coupled to careful preparation of surfaces, electronic shielding and background subtraction, results in improved analytical performance, compared to that at macroelectrodes, yielding a limit of quantification of ca. 0.6 pM, corresponding to the presence of ca. 2.5 million copies in the 7 μL assay droplet.

Physical Mapping of the 5S Ribosomal RNA Gene in Citreae of Aurantioideae Species using Fluorescence in situ Hybridization

The location of the 5S ribosomal RNA gene (rDNA) in species from six genera of the Citreae of Aurantioideae was determined using fluorescence in situ hybridization (FISH). A 5S rDNA probe was labeled with biotin-16-dUTP. The probe was detected using a fluorescein isothiocyanate (FITC)-avidin conjugate with chromosomes counterstained with propidium iodide (PI). When the chromosomes were observed under a G filter, PI-stained chromosomes were classified into the following five types based on the number and position of PI-positive (+) bands; B: one telomeric and one proximal band, C: two telomeric bands, D: one telomeric band, E: without bands and Dst: type D with a satellite chromosome. Two 5S rDNA sites were located in type D chromosomes in Citropsis gabunensis, whereas in Citrus reshni, Fortunella japonica, Clymenia polyandra and Swinglea glutinosa they were found in type E chromosomes. In Poncirus trifoliata, three rDNA sites were located together at the proximal PI (+) band of a type B chromosome, one site was located in the proximal region of a type D chromosome, and two sites were located together at the PI (+) band of a type D chromosome.

Fluorescent avidin-bound silver particle: a strategy for single target molecule detection on a cell membrane.

Cy5-avidin conjugate-bound silver nanoparticles were prepared as a fluorescence molecular reagent for the cell imaging. Compared with the metal-free avidin conjugate, the avidin-metal complex was observed to display a stronger emission intensity, shorter lifetime, and better photostability. The avidin-metal complexes were conjugated with the biotin-sites on the surfaces of PM1 cell lines, and the cell images were recorded using scanning confocal microscopy. It was noticed that the avidin-metal complexes bound on the cell surfaces could be identified as the isolated emission spots distinct from the cellular autofluorescence. The emission intensity over the cell image was increased with an increase of the number of avidin-metal complexes on the cell surface but the lifetime was decreased. A quantitative regression curve was achieved between the amount of avidin-metal complex on the cell surface and the emission intensity or lifetime over the entire cell image. On the basis of this curve, we expect to develop an approach that can be used to quantify the amount of target molecules on the cell surfaces using the cell intensity and lifetime images at the single cell level.

Ligase-based multiple DNA analysis by using an electrochemical sensor array

We herein report an electrochemical biosensor for the sequence-specific detection of DNA with high discrimination ability for single-nucleotide polymorphisms (SNPs). This DNA sensor was constructed by a pair of flanking probes that “sandwiched” the target. A 16-electrode electrochemical sensor array was employed, each having one individual DNA capture probe immobilized at gold electrodes via gold-thiol chemistry. By coupling with a biotin-tagged detection probe, we were able to detect multiple DNA targets with a single array. In order to realize SNP detection, a ligase-based approach was employed. In this method, both the capture probe and the detection probe were in tandem upon being hybridized with the target. Importantly, we employed a ligase that specifically could ligate tandem sequences only in the absence of mismatches. As a result, when both probes were complementary to the target, they were ligated in the presence of the ligase, thus being retained at the surface during the subsequent stringent washing steps. In contrast, if there existed 1-base mismatch, which could be efficiently recognized by the ligase, the detection probe was not ligated and subsequently washed away. A conjugate of avidin–horseradish peroxidase was then attached to the biotin label at the end of the detection probe via the biotin–avidin bridge. We then electrochemically interrogated the electrical current for the peroxidase-catalyzed reduction of hydrogen peroxide. We demonstrated that the electrochemical signal for the wild-type DNA was significantly larger than that for the sequence harboring the SNP.

