Glucose Oxidase Conjugates Research Articles

A platinum nanourchin-based multi-enzymatic platform to disrupt mitochondrial function assisted by modulating the intracellular H2O2 homeostasis

Endogenous H2O2 sacrifices for diversified therapeutic reactions against tumor. However, the treatment outcome is not always satisfactory owing to the unsustainable H2O2 supply from tumor microenvironment (TME). Herein, a platinum (Pt) nanourchin-based multi-enzymatic platform (referred to PGMA) is established by surface conjugation of glucose oxidase (GOx) capped with manganese carbonyl (MnCO) and loading 3-amino-1,2,4-triazole (3-AT). The mild acidic and H2O2-rich TME can render the degradation of MnCO, followed by triggering the release of CO gas, 3-AT and Mn2+/3+. The resultant GOx exposure initiates intratumoral glucose depletion, which is promoted by the O2 replenishment through Pt-catalyzed decomposition of H2O2. Meanwhile, intracellular reactive oxygen species (ROS) level is elevated through Mn2+/3+ couple-mediated Fenton-like reaction. Hence, CO release-initiated gas therapy, glucose exhaustion-induced tumor starvation and ROS-triggered chemodynamic therapy are committed to realizing a combinatorial disruption effect on mitochondrial function. Importantly, the released 3-AT can inhibit the activity of endogenous catalase, which effectively elevates the intracellular H2O2 level to compensate its consumption and provides incremental reactant for cascade utilizations. Taken together, this study aims to emphasize the importance of intracellular H2O2 balance during H2O2-depleted therapeutic process, and affords a prime paradigm of applying this strategy for tumor treatment via mitochondrial dysfunction.

Fusiform-Like Copper(II)-Based Metal-Organic Framework through Relief Hypoxia and GSH-Depletion Co-Enhanced Starvation and Chemodynamic Synergetic Cancer Therapy.

The therapeutic effect of traditional chemodynamic therapy (CDT) agents is severely restricted by their weakly acidic pH and glutathione (GSH) overexpression in the tumor microenvironment. Here, fusiform-like copper(II)-based tetrakis(4-carboxy phenyl)porphyrin (TCPP) nanoscale metal-organic frameworks (nMOFs) were designed and constructed for the first time (named PCN-224(Cu)-GOD@MnO2). The coated MnO2 layer can not only avoid conjugation of glucose oxidase (GOD) to damage normal cells but also catalyzes the generation of O2 from H2O2 to enhance the oxidation of glucose (Glu) by GOD, which also provides abundant H2O2 for the subsequent Cu+-based Fenton-like reaction. Meanwhile, the Cu2+ chelated to the TCPP ligand is converted to Cu+ by the excess GSH in the tumor, which reduces the tumor antioxidant activity to improve the CDT effect. Next, the Cu+ reacts with the plentiful H2O2 by enzyme catalysis to produce a toxic hydroxyl radical (•OH), and singlet oxygen (1O2) is synchronously generated from combination with Cu+, O2, and H2O via the Russell mechanism. Furthermore, the nanoplatform can be used for both TCPP-based in vivo fluorescence imaging and Mn2+-induced T1-weighted magnetic resonance imaging. In conclusion, fusiform-like PCN-224(Cu)-GOD@MnO2 nMOFs facilitate the therapeutic efficiency of chemodynamic and starvation therapy via combination with relief hypoxia and GSH depletion after acting as an accurate imaging guide.

Conjugation of glucose oxidase and bilirubin oxidase bioelectrodes as biofuel cell in a finger-powered microfluidic platform

In this study the development of a biofuel cell for potential application in microfluidics is described. The biocatalysts support is produced through vacuum-filtration of a Vulcan carbon black suspension resulting in flexible, paper-like electrodes. The glucose oxidase bioanode was assembled using an enzyme precipitate coating procedure whereas the biocathode was implemented by attaching bilirubin oxidase enzyme onto the carbon black surface via a pyrene intermediate. The bioelectrodes were first characterized by voltammetry and by chronoamperometry on their sensing ability towards the respective substrates, namely glucose and oxygen. Finally, bioanode and biocathode were integrated in a single-compartment of a heterogeneous poly (methyl methacrylate) - polydimethylsiloxane microfluidic platform and the power density produced was assessed. In 10 mM glucose solution at pH 7.0, the biofuel cell showed an open circuit potential (OCP) of 0.43 V and a maximum power density of about 28 μW cm−2 at 0.24 V. Also we demonstrate here the viability of human propelled fluidics by introducing in the microfluidic device a finger-pressure mechanism.

