Conjugated To Horseradish Peroxidase Research Articles

Immobilization of Thiol-Modified Horseradish Peroxidase on Gold Nanoparticles Enhances Enzyme Stability and Prevents Proteolytic Digestion.

The specificity and efficiency of enzyme-mediated reactions have the potential to positively impact many biotechnologies; however, many enzymes are easily degraded. Immobilization on a solid support has recently been explored to improve enzyme stability. This study aims to gain insights and facilitate enzyme adsorption onto gold nanoparticles (AuNPs) to form a stable bioconjugate through the installation of thiol functional groups that alter the protein chemistry. In specific, the model enzyme, horseradish peroxidase (HRP), is thiolated via Traut's reagent to increase the robustness and enzymatic activity of the bioconjugate. This study compares HRP and its thiolated analog (THRP) to deduce the impact of thiolation and AuNP-immobilization on the enzyme activity and stability. HRP, THRP, and their corresponding bioconjugates, HRP-AuNP and THRP-AuNP, were analyzed via UV-vis spectrophotometry, circular dichroism, zeta potential, and enzyme-substrate kinetics assays. Our data show a 5-fold greater adsorption for THRP on the AuNP, in comparison to HRP, that translated to a 5-fold increase in the THRP-AuNP bioconjugate activity. The thiolated and immobilized HRP exhibited a substantial improvement in stability at elevated temperatures (50 °C) and storage times (1 month) relative to the native enzyme in solution. Moreover, HRP, THRP, and their bioconjugates were incubated with trypsin to assess the susceptibility to proteolytic digestion. Our results demonstrate that THRP-AuNP bioconjugates maintain full enzymatic activity after 18 h of incubation with trypsin, whereas free HRP, free THRP, and HRP-AuNP conjugates are rendered inactive by trypsin treatment. These results highlight the potential for protein modification and immobilization to substantially extend enzyme shelf life, resist protease digestion, and enhance biological function to realize enzyme-enabled biotechnologies.

Polyethylene glycol fusion repair of severed rat sciatic nerves reestablishes axonal continuity and reorganizes sensory terminal fields in the spinal cord.

JOURNAL/nrgr/04.03/01300535-202507000-00030/figure1/v/2024-09-09T124005Z/r/image-tiff Peripheral nerve injuries result in the rapid degeneration of distal nerve segments and immediate loss of motor and sensory functions; behavioral recovery is typically poor. We used a plasmalemmal fusogen, polyethylene glycol (PEG), to immediately fuse closely apposed open ends of severed proximal and distal axons in rat sciatic nerves. We have previously reported that sciatic nerve axons repaired by PEG-fusion do not undergo Wallerian degeneration, and PEG-fused animals exhibit rapid (within 2-6 weeks) and extensive locomotor recovery. Furthermore, our previous report showed that PEG-fusion of severed sciatic motor axons was non-specific, i.e., spinal motoneurons in PEG-fused animals were found to project to appropriate as well as inappropriate target muscles. In this study, we examined the consequences of PEG-fusion for sensory axons of the sciatic nerve. Young adult male and female rats (Sprague-Dawley) received either a unilateral single cut or ablation injury to the sciatic nerve and subsequent repair with or without (Negative Control) the application of PEG. Compound action potentials recorded immediately after PEG-fusion repair confirmed conduction across the injury site. The success of PEG-fusion was confirmed through Sciatic Functional Index testing with PEG-fused animals showing improvement in locomotor function beginning at 35 days postoperatively. At 2-42 days postoperatively, we anterogradely labeled sensory afferents from the dorsal aspect of the hindpaw following bilateral intradermal injection of wheat germ agglutinin conjugated horseradish peroxidase. PEG-fusion repair reestablished axonal continuity. Compared to unoperated animals, labeled sensory afferents ipsilateral to the injury in PEG-fused animals were found in the appropriate area of the dorsal horn, as well as inappropriate mediolateral and rostrocaudal areas. Unexpectedly, despite having intact peripheral nerves, similar reorganizations of labeled sensory afferents were also observed contralateral to the injury and repair. This central reorganization may contribute to the improved behavioral recovery seen after PEG-fusion repair, supporting the use of this novel repair methodology over currently available treatments.

