CN113295599A

CN113295599A - Multifunctional cell-protein detection method and detection device

Info

Publication number: CN113295599A
Application number: CN202010108655.6A
Authority: CN
Inventors: 陶靖
Original assignee: Shanghai Nayan Biotechnology Co ltd
Current assignee: Shanghai Nayan Biotechnology Co ltd
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2021-08-24

Abstract

The invention relates to a multifunctional cell-protein detection method and a detection device, wherein the method integrates a sample to be detected by using a flow-type fluorescence analysis technology to realize blood cell analysis, microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis; and, the blood cell analysis provides basic data for microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis, and is used for correcting detection results and optimizing sample reaction. Compared with the prior art, the method has the advantages of multifunctional integration, rapid detection of the whole blood sample, accurate correction of the detection result, reaction optimization and the like.

Description

Multifunctional cell-protein detection method and detection device

Technical Field

The invention relates to the field of biological analysis and detection, in particular to a multifunctional cell-protein detection method and a detection device.

Background

In clinical disease diagnosis, blood cell analysis, soluble protein molecule analysis, and cell characteristic antigen molecule analysis are commonly used test items, and they provide important diagnostic information from different perspectives.

With the progress of medicine and the improvement of diagnosis level, comprehensive judgment is required to be carried out by combining a plurality of detection results for the diagnosis of many diseases. For example, infection and inflammation require the measurement of differential white blood cell count and the amount of characteristic proteins such as CRP and PCT, and diseases of the immune system require the measurement of differential white blood cell count, the absolute cell count of the cell surface characteristic antigen CD4 and the amount of certain characteristic proteins.

Today, blood cell analysis is usually performed on fully automated blood cell analyzers; cellular signature antigen molecular analysis is typically detected on a flow cytometer; the soluble protein molecule analysis is usually performed on devices such as a biochemical analyzer, a chemiluminescence analyzer or a specific protein analyzer, three detection items are respectively performed on different devices, and the detection items are respectively performed on the separate devices, so that the consumed sample amount is large, and the detection time is long.

At present, the soluble protein molecule analysis which is used in large quantities clinically usually uses a serum or plasma sample to detect on devices such as a biochemical analyzer, a chemiluminescence analyzer and the like, but blood coagulation and centrifugation operations are required for preparing the serum or plasma, and a lot of time and labor are consumed.

For acute diseases, a whole blood sample such as a specific protein analyzer is used for rapid detection of characteristic proteins, but the specific protein analyzer generally uses an immunoturbidimetry method such as an immunotransmission or an immunoscattering turbidimetry method, and due to the limitation of the detection principle, the detection sensitivity and precision of the turbidimetry method are not high.

In the immune serological detection, such as biochemical immune analysis and chemiluminescence immune analysis, the original whole blood sample needs to be firstly subjected to standing and centrifugal treatment to separate blood plasma and blood cells, so that the sample to be detected does not contain blood cells, and the detection result is the number of the target object to be detected/uL serum.

If a whole blood sample is used for detecting soluble protein molecules, the result is the amount of the target substance to be detected/uL whole blood, and thus the result cannot be compared with the result of an immunoserological test, and therefore, the detection result of the whole blood sample must be corrected by the volume information of blood cells. The proportion of blood cells in the whole blood sample is called the hematocrit, and as shown in fig. 11, the hematocrit is usually 40-50%, but the hematocrit of infants varies greatly, and some can reach 70%. Current instruments that use raw whole blood samples for immunoassays typically use 40% as a default value for blood cell volume correction, which is subject to large errors. In order to obtain accurate information on the volume of blood cells, a hematology analyzer is additionally used to obtain the data on the volume of cells.

At present, in clinical diagnosis, the absolute count analysis of the cell characteristic antigen CD4 is a core judgment index for detecting immune system diseases. Cellular signature antigen analysis, typically performed using a flow cytometer, gives information only on the proportion of signature antigen cells, such as the percentage of CD4 cells in whole lymphocytes, but not the absolute number of CD4 cells (number/uL whole blood sample). Usually, in order to obtain the absolute quantity information of the white blood cells or the lymphocytes, the absolute quantity information needs to be detected by using a blood cell analyzer.

In the cell characteristic antigen analysis, an antigen and an antibody perform a specific binding reaction. In order to achieve the best signal-to-noise ratio, the amount of antibody added has an optimal ratio range, preferably the sample has a white blood cell count of less than 10.0 x 10^9/L, and if the white blood cell count is greater than 10.0 x 10^9/L, the sample needs to be diluted, usually in order to obtain the information of the absolute amount of white blood cells or lymphocytes, a blood cell analyzer needs to be additionally used.