Enzymatic biosensing by covalent conjugation of enzymes to 3D-networks of graphene nanosheets on arrays of vertically aligned gold nanorods: Application to voltammetric glucose sensing.

The authors demonstrate efficient direct electron transfer from the enzyme glucose oxidase to vertically aligned gold nanorods with a diameter of ~160nm and a length of ~2μm that are covalently linkage to a 3-dimensional network of reduced graphene oxide nanosheets. The assembly can be prepared by a 2-step electrochemical procedure. This hybrid structure holds the enzyme in a favorable position while retaining its functionality that ultimately provides enhanced performance for enzymatic sensing of glucose without utilizing mediators. The nanorod assembly was applied to the voltammetric detection of glucose. Figures of merit include an electrochemical sensitivity of 12μA·mM-1·cm-2 (obtained from cathodic peak current at a voltage of -0.45V vs. Ag/AgCl), a 3μM detection limit (at signal/noise = 3), and a wide linear range (0.01-7mM). The hybrid nanostructure has a heterogeneous electron transfer rate constant (ks) of 2.9s-1. The high electrochemical activity is attributed to the synergistic effect of a large active surface and an enhanced electron transfer efficiency due to covalent amide linkage. Graphical Abstract Schematic of the procedure utilized for the fabrication of an electrochemical biosensor based on gold nanorods (AuNRs) modified with a reduced graphene oxide (rGO)/glucose oxidase (GOx) conjugate. The enzyme electrode was employed to the determination of glucose by differential pulse voltammetry.

Preparation and characterization of alkaline phosphatase, horseradish peroxidase, and glucose oxidase conjugates with carboxylated carbon nano-onions

ABSTRACTCarbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO–enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ∼0.5 mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.

Enhanced Phototherapy by Nanoparticle-Enzyme via Generation and Photolysis of Hydrogen Peroxide.

Light has been widely used for cancer therapeutics such as photodynamic therapy (PDT) and photothermal therapy. This paper describes a strategy called enzyme-enhanced phototherapy (EEPT) for cancer treatment. We constructed a nanoparticle platform by covalent conjugation of glucose oxidase (GOx) to small polymer dots, which could be persistently immobilized into a tumor. While the malignant tumors have high glucose uptake, the GOx efficiently catalyzes the glucose oxidation with simultaneous generation of H2O2. Under light irradiation, the in situ generated H2O2 was photolyzed to produce hydroxyl radical, the most reactive oxygen species, for killing cancer cells. In vitro assays indicated that the cancer cells were destroyed by using a nanoparticle concentration at 0.2 μg/mL and a light dose of ∼120 J/cm2, indicating the significantly enhanced efficiency of the EEPT method when compared to typical PDT that requires a photosensitizer of >10 μg/mL for effective cell killing under the same light dose. Furthermore, remarkable inhibition of tumor growth was observed in xenograft-bearing mice, indicating the promise of the EEPT approach for cancer therapeutics.

Biofabrication Using Pyrrole Electropolymerization for the Immobilization of Glucose Oxidase and Lactate Oxidase on Implanted Microfabricated Biotransducers.

The dual responsive Electrochemical Cell-on-a-Chip Microdisc Electrode Array (ECC MDEA 5037) is a recently developed electrochemical transducer for use in a wireless, implantable biosensor system for the continuous measurement of interstitial glucose and lactate. Fabrication of the biorecognition membrane via pyrrole electropolymerization and both in vitro and in vivo characterization of the resulting biotransducer is described. The influence of EDC-NHS covalent conjugation of glucose oxidase with 4-(3-pyrrolyl) butyric acid (monomerization) and with 4-sulfobenzoic acid (sulfonization) on biosensor performance was examined. As the extent of enzyme conjugation was increased sensitivity decreased for monomerized enzymes but increased for sulfonized enzymes. Implanted biotransducers were examined in a Sprague-Dawley rat hemorrhage model. Resection after 4 h and subsequent in vitro re-characterization showed a decreased sensitivity from 0.68 (±0.40) to 0.22 (±0.17) µA·cm-2·mM-1, an increase in the limit of detection from 0.05 (±0.03) to 0.27 (±0.27) mM and a six-fold increase in the response time from 41 (±18) to 244 (±193) s. This evidence reconfirms the importance of biofouling at the bio-abio interface and the need for mitigation strategies to address the foreign body response.

