CN103645332A - Multifunctional workstation for automatically detecting outflow of cholesterol and other analyte cells with high throughput - Google Patents
Multifunctional workstation for automatically detecting outflow of cholesterol and other analyte cells with high throughput Download PDFInfo
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Abstract
The invention discloses a multifunctional workstation for automatically detecting the outflow of cholesterol and other analyte cells with high throughput. The multifunctional workstation comprises a constant-temperature short-period incubator (1), a cell washing and sample loading platform (2), a sample filtering platform (3), a sampling and collecting platform (4), a mechanical hand multi-branch distribution system (5), and a software control system and a display interface of the workstation. The workstation is reasonable in structural design and high throughput and automation can be realized in a manual test so that a plurality of clinical samples can be treated and the working efficiency is improved; the aims of clinical applications and rapid screening of the samples are realized. The multifunctional workstation can be widely used for measuring cell outflow dynamic characteristics of other molecules, such proteins, lipids, RNA (Ribonucleic Acid), ions, saccharides, hormones, vitamins, and metabolites; the application range is wide.
Description
Technical field
The present invention relates to a kind of high throughput analysis equipment of biomolecule, be specifically related to a kind of activity that can detect the outflow of HDL cellular cholesterol, also can be used for other molecules/compounds, as in the cells such as protein, lipid, ion, hormone, metabolin, outflow quantitatively, can be widely used in the multifunctional analysis system of the high throughput automated biomolecule of biological study, clinical diagnosis and drug development.
Background technology
Angiocardiopathy is the chief threat of human health, has very high M & M, and occurs the trend of increase year after year, especially in the economy developing rapidly as China.According to the cardiovascular study on prevention of Ministry of Public Health center, along with China's growth in the living standard and aging population, cardiovascular disease incidence rate is every year with 30% speed increase.
Atherosclerotic is to cause cardiovascular disease incidence and main causes of death.The at present measure of conventional reduction hazards comprises and changes lifestyles and take lipid lowerers, but only can reduce by about 30% coronary artery adverse events, shows to need badly the new therapy of development and meets medical demand.Blood plasma HDL is based on it to angiocardiopathy, and particularly atherosclerotic natural protection capability, has become the important target in predicting cardiovascular class disease and treatment.But on domestic and international market, there is not yet at present similar provided by the invention can Efficient Evaluation HDL function with screening people at highest risk and evaluate the instrument of result for the treatment of.In a word, the technical restriction of HDL Function detection has become the bottleneck of medicament research and development and clinical testing, and develop new HDL diagnostic method and carry out functional analysis, be the R&D direction now and even in the quite a while, especially develop the high-precision detection technique of high flux.
The anti-atherosis effect of HDL is removed unnecessary cholesterol (being Cholesterol Efflux) from arterial wall to it relevant.The cholesterol that is transported to liver is finally drained from ight soil, and this process is called cholesterol antiport.The still measurement based on biomarker concentration of present so-called cardiovascular health test, and the mark of measuring comprises that plasma lipoprotein or the biomolecule relevant to inflammation and oxidative damage etc. can not meet current needs far away.Particularly multinomially in recent years studies show that its blood plasma level of function ratio of HDL is more important, the latter is no longer considered to the reliability index of disease forecasting, causes its dysfunction such as HDL can be modified.Clinical testing in recent years provides strong evidence to show the activity of patient's blood plasma or HDL Cholesterol Efflux and arterial wall thickens and atherosclerotic remarkable improvement is positive correlativity, thereby more can predict the generation of blood vessel and coronary artery class disease.
Cholesterol Efflux test is widely used with the mechanism of study of disease in laboratory, finds new drug candidate, and foundation and disease is associated.Conventionally be applied to Cardiovascular Disease Study, but also comprise metabolism (as diabetes) and neurogenic disease etc.At present this experiment can only be undertaken by craft in laboratory, and manual operations relates to a large amount of diluted plasmas, cell cleaning and adds the work such as spline filter, and this experimental technique is time-consuming and easily have a human error; In addition, repeated various cleanings and sample add the sample size that step has not only limited each test, and a large amount of consumptive material, increase testing cost.Due to above defect, HDL functional test is very limited in the application aspect of clinical diagnosis or biotechnology/drug development, and the high flux screening that cannot need for large sample.