Avidin conjugation to up-conversion phosphor NaYF4:Yb3+, Er3+ by the oxidation of the oligosaccharide chains

NaYF4:Yb3+, Er3+ nanoparticles were successfully prepared by a polyol process using diethyleneglycol (DEG) as solvent. After being functionalized with SiO2–NH2 layer, these NaYF4:Yb3+, Er3+ nanoparticles can conjugate with activated avidin molecules (activated by the oxidation of the oligosaccharide chain). The as-formed NaYF4:Yb3+, Er3+ nanoparticles, NaYF4:Yb3+, Er3+ nanoparticles functionalized with amino groups, avidin conjugated amino-functionalized NaYF4:Yb3+, Er3+ nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR), UV/Vis absorption spectra, and up-conversion luminescence spectra, respectively. The biofunctionalization of the NaYF4:Yb3+, Er3+ nanoparticles has less effect on their luminescence properties, i.e., they still show the up-conversion emission (from Er3+, with 4S3/2 → 4I15/2 at ~540 nm and 4F9/2 → 4I15/2 at ~653 nm), indicative of the great potential for these NaYF4:Yb3+, Er3+ nanoparticles to be used as fluorescence probes for biological system.

Colorimetric Detection of Anthrax DNA with a Peptide Nucleic Acid Sandwich-Hybridization Assay

Technologies for genomic detection most commonly use DNA probes to hybridize to target sequences. To achieve required sensitivity, the use of PCR to amplify target sequences has remained standard practice in many labs. Direct detection methods that eliminate the requirement for a PCR step could afford faster and simpler devices that can be used outside of a laboratory. We have developed a sandwich-hybridization DNA detection system in which the DNA detection probes are replaced with a class of synthetic nucleic acid mimics called peptide nucleic acids (PNAs). There are numerous advantages to using PNA instead of DNA probes in hybridization assays, including complete resistance to degradation by enzymes, increased sequence specificity to complementary DNA, and higher stability when bound with complementary DNA. In this communication, several different strategies are described to improve and fine-tune the properties of PNA so that anthrax DNA can be detected at a 10 zmol limit. Key to the success of these strategies is the ability to introduce chemical modifications into the PNA to improve thermal stability and to increase the number of biotin groups to which a horseradish peroxidase−avidin conjugate can bind.

Redox Polymer and Probe DNA Tethered to Gold Electrodes for Enzyme-Amplified Amperometric Detection of DNA Hybridization

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.

Cell Transduction Pathways of Transportans

Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 mum diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportan-streptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportan-protein complexes is temperature dependent and strongly inhibited at 8-10 degrees C and blocked at 4 degrees C when only interaction with the plasma membrane takes place.

A biotin–protein bond with stability in plasma

A nonradioactive label for peptide hormones would be useful for pharmacokinetic studies in infants, children, and pregnant women. Because the binding affinity between biotin and avidin is large ( K a = 10 15 M −1), biotin could also serve as a covalent label for subsequent detection using a variety of avidin conjugates. However, biotin labels produced by most commercially available biotinylating reagents are rapidly cleaved from protein in plasma. We sought to synthesize a stable biotin label for protein. With the use of immunoglobulin G (IgG) as a model protein, biotin was conjugated through a cysteine residue; a carboxylate group was positioned alpha to the biotinamide bond. Stability of the bond in the presence of plasma and buffer control was assessed by release of biotin. Released biotin was separated from biotinylated IgG by ultrafiltration and was quantitated by an avidin-binding assay. In plasma, less than 0.6% of bound biotin was released. This release rate is not significantly different from buffer and is less than 7% of the release rate for IgG biotinylated by N-hydroxysuccinimide-LC-biotin. We conclude that this biotin–protein bond is stable in plasma. We speculate that many uses of avidin–biotin technology could be improved by using this method for protein labeling.

Labeled avidin bound to water-soluble nanocrystals by electrostatic interactions

Semiconducting nanocrystals of three different sizes capped with 3-mercaptopropionic acid were synthesized in aqueous solutions. They can efficiently bind to an avidin biomolecule by the electrostatic attraction. The conjugation of avidin leads to a red shift and a decrease in the intensity of the fluorescence emission spectra of the nanocrystals. Moreover, the red shift of the fluorescence spectra of the bioconjugates depends strongly on the pH, ionic strength, quantity of avidin, and nanocrystal size.