Integration of molecular and enzymatic catalysts on graphene for biomimetic generation of antithrombotic species

The integration of multiple synergistic catalytic systems can enable the creation of biocompatible enzymatic mimics for cascading reactions under physiologically relevant conditions. Here we report the design of a graphene-haemin-glucose oxidase conjugate as a tandem catalyst, in which graphene functions as a unique support to integrate molecular catalyst haemin and enzymatic catalyst glucose oxidase for biomimetic generation of antithrombotic species. Monomeric haemin can be conjugated with graphene through π-π interactions to function as an effective catalyst for the oxidation of endogenous L-arginine by hydrogen peroxide. Furthermore, glucose oxidase can be covalently linked onto graphene for local generation of hydrogen peroxide through the oxidation of blood glucose. Thus, the integrated graphene-haemin-glucose oxidase catalysts can readily enable the continuous generation of nitroxyl, an antithrombotic species, from physiologically abundant glucose and L-arginine. Finally, we demonstrate that the conjugates can be embedded within polyurethane to create a long-lasting antithrombotic coating for blood-contacting biomedical devices.

β-cyclodextrin-ferrocene host–guest complex multifunctional labeling triple amplification strategy for electrochemical immunoassay of subgroup J of avian leukosis viruses

A novel sandwich-type electrochemical immunosensor was fabricated for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) by employing β-cyclodextrin-ferrocene (CD-Fc) host–guest complex multifunctional Fe3O4 nanospheres as labels and β-cyclodextrin functional graphene sheets (CD-GS) nanocomposite as sensor platform. The sensitivity was greatly improved based on the triple amplification strategy. Firstly, the CD-GS improved the electron transfer rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab1). Secondly, the CD on the Fe3O4 surface with strong recognition capability could form stable CD-Fc host–guest inclusion complex and provided larger free room for the conjugation of secondary antibodies (Ab2) and glucose oxidase (GOD). Finally, the conjugated GOD exhibited extraordinary electrochemical biocatalysis towards the reduction reaction of Fc+ by glucose. Under the optimized conditions, the electrochemical immunosensor exhibited a wide working range from 102.27–103.50 TCID50/mL (TCID50: 50% tissue culture infective dose) with a low detection limit of 102.19 TCID50/mL (S/N=3). The selectivity, reproducibility, and stability are acceptable. The assay was evaluated for real avian serum sample, receiving satisfactory results. This new type of triple amplification strategy may provide potential applications for the clinic application.

Abstract 2443: MR imaging in human glioma tumor xenograft models using enzyme-conjugated anti-EGFRvIII-specific antibody fragments

Abstract A truncated and constitutively active form of the EGF receptor variant III (EGFRvIII) is a major determinant of tumor growth and poor prognosis in glioblastoma multiforme [1]. To test a system for targeted imaging of EGFRvIII we investigated the retention of peroxidase-generated products of a paramagnetic substrate diTyr-DTPAGd in U87αEGFR orthotopic human glioma xenografts. F(ab’)2 fragments of EMD72000 mAb were conjugated to deglycosylated horseradish peroxidase (HRP) and glucose oxidase (GOX) and used as a self-complementing enzymatic signal amplification system [2] for EGFRvIII targeted imaging. We anticipated that receptor expression sites will exhibit a prolonged MR signal enhancement due to the formation of polymerized products of diTyr-DTPAGd oxidation by mAb conjugates [2]. F(ab’)2 fragments of mAb were linked to enzymes using bisaromatic hydrazone bonds and purified by HPLC. The conjugates were characterized in U87αEGFR cell culture and optimal ratios of HRP and GOX conjugates were determined to provide the maximum signal with low cytotoxicity. F(ab’)2-GOX and F(ab’)2-HRP were also modified with NHS-MAG3 for radiolabeling. U87αEGFR cells were stereotaxically implanted in the brains of RNU rats. Ten days after tumor implantation, each animal was imaged using T1-weighted spin-echo MRI (TR/TE=700ms/8.2ms) at 3T on two occasions: Day 1 - images were acquired after IV injection of 0.1 mmol/kg diTyr-DTPAGd over a 1.5h period; Day 2 - anti-EGFRvIII conjugates (100 µg mAb/animal) were injected IV, and then 4h later 0.1 mmol/kg diTyr-DTPAGd was injected IV followed by image acquisition for 2h. Both 99mTc-labeled conjugates showed specific binding to U87αEGFR cells in vitro and in vivo as demonstrated using SPECT/CT. Cell-binding and internalization studies showed that 80% conjugates were internalized at 37°C. MR T1-weighted images showed significantly higher enhancement and longer retention of the MR signal on Day 2 in rats pre-injected with conjugates compared to Day 1 over the same time period due to the conjugate co-localization at the EGFRvIII target sites. The washout of the contrast agent was best modeled using a biexponential decay as compared to Gli36αEGFR-bearing rats [4]. U87αEGFR tumors showed biexponential MR signal decay on both days. Administration of EGFRvIII-targeted mAb conjugates resulted in specific binding to U87αEGFR cells of which at least ∼20% remained on the surface enabling the reaction with the contrast agent. The biexponential decay constants on Day 2 for both Gli36αEGFR and U87αEGFR animals were not significantly different indicating a similarity of conjugate accumulation in both tumor models which resulted in a similar bimodal washout of the contrast agent.