Therefore, on the basis of existing technology necessary, a kind of automaticity of design research and development is high, can high flux, and fast, accurately detect HDL cellular cholesterol and flow out multifunctional analysis system active and that other biomolecule cell flows out.
Summary of the invention
Goal of the invention: the present invention is in order to solve in the deficiencies in the prior art aspect HDL functional analysis, a kind of reasonable in design is provided, easy to operate, there is high flux, high precision, low cost, the advantages such as high-level efficiency, and the high throughput automated HDL functional analysis workstation having wide range of applications.
Technical scheme: in order to realize above object, the technical solution used in the present invention is:
The multifunctional workstation that a kind of robotization high flux cholesterol detection and other analyte cell flow out, it comprises constant temperature short incubation device, the cell that is connected with constant temperature short incubation device cleans and application of sample platform, sample filtering platform, the sampling that is connected with sample filtering platform and collecting platform, the mechanical hand manifold delivery system that is all connected with collecting platform with application of sample platform, sample filtering platform and sampling with cell cleaning, the software control system of workstation and display interface.
As preferred version, above-described high throughput automated multifunctional analysis workstation, described mechanical hand manifold delivery system comprises hole rectangular slab by all kinds of means, be arranged on the metal joint below hole rectangular slab by all kinds of means, the disposable sterilized rifle head being connected with metal joint, described disposable sterilized rifle head can be by automatically controlled mechanical hand control along X-axis, Y-axis and Z-direction move, thereby can reach the object that fast accurate moves liquid from Tissue Culture Plate hole.The present invention is by utilizing the precise motion of automatically controlled mechanical hand control cleaning step and collect sample and analyze, and high flux is to repeat to realize the application of sample of Tissue Culture Plate multisample and cleaning by the automatic liquor-transferring system of central hyperchannel corresponding with 6 holes, 12 holes, 24 holes, 48 holes and 96 well culture plates.
As preferred version, above-described high throughput automated multifunctional analysis workstation, the function of described mechanical hand manifold delivery system is from a plurality of sample bottles/porous culture plate, to draw the solution of tool precise volumes (0.01-1 milliliter) by Automatic Control simultaneously, for flowing out the dilution of dynamic experiment serum and carrying out the cleaning of cell and medicine/chemical treatment at particular point in time.
As preferred version, above-described high throughput automated multifunctional analysis workstation, the temperature of described constant temperature short incubation device is 25 ℃ or 37 ℃, is provided with controlled automatic opening and closing door, receivability 6 holes, 12 holes, 24 holes, 48 holes or 96 well culture plates.Described cell clean and application of sample platform in dispose sliding panel, described sliding panel can insert in short-term constant-temperature incubator to be released culture plate cell cleaning and application of sample platform or culture plate is sent back in constant temperature short incubation device after task completes.
As preferred version, above-described high throughput automated multifunctional analysis workstation, described sample filtering platform base can be placed the porous filter plate having with culture plate identical standard size, utilizes gravity principle to filter and removes cell fragment and collect the adjustment nutrient culture media of cultivating cell.
On described sampling and collecting platform, be provided with multitube pipe support, multitube pipe support is provided with collects test tube (bottle);
After the porous filter plate collected specimens of described automatically controlled mechanical hand from sample filtering platform, move in the collection test tube (bottle) on sampling and collecting platform.
Multifunctional analysis system provided by the invention, wherein short incubation device can keep steady temperature (25
oc, 37
oc), maintain the monolayer in Tissue Culture Plate; Cell cleans and application of sample parts can carry out the high-efficiency washing that various intermediate steps need, for (in advance) processing cell and/or application of sample are prepared; Application of sample carries out in identical platform, comprises the biological sample of can dilution process cell using, as clinical serum, and by its simultaneously and rapidly application of sample in cultivating plate hole.The critical component of workstation is mechanical hand manifold delivery system, multisample is carried out cleaning, sample distribution, is filtered and collect simultaneously.At sample filtering platform, the nutrient culture media that Cell and organism sample is collected after cultivating (containing
3h ］ or fluorescently-labeled cholesterol) be filtered, then by mechanical hand, sampled and move to and collect test tube (bottle).All above operations are all set and are automatically switched by supporting software control system, and can programme voluntarily, thereby can meet different experiments requirement, and workstation can deposit the information such as the numbering of a large amount of samples and analysis result automatically in.