Multiple and simultaneous detection for cytokines based on the nanohole array by electrochemical sandwich immunoassay

Monitoring of cytokine storm markers is critical for determining the disease progression and treatment efficacy. In this study, we developed a novel fourplex electrochemical (EC) biosensor using nanohole array (NHA) structures containing several nanoholes on a single electrode for detecting cytokines with a low limit. A framework was developed for fabricating and optimizing multiplexed EC sandwich immunoassays using NHA electrodes because an NHA can enhance the EC detection of molecular binding by controlling the binding sites of captured biomolecules. For detecting multiple cytokines, the captured antibodies were immobilized on the surface-modified NHA and sandwich immunoassays with amperometric signal amplifications using poly-streptavidin horseradish peroxidase conjugates and 3,3′,5,5′-tetramethylbenzidine. Under the optimal conditions, the EC NHA immunosensors exhibited high sensitivity and specificity with a limit of detection of 1 pg/mL and a detection range of 1–300 pg/mL. The results revealed that the developed NHA sensor exhibits reliable EC characteristics and could be used to analyze multiple cytokines using small sample volume and subsequently developed for other applications.

Carbon nanofiber nanomaterial modified polystyrene enzyme linked immunosorbent assay substrate for detecting osteoporosis

Osteoporosis (OS) develops without any pain or symptom, and it is usually identified when the weakened bone causes fracture. To overcome this issue, measuring bone density (BMD) helps to identify osteoporosis and its conditions. It was identified that when the level of anti-cyclic citrullinated peptide (anti-CCP) increases, values of BMD were decreased. This research was focused to quantify the level of anti-CPP on nanomaterial modified polystyrene ELISA substrate. ELISA wells were modified into amine-carbon nanofiber and then glutaraldehyde used as a linker to attach CCP. Anti-CCP antibody level was quantified on PS plate by secondary-antibody conjugated horseradish peroxidase. Carbon nanofiber improves the immobilization of CCP on PS well and attract higher number of anti-CCP antibody, which helps to reach the limit of detection at 10 pg/mL, which was four times lower limit of detection with conventional ELISA (40 pg/mL). Further, specific experiment was conducted with serum spiked anti-CCP antibody shows the absorbance increment in all the concentrations of anti-CCP antibody and control performances with relevant molecules did not increase the absorbance level confirms the selective and specific detection. This carbon nanofiber modified ELISA well quantifies anti-CCP level and helps to identify the condition of bone for diagnosing osteoporosis.

Dual-Responsive Enzyme-Polysaccharide Conjugate as a Nanocarrier System for Enzyme Prodrug Therapy.

Biopolymer-based drug delivery systems have gained considerable attention in the field of nanomedicine. In this study, a protein-polysaccharide conjugate was synthesized by covalent conjugation of the enzyme horseradish peroxidase (HRP) with acetalated dextran (AcDex) via a thiol exchange reaction. The resulting bioconjugate shows a dual-responsive behavior in acidic and reductive environments to achieve a controlled release of drugs. The self-assembly of this amphiphilic HRP-AcDex conjugate allows the encapsulation of prodrug indole-3-acetic acid (IAA) into the hydrophobic polysaccharide core. Under slightly acidic conditions, the acetalated polysaccharide reverts to its native hydrophilic form, which triggers the disassembly of micellar nanoparticles and the release of the encapsulated prodrug. The conjugated HRP further activates the prodrug by oxidation of IAA into cytotoxic radicals, which leads to cellular apoptosis. The results indicate that the HRP-AcDex conjugate in combination with IAA has great potential to be used as a novel enzyme prodrug therapy for cancer treatment.

Validation of an ELISA test kit for the quantitative analyses of total Aflatoxins in spices marketed in Cameroon

