JP2002540401A - Automated two-dimensional analysis of biological and other samples - Google Patents

Abstract

(57) Abstract: A method and apparatus for separating and detecting components in a sample, particularly a biological sample, is provided. The sample is subjected to a first separation method, such as isoelectric focusing. After the first separation step, the sample is divided into a plurality of fractions, and each fraction is then subjected to a second separation method, for example, any type of chromatography, wherein the components leave the second separation step. Come and be detected. Thereby, a two-dimensional separation method in which the two separation methods are different, such as isoelectric focusing and SDS-gel capillary method, can be performed. Conveniently, the first and second separation methods are performed in the first and second capillaries. In another aspect of the invention, a manifold is provided for connecting a plurality of capillary pairs together, thereby providing a multiple bi-separation system in which, for example, 96 pairs of capillaries operate in parallel. The invention aims at providing high resolution separation and sensitive detection, especially for components in biological samples.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] TECHNICAL FIELD THE INVENTION The present invention relates to materials and methods chromatography. More particularly, the present invention relates to the high resolution separation and sensitive detection of proteins and other biological samples.

BACKGROUND OF THE INVENTION The proteome is a coined term in 1995 that describes the total complement protein of the genome. The human genome encodes approximately 100,000 genes, corresponding to similar protein numbers. Not all genes are expressed in all tissues: Approximately 10,000 proteins are found in any particular cell. The fraction of the proteome expressed by a living body varies between tissues and varies depending on the environment.

[0003] Single cell proteome analysis offers several important advantages. In particular, the distribution in the expression of the labeled protein that correlates with the cancer stage can be monitored. As with ploidy measurements, the distribution of expressed proteins can provide a useful prognostic assessment. Subpopulations of metastatic or therapy-resistant cells can be identified at an early stage, providing guidance for treatment.

Conventional proteome analysis is performed by two-dimensional gel electrophoresis,
Requires protein from 10,000 cells. Therefore, there is a need for improved devices and methods for improving resolution and sensitivity and increasing the number of samples that can be analyzed simultaneously.

[0005] In addition, there are new developing areas that are considered herein to be identical to "molecular cytometry". Cytometry is the analysis of individual cells and their contents, and is often used for tumor characterization. Molecular cytometry uses the chemical composition of cells to perform the above characterization. It has been suggested that marker proteins may eventually be found that "can identify not only the presence of cancer but also possibly the possibility that the cancer is malignant" (P. Peters, " Prognostic Cytometry and Cytopathology ", J.
P. Karr, DS Coffey and W. Gardner
Ed., Elsevier, New York, pp. 412-412 (19)
1988)). Also, in 1995, "From a therapeutic standpoint, it is highly desirable to increase the accuracy of diagnosis with respect to grading and sensitivity of tumor treatment. Analysis of total protein patterns of tumor cells from clinical material using two-dimensional electrophoresis. Will provide new information and will probably lead to the development of new signs. "(B. Franzen
(Franzen) et al., "Electropholysis", Vol. 16, pages 1087-1089 (1995)).

[0006] The present invention focuses on the identification of such markers. The identification of molecular changes that distinguish any particular cancer cell from normal cells is not only responsive to the treatment of that particular tumor, but is also the best way to characterize that cancer cell and predict pathological behavior Would be helpful. Understanding the molecular profile changes in any particular cancer will allow the phenotype obtained for that cancer to be correlated with molecular events. The knowledge gained is: a better understanding of cancer biology;
Discovery of new tools and biomarkers for detection, diagnosis, and prevention studies; and new goals for therapeutic development (National Institutes of Health: Program Announcement
No. PAR-98-067).

The inventors of the present invention have realized that single cell analysis offers several important advantages over the analysis of cell extracts. First, single cell proteomic analysis provides information on both the average and distribution of expressed proteins across a cell population. The distribution of DNA contained in cancer cells is used as a prognostic indicator, where aneuploidy is taken as evidence of aggressive cancer. Similar diagnostic and prognostic information can be obtained from the concentration distribution of protein markers.

Second, and similarly as above, the response to a therapeutic agent can be tracked across a cell population. It can be ensured that chemotherapy targets a specific subpopulation of the cell population. If the disappearance of the subpopulation is detected, treatment can proceed to target the surviving cell population.

Third, the ploidy of a cell can be correlated with the protein electropherogram of that cell. In the present invention, an imaging cytometry microscope is incorporated into our system to measure DNA contained in individual cells. Thus, cells containing abnormal DNA or having cytopathological abnormalities can be selected for the assay.

Fourth, single cell protein electropherograms can be correlated with the cell cycle of cultured cells. Understanding expressed proteins as a function of the cell cycle will provide information on expressed genes and protein turnover.

Fifth, the single-cell analysis makes it possible to evaluate and analyze the microdissected tissue without being contaminated by connective tissue, erythrocytes, fibroblasts and the like. The non-malignant tissues described above enlarge the protein electropherogram of the biopsy, making it more difficult to identify tumor-specific markers. Single cell analysis, combined with microselection of the cells, ensures analysis of only the tumor material.

Finally, the techniques used for single cell analysis can also be used to assay small samples made from fine needle aspiration biopsies. In many cases, sufficient material will be obtained for two-dimensional proteome analysis without the need for surgical biopsy.

