RU200301U1 - MICROFLUID CHIP FOR MULTI-PARAMETRIC IMMUNO ASSAY - Google Patents

Abstract

The claimed utility model relates to the field of biochemistry and molecular biology, namely, to microfluidic chips, which can be used as means of immunoassay, in particular for determining the level of cytokines. The technical result achieved when using the claimed utility model of a microfluidic chip is to ensure a complete process of sequential supply and removal of small volumes of reagent solutions for conducting fluorescence immunoassay on microspheres, due to the design features of this microfluidic chip, uncontrolled changes in the volumes of these solutions are excluded, including cross-mixing of samples and reagents, their concentrations and feed rates. The problem is solved by the fact that according to the technical solution, the claimed microfluidic chip, which is a body made of optically transparent material, equipped with technological holes for supplying sample solutions, buffer and reagents, inside which layers of optically transparent material are placed close to each other, equipped with slots and openings forming during assembly tanks and chambers located in a technological order in the direction of flow and interconnected by a system of microfluidic channels: - a flow-through sample filtration chamber equipped with a track membrane and a check valve, - a flow-through separator of air bubbles in the sample, made in the form of a chamber equipped with a microfiltration membrane and a check valve, - a reaction chamber designed for mixing the prepared sample with fluorescently labeled polymer microspheres, made with the possibility of feeding reagents into its volume, and containing a hydrophilic microfilter The second membrane, with a pore size smaller than the size of the microspheres, is a collection chamber for spent reagents connected by a channel to the reaction chamber, while a membrane with complete residual deformation is installed in the process hole intended for supplying the sample, which ensures the possibility of membrane injection of the sample into the chip. Check valves installed at the inlet to the flow-through air bubble separator and flow-through chamber for sample filtration are an elastic film placed with tension at the inlet to the corresponding chamber. 1 wp f-crystals, 24 ill.

Description

Technology area

The claimed utility model relates to the field of biochemistry and molecular biology, namely, to microfluidic chips, which can be used as means of immunoassay, in particular, to determine the level of cytokines.

State of the art

Microfluidic chips (or microfluidic systems) are widely used in various fields of science and technology.

Microfluidic technologies allow working with very small volumes of liquids, gases, bubbles and drops, with crystalline and polymer particles, even with individual biological cells. Moreover, in the process of research, one can observe the objects under study, manipulate them, control the ongoing processes.

Microfluidics is based on microfluidic chips that allow you to control micro-, nano- and even picoliter volumes of liquids. This is important for such operations as sample preparation, transportation, mixing, separation, detection, dosing, etc. The advantages of microfluidic technologies are that the reactions take place in closed systems, where contamination is excluded, and the volume of reagents is minimal. As a result, not only traditional studies can be performed in a miniature format, but also analyzes that were previously unrealizable.

Microfluidic chips are most effectively used in chemical, biological and medical research, for example, for cell cultivation, modeling of human tissues and organs.

The use of microfluidic technologies in combination with immunochemical analysis is a modern trend in the development of laboratory technologies.

A microfluidic system for carrying out an immunoassay ( RU2510509 ) is known from the prior art , which includes a channel for the analyzed fluid and four more channels located perpendicular to the channel for the analyzed fluid and one end connected to it, while one of these channels is measuring and receptors are placed in it in a liquid medium, the other channel is a reference channel and contains only a liquid medium, while the other two channels contain fluorescent labels with a substrate immobilized on them in a liquid medium. Viral antigens are sorbed directly on the wall of one of the capillaries. The functions of the capillaries are different. The result is assessed based on the fluorescence under a microscope. The reaction is carried out on the basis of free immunodiffusion. Loading is carried out with a syringe; the method of pumping the liquid is not described. Such a system is one of the simplest versions of a microfluidic diagnostic device.

