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Cholesterol Detection Device

#2212


Device for Noninvasive Detection of Cholesterol

Tech Area / Field

  • INS-DET/Detection Devices/Instrumentation
  • MED-DID/Diagnostics & Devices/Medicine
  • BIO-RAD/Radiobiology/Biotechnology

Status
3 Approved without Funding

Registration date
23.05.2001

Leading Institute
Russian Cardiology Research Center / Institute of Experimental Cardiology, Russia, Moscow

Supporting institutes

  • VNIIEF, Russia, N. Novgorod reg., Sarov

Collaborators

  • Trykor, USA, MA, Boston

Project summary

The purpose of this project is to provide a new method and equipment for the determination of cholesterol in the epidermis on the basis of fluorescence laser spectroscopy. Noninvasive diagnostic techniques have been extensively developed during recent decades. The use of new technologies based on nuclear-magnetic resonance, computer tomography, ultrasound, enzyme-substrate interactions and “dry chemistry” has opened new prospects in clinical and “home” diagnostic techniques.
The participants of this project have developed the following noninvasive methods for the determination of cholesterol in the epidermis: enzyme technique with the use of cholesterol oxidase and electrochemical biosensors and a “three drop test”, which were patented in Russia, the USA and Canada. Experiments on an autopsy material have shown that the cholesterol content in the epidermis strongly correlates with that in the aortic and coronary arterial walls and reflects these verity of atherosclerotic changes in arteries.
The amount of cholesterol in the lipid film of the epidermis reflects the intensity of cholesterol production by epidermal basal cells. The three-drop tests, as well as an improved one-drop test in the USA, provide sufficient information on the effectiveness of anticholesterol therapy (for example, when statins and plasmapherersis are prescribed).
Since this method is semiquantitative, sometimes leading to over- or underestimation of the results, depending on inpidual color perception, we decided to develop a portable fluorimeter that measures the intensity of fluorescence, which is directly proportional to the cholesterol content in the epidermis.

Major goals of investigation
1. Development of methods for conjugation of fluorescent dyes to digitonin.
2. Development of methods for immobilization of digitonin and fluorescent dyes on a polymer carrier.
3. Development of a portable fluorescent sensor.
4. Development and manufacture of a portable source for excitation of fluorescence.
5. Preclinical trials of the preparations and equipment.

Technical approach and methodology
A mixture of organic solvents will be used to develop a method for solubilization of insoluble (hydrophobic) fluorescent dye by steroid glycoside with subsequent removal of the solvent and suspension of the binary system in a water-salt solution, containing a stabilizer. The major parameter of the micelle formation process (critical concentration of micelle formation) will be determined from the relationship between surface tension and concentration of a surface-active substance (SAS) using Langmuir scales.
The effects of the solvent and SAS on the fluorophore microenvironment will be determined by fluorescence quenching using a fluorimeter. The size of micelles will be determined by diffusion in a photon-correlation spectrophotometer with a laser source.
Chemical modification of carbohydrates, insertion of a bifunctional “link” and attachment of a fluorescent agent to reactive group will be used to develop the method of covalent conjugation of fluorescent marker to a steroid glycoside. Modified steroid glycoside will be purified by precipitation, hydrophobic chromatography, gel-filtration, and ultra- filtration. The final product will be analyzed by infra-red spectroscopy, element analysis, fluorimetry, and thin-layer chromatography (TLC). The glycoside will be quantified in a fluorimeter using anthranole reagent, fluorescent dye.
The anhydride reaction with hydroxyl group of sugar residues in steroid glycoside yielding a complex ester bond will be used for the development of a simultaneous covalent conjugation of glycoside and fluorescent agent. Fluorescent agent will be modified by insertion of an alkaline group.
The obtained products will be tested in cholesterol-containing model systems, such as micelles, erythrocyte monolayers on amine-coated glass slides, and glutharaldehyde cross-linked erythrocytes. Cholesterol- and ceramide-containing polyvinyl acetate films, Whatman chromatography papers with liposomes and corneal layer negatives of varied thickness on a sticky band will be used as coatings.
A PZC matrix with appropriate sensitivity and signal-noise ratio for recording fluorescent spectrum in the 450-700 nm range will be used to develop a portable fluorimeter. Original software to convert the fluorescent spectrum into a computer file compatible with Excel and Origin software will be written.
The following methodology is proposed for the development of a portable sensor:
1. Choice of bright photodiodes and optimization of their characteristics for excitation of fluorescence in compliance with the sensitivity of recording photodiodes.
2. Choice of the method for measuring fluorescence and of operating algorithm for the portable fluorescence sensor.
3. Manufacturing and tests of portable fluorescence sensor to correct its design as well and the method of fluorescence measurement.
Preclinical trials of the equipment will be carried out in compliance with of the Health Ministry recommendation, GLP requirements and humane treatment of laboratory animals. Rabbits will be used in model experiments. The animals will be maintained in a vivarium according to Sanitary Regulations on Experimental Biological Clinics (Vivaria), USSR Health Ministry 6.03.1973, and USSR Health Ministry Order No. 755, 12.08.1977.

Expected results and their application
1. Of theoretical interest are new data on the properties of original diagnostic conjugates and their application.
2. Of practical interest is new diagnostic equipment, the wide use of which in the near future will allow noninvasive determination of the cholesterol content in human beings and early diagnosis, prevention and effective therapy of atherosclerosis.


3. New technology will be elaborated and further developed for industrial production after completion of the project.


Role of foreign collaborators

Joint trials and evaluations of the method and equipment developed within this project.

Meeting ISTC Goals and Objectives

This project will greatly contribute to the solution of an important social problem; it will allow the researchers and engineers of the Russian Federal Nuclear Center, who were previously employed by the Defense Ministry, to reorient the scope of their scientific interests toward applied investigations associated with international scientific problems in biophysics, medicine and biochemistry and to integrate these scientists into the international scientific community.


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