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Smart Biosensors for Analysis of Blood Esterases

#3130


Smart Biosensor Systems for Analysis of Blood Esterases, Assessment of Esterase Status of Organisms, and Biomonitoring

Tech Area / Field

  • MED-DID/Diagnostics & Devices/Medicine
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • ENV-EHS/Environmental Health and Safety/Environment
  • ENV-MIN/Monitoring and Instrumentation/Environment

Status
8 Project completed

Registration date
18.10.2004

Completion date
17.05.2010

Senior Project Manager
Visser H

Leading Institute
Institute of Physiologically Active Substances, Russia, Moscow reg., Chernogolovka

Supporting institutes

  • Moscow State University / Department of Chemistry, Russia, Moscow

Collaborators

  • University of Padova / Dipartamento di Medicina Ambientale e Sanita Pubblica, Italy, Padova\nInstitut de Pharmacologie et de Biologie Structurale/Centre National de la Recherche Scientifique, France, Toulouse\nUniversity of Michigan / School of Public Health, USA, MI, Ann Arbor\nMedical Research Council / Applied Neuroscince Group, UK, Nottingham\nUniversity of Leicester / MRC Toxicology Unit, UK, Leicester

Project summary

Organophosphorus compounds (OPs) are used worldwide. Some highly toxic OPs are chemical warfare agents, and have been used by terrorists. Defense against such agents requires rapid, sensitive and specific detection of them and their biological effects. Development of biomarkers of human exposures to OPs is crucial for prediction and early diagnosis of induced diseases. Real-time, sensitive and specific exposure assessment is vital for consequence management by national authorities and minimizing effects of terrorist acts on civilian populations.

The phosphorylating properties of OPs lead to differential interactions with various esterases. These enzymes include acetylcholinesterase (AChE, acute toxicity) and neuropathy target esterase (NTE, delayed neuropathy, OPIDN), as well as butyrylcholinesterase (BChE) and carboxylesterase (CaE), which act as scavengers of OPs. An important determinant of the toxicity of a given OP is its inhibitory preferences for different esterases (ki values). In addition, phosphotriester hydrolases (OPH) can hydrolyze and detoxify OPs. Determination of the activities of the above set of esterases constitutes the “esterase status” of an organism and may be used as a complex biomarker of exposure to OPs. This complex biomarker would be more effective and informative than separate determination of inpidual enzyme activities.

BChE hydrolyzes some ester prodrugs and metabolizes the muscle relaxant succinylcholine. People with genetic variants of BChE have prolonged postoperative paralysis from diminished hydrolysis of succinylcholine. CE can hydrolyze and detoxify OPs that contain carboxyl ester bonds. CaEs are also major determinants of the pharmacokinetic behavior of therapeutic agents containing ester or amide bonds, and alteration of the activity of these esterases has important clinical implications. OPH is known to be associated with high-density lipoproteins in human plasma and its activity may be important for prevention of atherosclerosis. Thus, assessing the “esterase status” before the use of muscle relaxants, local anesthetics, or treatment with ester-containing drugs, especially for people exposed to OPs, will enable therapy to be inpidualized, making it more effective and safe.

Currently, activities of blood esterases must be determined separately using corresponding substrates and specific inhibitors: this is a time-consuming process. In this project, we propose to create a smart biosensor system for assessing the esterase status of an organism with simultaneous analysis of five blood esterases. Fast and simultaneous analysis of esterases in human blood can be based on modern biosensor technologies and data processing of a multiparameter analytical response.

The aim of the Project is the development of a smart biosensor system for the simultaneous analysis of a set of esterases including AChE, BChE, NTE, CaE, and OPH. This will allow esterase status to be assessed quickly and exposure to chemical agents to be detected effectively. In addition, inpidualized therapy with esterase-metabolized medications can be developed.

The authors of the Project have considerable complementary expertise in the required fields, including biosensors for analysis of esterases and their OP inhibitors, biosensors for phenol detection, and the biochemistry and toxicology of OPs and their interactions with esterases. Furthermore, both Russian teams have successful experience in joint work on international projects, including ISTC project #1055.2 and CRDF projects #RB2-2035 and #RB2-2488.

Expected results and their application: A new highly sensitive smart biosensor for the simultaneous analysis of a set of esterases, including AChE, BChE, NTE, CaE, and OPH, in blood and other biological samples will be developed. Validation of biosensor measurements of blood esterases will be carried out and the “esterase status” of experimental animals and humans will be characterized. In vitro data on the efficiency of OP interactions with esterases will be obtained and analyzed using QSAR approaches. Validation of biosensor measurements of blood esterases as a complex biomarker for OP exposure will be carried out. A laboratory prototype of the smart biosensor device for automatic or semi-automatic rapid analysis of esterases will be created

It is expected that these new technological solutions will allow us to accelerate and simplify esterase assay and esterase status assessment, as well as to minimize manual participation in the procedure. These advances will be important in the development of consequence management strategies for chemical terrorist attacks and will enhance efforts to minimize the effects of chemical terrorist acts on civilian populations. Furthermore, the possibility to accelerate and simplify “esterase status” assessment will enable certain therapies to be more inpidualized and consequently more effective and safe. Moreover, these advances will increase the potential commercial value of the device in the medical and environmental protection marketplaces.