However, the known solution of the microfluidic system has the following disadvantages:

there are no valves in the microfluidic system that provide the possibility of primary filling of channels, without premature mixing of the reagents. In addition, such a system lacks a means for removing air bubbles that inevitably arise when the microfluidic system is connected to external pressure sources or flow devices. Thus, this system, like most other known microfluidic chips, is not suitable for fully automatic analysis. The use of such systems is limited only by experimental laboratory studies, including the selection of optimal parameters, the use of additional laboratory equipment, and additional sample preparation, as well as visual control of the processes occurring inside the chip.

US20150177233A1 discloses an automated "microfluidic" analytical test system . The disposable “smart cartridge”, which includes a microfluidic chip, carries both reagent tanks and reaction chambers. Reaction and detection chambers are coated with antibodies or contain lyophilized antibodies.

However, this solution has the following disadvantages. The buffer and a number of other reagents are charged into the system separately from the chip before the start of the analysis, in an amount calculated for a certain number of detection cycles. Moreover, the system provides automatic pipetting to dispense reagents into the system, which violates the concept of a closed microfluidic consumable chip.

Publication US2005 / 0221281A1 discloses a disposable disc cartridge for the analysis of biological samples , containing all the necessary reagents and microfluidic structure for carrying out multi-step reactions.

Publication US2004 / 0152200A1 discloses an automated (from sample injection to detection) microfluidic system for detecting a certain protein contained in a sample by ELISA (eg, http://www.bialexa.ru/technical-support/methods/elisa/). Such automation allows you to carry out all the work without the participation of specialists. Compressed air is used to move liquids.

Publication US2011 / 0020918A1 discloses multiple parallel ELISA tests performed by accurately separating reagents on a microfluidic chip. Each primary or secondary antibody is delivered via a separate delivery channel to the reaction chamber. The chip supports a high density of reaction chambers of 1,000-4,000 per square centimeter. This miniaturization makes it possible to operate with volumes of the order of nanoliters. The device involves the use of valves and pumps, membranes, has a complex topology and methods for controlling the movement of liquids. The order of placement of the reagents, including their covalent or non-covalent immobilization in the device, is described. Optical detection can be performed, in particular, using a microscope in special "detection zones" on the device. The device has a multilayer structure, it can be made of elastomeric materials (for example, polydimethylsiloxane PDMS) and stores the necessary reagents. Due to the use of hydrophobic materials, the stored reagents are placed in water droplets, being held in the right places without the use of chemical bonding. In this device, each of its layers has its own purpose and in this limited sense it is three-dimensional. The described system can have multiple applications, but it is not an independent device for bedside medicine.

Publication WO2016 / 140990A1 describes a hybrid multilayer microfluidic chip based on paper and polymethyl methacrylate. Such a chip is aimed at detecting common infections with low-cost methods. In such a chip, the paper part can make up a large three-dimensional surface, suitable for immobilizing antibodies or antigens, which significantly increases the ELISA sensitivity.

Probable designs of known microfluidic chips are also disclosed in the publication http://www.j-analytics.ru/files/article_pdf/6/article_6128_791.pdf.

The closest in technical essence to the claimed utility model is replaceable microfluidic module disclosed in Russian Patent №2380418, comprising a receiving chamber for inputting a biological sample to lyse reservoir containing membrane tanks for solutions of lysis buffer microcolumn containing solid sorbent for binding nucleic Acids, wash buffer and ethanol reservoirs to remove residual proteins and salt crystals from the microcolumn, elution buffer reservoir, outlet port compatible with standard microtube, reservoir to collect reaction waste.

All the devices described above do not have the necessary set of nodes and devices to ensure fully automatic analysis. So, for example, in the description of most of the given microfluidic systems, there is no integrated device for removing air bubbles. Air bubbles are a common problem in many microfluidic systems, as they act as pressure dampers, which impairs flow control in microchannels. Also, the presence of air bubbles changes the effective dosing volume, which entails a decrease in the accuracy of analyzes carried out using one or another microfluidic chip. In addition, air bubbles can clog hydrophilic microfiltration membranes or other components of the microfluidic system and impede fluid flow. It should be noted that air bubbles inevitably occur most often at the junctions of microfluidic chips with external devices and nozzles, at the places where the chip inlet holes are sealed. Therefore, the results of analyzes using microfluidic chips, which are not equipped with an efficient air bubble separation system, are poorly reproduced and cannot be used for fully automated medical diagnostic systems.