Multifunctional analysis system provided by the invention is the technology platform of an open to the outside world.Except activity that can the real time measure cholesterol efflux, also can measure cell flowing activity and its dynamics of other biomolecule, comprise protein, lipid, RNA, ion, carbohydrate, hormone, vitamin, metabolin etc.In addition, this workstation is taked compatible strong global design, can dock with other platform or add-on module.As the filtered fluid of above-mentioned collection can be sent to radic or fluorescence detector to quantize the concentration of labeled cholesterol; And can be group and learn the research of class Sample pretreatment is provided, apply very extensive.
The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analyte cell flow out, have wide range of applications, as can be used as based on take the experimental technique of cellular level be basis diagnostic application instrument, comprise that serum cholesterol flows out active level, to assess the risk of angiocardiopathy.
The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analyte cell flow out also can be used as the charge service platform that carries out cell-based assay and diagnostic test, comprises and uses blood serum sample process cell and reclaim described blood serum sample for subsequent analysis or detect the emiocytosis thing in it.
The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analyte cell flow out in addition, also can be used as and carries and/or screening natural products, the adjusting outflow of synthetic compound and/or the instrument of emiocytosis effect.As further preferred, workstation provided by the invention also can be used as carries and/or screens the instrument that cellular cholesterol flows out.
The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analyte cell flow out in addition, also can be used as and identifies for regulating and/or the instrument of the drug candidate of irritation cell Cholesterol Efflux.
The multifunctional workstation that a kind of robotization high flux cholesterol detection provided by the invention and other analyte cell flow out, can screen test-compound for the effect of various cell analysis things, as lipid, protein, polypeptide, nucleic acid, ion, vitamin and/or enter intracellular exogenous factor as medicine, microbiotic, and/or pesticide.
Beneficial effect: the multifunctional analysis workstation of high throughput automated and other biomolecule provided by the present invention compared to the prior art tool has the following advantages:
Multifunctional analysis workstation provided by the present invention, reasonable in design, easy to operate, automaticity is high, and testing cost is low, has wide range of applications.
The principal feature of multifunctional analysis system is:
(1) high flux: can carry out synchro measure to a large amount of biology/clinical samples, thereby eliminate manual retardation time, and can save experimental period, manpower and reagent, testing cost is low;
(2) high precision: multifunctional analysis workstation provided by the invention, detection accuracy is high and reproducible, thus error between can avoiding batch, the result of allow to compare better between different experiments chamber/different experiments batch, and carry out analysis and the contrast of large data;
(3) versatility: multifunctional analysis workstation provided by the invention, can be connected with other automation equipment or instrument, can be used for a lot of dissimilar experiments in cell and systems biology field, assist drug screening, the research of the aspect such as albumen and genomics, also can be widely used in hospital, clinic, company and research unit.
Accompanying drawing explanation
Fig. 1 is the structural representation of the multifunctional workstation of robotization high flux cholesterol detection of the present invention and the outflow of other analyte cell.
Fig. 2 is the structural representation of the mechanical hand manifold delivery system of multifunctional workstation of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, further illustrate the present invention, should understand these embodiment is only not used in and limits the scope of the invention for the present invention is described, after having read the present invention, those skilled in the art all fall within the application's claims limited range to the modification of the various equivalent form of values of the present invention.
As depicted in figs. 1 and 2, the multifunctional workstation that a kind of robotization high flux cholesterol detection and other analyte cell flow out, it comprises constant temperature short incubation device (1), the cell being connected with constant temperature short incubation device (1) cleans with application of sample platform (2), sample filtering platform (3), the sampling being connected with sample filtering platform (3) and collecting platform (4), cleans and application of sample platform (2) with cell, the mechanical hand manifold delivery system (5) that sample filtering platform (3) and sampling are all connected with collecting platform (4), the software control system of workstation and display interface.
