RU2327140C2 - Device and method for qualitative and quantative analysis of chemical compositions and biological bodies - Google Patents

Abstract

FIELD: chemistry; measurement.

SUBSTANCE: detecting element of the sensor is one or several consoles with complex of specifically interacting molecules and biological bodies immobilised on the surface. Cantilever can de used as a detecting element for atomic force microscopy. Determination of one of the components is achieved thanks to static deformations measurement of micromechanical device, which are appeared as a result of immobilised complex dissociation and binding of other element by analyte. Invention ensures absolute sensitivity increase.

EFFECT: it ensures increase of sensor absolute sensitivity for qualitative analysis of chemical compounds and biological bodies.

6 cl, 1 ex, 2 dwg

Description

The claimed device and method for the qualitative and quantitative determination of chemical compounds and biological objects belong to the field of sensor devices designed for the qualitative and quantitative determination of chemical compounds and biological objects that can specifically bind to each other with the formation of a stable complex, including biological materials: complementary nucleic acid molecules, cells and cellular receptors, components of the antigen-antibody complex and other protein 's and non-protein complexes, and methods of qualitative and quantitative determination of chemical compounds and biological objects. Determination of the concentration of biologically active substances is widely used in medicine for clinical diagnostics, in the pharmaceutical and food industries for the control of technological processes, analysis of raw materials and finished products.

A known method for determining the concentration of biotin, based on the immobilization of a conjugate of bovine serum albumin with destiobiotin on the surface of a piezoelectric crystal [1]. Upon incubation of the modified crystal in a solution of avidin, a metastable complex forms, which dissociates upon administration of the analyzed sample containing biotin. Avidin molecules are desorbed from the surface and in solution bind to a complex with biotin. The process of desorption of avidin is accompanied by an increase in the resonant frequency of the piezoelectric crystal, which is detected by the measuring system and serves as an analytical signal. The disadvantages of this method are the inapplicability of the method for determining the concentration of other biologically active compounds and the relatively large volume of the analyzed solution. The similarity of this method with the claimed one lies in the fact that to determine the concentration of a biologically active compound, an analytical signal is recorded that accompanies the dissociation of a specific complex formed by the analyte.

A device for the qualitative and quantitative determination of chemical compounds and biological objects, containing one or more consoles, at least part of the surface of at least one of them containing an immobilized compound or biological object [2]. During incubation of the modified cantilever in the analyzed solution, the analyte is adsorbed on the surface of the cantilever with the formation of a complex. The adsorption process is accompanied by a bending of the console, which is recorded by the optical system of an atomic force microscope, consisting of a laser and a photodiode. The disadvantage of this device is the decrease in sensitivity in the determination of low molecular weight compounds. The similarity of the described sensor device with the claimed one lies in the fact that the cantilever is the sensitive element of the device, the bend of which is an analytical signal and is recorded by an atomic force microscope optical detection system, as well as in the use of specific intermolecular complexes for the formation of biologically active compounds. In combination of essential features, the above-described sensor device is a prototype of the invention.

An object of the present invention is to increase the absolute sensitivity of a sensor device.

This object is achieved in that a device for the qualitative and quantitative determination of chemical compounds and biological objects, containing one or more consoles, at least on the surface of at least one of them containing an immobilized compound or biological object according to the invention as an immobilized compound or biological the object contains a complex of the determined compound or biological object with another compound or biological object.

In this case, at least one of the consoles is preferably made of materials selected from the group consisting of ceramic materials, polymeric materials, quartz, silicon nitride, silicon, silicon oxide, aluminum oxide, zinc oxide, tantalum oxide, germanium, germanium oxide and gallium arsenide .

Moreover, cantilever for atomic force microscopy is preferably used as at least one console. The length of the cantilever is preferably in the range of 0.1-200 microns, the width is in the range of 0.1-50 microns and the thickness is in the range of 0.03-30 microns.

The device preferably comprises an interferometer or laser optical system for measuring the deviation of at least one console, including a reflective region located on the console, a laser source and a photosensitive detector.

The goal is also achieved by the method of qualitative and quantitative determination of chemical compounds and biological objects. In a method comprising placing at least one console in a solution of a detectable chemical compound or biological object and comparing the deviation of the console with its deviation in this solution in the absence of a detectable chemical compound or biological object, according to the invention, such a console is used as at least one console on which the complex of the chemical compound or biological object being determined is immobilized with another chemical compound or biological object.

In this case, the deviation measurement of at least one console is preferably carried out using an interferometer or laser optical system, including a reflective region located on the console, a laser radiation source and a photosensitive detector.

Figure 1 presents a diagram of the proposed touch device. The sensor device according to the invention comprises a housing 1 provided with two holes 2 and metering devices 3 for introducing solutions, a mounting device 4 of one or more consoles 5 located in the housing. In case of using a laser detection system for bending the console, the housing further comprises a window 6 made of transparent to the used laser radiation material, and the console 5 on one of the surfaces contains an area covered by a layer reflecting laser radiation. The laser detecting system consists of a laser source 7, a photodiode 8, and a signal processing system from a photodiode 9. The laser detecting system may include a mirror 10, prisms, lenses, and other devices for converting an optical signal. Instead of a simple laser system, a precision optical interferometer can be used to measure console deformations. Figure 2 presents a diagram used in the touch device console. On the console there is a region 11 with the complex of the analyte 12 or biological object immobilized on it with another substance or biological object 13, while the detected substance or biological object is connected to the console surface. On the console there is also an area 14 covered with a layer reflecting laser radiation. Areas 11 and 14 may overlap or overlap. The console or its individual parts can be made of ceramic materials, polymeric materials, quartz, silicon nitride, silicon, gold, silver, silicon oxide, aluminum oxide, zinc oxide, tantalum oxide, germanium, germanium oxide and gallium arsenide. It is preferable to make the console of silicon or silicon nitride. Other materials may also be used in the manufacture of the console to provide the best mechanical properties for the sensor. The shape and size of the console are not essential for achieving a technical result.