Disclosure of the essence of the utility model

When automating the process of supplying and discharging small volumes of solutions, known chips, as a rule, are characterized by uncontrolled dilution of solutions, their cross mixing and the ingress of air into the volume.

The technical problem to be solved by the claimed utility model lies in the need to overcome the disadvantages inherent in analogues and prototypes by creating a microfluidic chip of a multilayer structure, which provides separation of the sample and reagents, their filtration, removal of air bubbles from microchannels, controlled mixing and incubation, which will allow to carry out fully automated manipulation of the sample and reagents necessary for the immunofluorescence analysis on microspheres.

The technical result achieved when using the claimed utility model of a microfluidic chip is to ensure complete automation of the process of sequential supply and discharge of small volumes of reagent solutions for conducting fluorescence immunoassay on microspheres, in which , due to the design features of this microfluidic chip, uncontrolled changes in the volumes of these solutions are excluded , including cross-mixing of samples and reagents, their concentrations and feed rates.

In this case, the design of the chip is made in such a way that when it is used at all stages of analysis, one external source of pressure or flow is required to manipulate the sample and reagents inside the microchannel system of the chip.

The chip's check valve system automatically prevents unwanted premature mixing of sample and reagents outside the reaction chamber. The original design of the check valves in this chip does not require external control - their position is controlled exclusively by the direction of flow, which is created by one external source of pressure. The microfluidic chip also contains a unit for automatic removal of air bubbles from the volume of the sample and reagents, due to which there is no need for preliminary degassing of the liquids used, or visual control to detect their presence.

The inventive microfluidic chip can be used for fully automated quantitative analysis, which makes it possible to measure the concentration of protein markers in small volumes of the test samples. At the same time, due to the complete exclusion of the human factor from the analysis process, such an analysis does not require the participation of a medical or laboratory employee. This automation improves the accuracy, reproducibility and reliability of the analyzes. The use of disposable personalized microfluidic chips of the claimed design allows for a study on an individual set of protein markers and excludes cross-contamination of samples.

The problem is solved by the fact that according to the technical solution , the claimed microfluidic chip, which is a body made of optically transparent material, equipped with technological holes for supplying sample solutions, buffer and reagents, inside which layers of optically transparent material are placed close to each other, equipped with slots and holes that form tanks and chambers during assembly, located in a technological order in the direction of flow and interconnected by a system of microfluidic channels:

- a flow-through sample filtration chamber equipped with a track membrane and a check valve,

- a flow-through separator of air bubbles in the sample, made in the form of a chamber equipped with a microfiltration membrane and a check valve,

- a reaction chamber intended for mixing the prepared sample with fluorescently labeled polymer microspheres, made with the possibility of feeding reagents into its volume, and containing a hydrophilic microfilter membrane with a pore size smaller than the size of the microspheres,

- a chamber for collecting spent reagents connected by a channel with a reaction chamber,

At the same time, a membrane with complete permanent deformation is installed in the technological hole intended for supplying the sample, which provides the possibility of membrane injection of the sample into the chip. Check valves installed at the inlet to the flow-through air bubble separator and flow-through chamber for sample filtration are an elastic film placed with tension at the inlet to the corresponding chamber.

Whole capillary blood or human blood serum diluted 5 times or any other biological fluid is used as a sample, and the sample volume is no more than 50 μl. The membrane material is chosen so that its fluorescence spectrum, if any, does not overlap with the fluorescence spectra of the dyes used in the implemented immunoassay technique.

Brief Description of Drawings

The claimed utility model is illustrated by the following drawings and schematic illustrations.

FIG. 1 shows the layout of the layers of the claimed chip.

Figures 2-16 show images of each of the layers forming the claimed chip.

Figure 17 shows a projection of the layering of the layers of the claimed chip

On Fig-19 schematically shows the device of the flow-through separator of air bubbles.

Figure 20 is an image of fluorescent microspheres on a SiO2 membrane.