Aflatoxins (mold-synthetized secondary metabolites) contamination associated with food such as spices continue to be a global problem in the world. The aim of this study was to measure the standard parameters of validation (Linearity, occuracy, repeatability, intermediate precision, reproducibility, limit of detection, limit of quantification and robustness) of the total Aflatoxins (AFt) low-matrix Enzyme-Linked ImmunoSorbent Assay (ELISA) Kit (Lot No. 062218, RENEKABIO, USA) and used this method to evaluate the contamination degree of AFt ( AFB1, AFB2, AFG1 and AFG2) in 4 different spice matrices [white pepper (Piper nigrum), black pepper (Piper nigrum), njansang (Ricinodendron heudelottii) and super épice mbongo ( Mix of Aframomum citratum, Monodora myristica and Afrostyrax lepidophyllus] marketed in Cameroon and commonly used as ingredients in Cameroonian sauces. The test was performed as a solid phase direct competitive ELISA using a horse radish peroxidase conjugate as the competing, measurable entity. Data obtained from validation showed linearity in the range between 0 mg kg-1 and 20 mg kg-1; an accuracy marked by a recovery rate ranging from 95.09 % to 110.29 %; a coefficient of variation (CV) of repeatability of 3.14 %, a coefficient of variation (CV) of intermediate precision of 6.82 % and a coefficient of variation (CV) of reproducibility of 13.96 %. Detection and quantification limits of 0.15 mg kg -1 and 0.50 mg kg -1 were determined, respectively. The robustness test revealed a critical point of the method as an incubation for 30 minutes at room temperature. The application of the method to our spices analyzed revealed 100 % contamination of 2.34 ± 0.25 µg kg -1 (white pepper), 2.37 ± 0.38 µg kg -1 (black pepper), 2.58 ± 0.87 µg kg -1 (Njansang) and 6.60 ± 5.21 µg kg -1 (Super épice mbongo). The RENEKABIO low matrix ELISA kit used for this research enables an accurate, reliable and faithful screening and quantification of AFt in spice matrices. This will be an asset in quality control of spices marketed in Cameroon.

Preparation of polyclonal anti-Schistosoma mansoni cysteine protease antibodies for early diagnosis

In many parts of the tropics, schistosomiasis is a major parasitic disease second only to malaria as a cause of morbidity and mortality. Diagnostic approaches include microscopic sampling of excreta such as the Kato-Katz method, radiography, and serology. Due to their vital role in many stages of the parasitic life cycle, proteases have been under investigation as targets of immunological or chemotherapeutic anti-Schistosoma agents. Five major classes of protease have been identified on the basis of the peptide hydrolysis mechanism: serine, cysteine, aspartic, threonine, and metalloproteases. Proteases of all five catalytic classes have been identified from S. mansoni through proteomic or genetic analysis. The study aimed to produce polyclonal antibodies (pAbs) against schistosomal cysteine proteases (CP) to be used in the diagnosis of schistosomiasis. This study was conducted on S. mansoni-infected patients from highly endemic areas and from outpatients’ clinic and hospitals and other patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids). In this study, the produced polyclonal antibodies against S. mansoni cysteine protease antigens were labeled with horseradish peroxidase (HRP) conjugate and used to detect CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA. The study involved 200 S. mansoni-infected patients (diagnosed by finding characteristic eggs in the collected stool samples), 100 patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids), and 100 individuals who served as parasite-free healthy negative control. The prepared pAb succeeded in detecting CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA with a sensitivity of 98.5% and 98.0% respectively. A positive correlation was observed between S. mansoni egg counts and both stool and serum antigen concentrations. Purified 27.5 kDa CP could be introduced as a suitable candidate antigen for early immunodiagnosis using sandwich ELISA for antigen detection.Key points• Detection of cysteine protease antigens can replace parasitological examination.• Sandwich ELISA has a higher sensitivity than microscopic examination of eggs.• Identification of antigens is important for the goal of obtaining diagnostic tools.

On-bead DNA synthesis triggered by allosteric probe for detection of carcinoembryonic antigen.

Sensitive quantification of protein biomarkers is highly desired for clinical diagnosis and treatment. Yet, unlike DNA/RNA which can be greatly amplified by PCR/RT-PCR, the amplification and detection of trace amount of proteins remain a great challenge. Here, we combined allosteric probe (AP) with magnetic bead (MB) for assembling an on-bead DNA synthesis system (named as APMB) to amplify protein signals. The AP is designed and conjugated onto the MB, enabling the protein biomarker to be separated and enriched. Once recognizing the biomarker, the AP alters its conformation to initiate DNA synthesis on beads for primary signal amplification. During the DNA synthesis, biotin-dATPs are incorporated into the newly synthesized DNA strands. Then, the biotin-labeled DNA specifically captures streptavidin (STR)–conjugated horseradish peroxidase (HRP), which is used to catalyze a colorimetric reaction for secondary signal amplification. By using carcinoembryonic antigen (CEA) as a protein model, the APMB can quantify protein biomarkers of as low as 0.01 ng/mL. The response values measured by APMB are linearly related to the protein concentrations in the range 0.05 to 20 ng/mL. Clinical examination demonstrated good practicability of the APMB in quantifying serum protein biomarker. The on-bead DNA synthesis could be exploited to improve protein signal amplification, thus facilitating protein biomarker detection of low abundance for early diagnosis.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05404-4.