[0013] Two-dimensional gel electrophoresis is usually used for proteome assays. Almost 25
A year ago O'Farrell developed this classical procedure (PH O'Farrell, J. Biol. Chem, 250 (1975), pp. 4007-4021). The sample is homogenized, the proteins are extracted and separated by in-tube isoelectric focusing. The gel is removed from the tube and SDS (sodium dodecyl sulfate)
The protein is separated on a PAGE (polyacrylamide gel) perpendicular to the in-tube isoelectric focusing gel. After separation, the protein is stained to create a 2D representation of the spot corresponding to the protein's pI value and molecular weight. This manual technique is redundant and somewhat artistic. The components separated by 2D electrophoresis
Edman protein amino acids can be identified by sequencing, by mass spectrometry, or by immunoassay. Partial amino acid sequences are often sufficient for protein identification by comparison to databases.

[0014] Column switching techniques are well established in both gas and liquid chromatography for the analysis of complex mixtures. In the simplest system, the fraction containing the component of interest is captured as it elutes from the first column, and this fraction undergoes several further chromatographic separations to obtain the desired level of purity.

The above multidimensional separation method can be extended so that the second column sequentially separates all of the plurality of fractions from the first column. These 2D methods are particularly useful when characterizing extremely complex samples. In an earlier report, two-step continuous chromatography was used to purify peptides made from proteolytic digests of human immunoglobulin (H. Yamamoto, T. Manabe, T. Okuyama, "J. Chromatogr. "Journal, No. 480 (1989), 2
77-283; N. Takahashi, Y. Takahashi, FW Putnam
, "J. Chromatogr." Magazine, No. 266 (1983), pp. 511-522).

[0016] Jorgenson and others have developed elegant multiple column separation methods for proteins and peptides (Bushey, MM).
And JW Jorgenson, "Anal. Chem.", Vol. 62 (1990),
Pp. 161-167; JP Larmann, A.V. Lemmo
, AW Moor, JW Jorgenson, "Electrophoresis",
14 (1993), 439-447; L.A. Holland
And JW Jorgenson, "Anal. Chem.", Vol. 67 (1995), 3.
Pages 275-3283; AW Moore and JW Jorgenson, "Anal.
Chem., Vol. 67 (1995), pp. 3448-3455; A. W. Moore and J. W. Jorgenson, "Anal. Chem.", Vol. 67 (1995).
, Pp. 3456-3463; D.J. Rose and G.J.
Opiteck), "Anal. Chem.", Vol. 66 (1994), 2529-253.
6 pages). These systems rely on various combinations of exclusion chromatography, reverse phase chromatography and zone electrophoresis to characterize amines, peptides and proteins. In the most sophisticated models,
A mass spectrometer is used to identify components separated by an ion exchange / reverse phase chromatography coupled system or an exclusion / reverse phase chromatography coupled system (GJ Opitek, KC Lewis, J. Mol. W. Jorgenson, R
J. Anderegg, “Anal. Chem.”, Vol. 69 (1997).
Pp. 1518-1524; GJ Opitek, JW Jorgenson,
RJ Andereg, "Anal. Chem." Magazine, Vol. 69 (1997), 228.
Pages 3 to 2291; YM Lie and JV Sweedler
r), "Anal. Chem.", Vol. 68 (1996), pp. 3928-3933).

In Jorgenson's experiment, multiple fractions eluted from a micro-bore chromatography column are subsequently injected into an electrophoretic capillary to separate overlapping components. Since electrophoresis is faster than the elution time of the chromatographic peak, all components from the chromatographic column are sampled in the electrophoretic capillary. By plotting the spots separated by electrophoresis side by side, a 2D electropherogram is resynthesized. The appearance of this electropherogram closely resembles a classic 2D electropherogram, despite being made with a combination of liquid chromatography and capillary electrophoresis.

The above sequential separation offers several advantages over conventional 2D electrophoresis, in which fragments are separated simultaneously. First, since multiple fractions are detected sequentially, a sensitive detector can be incorporated into the device. Second, the separation is automated. Once the sample has been injected, no further operator intervention is required.

The interface between the two columns is key to system performance. Jorgenson has demonstrated an elegant flow-gate interface for joining a liquid chromatography column with a capillary electrophoresis column (TF Hooker).
(Hooker) and JW Jorgenson, "Anal. Chem.", Vol. 69 (199).
7), pp. 4134-4142; A.V. Lemmo and JW Jorgenson, "Anal. Chem.", 65 (1993), 1576-1584.
page). This interface uses a buffer cross-flow to control the injection of the eluted chromatographic fraction onto a capillary zone electrophoresis (CZE) column. The effluent moves continuously from the HPLC capillary and is swept away by crossflow at the interface into waste. In order to inject the fraction into the CZE capillary, the buffer cross-flow is stopped and one HPLC effluent mass is formed at the interface. A voltage is applied across the CZE capillary from the buffer container to the detection end of the capillary, thereby injecting the HPLC effluent into the electrophoresis column. An electric field is applied across the electrophoretic capillary to separate the injected components. To avoid continuous injection of HPLC components during the electrophoresis step, a buffer flows through the interface and flushes the HPLC effluent to waste. Note that the flow gated detector discards most of the HPLC effluent, and the discarded HPLC effluent is swept downstream to waste, and thus most of the sample is wasted.