Conjugation of Glucose Oxidase onto Mn-Doped ZnS Quantum Dots for Phosphorescent Sensing of Glucose in Biological Fluids

Integrating various enzymes with nanomaterials provides various nanohybrids with new possibilities in biosensor applications. Furthermore, the enzymatic activity and stability are also improved due to the large surface area of nanomaterials. Here we report the conjugation of glucose oxidase (GOD) onto phosphorescent Mn-doped ZnS quantum dots (QDs) using 1-ethyl-3-(3-dimethylaminopropy)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as coupling reagents for glucose biosensing based on the effective quenching of the room temperature phosphorescence (RTP) of Mn-doped ZnS QDs by the H(2)O(2) generated from GOD-catalyzed oxidation of glucose. The obtained bioconjugate not only provided improved enzymatic performance with Michaelis-Menten constant of 0.70 mM but also favored biological applications because the phosphorescent detection mode avoided the interference from autofluorescence and scattering light from the biological matrix. In addition, the GOD-conjugated Mn-doped ZnS QDs showed better thermal stability in the temperature range of 20-80 degrees C. The GOD-Mn-doped ZnS QDs based RTP sensor for glucose gave a detection limit of 3 microM and two linear ranges from 10 microM to 0.1 mM and from 0.1 to 1 mM. The developed biosensor was successfully applied to the determination of glucose in real serum samples without the need for any complicated sample pretreatments.

Amperometric Monitoring of the Neuropeptide “Substance-P” Level in Biological Fluids

Presented here is a capillary enzyme immunoassay constructed on a foil-based microfluidic chip for rapid determination of Substance-P (SP) level in human plasma, serum and saliva. Using a competitive ELISA implemented into the capillary, the amperometrically detected SP by flow injection analysis was related to the percentage amount between SP standard (or sample) and SP-labelled glucose oxidase conjugate bound to the capture antibody. A detection limit of 10 pg/ml was obtained. The assay allows fast reading of diagnostically relevant levels. Producing reliable results comparable to microplate format, the immunochip sensor may have a significant impact in point of care diagnostics.

Ultrasonic Processing of Single-Walled Carbon Nanotube–Glucose Oxidase Conjugates: Interrelation of Bioactivity and Structure

Supramolecular conjugates of single-walled carbon nanotubes and glucose oxidase were prepared in aqueous solution using ultrasonication processing and then isolated by high-speed centrifugation. The conjugates of the single-walled carbon nanotubes and the pristine glucose oxidase, serving as control, were investigated for their enzymatic bioactivity. In addition, the effect of the extent of ultrasonication was studied. The conjugates were also characterized by UV–VIS and circular dichroism spectroscopy as well as by high-resolution transmission electron microscopic and thermogravimetric analysis. Ultrasonication is shown to reduce catalytic activity by ca. 30% (10 min) and that prolonged ultrasonication (up to 60 min) further reduces V max by 40%. However, most of this decrease arises from ultrasonication itself. The presence of carbon nanotubes (CNTs), while not eliminating changes in catalytic activity, mitigates the magnitude of these changes and is effectively de-bundled by the presence of the surfactant properties of the protein. The enzymatic activity and conformation were found to be predominantly retained after the supramolecular conjugation process assisted by ultrasonication in the presence of the CNTs.

A doubly amplified electrochemical immunoassay for carcinoembryonic antigen

An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody–glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.