The expected results of the project should help advance research in medicine, medicinal chemistry, (especially metabolism-based drug design and drug targeting), toxicology, occupational health, and OP chemistry and biochemistry. This work will also extend electrochemical sensor research, which has multiple potential applications.

Utility. We anticipate that the development of smart biosensor device for rapid assessment of esterase status will improve our capability to determine exposure to OPs and assist in the diagnosis of poisoning in man and other species. The speed, sensitivity and integrated approach of the method will allow accurate interpretation of the hazard and appropriate intervention before overt toxic damage has occurred.

The development of the proposed biosensor device and methodology of esterase status determination will improve risk assessments relating to human health effects arising from incidental or occupational exposures, or ecological effects arising from discharges of OPs into the environment. It also provides a tool for carrying out epidemiological studies and acquiring a baseline database for these important esterase levels in human populations. Other potential uses include emergency response in the field or hospitals, and clinical management of occupational pesticide exposure scenarios.

Other important possibilities for using the smart biosensor are its broader clinical applications. Because activities of esterases may be biomarkers of different diseases and indicators of the metabolic status of patients who are prescribed certain medications, the capability for routine assay of a set of blood esterases will improve diagnostics and allow better selection of medications and their doses.

The scope of activities of the Project meets the main Goals and Objectives of ISTC. More than half of the Project participants have expertise related to chemical weapon agents and their detection; they are also skilled in studies of chemical weapon agents effects on human systems. In this Project, their knowledge will be used for peaceful applications of benefit to human health and environmental protection.

Four foreign collaborators from four different US institutions have agreed to participate in this Project. Direct contacts with collaborators were established during visits to the USA by members of Russian team, joint seminars and working contacts connected with former ISTC Project Development Grant #1055, ISTC Project #1055.2, CRDF, and IPP Projects. In addition to furthering the aims of the proposed Project, these working contacts undoubtedly promote integration of Russian scientists into the international scientific community.

The present Project supports applied research and technology development for human health care. The Project is focused on the assessment of the “esterase status”, that may be used as a complex biomarker of exposure to OPs, an indicator of disease states, and an enabler of inpidualized therapy. The development of the biosensor technologies proposed in this Project meets civil needs for health and environmental protection, ecology, and medicine.

Consistent with the scope of activities of the Project, the following main aspects of collaboration are planned: participation in Project elaboration and development of the working scheme, information exchange during Project implementation, information support, provision of comments on technical reports submitted to ISTC, testing and evaluation of equipment/technologies developed during the Project, sharing of materials and samples, joint presentation of results at scientific meetings, and joint publication of results in peer-reviewed journals. Joint seminars, workshops, and consultative visits to the collaborators are also planned during the Project.

To achieve the Project aims we plan to perform the following tasks:


· Based on new biosensor technologies, develop a universal biosensor analysis of several esterases using no more then two unified substrates; selection of parameters controlling hydrolysis of the unified substrate by each esterase.
· Create a data array from multiparameter biosensor responses of test mixtures of esterases in the presence of discriminative agents; develop data processing methods for multiparameter biosensor responses (e.g., modern chemometric assay).
· Adapt the biosensor for work in biological samples and validate biosensor measurements.
· Assess the “esterase profile” for a model series of OPs based on kinetic constants that characterize OP interactions with AChE, NTE, BChE, CaE, and OPH. Construct corresponding QSAR models.
· Investigate the smart biosensor system for assessment of esterase status and complex monitoring of OP exposures.
· Create a prototype of a smart biosensor device for automatic or semi-automatic rapid analysis of a set of esterases.

The following technical approaches and methodologies will be used: irreversible inhibition kinetics and classical enzyme kinetics with current methods for data processing and analysis; new approaches for design of biosensors with specific characteristics—layer-by-layer (LBL) technology; quality control of electrode surfaces by scanning probe microscopy; contemporary approaches for data processing of multiparameter analytical response (e.g., modern chemometric assay); different methods of esterase assay (spectrophotometric and biosensor) for validiation of biosensor measurements; esterase profiles of OPs determination by assessment of inhibitory potencies against 4 esterases; QSAR analyses of structure versus inhibitor potency and selectivity using different programs (TSAR, CODESA, TRAIL, and CLASSFORMER) to assess adequacy and predictive power of the models; molecular modeling of OP interactions with different esterases using CAChe 6.1 and SYBYL; esterase status as a complex biomarker of OP exposures using in vivo experiments on animals treated with OPs possessing different esterase profiles.


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