SUMMARY OF THE INVENTION The present invention provides an apparatus and method for achieving high resolution separation and sensitive detection of proteins and other components contained in a biological sample.

[0021] Other features and advantages of the invention will be apparent from the following detailed description. However, the detailed description and specific examples, which set forth preferred embodiments of the present invention, will be apparent to those skilled in the art from the detailed description that various changes and modifications that fall within the spirit and scope of the present invention. It is, of course, given only for explanation.

According to a first aspect of the present invention there is provided an apparatus for providing separation and detection of components in a sample, the apparatus comprising a first separation means, an interface means, a second separation means and the apparatus And a detector for detecting the component provided to the first and second means, and the interface means connects the first and second separation means.

The device comprises a first high voltage power supply connected to the first separating means, and a second
A second high-voltage power supply connected across the separation means. Further, the first separation means can be a capillary electrophoresis system, and the second separation means can be a molecular sieve electrophoresis system.

Another aspect of the invention provides an apparatus for providing sensitive detection of components of a biological sample, the apparatus comprising: an isoelectric focusing system, an SDS-polyacrylamide gel electrophoresis system, First and second separation means, each selected from the group consisting of a free solution electrophoresis system, a micellar electrokinetic chromatography system, a reversed phase liquid chromatography system, a normal phase chromatography system, an ion exchange chromatography system, and an exclusion chromatography system An interface chamber in which components separated according to the first means are mixed with an inducing agent therein before being applied to the second separation means; a first power supply for performing the first separation means and a first power supply for performing the first separation means; A second pump for implementing one of the first pumps and a second separating means; One of the second power supplies; and a detector.

The apparatus comprises a plurality of first separating means, a plurality of second separating means, and a plurality of interface areas, wherein each of the interface areas corresponds to one of the first separating means and the second separating area. It is beneficial to have a manifold that provides an interface between one of the separation means.

The manifold may include an inlet for connection to a buffer reservoir and valve means for allowing selective connection to a desired buffer reservoir, and a channel network connecting the inlet to a plurality of interface areas. Each interface area has a port for connection with one of the corresponding first separation means, a port for connection with one of the corresponding second separation means, and a third, waste liquid. It has a port.

[0027] Yet another aspect of the present invention provides a method for separating and detecting components in a sample,
The method includes providing the sample to an apparatus that separates components in the sample with high resolution and detects with high sensitivity; the apparatus is provided with a first separation means, an interface means, a second separation means, and an apparatus. The detector means for detecting the component; the interface means connecting the first separation means and the second separation means; the method comprises applying a first separation to the biological sample to achieve the first separation. Passing through the separation means, passing the sample from the first separation means through the interface means, dividing the sample into a plurality of fractions by the interface means, and passing each fraction through the second separation means individually. Process.

The method may include the step of applying an electric field by the high voltage power supply to each of the first separating means and the second separating means.

Another method provided by the present invention comprises providing high resolution separation and sensitive detection of components in a biological sample, the method comprising: (a) applying to a biological sample, etc. Consists of electrofocusing system, SDS-polyacrylamide gel electrophoresis system, free solution electrophoresis system, micellar electrokinetic chromatography system, reversed phase liquid chromatography system, normal phase chromatography system, ion exchange chromatography system, and exclusion chromatography system (B) passing the sample from the first separating means through the interface means and dividing the sample into a plurality of fractions; (c) isoelectrically separating each of the fractions. Point electrophoresis system, SDS-polyacrylamide gel electrophoresis Individually passing through a second separation means selected from the group consisting of a stem, a free solution electrophoresis system, a micellar electrokinetic chromatography system, a reversed phase liquid chromatography system, a normal phase chromatography system, an ion exchange chromatography system, and an exclusion chromatography system And d) detecting a component of the sample coming out of the second separation means with a detector. The method comprises applying a voltage across the first separation means and the second separation means. including.

The method further comprises the step of providing a plurality of first separating means and a plurality of second separating means and a plurality of interface means; each of the interface means comprises a corresponding one of the first separating means. Connecting between one of the first and second separating means; the method comprises connecting the first to the first separating means by a corresponding interface means.
And for each of the second separation means, passing the biological sample through the first separation means to achieve a first separation, transferring the sample from the first separation means to a corresponding interface means. The method further comprises a step of passing the sample, a step of dividing the sample into a plurality of fractions by the interface means, and a step of individually passing each fraction through the second separation means.

Next, the method comprises providing all interface means in a common manifold; an inlet for connection to a buffer reservoir, allowing selective connection of the desired buffer reservoir to the manifold. Providing a channel network connecting the valve means and the inlet to the plurality of interface areas; a port in each interface area connected to a corresponding first separating means, a port connected to a corresponding second separating means; And a third step of providing a waste port, the method comprising providing a plurality of buffer reservoirs connected to the inlet of the manifold and connecting the selected buffer reservoir to the manifold. Operating the valve means to ensure that the same buffer reservoir is connected to all interface areas,
The same processing steps are performed simultaneously in a plurality of interface areas.