Disclosure of Invention

The present invention aims at providing multifunctional cell-protein detecting method and device to overcome the demerits of available technology.

The purpose of the invention can be realized by the following technical scheme:

a multifunctional cell-protein detection method is characterized in that a flow fluorescence analysis technology is applied to integrate a sample to be detected to realize blood cell analysis, microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis;

moreover, the blood cell analysis provides basic data for microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis, and is used for correcting detection results and optimizing sample reaction, and the method specifically comprises the following steps:

correcting the results of the protein molecule analysis in a microsphere-based protein molecule analysis by the volumetric information of the cells in the whole blood sample obtained by the blood cell analysis;

the absolute quantity information of the white blood cells or the lymphocytes obtained by blood cell analysis is combined with the proportion of the characteristic antigen cells obtained by cell characteristic antigen molecule analysis, and the absolute quantity of the characteristic antigen cells is accurately and quantitatively calculated;

the absolute quantity information of the white blood cells or the lymphocytes obtained by the blood cell analysis is used for optimizing the sample reaction in the cell characteristic antigen molecule analysis so as to achieve the optimal antigen-antibody reaction ratio.

The sample to be tested for microsphere-based protein molecule analysis is whole blood.

A multifunctional cell-protein assay device, the device comprising:

a sample sucking needle assembly: the sample suction device is arranged on the detection device main body and used for sucking a sample to be detected from a sample introduction position and moving the sample to be detected to a set position to realize the suction, distribution and reaction of the sample to be detected;

reaction components: the sample sucking needle assembly is arranged at a first set position on the detection device main body and used for receiving a quantitative sample to be detected, which is discharged by the sample sucking needle assembly, and automatically realizing the reaction between the sample to be detected and a reagent;

storing the assembly: the device is arranged at a second set position on the detection device body, is provided with a plurality of containers in different shapes, is used for storing a plurality of reagents, reaction intermediate substances and samples to be detected, and is matched with a sample sucking needle assembly for analysis reaction;

an optical flow cell: the detection device is arranged on the detection device main body and is respectively communicated with the reaction component and the sample sucking needle component through connecting pipelines, and detected substances are respectively sent into the optical flow chamber for flow type optical detection;

an optical detection assembly: the device comprises a one-color or multi-color light source and a plurality of scattered light and fluorescence detectors, wherein the light source and the detectors are configured for blood cell analysis, microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis according to detection items to obtain characteristic detection signals.

The device also includes:

detecting components by an electrical impedance method and a colorimetric method: the sample sucking needle assembly is arranged at a third set position on the detection device body and used for receiving a quantitative sample to be detected, which is discharged by the sample sucking needle assembly, and carrying out electrical impedance detection and colorimetric detection.

The storage assembly comprises a container fixing frame, a fixing frame moving mechanism and containers of various shapes and systems, wherein the containers are placed in hole sites on the container fixing frame, and each container comprises a reaction tube, a multi-connection tube and a multi-tube reagent strip packaged with various reagents.

The scattered light and fluorescence detectors include a forward scattered light FSC detector, a side scattered light SSC detector and a polychromatic fluorescence FL detector.

Compared with the prior art, the invention has the following advantages:

firstly, multifunctional integration: the invention integrates and realizes blood cell analysis, protein molecule analysis based on microspheres and cell characteristic antigen molecule analysis in one detection device by applying a flow type fluorescence analysis technology, and the detection sensitivity and precision of the flow type fluorescence analysis technology are good.

Secondly, the whole blood sample is rapidly detected: the soluble protein molecule analysis can directly use a whole blood sample, and is suitable for the rapid detection of acute diseases.

Thirdly, accurately correcting the detection result and optimizing the reaction: the sample to be detected for microsphere-based protein molecule analysis is whole blood, and the result of protein molecule analysis is corrected in microsphere-based protein molecule analysis through the volume information of cells in the whole blood sample obtained by blood cell analysis. The absolute quantity information of the leucocytes or the lymphocytes obtained by analyzing the blood cells is combined with the proportion of the characteristic antigen cells obtained by analyzing the cell characteristic antigen molecules, the absolute quantity of the characteristic antigen cells is accurately and quantitatively calculated, and the sample reaction is optimized in the analysis of the cell characteristic antigen molecules by the absolute quantity information of the leucocytes or the lymphocytes obtained by analyzing the blood cells, so that the optimal antigen-antibody reaction proportion is achieved.

Drawings

FIG. 1 is a schematic diagram of the detection of an optical flow cell.

FIG. 2 is a schematic block diagram of the detection apparatus and detection method of the present invention.

Fig. 3 is a schematic structural diagram of the present invention.

Fig. 4 is a schematic diagram of an optical detection system.

Fig. 5 is a system configuration diagram of the present invention.