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Conjugates of Aminopenicillins with Proteins: Synthesis, Immunogenic Properties, and Binding to the \u03b2-Lactam Receptor and Antibodies

A new approach to aminopenicillin modification and conjugation with proteins was developed using di-N-hydroxysuccinimide esters of dicarboxylic acids as crosslinkers. Acylation of ampicillin (Amp) and amoxicillin (Amox) with di-N-hydroxysuccinimide esters of adipic or terephthalic acids was carried out in an organic solvent. Subsequent conjugation of the resulting aminopenicillin derivatives with proteins was done in an aqueous medium at pH 8.3 to produce immunogenic and enzymatic conjugates of Amp and Amox. The β-lactam cycle of Amp was shown to remain intact after chemical modification and synthesis of linker conjugates. An immunogenic Amp–thyroglobulin conjugate containing an aromatic linker was used for long-term immunization of rabbits, and polyclonal antibodies thus obtained were found to bind Amp, Amox, and penicillin G with extremely high sensitivity. Amp and Amox conjugates with horseradish peroxidase (HRP) were synthesized and characterized in a competitive protein-binding (receptor) assay and a direct competitive enzyme-linked immunosorbent assay (ELISA). Of the model immunoassay systems tested, the best characteristics were observed for heterologous direct ELISA with polyclonal antibodies and the Amp–HRP conjugate that contained an adipic acid fragment as a linker: the Amp sensitivity was 0.03 ng/mL and IC50 = 0.20 ng/mL.

Microwave-assisted rapid conjugation of horseradish peroxidase-dextran aldehyde with Schiff base reaction and decolorization of Reactive Blue 19.

Microwave irradiation has become a routine technique in homogeneous and effective heating in organic synthesis. However, its application in enzyme-containing reactions is limited since it can cause denaturation of the enzyme. In this study, we have briefly investigated the effect of microwave heating on the conjugation reaction of horseradish peroxidase (HRP) with aldehyde derivative of dextran (D-CHO). The reaction was irradiated by microwave at 50 °C for 5 min. The conjugate was confirmed via GPC, in which the conjugates of HRP and D-CHO coexist with free unbound HRP molecules. Activity studies of HRP revealed that there is a small decrease in conjugate activity relative to the free enzyme after a short bioconjugation reaction with microwave irradiation. In decolorization studies of the textile dye Reactive Blue 19 (RB19), 99% of RB19 was decolorized through the free enzyme at 35 °C while the decolorization of the dye was 96% at 25-35 °C by the conjugate, which is a critical result showing clearly that the HRP conjugated via D-CHO is not denatured and still active after microwave-assisted reaction. This phenomenon is due to the multiple point conjugation of D-CHO on the surface of HRP and locking the 3D structure which may prevent changes in the secondary or tertiary structure of the enzyme. The results reveal that microwave irradiation can be used in production of covalently modified enzymes.

Reversible Lectin Binding to Glycan-Functionalized Graphene.

The monolayer character of two-dimensional materials predestines them for application as active layers of sensors. However, their inherent high sensitivity is always accompanied by a low selectivity. Chemical functionalization of two-dimensional materials has emerged as a promising way to overcome the selectivity issues. Here, we demonstrate efficient graphene functionalization with carbohydrate ligands—chitooligomers, which bind proteins of the lectin family with high selectivity. Successful grafting of a chitooligomer library was thoroughly characterized, and glycan binding to wheat germ agglutinin was studied by a series of methods. The results demonstrate that the protein quaternary structure remains intact after binding to the functionalized graphene, and that the lectin can be liberated from the surface by the addition of a binding competitor. The chemoenzymatic assay with a horseradish peroxidase conjugate also confirmed the intact catalytic properties of the enzyme. The present approach thus paves the way towards graphene-based sensors for carbohydrate–lectin binding.