The method includes providing a flat surface having a plurality of immobilization sites for capturing cells, providing a first separation means having a plurality of capillaries having an inlet end, and a plurality of inlet ends. May be arranged in the form of an array corresponding to the location of the immobilizing agent on the flat surface, the method further comprising the step of providing a biological sample on the flat surface, thus providing at least one Cells are captured at each of the immobilizer sites, aligning the plurality of capillary inlet ends of the first separation means with the immobilizer sites;
To perform the first separation in each of the plurality of capillaries, cells are drawn into the capillaries of the first separation means.

Finally, another aspect of the invention provides a manifold for use in separating and detecting components on a sample, the manifold comprising an inlet for connecting to a buffer reservoir,
A valve means for allowing selective connection with a desired buffer reservoir, and a channel network connecting the inlet to a plurality of interface areas, each interface area having a port for connection to a first separation means; A port for connection to a second separation means and a third, waste port.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be better understood with reference to the drawings, which show preferred embodiments of the present invention.

The present invention provides devices and methods for achieving high resolution separation and sensitive detection of proteins and other components contained in a biological sample. According to this embodiment of the invention, there is provided a first dimensional separation, which is preferably performed by capillary electrophoresis or by chromatographic means. According to the present invention,
This first separation divides the sample into many fractions, each of which can contain one or more components.

The invention further provides that each fraction is directed to the interface chamber. As the fractions are introduced into the interface, the flow is cut off from the first dimension separation means and one or more components in each fraction are mixed with an inducing reagent, which is preferably a fluorescent labeling reagent. Once labeled, the one or more components are then subjected to a second dimension separation. The second dimension separation means further separates the components in the fraction from the first dimension separation means. According to a preferred embodiment, the two separating means should separate the components based on different properties of the components. Classically, isoelectric focusing and SDS-
Polyacrylamide gel electrophoresis is used to separate proteins. Isoelectric focusing separates proteins based on differences in their isoelectric points, while gel electrophoresis separates components based on size. Alternatively, an electrophoresis method such as SDS-polyacrylamide gel electrophoresis, isoelectric focusing, isotachophoresis, or free solution electrophoresis may be replaced by chromatography such as reverse phase chromatography, normal phase chromatography, or ion exchange chromatography. It can be combined with the separation method. In general, for first dimension separation, electrophoresis is preferred because the flow can be quickly and easily stopped by removing the electric field across the capillary. In contrast, chromatography systems take a long time to decompress and it is much more difficult to stop the flow.

Components are detected when they migrate or elute from the end of the second dimension separation means, which is preferably a column. Although laser-induced fluorescence spectroscopy is preferred, other techniques such as absorbance spectroscopy, chemiluminescence spectroscopy, electrochemical spectroscopy or mass spectroscopy can be used. Fluorescence spectroscopy and mass spectroscopy are considered to be a useful combination. Fluorescence spectroscopy provides high sensitivity, and mass spectrometry provides additional information useful for identifying unknown components. One or more components that migrate or elute from the first dimension separation means are induced (preferably fluorescently labeled) for detection before being analyzed by the second separation means.
This approach is most useful when the second dimension separation means does not significantly discriminate between one or more labeled components. It is difficult to control the degree of the sign with high precision. As an alternative, a labeling reagent can be added after the second dimension separation column and before detection. Post-column reactions are less desirable because more components are required and more reagents are needed because re-mixing of the components separated by the second dimension separation means may occur in the post-column reaction chamber. Also, the reaction time in the post-column reactor is often limited because the flow is not interrupted, which results in the reaction not proceeding sufficiently, less fluorescent product being produced and lower sensitivity. Finally, the reaction reagents are present in the detector with the sample, raising the background signal. The second dimension separation column separates a reagent and a product by the method described above.

In this preferred embodiment of the present invention, isoelectric focusing and molecular sieving electrophoresis are used as two separation means. Other means can be used, but this combination is most familiar to the biochemist community in that it is a classic slab-gel format for the analysis of protein mixtures. One skilled in the art will readily appreciate that other separation methods may be used. For example, SDS-gel, free zone electrophoresis, isoelectric focusing, normal phase chromatography, reverse phase chromatography, ion exchange chromatography, or exclusion chromatography, preferably in any combination with the first dimension by electrophoresis. Can be used together.

Single Cell Analysis The inventors have been involved in single cell analysis for the past three years. Most of the research has been concentrated on oligosaccharide metabolizing enzymes (JY Zhao et al., Glycobiology, 4
Vol. (1994), pp. 239-242; Y. Tsang et al., "Anal. Biochem",
227 (1995), pp. 368-376; XC Lie et al., "J. Ch.
roma-togr. ", No. 716 (1995), pp. 215-220; NWC.
Chan et al., "Glycobiology", Volume 5 (1995), pp. 683-688; XC Lie et al., J. Chromatogr., Magazine, No. 781 (1997), 5
15-522; XC Lie et al., "Glycobiology", in print). In these experiments, the inventors have developed an efficient technique for injecting a single eukaryotic cell into a fused silica capillary followed by lysis and electrophoretic analysis of cell contents by sensitive laser-induced fluorescence detection.