Fig. 6 is a first perspective structural view of the present invention.

Fig. 7 is a second perspective view of the present invention.

FIG. 8 is a scatter plot of blood cell analysis.

FIG. 9 is a scatter plot of microsphere-based protein molecule analysis.

FIG. 10 is a scatter plot of the cellular signature antigenic molecule analysis.

FIG. 11 is a schematic composition of a whole blood sample.

The notation in the figure is:

31. sample introduction position, 32, reaction component, 33, storage component, 34, sample sucking needle component, 35, optical detection system, 351, optical flow chamber, 352, detection laser beam, 36, electrical impedance method and colorimetric detection component, 37, connecting pipeline, 38, container, 39, connecting pipeline, 41, first light source, 42, second light source, 43, forward scattering light FSC detector, 44, side scattering light SSC detector, 45, multicolor fluorescence FL detector, 71, sample sucking needle, 72, first driving motor, 73, swab, 74, Z-axis guide rail, 75, X-axis guide rail, 76, electromagnetic valve, 77, precision injection pump, 78, display, 55, liquid storage tank, 56, pump, 701, container fixing frame, 702, second driving motor, 703, Y-axis guide rail, 704, driving screw.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

The objects detected by the flow cytometer are cells and microparticles suspended in a liquid in an independent state, the flow cytometer uses a hydrodynamic focusing principle (as shown in fig. 1), a sheath fluid in an optical flow chamber 351 arranges sample cells or microspheres to be analyzed in a sample suspension into a line, the sample cells or microspheres rapidly flow through a detection laser beam 352 one by one, and a high-precision optical system collects and detects excited multi-angle scattered light and fluorescence with multiple wavelengths; through complex electronic signal processing and computer data analysis, information of a plurality of physical structural characteristics and biological expression characteristics of tens of thousands of cells or microspheres can be obtained in a short time, and a flow cytometry can be provided with a multicolor laser, a multipath scattered light and a fluorescence detector to realize powerful analysis functions of the cells and the microspheres.

Scattered light from different angles reflects the physical structural characteristics of the cells or microspheres, and the white blood cells can be classified by combining a scatter diagram of forward scattered light and a scatter diagram of side scattered light.

The detection of the lateral fluorescence with different wavelengths can reflect the characteristics of the expression of biological molecules, for example, in order to detect specific cell surface antigens, RNA or intracellular specific molecules, a proper fluorescent marker is required, such as CD4 monoclonal fluorescent antibody connected with PE fluorescent molecules can be specifically combined with the CD4 surface antigens of cells, the PE fluorescent molecules can detect emitted fluorescence at 585nm under the excitation of 532nm laser, the quantitative information of CD4 characteristic antigen cells can be obtained, and important bases are provided for the diagnosis of diseases.

The flow analysis method is used for blood cell analysis, and can classify and count white blood cells, red blood cells and platelets in a blood sample. A common clinical diagnostic is a differential analysis of leukocytes, which include lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The method combines scattered light and fluorescence detection to perform differential counting analysis on the white blood cells, the differential counting analysis of the white blood cells is an important conventional detection item, can reflect the basic condition of diseases, and can also perform differential counting on red blood cells and platelets in a blood sample by applying a flow analysis method.

In conjunction with electrical impedance methods, the blood analysis apparatus can analyze the classification and counting of red blood cells, platelets, and the total number of white blood cells, or the number of certain types of cells in white blood cells, such as basophils, and in conjunction with colorimetry, the blood analysis apparatus can measure the hemoglobin content.

The flow analysis method is used for analyzing cell characteristic antigen molecules, such as characteristic expression of cell surface antigens or nucleic acid analysis, and the like, for example, leukocyte differentiation antigens CD4, CD8, CD3, CD45, CD19 and platelet characteristic antigen CD61, ankylosing spondylitis characteristic antigen HLA-B27, intracellular substances and nucleic acid, and the like. The lymphocyte subpopulation analysis can be realized by combining the detection of a plurality of cell surface antigens, the immune status of a human body can be accurately reflected, and the lymphocyte subpopulation analysis method has important reference significance for clinical disease diagnosis, such as infectious diseases, blood diseases and the like. Particularly, the CD4 cell subset in the T lymphocyte has important assistance and regulation effects on humoral immunity and cellular immunity, the CD4 cell absolute count analysis is a core judgment index for detecting immunodeficiency diseases, and the cell characteristic antigen analysis usually needs a plurality of fluorescent markers and is analyzed by flow multi-color fluorescence detection.

The flow analyzer can analyze not only cells but also microparticles, and one typical application is microsphere-based soluble protein molecular analysis, i.e., flow liquid phase multiple protein quantification.