Figures 21-22 show a diagram of the check valve operation.

Figure 23 shows a diagram of the immunodetection of a microfluidic chip using microspheres.

Figure 24 shows a photo of the claimed chip.

Positions in the drawings indicate:

1- layer 1,

2- layer 2,

3- layer 3,

4- layer 4,

5- layer 5,

6- layer 6,

7- layer 7,

8- layer 8,

9- layer 9,

10- layer 10,

11- layer 11,

12- layer 12,

13- layer 13,

14- layer 14,

15- layer 15,

16- reaction membrane,

17- teflon membrane,

18- track membrane,

19- device body,

20- sample storage tank (P1)

21- reagent storage tank (P2)

22- reagent storage tank (P3)

23- sample filtration chamber (FC),

24- flow-through separator of air bubbles (F),

25- reaction chamber (RC),

26- waste reagents collection chamber (KOR),

27- pressure relief hole from the waste reagent collection tank,

28- port for connecting an external pressure source with a solution for washing the reaction chamber,

29- port for connecting an external pressure source for moving the sample from the reservoir P1 along the microfluidic system of the chip up to the reaction chamber of the RK.

30 - ports for loading the sample into the reservoir P1.

31- port for connecting an external pressure source for flushing the lower part of the FC sample filtration chamber.

32- port for connecting an external pressure source to create a flow and move the reagent from the reservoir P2 through the microfluidic system of the chip to the reaction chamber of the RK.

33- port for connecting an external pressure source for creating a flow and moving the reagent from the reservoir P3 along the microfluidic system of the chip to the reaction chamber of the RK.

34- port for connecting an external vacuum line, to ensure the operation of the air bubble separator F.

35- check valve K1 of the channel for supplying the flushing solution from hole 28 to the reaction chamber of the RK,

36- check valve K2 of the channel connecting the reservoir with the P1 sample to the upper part of the FC sample filtration chamber,

37- check valve K3 of the channel for supplying the flushing solution from hole 31 to the bottom of the filter chamber FK,

38- check valve K4 of the channel connecting the lower part of the filtering chamber of the FC sample with the upper part of the reaction chamber of the PK,

39- check valve K5 of the channel connecting the reservoir P2 with the reagent solution to the upper part of the reaction chamber,

40- check valve K6 of the channel connecting the reservoir P3 with the reagent solution to the upper part of the reaction chamber.

41- inlet of the flow-through air bubble separator (F1),

42- outlet of the flow-through air bubble separator (F2),

43- bioconjugated microsphere,

44- biological marker,

45- fluorescent label,

46- optical detection system.

Implementation of the utility model

The inventive microfluidic chip is a disposable sensor designed for immunological multivariate analysis.

The microfluidic chip is made in the form of a transparent body, in which thin layers of different optically transparent materials glued together under pressure are placed with through-slots of various thicknesses applied to them and fixed membranes and valves. The external dimensions of the chip are dictated by the dimensions of the cameras and equipment on which it operates, as well as aesthetic and practical considerations. Technological holes (ports) are made in the layers and in the body for connecting the means for supplying sample solutions, buffer and reagents. All layers have their own functional meaning.

The chip consists of: five layers made of polymethyl methacrylate (PMMA) with a thickness of no more than 1 mm, 2 layers of polycarbonate film with a thickness of 0.2 mm and 8 adhesive (adhesive) layers of baseless two-layer adhesive tape 3M 468MP, with 6 and 7 are about 0.05 mm thick and the rest are about 0.1 mm thick. Adhesive layers are intended for bonding other layers and membranes. The following is a description of all the layers of the claimed chip, starting with the top layer.

Layer 15 (see figure 2) is made of polymethyl methacrylate with a thickness of not more than 1 μm. This layer is the upper wall of the entire microfluidic chip; all holes 27-34 are located in it for connecting external sources of pressure and vacuum, filling tanks.