The inventors used an Olympus inverted microscope equipped with a set of xyz hydraulic micromanipulators for the experiments. Assemble the transparent capillary holder,
Interfaced with manipulator. This holder allows observation of the cells with a transmission microscope while the capillaries are placed above the cells. This capillary holder allows for simple injection of cells into the capillary, a buffer reservoir for electrophoresis and a flushing of the capillary with sodium hydroxide solution between one separation operation and the next, The feature is that a pressurized chamber can be accommodated.

A drop of cell suspension phosphate buffered saline is placed on a microscope slide. Cells are viewed in inverted contrast microscope in phase contrast mode. Once one cell is selected for analysis, a micromanipulator is used to lower a capillary (20 mm diameter) with a flat tip over the selected cell. A computer controlled vacuum pulse is applied to the distal end of the capillary, and cells and a small amount of supernatant are drawn into the capillary in a reproducible manner. Based on the transit time of the fluorescent component of the cells, it is estimated that the cells are drawn into the capillary approximately 0.5 mm with a relative accuracy of 0.05 mm by injection.

Once the cells have been sucked into the capillary, the cells must be lysed to release their contents for subsequent analysis. The capillary tip is placed in a container filled with buffer, and the container is then placed in an ultrasonic bath. This method is extremely efficient in lysing cells. However, the transfer of capillaries and vessels from the microscope to the ultrasonic bath is time consuming and reproducibility is not very good. It is difficult to avoid the formation of a siphon during transfer, which can change the location of cells and their contents within the capillaries.

Human cancer cells lyse within 30 seconds when kept in contact with SDS buffer. The action of the surfactant is rapid and reproducible. SDS 10 mM, phosphoric acid 50 mM
Buffer was used for separation. Capillaries are filled with this buffer prior to injection of single cells. After injection of the cells, during incubation, the detergent rapidly diffuses into contact with the cells, resulting in efficient lysis.

[0044] Single cell proteome analysis has one disadvantage compared to flow cytometry.
That is, analysis of thousands of cells would be difficult. On the other hand, the huge amount of data generated from each cell would be too much to follow at a reasonable data collection rate. To optimize the value of the information generated, microscopy of the biopsy is performed,
It will be selected for subsequent proteome analysis in the first generator.

The microneedle aspiration biopsy was spread on a microscope slide and examined with an inverted microscope. Cells are often aggregated when viewed under a microscope. Cells are dispersed as a dilute suspension. So that the location of nearby cells is not disturbed by the selection process
It is important that the cells are not too close. Also, cellulose will be added to the suspension buffer to ensure that cells do not migrate prior to injection.

The microscope stage is equipped with a step motor driven micrometer. The micrometer is controlled by a joystick, so that the user can scan while precisely stepping the image field. A foot button will be pressed to indicate the location of the cell to be analyzed. The computer will record the location of the cell. Once a set of cells has been selected for analysis, an injection / electrophoresis program will be invoked. This program will drive the micrometer so that each cell is in turn centered in the microscope field of view. The cells are photographed with a CCD camera attached to the microscope and the images are digitized and stored so that they can be used later for comparison with proteomic data.

The cells will then be DNA stained prior to testing and the DNA content of the cells will be assessed based on the fluorescence from the dye. Hoesch 33342 fluorescent dye (Molecular Probes (Mouse Probe), which penetrates live cells and does not require fixing cells prior to staining.
-Lecular Probe), Eugene, Oregon, USA. Also,
This dye is excited only by UV (ultraviolet light), so that no bleaching occurs during preliminary inspection with phase contrast illumination. The microscope would be equipped with a photomultiplier tube for measuring fluorescence in a region of 10 μm diameter from the center of the microscope field of view.

The position of the micrometer is automatically adjusted to ensure that the fluorescence from the cells is maximized and that the cells are precisely centered in the field of view. In our microscope, the DNA stain is photobleached when illuminated for 1 minute. We anticipate that it will take 10 seconds to adjust the position of the cells to maximize the fluorescent signal and capture the peak intensity. DNA fluorescence will be used to determine cell ploidy. If done properly, only cells with a particular ploidy will be taken for subsequent injection into the electrophoretic capillary.

[0049] Each of the cells that meet the specified criteria is injected into a separate capillary. Both flat tip and etched tip capillaries were investigated for injection. The location of the capillary will be precisely located above the center of the microscope field, which should correspond to the location of the cell of interest. The capillaries will be lowered towards the microscope slide and will surround the cells. The solenoid at the far end of the capillary is opened for one second, drawing the capillary tip with an in-house vacuum to draw cells into the capillary. With multiple capillaries (described in more detail below), it is natural that at this point all other capillaries and reservoirs will be shut off to ensure successful cell injection.

The individual capillaries or capillaries will then be moved to a set of buffer reservoirs. If the cells are subjected to 2D electrophoresis, if the cells have been treated with FQ to label the cytoplasmic proteins with a label for 1D electrophoresis, the first reservoir will contain a tween ( TWEEN) -20 solution would be included. Unlike SDS, non-ionic detergents do not interfere with subsequent isoelectric focusing. If all of the cell's proteins were to be labeled on the column prior to 1D separation, the first reservoir would contain both lysis and induction reagents. One nanoliter of the reagent solution is injected into the capillary and cell lysis is expected to be completed within 30 seconds. The capillary tip will then be placed in a suitable working buffer. If 1D free solution electrophoresis is performed, this buffer should be basic SD
Will be S-phosphate buffer. If capillary isoelectric focusing is the first dimension of the 2D analysis, an acidic anolyte would be used as a buffer.