The special microsphere is provided with a feature or a marker for identifying the type, such as fluorescent dyes with various intensities, and after being excited by a light source with a specific wavelength, the type of the microsphere can be identified by detecting a feature signal, such as fluorescence with a specific wavelength or specific wavelengths, namely the type of the soluble protein molecule to be detected. The method comprises the steps of coating specific antigen, antibody or nucleic acid probes on different types of microspheres respectively, reacting with a blood sample, performing specific binding with soluble protein molecules to be detected in the sample respectively, and connecting with a fluorescent label. In the flow-type fluorescence analysis system, various soluble proteins, cytokines, autoantibodies, specific nucleic acid sequences, and the like can be quantitatively analyzed and detected by a multicolor light source and a plurality of scattered light and fluorescence detections. By means of the microspheres and specific markers, dozens of proteins can be simultaneously detected in a trace sample (25-50ul), such as multiple characteristic proteins related to immunity, the sample amount used is greatly reduced compared with other immunoassay methods, and the sensitivity and precision of flow-type fluorescence detection are higher compared with an immunity transmission or immunity scattering turbidimetry.

Based on the flow-type fluorescence analysis technology, the sample to be detected for soluble protein molecule analysis is whole blood, serum and plasma, and the whole blood sample can be used for rapid detection of characteristic protein aiming at acute diseases, such as point-of-care testing (POCT).

Based on the above, the invention provides a multifunctional cell-protein detection device aiming at the requirements of multifunctional and rapid detection in clinical diagnosis, which integrates a sample to be detected to realize blood cell analysis, microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis by applying a flow-type fluorescence analysis technology;

the volume information of the cells in the whole blood sample obtained by the blood cell analysis is corrected for the results of the protein molecule analysis in the microsphere-based protein molecule analysis.

The absolute quantity information of the white blood cells or the lymphocytes obtained by blood cell analysis is combined with the proportion of characteristic antigen cells obtained by cell characteristic antigen molecule analysis, and the absolute quantity of the cell characteristic antigen cells is accurately and quantitatively calculated;

As shown in fig. 2, 3 and 5, the detection device includes a sample processing system, an optical detection system, an electronic processing system, a computer control and analysis system.

The sample processing system includes:

and a sample feeding position 31, wherein a sample to be detected is loaded by a manual or automatic device.

The sample sucking needle mechanism 34 includes a sample sucking needle and a moving mechanism, and the sample sucking needle sucks the sample to be detected and moves to a designated space position to realize quantitative sucking, distribution and reaction of the sample to be detected. Specifically, the pipette tip is provided with a moving mechanism, as shown in FIG. 3, which can be moved accurately in the X-direction and the Z-direction as shown. According to the detection items, the sample to be detected is distributed to blood cell analysis, cell characteristic antigen molecule analysis and microsphere-based soluble protein molecule analysis by a sample suction needle. Depending on the speed of the test, two pipette tips may be provided, one for aspirating and dispensing the sample to be tested and for operation in the reaction module 32 and one for operation in the storage module 33. The sample suction needle is not limited to a linear moving mode, and the sample suction needle can also be driven by the moving mechanism to rotate.

A connection line 39 is provided between the sample-aspirating needle and the optical flow cell 351, and the substance to be detected is aspirated by the sample-aspirating needle, sent into the optical flow cell 351, and subjected to flow-type optical detection.

The reaction assembly 32, the sample sucking needle can move to the reaction cell of the reaction assembly 32 to discharge quantitative samples, the reaction cell is connected with a plurality of reagent containers, and various automated reactions of the samples and the reagents, such as dilution, hemolysis and the like, can be carried out in the reaction cell; the reaction assembly 32 is provided with a temperature control component to keep the reaction at a proper temperature; a connecting pipe 37 is provided between the reaction cell and the optical flow chamber 351, and a substance to be detected is sent into the same optical flow chamber 351 by an electromagnetic valve and an injection pump to perform flow type optical detection.

The storage assembly 33 is used for placing various containers 38, and is provided with a moving mechanism for storing various reagents, reaction intermediate substances and substances to be tested, and is used for various reactions such as cell characteristic antigen molecule analysis, microsphere-based soluble protein molecule analysis and the like.

The holder of the storage module 33 has a plurality of holes, particularly arranged in a matrix form, or arranged in other forms, for holding and fixing containers 38 of different forms, for storing various reagents, reactive intermediate substances and substances to be tested, and for cellular characteristic antigen molecule analysis and microsphere-based soluble protein molecule analysis. The pipette needle may be moved into the container 38 of the storage assembly 33 to discharge a quantitative sample, and perform automated various reactions of the sample and reagents, such as dilution, washing, and labeled incubation.

The container 38 may comprise a single reaction tube, or multiple tubes, or a multi-tube reagent strip enclosing multiple reagents.