Morphine can be used as a detectable chemical compound. As defined biological object can be used poly - and monoclonal antibodies.

The device operates as follows: the housing 1 is filled with a solution that does not contain a detectable compound or object, using a laser or other detecting system, the console bends are measured, then the housing is filled with a solution containing a detectable compound or biological object. In this case, the introduced determined compound or biological object competes with the complex immobilized for the other component, part of the complex dissociates and the complex component associated with the determined substance passes from the console surface into the solution. The dissociation of the complex is accompanied by a change in surface tension at the interface of the console with the solution and, as a consequence, the bend of the console. For a qualitative or quantitative determination of a chemical compound or biological object, the speed of the initial bending of the cantilever from its position in a solution that does not contain a detectable compound or object, which depends on the concentration of the determined compound or object, is measured. Another way to determine a chemical compound or biological object is to compare the bend of the cantilever in the absence of a detectable compound or object with a stationary bend of the cantilever established in the solution of the determined compound or object. Instead of the stationary bending value, the bending value achieved after a predetermined time has elapsed can be used.

The method is illustrated by the following examples.

Example 1. To determine the concentration of morphine in an aqueous solution, a sensor device is used, which includes a housing made of Teflon, a glass window and a console made of Teflon, a laser detecting system of the FemtoScan ™ atomic force microscope (manufactured by Academy of Biosensors LLC). The cantilever CSG01 ™ (manufactured by the F.V. Lukin State Research Institute of Physical Problems) with the morphine complex with polyclonal rabbit antibodies to morphine immobilized on it is used as a console. The immobilization of the complex of morphine and antibodies on the surface of the complex is as follows: to clean the surface, the cantilever is immersed in a mixture of equal volumes of concentrated sulfuric acid and a 30% aqueous solution of hydrogen peroxide. The cantilever is then treated sequentially with a 50 mM solution of 4-aminothiophenol in methanol for 17 hours, a 5% aqueous solution of glutaraldehyde for 2 hours and a 100 μg / ml solution of the ovalbumin conjugate with morphine in 0.03 M phosphate buffer solution with a pH of 7.0 for 1 hour, with a 0.05 M solution of tris-hydroxymethylaminomethane in a phosphate buffer solution for 30 minutes and a rabbit blood serum solution containing specific antibodies to morphine with a 1:50 dilution in phosphate buffer solution for 2 hours.

The cantilever is fixed in the holder and the housing is filled with 0.03 M phosphate buffered saline. For qualitative and quantitative determination of morphine, the casing is filled with the analyzed solution and the cantilever bend is measured relative to its position in the phosphate buffer solution 5 minutes after the injection of the test solution. The morphine content is determined by the previously constructed calibration dependence of the cantilever bend on the concentration of morphine in the solution. The method allows to determine morphine at a concentration of 1 μg / ml.

Example 2. To determine the concentration of morphine, a sensor device similar to that used in example 1 is used, except that human antibodies to morphine are used as one of the components of the complex. The concentration of morphine is measured in the same manner as in example 1.

From the literature [3] it is known that the device for the quantitative determination of antibodies to 2,4-dichlorophenoxyacetic acid, implemented according to [2], allows the determination of antibodies at a concentration of 5 μg / ml, thus, the goal of increasing the absolute sensitivity of the sensor device is achieved.

Information sources

1. Masson M., Yun K.S., Haruyama T., Kobatake E., Aizawa M. // Anal. Chem. 1995. V. 67. No. 13. P.2212.

2. Thundat T.O., Jacobson K.B., Doktycz M.J., Kennel S.J., Warmack R.J. // United States Patent No. 6289717.

3. Raiteri R., Nelles G., Butt H.-J., Knoll W., Skladál P. // Sensors and Actuators B. 1999. V. 61. P.213.

Claims (6)

1. A device for the qualitative and quantitative determination of chemical compounds and biological objects, containing one or more consoles, at least on the surface of at least one of them containing an immobilized compound or biological object, characterized in that it contains a detection system for measuring the deflection of the console comprising an interferometer or laser detecting system for measuring the deviation of at least one console, including a reflection region located on nsoli, the laser source and the photosensitive detector, and as the immobilized compound or biological object comprises a set of defined compound or biological object with another compound or biological object, 2. The device according to claim 1, characterized in that at least one of the consoles is made of materials selected from the group comprising ceramic materials, polymeric materials, quartz, silicon nitride, silicon, silicon oxide, alumina, zinc oxide, oxide tantalum, germanium, germanium oxide and gallium arsenide. 3. The device according to claim 1, characterized in that the cantilever for atomic force microscopy is used as at least one console. 4. The device according to claim 3, characterized in that the cantilever length is in the range of 0.1-200 μm, the width is in the range of 0.1-50 μm and the thickness is in the range of 0.03-30 μm. 5. A method for the qualitative and quantitative determination of chemical compounds and biological objects, comprising placing at least one console in a solution of a defined chemical compound or biological object and comparing the deviation of the console with its deviation in this solution in the absence of a determined chemical compound or biological object, characterized in that as at least one console use a console on which the complex of the chemical compound or biological object with another chemical compound or a biological object, and the determination is carried out by preliminarily constructed calibration curve bending from the console of the analyte content. 6. The method according to claim 5, characterized in that the measurement of the deviation of at least one console is carried out using an interferometer or a laser optical system that includes a reflective region located on the console, a laser radiation source and a photosensitive detector.

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