Tunable Dual-Effector Allostery System for Nucleic Acid Analysis with Enhanced Sensitivity and an Extended Dynamic Range

In the last few years, studies have demonstrated the existence of dual-effector allosteric cooperativity in nature and the mechanism underlying enhanced activation/inhibition performance. In this work, we design an artificial dual-effector allostery system for the construction of a dynamic biosensor that can achieve nucleic acid detection with superior sensitivity and across an extraordinary broad detection range. Our dual-effector allostery-regulated biosensor is based on the multibranched hybridization chain reaction (mHCR) involving three hairpins (H1, H2, and H3). In the presence of the target nucleic acid, the mHCR is initiated via cascading strand displacement events. The products of mHCR are then captured on the electrode surface based on the mechanism of the multivalent proximity ligation assay (mPLA) and the multivalent binding assay (mBA). The subsequent conjugation of streptavidin-modified horseradish peroxidase (SA-HRP) can lead to an increase in the electrochemical signal. Importantly, two distinct allosteric activation sites and two distinct allosteric inhibition sites in H1 are designed to fine-tune the nucleic acid detection sensitivity and the dynamic range. Using this new dual-effector allostery tool, we report the detection of nucleic acid at a dynamic range spanning 10-1012 aM, 11 orders of magnitude showing the broadest dynamic range reported to date with an allosteric regulation biosensor construct.

Protecting Consumer Health by Developing an Electrochemical Immunosensor for the Detection of Banned Antibiotic Residues in Honey

IntroductionChemical (eg pesticides, veterinary drugs, etc.) and bacteriological contaminants (eg. foodborne pathogens) could contaminate animal and plant derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites, dyes) are banned in foodstuffs of animal origin (eg. milk, meat, eggs, etc.) in European Union because of toxicological risks for the consumer. The European Regulation has set Minimum Required Performance Limits (MRPL) [1] or Reference Point for Action (RPA) for banned substances [2]. Food containing residues of substances at or above the MRPL or RPA are declared non-compliant and consignments are rejected from the consumer's market.Screening methods are the first stage of food control and so are essential for food safety monitoring. Conventional screening methods are microbiological methods (eg. plate tests, tube tests), immunological methods (eg. ELISA, radioimmunoassays) or physico-chemical methods (Thin Layer Chromatography, High Performance Liquid Chromatography (HPLC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). These methods sometimes lack of sensitivity or specificity; they also could be time and money consuming. There is thus a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach.Electrochemical immunosensorAn innovative electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [3]. This technique presents major advantages: low cost (eg. disposable electrodes, potentiostat), promising detection limits, portability, and possible automatisation. Our laboratory has evaluated the transferability of the method. An electrochemical immunosensor has been developed for the detection of chloramphenicol residues in honey as a proof of concept. Honey composition and colour varies considerably depending on the botanical origin. Moreover some honey ingredients can interfere with the electrochemical detection, especially substances with antioxidant activities (eg. polyphenols). Therefore a lot of work had to be done to improve sample extraction to reduce matrix effects.The objective is to develop an electrochemical bead-based immunosensor for the multiplex detection of banned antibiotics (eg. chloramphenicol, nitrofuran metabolites, dyes) in bovine milk.MethodAntibodies (Abs) against antibiotics are grafted on the surface of magnetic beads (MBs). Milk samples and antibiotic conjugated with Horseradish peroxidase (HRP) are mixed with MBs-Abs. A competition occurs between the HRP conjugates and the antibiotic residues if present in the sample, for the binding to the antibody. The MBs are washed to remove free antibiotics and conjugates. Then, a Screen Printed Carbon Electrode (SPCE) with MBs on its surface (maintained by a magnet) is soaked into a buffer solution containing hydroquinone; when adding hydrogen peroxide (H2O2) to the solution, an electrochemical signal is produced, due to the enzymatic activity of HRP and measured. The electrochemical signal is inversely proportional to the antibiotic concentration in the sample.Results and ConclusionsScreening methods for the detection of veterinary drugs in food products have to be validated according to the European regulation [1] and to the European guideline for the validation of screening methods [4]. After the development and the optimization of the analytical parameters (eg. sample preparation, HRP concentration, incubation time, applied potential, etc), the methods developed for single compounds will be evaluated and validated according to the European regulations. Then the single compound methods will be merged into one multiplex method if possible. The results will be presented to the conference, discussing the advantages and drawbacks of electrochemical biosensors for the screening of antibiotic residues in food products.