The storage assembly is provided with a temperature control component, so that the storage and reaction are kept at proper temperature.

The storage unit 33 is provided with a one-dimensional or two-dimensional moving mechanism that can move or rotate in the illustrated X direction and Y direction, and particularly, with a one-dimensional moving mechanism that can move in the illustrated Y direction for loading the containers 38; and the sample sucking needle moving mechanism is matched for positioning, sucking and reacting different containers.

According to the detection items, the reaction of the sample to be detected and the reagent is automatically carried out in the container of the storage assembly 33 under the control of a computer, such as the reactions of dilution, washing, labeling incubation and the like which are sequentially carried out; or in the small batch detection, after the reaction between the sample to be detected and the reagent is manually completed, the storage part is moved out, and the container is loaded into the storage part.

The sample probe aspirates the analyte in the container 38, and the analyte is sent to the optical flow cell 351 through the connecting line 39 between the sample probe and the optical flow cell 351 to perform flow optical detection. Or the sample sucking needle sucks the reaction intermediate substance or the substance to be detected in the container 38, the sample sucking needle moves to the reaction cell of the reaction assembly 32 to discharge the reaction intermediate substance or the substance to be detected, the reaction cell is connected with a plurality of reagents, and various automated reactions of the sample and the reagents, such as dilution, hemolysis and the like, can be performed in the reaction cell. The substances to be detected are sent to the same optical flow cell 351 through the reaction cell and the connecting line 37 of the optical flow cell 351, respectively, and flow optical detection is performed.

The sample processing system also comprises a sample sucking needle cleaning device and a pipeline cleaning function.

The reaction assembly 32, the storage assembly 33, the electrical impedance method and colorimetric detection assembly 36 may be arranged on the detection device body along the X-axis in a straight line, or may be arranged along the circumference, and the corresponding sample suction needle mechanism may be provided with one or more sets.

The optical detection system includes:

the optical flow chamber 351 is connected with the same optical flow chamber 351 through a pipeline, and the substances to be detected are respectively sent into the optical flow chamber for flow analysis and detection;

an optical detection module, as shown in fig. 4, the analyzed cells or microspheres are wrapped by sheath fluid and focused in a row in an optical flow chamber 351, and rapidly flow through the detection laser beams one by one; the spots where the cells flowed past the laser beam were M and N, respectively. The optical detection system comprises one or more

color light sources

41 and 42, a plurality of scattered light and fluorescence detection means, including a forward scattered light FSC detector 43 and a side scattered light SSC detector 44, and a multi-color fluorescence FL detector 45. The scattered light and the fluorescence detector respectively correspond to the excitation light source, and a wavelength selection filter is arranged in front of the detector. According to the detection items, the light source and the detector are configured for blood cell analysis, microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis to obtain characteristic detection signals.

The electronic processing system comprises parts of electronic signal processing, digital sampling, data transmission and the like, and detection data of blood cell analysis, microsphere-based protein molecule analysis and cell characteristic antigen molecule analysis are obtained.

And the computer control and analysis system controls the operation of the detection process and processes the detection data to obtain the detection results of blood cell analysis, protein molecule analysis based on microspheres and cell characteristic antigen molecule analysis.

The detection device is also provided with an electrical impedance method and colorimetric method detection component 36, the sample suction needle can be moved into a reaction tank of the electrical impedance method and colorimetric method detection component 36 to discharge quantitative samples, the reaction tank is connected with a plurality of reagents, and various automatic reactions of the samples and the reagents, such as dilution, hemolysis and the like, can be carried out in the reaction tank; the electrical impedance detector analyzes the classification and counting of red blood cells and platelets, the total number of white blood cells, and the number of certain types of cells in the white blood cells, such as basophils. And a colorimetric detector for measuring the hemoglobin content.

Example 1:

blood cell analysis: the present invention is exemplified by the classification and counting of cells from a whole blood sample.

As shown in the detection device shown in FIGS. 6 and 7, the whole blood sample to be detected is loaded by a manual or automatic device at the sample injection position 31, the sample suction needle 71 is pulled by the driving motor 72, moves along the Z-axis guide rail 74, and is matched with the electromagnetic valve 76 and the precision injection pump 77 to precisely and quantitatively suck the sample, and after the sample is sucked, the sample suction needle assembly moves to the reaction assembly 32 along the X-axis guide rail 75.