The multifunctional workstation that above-described robotization high flux cholesterol detection and other analyte cell flow out, described mechanical hand manifold delivery system (5) comprises hole rectangular slab (5-1) by all kinds of means, be arranged on hole rectangular slab (5-1) metal joint (5-2) below by all kinds of means, the disposable sterilized rifle head (5-3) being connected with metal joint (5-2), described disposable sterilized rifle head (5-3) is controlled by automatically controlled mechanical hand, can be along X-axis, Y-axis and Z-direction move, described metal joint (5-2) is connected with manifold system (5-4), described manifold system (5-4) top is provided with quantitative liquid-feeding pump (5-5), and manifold system (5-4) connects solution bottle (5-6), diverting valve (5-7) is installed on solution bottle (5-6), described solution bottle (5-6) is 5, between manifold system (5-4) and solution bottle (5-6), five-way valve (5-8) is installed.
Described disposable sterilized rifle head (5-3) to move that liquid amasss be 0.01 to 1 milliliter, wherein the rifle head of 1 milliliter is used for carrying out the cleaning of sample, and the rifle head of 10 ~ 200 microlitres is used for accurately moving liquid to send blood plasma to be tried or other sample to the porous of Tissue Culture Plate.
The multifunctional workstation that above-described robotization high flux cholesterol detection and other analyte cell flow out, the function of described mechanical hand manifold delivery system (5) is from a plurality of sample bottles/porous culture plate, to draw the solution of 0.01 ~ 1 milliliter of precise volumes by Automatic Control simultaneously, for flowing out the dilution of dynamic experiment serum and carrying out the cleaning of cell and medicine/chemical treatment at particular point in time.And described mechanical hand manifold delivery system (5) can accurately be carried out following functions: clean and cultivate cell in plate hole, and dilute serum, quick application of sample, filtered sample, collects for the nutrient solution of analyzing.
The multifunctional workstation that the above robotization high flux cholesterol detection and other analyte cell flow out, the temperature of described constant temperature short incubation device (1) is 25 ℃ or 37 ℃, be provided with controlled automatic opening and closing door, receivability 6 holes, 12 holes, 24 holes, 48 holes or 96 well culture plates;
And described cell cleans and application of sample platform (2) in dispose sliding panel, described sliding panel can insert in short-term constant-temperature incubator (1) to be released culture plate cell cleaning and application of sample platform (2) or culture plate is sent back in constant temperature short incubation device (1) after task completes.
The multifunctional workstation that above-described a kind of robotization high flux cholesterol detection and other analyte cell flow out, the base of described sample filtering platform (3) is provided with the porous filter plate with culture plate identical standard size;
Described sampling and collecting platform are provided with multitube pipe support on (4), and multitube pipe support is provided with collection test tube;
After the porous filter plate collected specimens of described automatically controlled mechanical hand (5) from sample filtering platform (3), move to sampling and collecting platform (4) on collection in vitro.
The multifunctional workstation that robotization high flux cholesterol detection provided by the invention and other analyte cell flow out, because of its quick and high-throughout feature, the range of application of platform comprises:
1) automated analysis Cholesterol Efflux
A. for the research of fundamental research-atherosclerotic, lipid and lipoprotein metabolism, HDL function.Platform is by providing desirable laboratory facilities for take detection method and the research that dynamics is object, to promote biomedical research and drug development.
B. for medicament research and development and the screening of clinical detection-high flux lead compound and clinical detection.
2) cell of automated analysis various biomolecules flows out
For measuring the interior outflow of cell and the dynamics thereof of other biomolecule, comprise protein, lipid, RNA, ion, carbohydrate, hormone, vitamin, metabolin etc.
3) platform is cultivated/dealt with the work to general cell
Can be connected with many biological experiment operating systems and carry out dissimilar cell and systems biology experiment, as the research of protein science and genomics aspect.