Biofunctionalization of Fluorescent Rare‐Earth‐Doped Lanthanum Phosphate Colloidal Nanoparticles

Approximately eight avidin molecules are conjugated to each Ce/Tb-doped lanthanum phosphate nanoparticle (NP) through the formation of amide bonds (see scheme). This biofunctionalization process comprises modification of the LaPO4 NPs with 6-aminohexanoic acid (AHA) to confer colloidal stability, activation of the AHA carboxy groups by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, and avidin conjugation.

Neurotransmitter analog tethered to a silicon platform for neuro-BioMEMS applications.

The design of chemically well-defined, machinable surfaces containing neuroactive molecules offers potential for fundamental neuroscience and clinical neural engineering applications. Here we report the assembly and characterization of silicon platforms containing a tethered form of muscimol. Muscimol, an analog of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), is a potent agonist at postsynaptic GABA(A) and GABA(C) receptors. Surfaces were assembled using covalent avidin conjugation to silanized silicon followed by high-affinity avidin-biotin binding of a biotinylated derivative of muscimol (muscimol-biotin). Contact angle measurements, ellipsometry, and X-ray photoelectron spectroscopy (XPS) were conducted to characterize the wettability, thickness, and chemical composition of progressively deposited surface layers. The data demonstrate successful incorporation of a neurotransmitter analog as part of a layered, silicon-based structure possessing robust and specific biomolecular composition. These findings represent a step toward the design of platforms for applications involving control and modulation of neural signaling.

Parasol cells of the hemiellipsoid body in the crayfish Procambarus clarkii: dendritic branching patterns and functional implications.

Multimodal, higher-order sensory integration in decapod crustaceans occurs in local interneurons (parasol cells) within a structure in the lateral protocerebrum, the hemiellipsoid body, which is located dorsal to the terminal medulla. The hemiellipsoid body is targeted by projection neuron inputs by means of the olfactory globular tract from bilateral deutocerebral neuropils, the accessory lobes, which receive secondary visual, mechanosensory, and olfactory inputs. Parasol cell dendrites arborize extensively within the two neuropils of the hemiellipsoid body and possibly have some neurites within another neuropil at its base. The two neuropils of the hemiellipsoid body, neuropils I and II, are known to receive asymmetrical inputs from the contralateral and ipsilateral accessory lobes, and our current study addresses the question of the distribution of parasol cells within these two neuropils. Three anatomic methods were used to analyze this distribution: intracellular filling of cells with neurobiotin and visualization of the cells by using either a fluorescent or a peroxidase avidin conjugate, or placement of a fluorescent lipophilic tracer within a lobe of the hemiellipsoid body. All of these methods demonstrated that single parasol cells exclusively arborize within one of the two lobes of the hemiellipsoid body, but not in both lobes. Electrophysiological recordings from pairs of parasol cells with dendrites in the same or different lobes confirm a functional separation between neuropils I and II. Comparisons are made between insect and crustacean systems, emphasizing the inputs to the hemiellipsoid body and the mushroom body and similarities between extrinsic cells in insects and parasol cells in decapod crustaceans.

Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase–avidin conjugate

A new amperometric DNA sensor was constructed using a pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH) conjugated with avidin. Our aim was to specifically detect the DNA sequence of the invA virulence gene from the pathogenic bacterium Salmonella. Probe DNA with a sequence complementary to that of a specific fragment of the invA gene was immobilized onto a carbon paste electrode. After hybridization with biotinylated target DNA, (PQQ)GDH–avidin conjugate was added and the resulting electric current was measured. The electric current is generated from glucose oxidization catalyzed by (PQQ)GDH via 1-methoxyphenazine methosulphate (m-PMS) electron mediator. The sensor response increased with the addition of glucose and in the presence of 6.3 mM glucose the response increased with increasing DNA in the range 5.0×10 −8–1.0×10 −5 M.