The whole blood sample is sucked and distributed to the reaction assembly 32 by the sample sucking needle assembly 34, the sample sucking needle 71 can move to a reaction cell of the reaction assembly 32 to discharge a quantitative sample, the reaction cell is connected with a plurality of reagent containers, automatic reactions of the sample and various reagents, such as dilution, hemolysis classification and the like are carried out in the reaction cell, a temperature control component is arranged in the reaction assembly 32 to keep the reaction at a proper temperature, in this case 35 ℃, a connecting pipeline 37 is arranged between the reaction cell and the optical flow chamber 351, a detected substance after the reaction is sent to the optical flow chamber 351 by an electromagnetic valve 76 and a syringe pump 77, flow type optical detection is carried out in the optical detection system 35, and the detection device further comprises a liquid storage pool 55 and a pump 56 to perform functions of pipeline cleaning and the like.

Blood cell analysis uses a light source such as a 635nm laser as an excitation light source, a multi-angle forward scattering light and side scattering light detector, or a side scattering light and fluorescence detector, and obtains a two-dimensional or multi-dimensional scatter diagram through electronic signal processing and sampling, as shown in fig. 8, white blood cell classification and counting are performed, and the detection result is displayed on a display screen 78.

The detection device is also provided with an electrical impedance method and colorimetric method detection component 36, the sample sucking needle 71 can be moved into a reaction tank of the electrical impedance method and colorimetric method detection component 36 to discharge quantitative samples, the reaction tank is connected with a plurality of reagent containers, and various automatic reactions of the samples and the reagents, such as dilution, hemolysis and the like, can be carried out in the reaction tank; the electrical impedance detector analyzes the classification and counting of red blood cells, platelets, as well as the total number of white blood cells, and the number of basophils. And a colorimetric detector for measuring the hemoglobin content.

Example 2:

microsphere-based molecular analysis of soluble proteins: the invention takes the detection of multiple characteristic proteins related to immunity as an example.

The special microsphere is provided with a feature or a marker for identifying the type, such as fluorescent dye, and after being excited by laser with the wavelength of 635nm, the type of the microsphere, namely the type of the soluble protein molecule to be detected, can be identified by detecting the fluorescence with the wavelength of about 660nm and the wavelength of about 780 nm. Specific capture antibodies such as CRP (C Reactive protein) antibodies and the like are respectively coated on different types of microspheres and react with a blood sample, the antibodies on the different types of microspheres are respectively specifically combined with a plurality of soluble proteins to be detected in the sample, and then PE fluorescent markers are connected. For quantitative analysis of protein, a 532nm laser is used as an excitation light source, a PE fluorescent dye connected with the protein to be detected is excited by the 532nm laser, the fluorescence intensity with the wavelength of about 585nm is detected, and the concentration of various characteristic proteins can be accurately and quantitatively analyzed and detected.

With the help of various microspheres and various specific markers, dozens of characteristic proteins can be simultaneously detected in a trace sample (25-50ul), and compared with other immunoassay methods, the used sample amount is greatly reduced. Moreover, the sensitivity and precision of flow-type fluorescence detection are higher than those of the immuno-transmission or immuno-scattering turbidimetry.

Based on the flow-type fluorescence analysis technology, the samples to be detected for soluble protein molecule analysis are whole blood, serum and plasma. Using whole blood samples, rapid detection of characteristic proteins, such as point-of-care POCT, can be performed for acute conditions.

The reagent used for the microsphere-based soluble protein molecule analysis comprises buffer solution, microsphere mixed solution with specific capture antibody, PE labeled antibody solution, cleaning solution and the like. The buffer is used for diluting the blood sample and contains a component capable of suppressing nonspecific reactions.

As shown in the detecting unit of FIGS. 6 and 7, various reagents are stored in a container 38, such as a single reaction tube, or multiple tubes, or a strip of reagent strips with multiple tubes packaged. Prior to testing, the containers holding the reagents are loaded into a plurality of wells of the storage module 33, arranged in a matrix, in this case in a 5 x 6 arrangement, and the samples are aspirated and dispensed by the pipette tip mechanism 34 into the storage module 33.

The whole blood sample to be measured is loaded by a manual or automatic device at the sample injection position 31, the sample suction needle 71 is pulled by a first driving motor 72, moves along a Z-axis guide rail 74, is matched with an electromagnetic valve 76 and a precision injection pump 77 to precisely and quantitatively suck the sample, and is also provided with a swab 73 for cleaning the sample suction needle, and after the sample is sucked, the sample suction needle mechanism 34 moves to the storage assembly 33 along an X-axis guide rail 75.

The pipette needle 71 moves to each container of the storage unit 33 to discharge a predetermined amount of the sample, and the automated reactions of diluting the sample and the reagent, labeling incubation, and washing are sequentially performed.

The second driving motor 702 is transmitted to the transmission screw 704 to drive the container fixing frame 701 to move along the Y-axis guide rail 703 along the Y direction, and the container fixing frame is matched with the movement of the sample sucking needle 71 in the X direction and the Z direction for positioning, sucking and reacting different containers, and the storage group 33 can be provided with a two-dimensional moving mechanism which can move in the X direction and the Y direction.