Non-Invasive Colorimetric Magneto Loop-Mediated Isothermal Amplification (CM-LAMP) Method for Helicobacter pylori Detection.

More than half the world's population is thought to be infected with Helicobacter pylori. Although the majority of infected people are asymptomatic, H. pylori infection may cause gastric ulcers and deadly gastric cancer. Owing to the difficulty and invasiveness of current routine culture and diagnostic methods, a highly sensitive and specific noninvasive assay for H. pylori is of interest. This study highlighted the design and performance of a colorimetric magneto loop-mediated isothermal amplification (CM-LAMP) assay to detect H. pylori in spiked saliva samples. LF primers were coated on magnetic nanoparticles by carbodiimide-induced immobilization and functionally used for solidphase amplification. During the LAMP reaction at 66°C, biotin-tagged FIPs were incorporated into LAMP amplicons. The colorimetric signal developed after the addition of NeutrAvidin horseradish peroxidase conjugate (NA-HRP) and ABTS. None of the tested microorganisms, including closely related bacteria, was shown positive by the CM-LAMP assay except H. pylori isolates. This novel platform was highly specific and 100-fold more sensitive (40 CFU/ml or 0.2 CFU per reaction) than the PCR and conventional LAMP assays for the detection of H. pylori in spiked saliva. Our results demonstrated the feasibility of using this noninvasive molecular diagnostic test to detect H. pylori in saliva samples.

A fast and ultrasensitive ELISA based on rolling circle amplification.

A highly sensitive ELISA is critical for early diagnosis and biomarker discovery of various diseases. Although various ELISA technologies have been developed with high sensitivity, they are limited by poor repeatability, high cost, the dependence on complex equipment and/or a prolonged reaction time. To this end, we developed a fast and ultrasensitive ELISA (termed RELISA) based on rolling circle amplification (RCA) and enzymatic signal amplification. The RELISA is established on the traditional ELISA, with only one more RCA step that can be accomplished within 10 minutes. The prolonged single strand DNA (ssDNA) from RCA is able to enrich abundant horseradish peroxidase conjugate (HRP) modified detection probes. Consequently, the intensive HRP is able to catalyze TMB-H2O2 to produce significantly enhanced colorimetric signals. With CEACAM-7 as a model biomarker, the RELISA achieves the limit of detection as low as 2.82 pg mL-1, which is ∼50 times higher than that of the traditional ELISA. Therefore, we envision that the developed RELISA would be a powerful tool for the early diagnosis of various major diseases.

Enzyme immunoassay of vitamin d3 metabolites

We report herein improved version of the synthesis of hapten based on cholecalciferol and its active metabolite 25-hydroxycholecalcalferol. The methodology of obtaining high-molecular immunogenic conjugates of vitamin D3 derivatives with bovine serum albumin and horseradish peroxidase conjugates for direct ELISA was optimized. Immunisation of rabbits was carried out and polyclonal antibodies to 25-hydroxycholecalceferol were obtained and tested in an enzyme-linked immunosorbent assay. To improve the accuracy of the method, the sample preparation procedure was optimized, including the release of vitamin D3 and its active metabolites from complexes with vitamin D-binding protein.

Protecting Consumer Health By Developing a Multiplex Amperometric Immunosensor for the Detection of Banned Antibiotic Residues in Milk