Layer 14 (see Fig. 3) is made of an adhesive material, for example, 3M 468MP adhesive tape with a thickness of no more than 120 μm. This layer contains the upper chambers of the check valves K1, K2, K3, K4, K5 and K6, reservoirs for storing the P1 sample with a volume of ~ 50 μL, and reagents P2, P3 with volumes of ~ 30 μL, and channels connecting these reservoirs with the corresponding holes 27 -34.

Layer 13 (see Fig. 4), similar to Layer 1, is made of polymethyl methacrylate with a thickness of no more than 1 μm. This layer is intended to give the upper chambers of the check valves K1, K2, K3, K4, K5 and K6 the required thickness to ensure their correct operation. In addition, this layer also contains the bulk of the reservoirs P1, P2, P3. This layer is also the upper wall of the RC reaction chamber, through which fluorescent signals from microspheres are detected.

Layer 12 (see Fig. 5) is made of an adhesive material, for example, 3M 468MP with a thickness of no more than 120 μm. This layer contains the upper chambers of the check valves K1, K2, K3, K4, K5 and K6, as well as the upper part of the reaction chamber RK, reservoirs for storing sample P1 and reagents P2, P3, and channels connecting these nodes with the corresponding holes 27- 28, 30, 34.

Layer 11 (see Fig. 6) is made of a polyethylene terephthalate (PET) film with a thickness of not more than 120 μm. This layer contains the upper chambers of the check valves K1, K2, K3, K4, K5 and K6, as well as the upper part of the reaction chamber RK. PET film is designed to add depth to the reaction chamber of the RK, to equalize the flow through the SiO2 membrane. In addition, this layer is the lower wall of the reservoirs for storing samples P1 and reagents P2, P3, and separates them from the rest of the microfluidic channel system.

Layer 10 (see Fig. 7) is made of an adhesive material, for example, 3M 467MP with a thickness of not more than 60 μm. This layer contains the upper chambers of the check valves K1, K2, K3, K4, K5, and K6, as well as the upper part of the reaction chamber RK, the upper part of the FC sample filtration chamber and the upper part of the air bubble separator F, which is an elongated flowing microfluidic cell.

Layer 9 (see Fig. 8) is made of an adhesive material, for example, 3M 467MP with a thickness of no more than 60 μm. This layer contains holes for fastening membranes 16 and 18, as well as the upper part of the filtration chamber of the PK sample.

Layer 8 (see Fig. 9) is made of an adhesive material, for example, 3M 468MP with a thickness of not more than 120 μm. This layer contains the lower chambers of the check valves K1, K2, K3, K4, K5 and K6, as well as the lower part of the reaction chamber RK, the lower part of the FC sample filtration chamber with supporting lamellas.

Layer 7 (see Fig. 10) is made of a polyethylene terephthalate (PET) film with a thickness of not more than 120 μm. This layer contains the lower chambers of the check valves K1, K2, K3, K4, K5 and K6, as well as the lower part of the reaction chamber RK and flow bubble separator F. The PET film is intended to give depth to the reaction chamber RK, to equalize the flow through the SiO2 membrane.

Layer 6 (see Fig. 11) is made of an adhesive material, for example, 3M 468MP with a thickness of not more than 120 μm. This layer contains the lower chambers of the check valves K1, K2, K3, K4, K5 and K6, as well as the lower part of the reaction chamber PK, and channels connecting these units with the corresponding holes.

Layer 5 (see Fig. 12) is made of polymethyl methacrylate with a thickness of not more than 1 μm. This layer is the upper wall of the KOR waste reagent collection chamber and separates this reservoir from the rest of the channel system. In this layer, there are holes 27, 34, and a flow bubble separator F.

Layer 4 (see Fig. 13) is made of an adhesive material, for example, 3M 468MP with a thickness of not more than 120 μm. This layer follows the geometry of Layer 14 and is designed to connect Layer 13 and Layer 11 together.

Layer 3 (see Fig. 14) is made of polymethyl methacrylate with a thickness of not more than 1 μm. This layer repeats the geometry of Layer 14 and is designed to give the KOR spent reagents collection chamber the required volume of ~ 1 cm ³ . Also, with the help of this layer, the required depth is given to the channel connecting the opening 34 with the air bubble separator F, for efficient transmission of the vacuum to the lower evacuated chamber.