After the labeling incubation reaction is completed, the sample aspirating needle 71 aspirates the substance to be detected in the container, and the substance to be detected is sent to the optical flow cell 351 through the connecting pipeline 39 of the sample aspirating needle 71 and the optical flow cell 351, and is subjected to flow type optical detection in the optical detection system 35.

Alternatively, the pipette needle 71 sucks the reaction intermediate substance in the container, moves the container in the X direction to the reaction cell of the reaction module 32, and discharges the reaction intermediate substance, the reaction cell is connected to a plurality of reagent containers, and various automated reactions of the sample and the reagent, such as dilution, hemolysis, and the like, can be performed in the reaction cell. After the reaction, the substance to be detected is sent to the optical flow cell 351 through the connection line 37 between the reaction cell and the optical flow cell 351 by the electromagnetic valve 76 and the syringe pump 77, and is subjected to flow-type optical detection in the optical detection system 35.

In this example, the optical detection system for microsphere-based analysis of soluble protein molecules uses a polychromatic light source, such as 532nm and 635nm dichroic lasers, as the excitation light source. The 635nm laser is used for exciting fluorescent dyes which are carried by the microspheres and used for identifying types, the types of the microspheres, namely the types of soluble protein molecules to be detected, are identified by detecting fluorescence with the wavelength of about 660nm or/and about 780nm, the 532nm laser is used for exciting the PE marked fluorescent dyes, the fluorescence intensity with the wavelength of about 585nm is detected, the quantity of the proteins with multiple characteristics can be quantitatively detected, and the detection result is shown in figure 9.

The method can be used for quickly detecting the characteristic protein aiming at acute diseases by using a whole blood sample for soluble protein molecular analysis, but the detection result of the whole blood sample must be corrected by using the volume information of blood cells to obtain accurate quantitative information of the characteristic protein.

Therefore, based on the soluble protein molecular analysis of the microspheres, the whole blood sample to be tested is subjected to blood cell analysis on the device of the invention to obtain accurate blood cell volume information, such as a sample to be tested, and the blood cell analysis gives 55% of hematocrit; the result obtained by the microsphere-based soluble protein molecule analysis is the number of the target object to be detected/uL whole blood, and then the result of the number of the target object to be detected/uL serum can be accurately calculated.

Example 3:

cell characteristic antigen molecule analysis: the present invention is exemplified by the absolute count analysis (number/uL whole blood sample) of CD4 lymphocyte subpopulation.

CD4-PE and CD45-PerCP monoclonal fluorescent antibody reagents are used to label the test sample.

As shown in FIGS. 6 and 7, the monoclonal fluorescent antibody reagent is stored in a container 38, such as a single reaction tube, or multiple tubes, or a strip with multiple tubes enclosed therein. Prior to testing, containers 38 holding reagents for monoclonal fluorescent antibodies are loaded into a plurality of wells of storage assembly 33, arranged in a matrix.

The sample is aspirated and dispensed by the aspirating needle mechanism 34 to the storage assembly 33.

The whole blood sample to be detected is loaded by a manual or automatic device at the sample injection position 31, the sample suction needle 71 is pulled by a first driving motor 72, moves along a Z-axis guide rail 74, is matched with an electromagnetic valve 76 and a precision injection pump 77 to precisely and quantitatively suck the sample, and is also provided with a swab 73 for cleaning the sample suction needle, after the sample is sucked, the sample suction needle mechanism 34 moves to the storage assembly 33 along an X-axis guide rail 75, the sample suction needle 71 moves to the container 38 of the storage assembly to discharge the quantitative sample, and the automatic marking incubation reaction of the sample and the reagent is executed.

The rotation of the second driving motor 702 is transmitted to the driving screw 704 to drive the container holder 701 to move along the Y-axis guide 703 in the Y direction. In cooperation with the movement of the aspirating needle 71 in the X-direction and the Z-direction for positioning, aspirating, and reacting different containers, the storage module 33 may also be equipped with a two-dimensional moving mechanism that can move in the X-direction and the Y-direction.

After the labeling incubation reaction is completed, the sample sucking needle 71 sucks the reaction intermediate substance in the container 38, the sample sucking needle 71 moves in the X direction to the reaction cell of the reaction module 32 to discharge the reaction intermediate substance, the reaction cell is connected with a plurality of reagent containers, various automated reactions of the sample and the reagent, such as dilution, hemolysis and the like, can be performed in the reaction cell, after the reaction, the substance to be detected is sent to the optical flow cell 351 through the connecting pipeline 37 of the reaction cell and the optical flow cell 351 by an electromagnetic valve and an injection pump, and the flow type optical detection is performed in the optical detection system 35.