Introduction Chemical (eg pesticides, veterinary drugs, etc.) and bacteriological contaminants (eg. foodborne pathogens) could contaminate animal and plant derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites, dyes) are banned in foodstuffs of animal origin (eg. milk, meat, eggs, etc.) in European Union because of toxicological risks for the consumer. The European Regulation has set Minimum Required Performance Limits (MRPL) [1] or Reference Point for Action (RPA) for banned substances [2]. Food containing residues of substances at or above the MRPL or RPA are declared non-compliant and consignments are rejected from the consumer’s market.Screening methods are the first stage of food control and so are essential for food safety monitoring. Conventional screening methods are microbiological methods (eg. plate tests, tube tests), immunological methods (eg. ELISA, radioimmunoassays) or physico-chemical methods (Thin Layer Chromatography, High Performance Liquid Chromatography (HPLC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). These methods sometimes lack of sensitivity or specificity; they also could be time and money consuming. There is thus a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach. Electrochemical immunosensor An innovative electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [3]. This technique presents major advantages: low cost (eg. disposable electrodes, potentiostat), promising detection limits, portability, and possible automatisation. Our laboratory has evaluated the transferability of the method. An electrochemical immunosensor has been developed for the detection of chloramphenicol residues in milk as a proof of concept. The matrix effect (milk samples) was high and so sample preparation has to be improved to reduce matrix effects.The objective is to develop an amperometric bead-based immunosensor for the multiplex detection of banned antibiotics (eg. chloramphenicol, nitrofuran metabolites, dyes) in bovine milk. Method Antibodies (Abs) against antibiotics are grafted on the surface of magnetic beads (MBs). Milk samples and antibiotic conjugated with Horseradish peroxidase (HRP) are mixed with MBs-Abs. A competition occurs between the HRP conjugates and the antibiotic residues if present in the sample, for the binding to the antibody. The MBs are washed to remove free antibiotics and conjugates. Then, a Screen Printed Carbon Electrode (SPCE) with MBs on its surface (maintained by a magnet) is soaked into a buffer solution containing hydroquinone; when adding hydrogene peroxide (H2O2) to the solution, an amperometric signal is produced, due to the enzymatic activity of HRP and measured. The amperometric signal is inversely proportional to the antibiotic concentration in the sample. Results and Conclusions Screening methods for the detection of veterinary drugs in food products have to be validated according to the European regulation [1] and to the European guideline for the validation of screening methods [4]. After the development and the optimization of the analytical parameters (eg. sample preparation, HRP concentration, incubation time, applied potential, etc), the methods developed for single compounds will be evaluated and validated according to the European regulations. Then the single compound methods will be merged into one multiplex method if possible. The results will be presented to the conference, discussing the advantage sand drawbacks of amperometric biosensors for the screening of antibiotic residues in food products.

Multifunctional Photosensitizing and Biotinylated Polystyrene Nanofiber Membranes/Composites for Binding of Biologically Active Compounds

A three-step postprocessing functionalization of pristine electrospun polystyrene nanofiber membranes was used for the preparation of nanostructured biotinylated materials with an externally bonded porphyrin photosensitizer. Subsequently, the material was able to strongly bind biologically active streptavidin derivatives while keeping its photosensitizing and antibacterial properties due to the generation of singlet oxygen under the exclusive control of visible light. The resulting multifunctional materials functionalized by a streptavidin-horseradish peroxidase conjugate as a model bioactive compound preserved its enzymatic activity even in the presence of a porphyrin photosensitizer with some quenching effect on the activity of the photosensitizer. Prolonged kinetics of both singlet oxygen luminescence and singlet oxygen-sensitized delayed fluorescence (SODF) were found after irradiation by visible light. The above results reflected less effective quenching of the porphyrin photosensitizer triplet state by ground state oxygen and indicated hindered oxygen transport (diffusion) due to surface functionalization. We found that SODF could be used as a valuable tool for optimizing photosensitizing efficiency as well as a tool for confirming surface functionalization. Full photosensitizing and enzyme activity could be achieved by a space separation of photosensitizers and enzyme/biomolecules in the nanofiber composites consisting of two layers. The upper layer contained a photosensitizer that generated antibacterial singlet oxygen upon irradiation by light, and the bottom layer retained enzymatic activity for biochemical reactions.

Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot.

Chemiluminescent western blot (WB) is often performed sequentially for detection of overlapping proteins; in between, prior antibodies must be stripped or the conjugated horseradish peroxidase (HRP) inactivated. However, often, stripping either is insufficient to remove all the bound antibodies or causes protein loss, whereas treatment with hydrogen peroxide, a popular way to inactivate HRP, may affect epitope recognition as the authors previously reported. To date, an ideal method for sequential chemiluminescent WB is still missing. Here it is demonstrated that acid equivalent to 10% acetic acid can efficiently inactivate HRP, allowing sequential probing without protein loss or epitope damage.