Layer 2 (see Fig. 15) is made of an adhesive material, for example, 3M 468MP with a thickness of not more than 120 microns - contains a chamber for collecting spent reagents KOP, which is connected to the hole 27 to relieve pressure from this reservoir. In addition, this layer contains a lower evacuated chamber of the air bubble separator F, connected to the hole 34 for connecting an external vacuum line.

Layer 1 (see Fig. 16) is made of polymethyl methacrylate with a thickness of no more than 1 micron and is the base of the chip, contains only technical centering holes at the corners necessary for assembling the chip and fixing the layers during assembly.

The chamber for collecting spent reagents (KOR) is a rectangular reservoir with a volume of ~ 1 ml, the main volume of which is in Layer 3. The reservoir is connected to the lower part of the reaction chamber of the RK from where the spent reagents come and has an outlet for pressure relief.

The flow-through separator of air bubbles F is a chamber horizontally divided by a hydrophobic membrane No. 17 made of polytetrafluoroethylene (PTFE), which has a pore diameter of ~ 1 micron. The lower part of the air bubble separator is an evacuated chamber into which air bubbles from solutions are pumped out through a membrane. The upper part of the air bubble separator is a flow-through microfluidic cell, the lower wall of which is a PTFE membrane. All solutions that are sequentially supplied to the reaction chamber are pumped through the flow cell. At the same time, due to the hydrophobic properties of the membrane, the liquid does not pass through its pores and does not enter the evacuated chamber, and air bubbles are removed by vacuuming the lower evacuated chamber. The absence of air bubbles eliminates their influence on the pumping rate and changes in the effective volume of the sample or reagent. PTFE membrane # 17 is located in Layer 9 and is a 4 * 8 mm rectangle. A sectional diagram of the operation of the air bubble separator is shown in Fig. 18-19.

The reaction chamber (RC) is a chamber divided horizontally by a hydrophilic membrane No. 16 made of SiO ₂ fibers. The effective pore diameter of such a membrane is ~ 0.5 μm. When assembling a microfluidic chip in the upper part of the RC, fluorescent conjugated microspheres with immobilized antibodies are applied to the detection membrane. When processing a chip, all reagents pass sequentially through the membrane and through the layer of microspheres on its surface. In this case, specific binding of protein markers from the sample and their subsequent development occurs on the surface of microspheres. This unit makes it possible to ensure the reaction of the entire volume of the sample or reagent with the surface of the microspheres. After pumping the solutions through the membrane, they enter the lower part of the reaction chamber, which is connected by a channel to the chamber for collecting spent reagents. The SIO2 # 16 membrane is located between Layer 9 and Layer 10 and is a 5 * 5 mm square. Such a constructive solution of the RK makes it possible to overcome diffusion limitations in the process of binding of protein markers from a sample to microspheres. The fluorescence image of microspheres on this membrane is shown in Fig. 20 (field of view 1 mm).

Each of the K1-K6 check valves is a chamber divided horizontally by an elastic membrane No. 19, which can be made of rubber 0.1 mm thick and has a single perforated hole in the center with a diameter of 0.7 mm. The lower part of the check valve chamber under the diaphragm has a depth of 0.2 mm; the upper part of the check valve chamber has a depth of 1.2 mm. Thus, when the flow of fluid enters from the bottom up, the elastic membrane flexes, the perforated hole expands and the fluid flows through the valve. In the opposite case, when the liquid flow enters from top to bottom, the elastic membrane is pressed against the bottom wall of the check valve chamber, the perforated hole remains closed and the liquid does not flow through the valve. Thus, check valves of this design do not require additional external control, their capacity depends only on the direction of fluid flow through them. Check valves prevent uncontrolled dilution of sample and reagent solutions and their premature displacement outside the reaction chamber. Elastic membrane # 19 is located in Layer 9 and is a 5 * 5 mm square. A cross-sectional diagram of the check valve operation is shown in Figs. 21-22, arrows indicate the directions of fluid flow.