When the separation is complete, the next cell is moved to the microscope field, a picture is taken,
The fluorescence will be measured and the cells will be analyzed by electrophoresis. This procedure will be repeated for all of the cells specified by the pathologist. The cell chamber will be kept in a constant humidity chamber to prevent evaporation of the cell suspension buffer.

Single Capillary Embodiment Referring to FIG. 1, there is shown an apparatus 10 having a first dimension separation column 12 and a second dimension separation column 14. In a known embodiment, two separation columns 12, 1
Reference numeral 4 denotes a capillary having an inner diameter of 10 to 75 μm.

As described above, isoelectric focusing is used for the first dimensional separation 12. For this purpose, the column 12 is filled with a mixture of ampholytes. A first high voltage power supply 16 is connected to one end of the column 12 as shown at 18 and to the interface 20 as shown at 20. Interface 22 is described in further detail below, and is connected to both columns 12 and 14 as shown.

A second high voltage power supply 24 is connected to the interface 22 as shown at 26 and to the end of the second dimension column 14 opposite the interface 22 side as shown at 28.

The interface 22 is shown in detail in FIG. 2 and is made of an inert, non-conductive material, such as a high quality plastic. Four holes 30 are machined into the plastic and connected by a lumen 32 to form an intersecting structure. As shown diagrammatically, the holes are preferably provided with a ferrule system or some other means of sealing the capillaries in place. Hole 30 and lumen 32 should be small, with hole 30 having a diameter less than 1 mm and lumen 32 preferably having a diameter less than 0.2 mm. Capillary columns 12 and 14, as shown schematically in FIG.
Connected to high voltage power supplies 16 and 24. The connections 20, 26 for the high voltage power supply are shown here connected to the drain or buffer solution waste line 34, also shown in FIG.

The buffer solution inlet 36 is connected to an air pressure control switching valve 38 capable of switching between four positions. Three of the four locations provide a connection to one of the three buffer reservoirs 40, 42 and 44. The fourth position connects valve 38 to cutoff port 46.

The buffer reservoirs 40, 42 and 44 are located above the air valve 38 so that the flow from the reservoirs can pass by gravity through the interface 22. Valve 38
Are switched pneumatically to avoid the danger of electric shock due to the voltage applied during the electrophoresis step. Similarly, buffer reservoirs 40, 42 and 44 should be safely housed in interlocked chambers to avoid accidental contact during the electrophoresis process.

In the apparatus 10, the sample is injected by placing the sample in a solution in contact with one end of the capillary 12 and applying a short electric field pulse. Alternatively, pressure can be used to fill a portion of the capillary 12 with the sample. When the sample is provided, the sample is transferred to a working buffer (not shown), and an electric field is applied to the first dimension separation column 12 by the power supply 16. Thereby, separation is performed. According to isoelectric focusing techniques known to those skilled in the art, the capillary 12 is filled with an amphoteric electrolyte mixture, which is a multiple mixture of compounds having both weak acid and weak base functional groups. The ampholyte can be mixed with the sample, and the resulting mixture is used to fill the capillary 12. Alternatively, an amphoteric electrolyte mixture can be used to fill most of the capillaries and the sample can be injected later.

When the capillary 12 is filled with the sample and the ampholyte, the sample end of the capillary is transferred to a sodium hydroxide (or other suitable base) solution as a working buffer, and the interface 22 is connected to the reservoir 40, Fill with a phosphoric acid (or other suitable acid) solution from a suitable one of 42 and 44. The anode of the high voltage power supply 16 is connected to the acid end, and the cathode is connected to the base end. A voltage large enough to form a pH gradient in the capillary 12 is applied. The protein contained in one or more samples moves to a position on the capillary where the pH is equal to the isoelectric point of the protein. Completion of isoelectric focusing correlates with a drop in current through the capillary. When the current reaches 10% or less of the initial value, the power supply 16 is turned off.

During the isoelectric focusing step, interface 22 is filled with an acid solution from reservoir 40 (FIG. 2). The acid solution can be run continuously at a low flow rate (less than 10 milliliters / hour) during the isoelectric focusing process to minimize the accumulation of electrolytic products. An electric field is applied across the isoelectric focusing capillary tube 12.

Fractionation Fluid to Interface The separated fraction is subsequently flowed from the first dimension separation capillary to the interface. Although several methods of flowing isoelectrically concentrated proteins are described herein, it will be readily apparent to those skilled in the art that such methods are in no way limited to the methods described. Other effective fluidization methods are within the scope of the present invention.