In the case of the small-lot sample test, the monoclonal fluorescent antibody reagent is stored in the container 38, and the labeled incubation reaction of the whole blood sample to be tested and the monoclonal fluorescent antibody reagent is performed externally by hand. After completion, the storage module 33 is removed and the containers 38 storing the reaction intermediate substances are loaded into a plurality of well sites of the storage module 33. Then the reaction and detection are carried out in the same way as the steps.

Cellular signature antigen molecule analysis uses a light source, in this example a 532nm laser as the excitation light source, to detect forward scattered light, side scattered light, fluorescence at wavelengths around 585nm and 670 nm. The CD45-PerCP monoclonal fluorescent antibody can effectively distinguish white blood cells, the side scattered light and the CD4-PE monoclonal fluorescent antibody can distinguish CD4 cell subsets, and as shown in FIG. 10, the proportion information of characteristic antigen cells, namely the percentage of CD4 cells in the whole lymphocytes, is obtained.

To obtain an absolute count of characteristic antigen cells, e.g., the absolute number of CD4 cells (counts/uL whole blood sample). The whole blood sample to be detected is subjected to blood cell analysis on the device to obtain the absolute quantity information of white blood cells or lymphocytes; the absolute number of the characteristic antigen cells can be accurately and quantitatively calculated by combining the proportion information of the characteristic antigen cells given by the flow-type multicolor fluorescence detection. For example, if a test sample is obtained, the blood cell analysis gives a whole blood sample with 1600 lymphocytes/uL, and the cell characteristic antigen analysis gives a total lymphocyte proportion of 40% CD4 cells, then the absolute number of CD4 cells is 640/uL whole blood sample.

In the cell characteristic antigen analysis, an antigen and an antibody perform a specific binding reaction. In order to achieve the best signal-to-noise ratio, the amount of antibody added has an optimal ratio range, and the white blood cell count of the whole blood sample to be tested should preferably be less than 10.0 x 10^ 9/L. If the white blood cell count is >10.0 x 10^9/L, the sample needs to be diluted with reagents.

Therefore, the whole blood sample to be detected is subjected to blood cell analysis on the device to obtain the absolute quantity information of the white blood cells or the lymphocytes; in the cell characteristic antigen analysis of the same sample, the sample can be diluted according to the absolute quantity information of white blood cells or lymphocytes and the requirement, so as to achieve the optimal antigen-antibody reaction ratio.

Claims

1. A multifunctional cell-protein detection method is characterized in that a flow fluorescence analysis technology is applied to integrate a sample to be detected to realize blood cell analysis, protein molecule analysis based on microspheres and cell characteristic antigen molecule analysis;

2. The multifunctional cell-protein assay method of claim 1, wherein the sample to be assayed for the microsphere-based protein molecule assay is whole blood.

3. A test device for carrying out the multifunctional cell-protein test method according to any one of claims 1 to 2, which comprises:

pipette tip assembly (34): the sample suction device is arranged on the detection device main body and used for sucking a sample to be detected from a sample introduction position (31) and moving the sample to be detected to a set position to realize the suction, distribution and reaction of the sample to be detected;

reaction assembly (32): the sample sucking needle is arranged at a first set position on the detection device body and used for receiving a quantitative sample to be detected discharged by the sample sucking needle component (34) and automatically realizing the reaction between the sample to be detected and a reagent;

storage assembly (33): a plurality of containers (38) which are arranged at a second set position on the detection device body and are loaded with a plurality of reagents, reaction intermediate substances and samples to be detected, and are matched with the sample sucking needle assembly (34) for analysis reaction;

optical flow cell (351): the detection device is arranged on the detection device main body and is respectively communicated with the reaction component (32) and the sample sucking needle component (34) through connecting pipelines, and the substances to be detected are respectively sent into the optical flow chamber for flow optical detection;

4. A testing device according to claim 3, further comprising:

electrical impedance and colorimetric detection assembly (36): the sample suction needle is arranged at a third set position on the detection device body and is used for receiving a quantitative sample to be detected discharged by the sample suction needle component (34) to carry out electrical impedance detection and colorimetric detection.

5. The detecting device according to claim 3, wherein the storing assembly (33) comprises a container holder (701), a holder moving mechanism and a plurality of shaped containers (38) arranged in holes on the container holder (701), and the containers (38) comprise reaction tubes, multi-connection tubes and multi-tube reagent strips packaged with a plurality of reagents.

6. A testing device according to claim 3, wherein said scattered light and fluorescence detectors comprise a forward scattered light FSC detector (43), a side scattered light SSC detector (44) and a polychromatic fluorescence FL detector (45).