The sample filtration chamber (FC) is a chamber divided horizontally by a track membrane No. 18 (the method of making a track membrane is disclosed, for example, in patent RU 2325944), which can be made of lavsan with a thickness of 0.012 mm and has pores with a diameter of ~ 480 nm. A sample from reservoir P1 enters the upper part of the FC, the sample passes through the membrane, while objects larger than the pore diameter remain on the membrane. The filtered sample enters the lower part of the FC, which communicates with the reaction chamber of the FC. Membrane # 18 is located between Layer 8 and Layer 9 and is a rectangle of approximately 17 * 22 mm. At the same time, in Layer 8 there are lamellas that support membrane # 18 to avoid sagging.

The claimed chip is manufactured as follows.

With the help of the programmable laser engraver Laser Pro GCC Spirit GLS 100, the layers (parts) of the chip are manufactured (cut out) from the appropriate materials and prepared for assembly. Partial alignment of layers (parts of the chip) is carried out, as a result of which 4 parts of the chip are obtained, including the corresponding layers and membranes: layers 15-12; layers 11-8, layers 7-5, layers 4-1.

A solution containing microspheres is applied to a SiO2 microfiltration membrane located in the third part of the chip using a laboratory dispenser. Then the membrane is dried in a stream of air. The 4 parts of the chip are individually compressed under vacuum using a hydraulic press with a pressure of about 800 kg per part. After that, 4 parts of the chip are combined and pressed under vacuum using a hydraulic press with a pressure of about 800 kg per chip. Tanks P2 and P3 are filled with the necessary reagents through holes 32 and 33 using a laboratory dispenser. All holes of the chip are sealed with adhesive tape. In this form, the chip is stored until the start of analysis at a temperature of +4 degrees Celsius.

The combined holes and slots in the layers form ports and channels in which both lateral and transverse fluid movement occurs.

Valves redirect flows, and reservoirs serve to slow them down or store fluid.

The microfluidic chip performs the task of delivering reagents from various internal chambers and blood serum to the reaction chamber; membranes serve both to separate blood into serum and shaped elements, and to retain microspheres with molecular complexes attached to them.

The internal dimensions of channels, valves, chambers, as well as the entire architecture of the microfluidic chip are experimentally selected in accordance with the requirement for the flow rate, fluid pressure and the fulfillment of the tasks assigned to it - the delivery of reagents from various internal chambers and blood serum to the reaction chamber. Membranes are intended both for separating blood into serum and shaped elements, and for holding microspheres with enzyme immunoassays attached to them. The composition and materials of the chip layers are dictated by the tasks to be performed - optical transparency - and the capabilities of the existing equipment for the production of chips - suitability for laser and mechanical processing.

The inventive microfluidic chip works as follows.

Through hole 30, a sample of 50 μL is introduced into the reservoir P1 of the claimed chip, after which the port is sealed, for example, using adhesive tape. The chip is placed in the analyzer, after which the immune analysis process is carried out in an automatic mode. An external vacuum line is connected to the external port 34 to operate the air bubble separation system. Further, through port 28, the initial filling of the reaction chamber with a volume of 60 μL with a washing solution at a linear rate of about 50 μL / sec takes place, which passes through the following points of the chip (see layer diagrams): K1 - K5 - K6 - F1 - F2 - RK and then enters into the chamber for collecting spent reagents (KOR).

Through port 29, by pumping a buffer solution, a pressure of 0.12 -0.2 MPa is supplied and the blood sample from the reservoir P1 is moved to the K2 check valve, and then to the blood filtration chamber (FC), then through the valve K4, then points F1 - F2 enters the reaction chamber of the RK.

Port 31 is supplied with buffer for FC washing. It passes through the K3 check valve, then through FK, K4, F1 - F2 - RK, and is discharged into the container for spent reagents (KOR).

Through port 32, by pumping a buffer solution, a pressure of 0.12 -0.2 MPa is supplied to the reservoir with the reagent (P2). Liquids pass through K5 and K6, F1 - F2 - RK and are disposed of in the KOR.