The basic mechanical design feature index of the multifunctional workstation instrument that robotization high flux cholesterol detection provided by the invention and other analyte cell flow out
:
Each sample size: 1-96(can many plates)
Move liquid flux: 0.01-1 ml, high precision is moved liquid head (error
+2%)
Measuring Time: (0,5,10 minutes to 10 hours)
Measure temperature: 25
oC, 37
oc
System sterilization: ultraviolet ray
Power supply: 220/110 AC; 50-60HZ
Operating temperature: 18-37
oC
Multifunctional analysis system instrument provided by the invention can detect a large amount of biology/clinical samples simultaneously, and be a few minutes to arrive several hours detection time, and workstation operation is accurately high with repeatability, thereby can reduce/eliminate personal error, improves the accuracy detecting.
The clinical detection HDL Cholesterol Efflux of now take is embodiment, and the principle of work of this multifunctional workstation and concrete operation steps process are described.Test cell line can adopt the laboratory mammalian macrophage (as J774 cell) of application conventionally, include [
3h] or other tracer agent, as fluorescently-labeled cholesterol.Cell can carry out pre-service with biochemical reagents conventionally, as passed through cAMP active cell film cholesterol transport body ABCA1.The process operation of above mark and active cell is relative simple and easy, not within the scope of robotization of the present invention.
Test cell line is inoculated in 6,12,24,48 or 96 well culture plates as required, in serum free medium, grow, and allow it be exposed in the air of constant temperature short incubation device (1), nutrient culture media contains stable buffer solution, as HEPES or other damping fluid, the time that cell is grown in cultivating plate hole can from 0 to 10 hour not etc., and can spend the night as required or hatching in 24 hours.
When experiment starts, the sliding panel of cleaning and application of sample platform (2) configuration inserts short incubation device (1), experiment culture plate is moved out to and is cleaned and application of sample platform (2), 1 ml by automatically controlled mechanical hand moves liquid head, with Hank balanced salt solution, rinses and cultivates the cell in plate hole and repeat twice; Then in hole, add 0.2% bovine serum albumin(BSA) (BSA) solution and place and be used as to seal for 1 hour, to reduce background signal; Then with serum free medium, rinse cell in culture plate (6) hole three times.Will be by functional unit software control with the quantity of the mechanical hand aseptic rifle head (5-3) that hole rectangular slab (5-1) is connected by all kinds of means, the hole count of maintenance and culture plate matches.
At mechanical hand manifold platform, move the aseptic rifle head (5-3) that liquid head automatically switches and varies in size under software control, clinical blood serum sample to be tried is diluted to variable concentrations as 10%, 5%, 2% and 1% with the nutrient culture media containing HEPES damping fluid.
The plasma sample diluting is divided and delivered in Tissue Culture Plate (6) hole of cleaning and application of sample platform (2), and by sliding panel, culture plate (6) is sent into constant temperature short incubation device (1), close chamber's door shares and hatches 4 hours (37
oc).If experiment purpose is dynamics (time dependence) research, need as 0,1, within 2,4 and 8 hours, culture plate be taken out and be drawn the supernatant nutrient solution in respective aperture at different time points, move to the porous filter plate filtering on platform (3).
After cultivation completes, culture plate is put back to and cleaned and application of sample platform (2), by aseptic rifle head (5-3), draw the supernatant nutrient solution in every hole and move to the porous filter plate filtering on platform (3), porous filter plate has the standard size identical with culture plate and hole count, utilizes gravity principle to filter and removes possible cell fragment in supernatant.
Automatically controlled mechanical hand is from filtering the quantitative filtration nutrient culture media of porous filter plate collection platform (3), then move to the microcentrifugal tube that is placed on collecting platform (4) on pipe support or other is collected in test tube (bottle), confession radiological measuring or fluorescence detector in order to quantize the concentration of labeled cholesterol.This concentration value is used for calculating the Cholesterol Efflux rate of macrophage.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (11)
1. the multifunctional workstation that a robotization high flux cholesterol detection and other analyte cell flow out, it is characterized in that, it comprises constant temperature short incubation device (1), the cell being connected with constant temperature short incubation device (1) cleans with application of sample platform (2), sample filtering platform (3), the sampling being connected with sample filtering platform (3) and collecting platform (4), cleans and application of sample platform (2) with cell, the mechanical hand manifold delivery system (5) that sample filtering platform (3) and sampling are all connected with collecting platform (4), the software control system of workstation and display interface.