Determination of the herbicide chlorsulfuron by amperometric sensor based on separation-free bienzyme immunoassay

A separation-free bienzyme immunoassay system has been developed for the electrochemical determination of the herbicide chlorsulfuron. Screen printed electrode with horseradish peroxidase as an integral component of the carbon ink was used as the detector. A membrane with immobilised anti-chlorsulfuron antibodies was attached to the electrode. Free chlorsulfuron in the sample under test and a chlorsulfuron–glucose oxidase conjugate competed for the available binding sites of the membrane-immobilised antibodies. Addition of glucose induced the generation of hydrogen peroxide by the glucose oxidase conjugate, which was in turn reduced by the peroxidase. The latter process caused an electrical current change, due to direct re-reduction of peroxidase by a direct electron transfer mechanism, which was measured to determine the chlorsulfuron content in the sample. Any H 2O 2 formed in the bulk solution by unbound chlorsulfuron–glucose oxidase conjugate was scavenged by catalase thus removing the need for a washing step. The optimal protocol for chlorsulfuron detection by the method described was found to be: Biodyne A membrane as a carrier, concentration of IgG during adsorption on the membrane—1 ng ml −1, concentration of the chlorsulfuron–glucose oxidase conjugate—2.5 μg ml −1 (by enzyme), working potential +50 mV versus Ag/AgCl. The total assay time was 15 min. The measuring range for chlorsulfuron detection was 0.01–1 ng ml −1. The method was suitable application for on-site ecological monitoring.

Immunochemical rapid test for multiresidue analysis of antimicrobial drugs in milk using monoclonal antibodies and hapten–glucose oxidase conjugates

Based on monoclonal antibodies (Mabs) against streptomycin, sulfadiazine, sulfamethazine and cloxacillin, a semiquantitative immunochemical rapid test for multiresidue analysis has been developed. The test format is that of an indirect competitive enzyme immunoassay. Glucose oxidase conjugates of the individual compounds were found to be suitable coating antigens. Individual immunoassays exhibited sensitivities far below the respective maximum residue limits (MRLs) in milk, i.e. 3.2 ng ml−1 for streptomycin, 9.0 ng ml−1 for sulfadiazine, 7.0 ng ml−1 for sulfamethazine and 0.8 ng ml−1 for cloxacillin. By optimising the assay conditions, total assay time was reduced to 15 min. The individual tests were then integrated onto a single microtitre plate array, in which the milk samples were simultaneously incubated with a mixture of the specific Mabs. The detection limits obtained with the multitest system again were below the respective MRLs, i.e. 14, 20, 13 and 1.7 ng ml−1, respectively, for streptomycin, sulfadiazine, sulfamethazine and cloxacillin. The coefficients of variation were less than 10%. Cross-reactivities with structurally related substances have been determined for both the individual assays and the multitest system.

Immunochemical rapid test for multiresidue analysis of antimicrobial drugs in milk using monoclonal antibodies and hapten–glucose oxidase conjugates

Based on monoclonal antibodies (Mabs) against streptomycin, sulfadiazine, sulfamethazine and cloxacillin, a semiquantitative immunochemical rapid test for multiresidue analysis has been developed. The test format is that of an indirect competitive enzyme immunoassay. Glucose oxidase conjugates of the individual compounds were found to be suitable coating antigens. Individual immunoassays exhibited sensitivities far below the respective maximum residue limits (MRLs) in milk, i.e. 3.2 ng ml −1 for streptomycin, 9.0 ng ml −1 for sulfadiazine, 7.0 ng ml −1 for sulfamethazine and 0.8 ng ml −1 for cloxacillin. By optimising the assay conditions, total assay time was reduced to 15 min. The individual tests were then integrated onto a single microtitre plate array, in which the milk samples were simultaneously incubated with a mixture of the specific Mabs. The detection limits obtained with the multitest system again were below the respective MRLs, i.e. 14, 20, 13 and 1.7 ng ml −1, respectively, for streptomycin, sulfadiazine, sulfamethazine and cloxacillin. The coefficients of variation were less than 10%. Cross-reactivities with structurally related substances have been determined for both the individual assays and the multitest system.