First, according to a preferred embodiment, the inner surface of the first dimension separation capillary 12 is coated with a neutral material to reduce electroosmosis and reduce adsorption of proteins to the inner surface of the capillary. . In this case, the protein can be caused to flow from the first dimension separation capillary by several means. For example, a low pressure can be applied to the sample side end of the first dimension separation capillary. The basic approach is to raise the sample side end of the first dimension separation capillary several cm above the level of interface 22 and create a siphon that causes the flow of sample from the first dimension separation capillary to interface 22 by gravity. be able to. After the appropriate fraction of the contents of the first dimension separation capillary 12 flows into the interface 12, the sample end of the first dimension separation capillary can be lowered to stop the flow. During this step, it is desirable to apply an electric field to keep the protein concentrated in a distinct band. Once the aliquot has been delivered to the interface 22, the electric field can be removed.

According to another embodiment, chemical means can be used to drive certain fractions of the first dimension separated capillary contents to the interface. According to this embodiment, the acid-containing solution at the interface end of the first dimension separation capillary 12 is replaced with a suitable basic buffer, for example, made with dilute sodium hydroxide solution. A change in the pH of the solution causes a migration of the capillary contents towards the interface when an electric field is applied across the first dimension separation capillary.

According to another embodiment, if the inner surface of the first dimensional separation capillary 12 has not been subjected to a neutral coating treatment, the analyte may be electro-osmotically condensed during the isoelectric focusing step. Be kicked out of According to this embodiment, the pH of the interface
The polarity is reversed from that of FIG. 1 to ensure that is basic.

When the isoelectric focusing is completed, it is necessary to flow the fraction from the isoelectric focusing capillary and label it. A basic buffer is mixed with the fluorescence inducing reagent (and most of the inducing reagents require a basic buffer) and placed in a buffer reservoir 42. The interface should be flushed with a basic buffer / labeling reagent, and then the flow from reservoirs 40, 42, 44 should be shut off. It is desirable that there be no flow during the process where the fraction is flowing from the isoelectric focusing capillary. An electric field is briefly applied across the isoelectric focusing capillary (500-5000 V, 1-10 seconds), which drives the fraction out of the isoelectric focusing capillary to the interface and mixes with the inducing reagent. When the fraction is introduced into the interface, the electric field is turned off.

Regardless of the method used, when a predetermined portion of the first dimension separation column moves to the interface 22, the flow will be terminated by removing the electric field from the high voltage power supply 16. Each volume of the aliquot or fraction transferred to the interface 22 can be from about 0.1% to about 5% of the volume of the capillary.
%. The larger the volume, the more components that migrate from the first dimension separation column will mix, while the smaller the volume, the more samples can be analyzed, but the corresponding total analysis time will be longer.

Labeling of Fractions Once the fraction of the first dimensional separation capillary has been introduced into the interface, a plug of a suitable inducing reagent is introduced into the interface. A suitable inducing reagent means that the reagent reacts with the material separated in the first dimension separation capillary. For protein analysis, the reagent can be 3- (2-furoyl) quinoline-2-carboxaldehyde (FQ), fluorescein sulfonyl chloride, ortho-phthaldialdehyde, and the like. Alternatively, if the compound is fluorescent, induction would not be necessary. Usually, it is convenient to control the temperature of the interface. Often the interface should be heated to facilitate the reaction. Other inducing agents will be known to those skilled in the art.

The interface temperature can be increased to 50 ° C. to increase the rate of the fluorescence induction reaction. The entire interface or only the part corresponding to the intersection of the two holes can be heated.

When the fluorescence induction reaction is completed, the labeled sample has to be injected into the SDS-gel capillary 14. An electric field is applied to the detection end of the SDS-gel capillary via an electrode or connection 28. A second electrode or connection 26 connects to the waste outlet 34 of the interface 22. Deposition occurs during implantation. Most, if not all, of the labeled fraction from isoelectric focusing capillary 12 is injected into SDS-gel capillary 14 as a narrow plug.

Upon completion of the induction reaction, the contents of the interface must be provided to the second dimension separation capillary by injection or other suitable means.

When the second-dimensional separation sample is provided to the second-dimensional separation capillary 14, the interface 22
It is preferred to flush with a suitable separation buffer used for dimensional separation.
According to a preferred embodiment, this buffer is provided in buffer reservoirs 40, 42 and 44.
Containing 5 mM SDS (pH 8), also supplied from a suitable one of
5 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid).

The second dimension separation is performed by molecular sieve electrophoresis. According to this embodiment, the capillary 14 is filled with an inert polymer material. Those skilled in the art will appreciate what is a suitable polymeric material and that suitable polymeric materials include polymeric materials such as polyacrylamide, dextran, and the like. Dextran has a relatively low viscosity,
This is preferred because it can be easily pumped into and removed from the second dimensional separation capillary to renew the column if necessary. Molecular sieving electrophoresis is performed in the presence of a charged surfactant such as sodium dodecyl sulfate (SDS) or the like. Such surfactants are approximately SDS / g protein
It binds to proteins at a rate of 1.4 g. This detergent soaks the charge on the protein and creates a uniform charge along the length of the protein. When a protein passes through a capillary filled with molecular sieves, the translocation time of the protein is proportional to the logarithm of the molecular weight, with smaller proteins moving first and larger proteins moving more slowly.