This is followed by flushing the chip with a buffer along the route K1 - K5 - K6 - F1 - F2 - RK.

Through port 33, a pressure of 0.12 -0.2 MPa is supplied to reservoir P2 by pumping a buffer solution, liquids pass through K6, F1 - F2 - RK and are disposed of in the KOR.

This is followed by the final flushing of the K1 - K5 - K6 - F1 - F2 - RK chip.

In this case, the pressure is applied in such a way that the speed of movement of liquids in the chip is 0.08 µl / s. The wash volume is 80 μL. The volume of the introduced sample is 50 μl. The volumes of reagents placed on the chip (P2 and P3) are 30-40 μl each.

Check valves K1 - K6 prevent serum from entering adjacent microchannels and mixing reagents on the chip.

A hydrophobic semi-permeable membrane is designed to separate air microbubbles from reagents and prevent bubbles from entering the RC.

An example of a specific implementation

The microfluidic chip of the claimed design was made for the simultaneous determination of the concentrations of 4 interleukins IL1b, IL6, IL8, IL10 and human heat shock protein HSP70 in blood serum.

The main technical characteristics of the manufactured chip are presented in Table 1.

Table 1 - Main technical characteristics

No.
p / p Name Value 1 Overall dimensions of the device (LxWxH), mm 74x50x6.1 (dimensions after pressing) 2 Device weight, kg 0.020 3 Materials in layers: PMMA Polymethylmethacrylate GOST 10667-90 Adhesive layer Baseless two-layer adhesive tape 3M 468MR GOST 20477-86 Film Polycarbonate GOST 25288-82 4 Crimping load 800kg

When assembling the chip, 2 μl of magnetic microspheres of each type with immobilized monoclonal antibodies to IL1b, IL6, IL8, IL10 (BioPlex ProTM Hu Cyto Grp I, Bio-Rad) and HSP70 were added to the reaction membrane. A solution of a mixture of biotinylated detecting antibodies to the corresponding proteins was used as reagent P2. Streptavidin conjugated with phycoerythrin was used as a P3 reagent for the detection of complexes of antigens and biotinylated antibodies. Thus, the higher the concentration of these protein markers in the test sample, the more fluorescent dye is bound to the surface of the microspheres. The fluorescent signal from the microspheres is detected, for example, by means of an epifluorescence microscope and, by means of calibration, is converted into the value of the concentration of protein markers.

Claims (8)

1. A microfluidic chip, characterized in that it is a body made of optically transparent material and equipped with ports for supplying sample solutions, buffer and reagents, and made with the possibility of connecting through these ports to a single external pressure source providing flow direction to the appropriate chambers, while inside the body, layers of optically transparent material are placed close to each other, provided with slots and holes, forming the following reservoirs and chambers during assembly, located in a technological order in the direction of the sample flow and interconnected by a system of microfluidic channels: - flow-through chamber for sample filtration, equipped with a track membrane and a check valve, as well as a port for connection to a single external pressure source, - a flow-through separator of air bubbles in the sample, made in the form of a chamber equipped with a microfiltration membrane and a check valve, - a reaction chamber designed for mixing the prepared sample with fluorescently labeled polymer microspheres, made with the possibility of feeding reagents into its volume and containing a hydrophilic microfilter membrane with a pore size smaller than the size of the microspheres, and also connected through channels with ports for connection to a single external a pressure source for moving the sample, reagents and rinsing the chamber, - a chamber for collecting spent reagents connected to the reaction chamber through a channel, at the same time, a membrane with complete permanent deformation is installed in the technological hole intended for supplying the sample, which makes it possible to inject the sample into the chip, check valves are chambers with a perforated elastic membrane placed with tension at the chamber inlet in a plane perpendicular to the direction of sample flow. 2. The microfluidic chip according to claim 1, characterized in that the material of the membrane of the reaction chamber is selected so that its fluorescence spectrum, if any, does not overlap with the fluorescence spectra of the dyes used in the implemented immunoassay technique.

Images