A glucose oxidase electrode based on polypyrrole with polyanion/PEG/enzyme conjugate dopant

This study investigated a new glucose sensor prepared by electrochemical polymerization of pyrrole with polyanion/poly(ethylene glycol) (PEG)/glucose oxidase (GOD) conjugate dopants. GOD was coupled to a strong polyanion, poly(2-acrylamido-2-methylpropane sulfonic acid) (AMPS) via PEG spacer to effectively and reproducibly immobilize GOD within a polypyrrole matrix onto a Pt electrode surface. PEGs with four different chain lengths (1000, 2000, 3000, and 4000) were used as spacers to study the spacer length effect on enzyme immobilization and electrode function. After conjugation, more than 90% of the GOD bioactivity was preserved and the bioactivity of the conjugated GOD increased with longer PEG spacers. The resulting polyanion/PEG/GOD conjugate was used as a dopant for electropolymerizing pyrrole. The activity of the immobilized enzyme on the electrode ranged from 119 to 209 mU cm −2 and the bioactivity increased with the use of longer PEG spacers. The amperometric response of the enzyme electrode was linear up to 20 mM glucose concentration with a sensitivity ranging from 180 to 270 nA mM −1 cm −2. The kinetic parameters Michaelis–Menten constant (K M app) and maximum current density ( j max) depended on the amount of active enzyme, level of substrate diffusion, and PEG spacer length. An increase in the electrical charge passed during polymerization (thus, increasing polypyrrole thickness) to 255 mC cm −2 increased the sensitivity of the enzyme electrode because of the greater amount of incorporated enzyme. However, although the amount of incorporated GOD continued to increase when the charge increased above 255 mC cm −2, the sensitivity began to decline gradually. The condition for preparing the enzyme electrode was optimized at 800 mV potential with a dopant concentration of 1 mg ml −1.

A highly sensitive flow-through amperometric immunosensor based on the Peroxidase chip and enzyme-channeling principle

A concept based on the Peroxidase-chip (P-chip), antibody co-immobilization, competitive and enzyme-channeling principle was exploited to develop an integrated flow-through amperometric biosensor for detection of environmental pollutants such as s-triazine herbicides. In this concept, recombinant peroxidase is immobilized on the gold electrode (P-chip) in such a way that direct electron transfer is achieved. The recognition and quantitation the target analyte is realized through the competition between the simazine–glucose oxidase (GOD) conjugate and free simazine for the binding sites of the monoclonal antibody co-immobilized with peroxidase on the gold electrode. The arrangement allows to generate a specific signal in the presence of glucose through the channeling of H 2O 2 produced by GOD conjugate bound to the antibody. The immunosensor exhibited 50% signal decrease (IC 50 value) at aproximately 0.02 μg l −1. A concentration of 0.1 ng l −1 gave a signal clearly distinguishable from the blank whereas the ELISA using the same antibody had a typical detection limit of about 1 μg l −1, which is four orders of magnitude higher compared to the presented biosensor system. The results demonstrated that gene engineering biomolecules, in this case recombinant peroxidase, might be attractive reagents for the development of electrochemical immunosensors.

Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells

AbstractCell-selective intracellular targeting is a key element of more specific and safe enzyme, toxin, and gene therapies. Endothelium poorly internalizes certain candidate carriers for vascular immunotargeting, such as antibodies to platelet endothelial cell adhesion molecule 1 (PECAM–1). Conjugation of poorly internalizable antibodies with streptavidin (SA) facilitates the intracellular uptake. Although both small and large (100-nm versus 1000-nm diameter) anti-PECAM/SA–beta galactosidase (SA–β-gal) conjugates bound selectively to PECAM-expressing cells, only small conjugates showed intracellular accumulation of active β-gal. To study whether size of the conjugates controls the uptake, a series of anti-PECAM/SA and anti-PECAM/bead conjugates ranging from 80 nm to 5 µm in diameter were produced. Human umbilical vein endothelial cells and PECAM-transfected mesothelioma cells internalized 80- to 350-nm anti-PECAM conjugates, but not conjugates larger than 500 nm. Further, size controls intracellular targeting of active therapeutic cargoes in vitro and in vivo. Small anti-PECAM/DNA conjugates transfected target cells in culture 5-fold more effectively than their large counterpart (350- versus 4200-nm diameter). To evaluate the practical significance of the size-controlled subcellular addressing, we coupled glucose oxidase (GOX) to anti-PECAM and antithrombomodulin. Both types of conjugates had equally high pulmonary uptake after intravenous injection in mice, yet only small (200- to 250-nm), not large (600- to 700-nm), GOX conjugates caused profound oxidative vascular injury in the lungs, presumably owing to intracellular generation of H2O2. Thus, engineering of affinity carriers of specific size permits intracellular delivery of active cargoes to endothelium in vitro and in vivo, a paradigm useful for the targeting of drugs, genes, and toxins.