2. the multifunctional workstation that robotization high flux cholesterol detection according to claim 1 and other analyte cell flow out, it is characterized in that, described mechanical hand manifold delivery system (5) comprises hole rectangular slab (5-1) by all kinds of means, be arranged on hole rectangular slab (5-1) metal joint (5-2) below by all kinds of means, the disposable sterilized rifle head (5-3) being connected with metal joint (5-2), the position of described disposable sterilized rifle head (5-3) is controlled by automatically controlled mechanical hand, can be along X-axis, Y-axis and Z-direction move, described metal joint (5-2) is connected with manifold system (5-4), described manifold system (5-4) top is provided with quantitative liquid-feeding pump (5-5), and manifold system (5-4) connects solution bottle (5-6), diverting valve (5-7) is installed on solution bottle (5-6), described solution bottle (5-6) is 5, between manifold system (5-4) and solution bottle (5-6), five-way valve (5-8) is installed.
3. the multifunctional workstation that robotization high flux cholesterol detection according to claim 2 and other analyte cell flow out, it is characterized in that, the function of described mechanical hand manifold delivery system (5) is from a plurality of sample bottles/porous culture plate, to draw the solution of 0.01 ~ 1 milliliter of precise volumes by Automatic Control simultaneously, for flowing out the dilution of dynamic experiment serum and carrying out the cleaning of cell and medicine/chemical treatment at particular point in time.
4. according to the multifunctional workstation of the robotization high flux cholesterol detection described in claim 1-3 and the outflow of other analyte cell, it is characterized in that, mechanical hand manifold delivery system (5) can accurately be carried out following functions: clean cell in culture plate (6) hole, dilute serum or other biological sample to be tried, quick application of sample, filtered sample, collects for the nutrient solution of analyzing.
5. the multifunctional workstation that robotization high flux cholesterol detection and other analyte cell flow out according to claim 1, it is characterized in that, the temperature of described constant temperature short incubation device (1) is 25 ℃ or 37 ℃, be provided with controlled automatic opening and closing door, receivability 6 holes, 12 holes, 24 holes, 48 holes or 96 well culture plates;
Described cell clean and application of sample platform (2) in dispose sliding panel, described sliding panel can insert in short-term constant-temperature incubator (1) to be released culture plate cell cleaning and application of sample platform (2) or culture plate is sent back in constant temperature short incubation device (1) after task completes.
6. the multifunctional workstation that robotization high flux cholesterol detection according to claim 1 and other analyte cell flow out, it is characterized in that, the base of described sample filtering platform (3) is provided with the porous filter plate with culture plate identical standard size;
Described sampling and collecting platform are provided with multitube pipe support on (4), and multitube pipe support is provided with collection test tube; After the porous filter plate collected specimens of described automatically controlled mechanical hand (5) from sample filtering platform (3), move to sampling and collecting platform (4) on collection in vitro.
7. the multifunctional workstation that the robotization high flux cholesterol detection described in claim 1-6 any one and other analyte cell flow out as based on take the experimental technique of cellular level be basis diagnostic application instrument, comprise that serum cholesterol flows out active level, to assess the risk of angiocardiopathy.
8. the multifunctional workstation that the robotization high flux cholesterol detection described in claim 1-6 any one and other analyte cell flow out, as the charge service platform that carries out cell-based assay and diagnostic test, comprises that the serum/media samples after the described processing of use blood serum sample processing cell and recovery supplies subsequent analysis or detects its interior emiocytosis thing.
9. the multifunctional workstation that the robotization high flux cholesterol detection described in claim 1-6 any one and other analyte cell flow out is as carrying and/or screening natural products, the adjusting outflow of synthetic compound and/or the instrument of emiocytosis effect.
10. the multifunctional workstation that the robotization high flux cholesterol detection described in claim 1-6 any one and other analyte cell flow out is as the instrument of carrying and/or screening cellular cholesterol flows out.
The multifunctional workstation that robotization high flux cholesterol detection described in 11. claim 1-6 any one and other analyte cell flow out is as identifying for regulating and/or the instrument of the drug candidate of irritation cell Cholesterol Efflux.
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