As the sample is injected into the SDS-gel capillary, SDS-gel buffer from buffer reservoir 44 passes through the interface. This buffer is used at low flow rates (10 ml / min) to minimize the accumulation of electrolytic products during SDS-gel electrophoresis separation.
Less than an hour). A high voltage is applied across the SDS-gel capillary to separate the fluorescently labeled proteins.

Separation is achieved by applying a voltage from the high voltage power supply 24 to the second dimensional separation capillary 14. At this time, it is preferable that there is no voltage applied to the first capillary 12.
Proteins or other components migrating from the second dimension separation capillary 14 are detected by a sensitive fluorescence detector (not shown) according to a preferred embodiment. Other detection methods include absorbance, electrochemical, radiochemical, or mass spectrometric means.

In the preferred embodiment described above, detection will be achieved by a sensitive laser-induced fluorescence detector. This detector is assembled from a sheath flow cuvette (S. Wu, NJ Dovich, "Journal of Chromatography").
Journal, No. 480 (1989), pp. 141-155). Preferably, the laser wavelength should match the excitation wavelength of the labeled analyte, and the emission wavelength should match the emission characteristics of the labeled analyte.

At the completion of the SDS-gel separation, the capillary 14 should be flushed with fresh molecular sieve medium. The air valve 38 is changed to the shut off position 46 and fresh medium should be flushed from the detector through the capillary.

A new fraction is flowed from the isoelectric focusing capillary 12 to the interface, labeled and separated by SDS-gel electrophoresis. Isoelectric focusing capillary tube 1
This process is repeated until all of the contents of 2 are analyzed.

Computer Resynthesis The data obtained in the present invention consists of a set of SDS-gel electropherograms, each electropherogram from successive fractions of an isoelectric focusing capillary.
According to a preferred embodiment, these data can be represented as raster images to generate a two-dimensional map of proteins from the sample.

The above is illustrated in FIG. 3, where the sample injection is indicated at 50 and the detector is indicated at 52. Columns 12 and 14 and interface 22 have the same reference numerals. At 54, a two-dimensional computerized resynthesis is schematically illustrated.

A laser-induced fluorescence detector is used. This detector based on a sheath flow cuvette provides excellent sensitivity. We used 488 nm for excitation.
10mW argon ion laser is used. This laser is reasonably robust and provides excellent beam quality.

The device will be equipped with a two-color fluorescence detector. This detector uses a dichroic filter to split the fluorescence into two spectral bands. An interference filter is used to separate fluorescence in the 30 nm band centered at 630 nm from the FQ-labeled protein. A second spectral channel will monitor fluorescence from the fluorescein labeled protein in the 25 nm band centered at 525 nm. Fluorescence from a fluorescein-labeled internal standard protein will be used as a marker in two-dimensional separations to correct for drift in transit time during and between analyses.

Informatics (informatics) will yield meaningful results. Flicker, supplied by the United States National Institutes of Health, is public domain software developed to visually compare and compare 2D electropherograms. For preliminary identification of proteins with significantly different expression levels in various samples, we will correct the migration pattern based on the appearance of fluorescein-labeled standard proteins. We will then convert the data to GIF format and compare the files using flicker.

QUEST (PJ Monardo, T. Boutell, J. Garrels, G. Latter, “Computer Application in the Bios
ciences ", Vol. 10 (1994), pp. 137-143) and (MAYA Design Grove, Pittsburgh, PA, USA).
The Visage system (distributed from up) is also developed for 2D gel analysis. However, for quantitative identification of these components that differ between samples, the Mathwork, Natick, Mass., USA
rk Inc.) is used to build our own image processing software. After the migration pattern has been corrected based on the standard protein migration, a peak detection algorithm will be used to locate and quantify each protein peak.

The concentration of each component will be measured and recorded from several samples, and the mean and standard deviation of the concentration of each protein will be determined. Finally, a simple Student's t-test or Behrens-Fisher's t-test would be used to identify proteins whose concentrations vary significantly between samples (Robinson et al. "Electrophoresis", Vol. 16, 1995
), Pp. 1176-1183).

A 2D capillary electrophoresis system will be used to analyze small amounts of proteins. Mass spectroscopy of the fraction would be difficult. To identify fractions, classical 2D electrophoresis on a large amount of cell extract is performed simultaneously.
The same markers as the fluorescent protein markers used in the capillary system will be added to the sample. These fluorescent proteins will be used to normalize the transit time so that capillary 2D and slab 2D electrophoresis data can be directly compared. The component of interest will be isolated from the slab 2D gel, analyzed by mass spectrometry, and injected with the sample into the capillary system to confirm the identity of the component in the capillary data.

Multiple Capillary Embodiment Referring to FIG. 4, a second embodiment of the device of the present invention is generally designated by the reference numeral 60.
Indicated by This embodiment includes storage tanks 40, 42, and 44, an air valve 38, and a shutoff port 46, as in the first embodiment.

In the second embodiment 60, a manifold 62 is provided that replaces the interface 22 for single capillary pair operation of FIGS. Like interface 22, manifold 62 can be formed of plastic, glass, silicone, or other suitable material. In order to create a set of channels for dispensing reagents, indicated at 64, it is appropriate to use photolithography to form the manifold 62. The width and depth of the channel 64 are both
It can be 10 to 200 μm.

The manifold 62 is provided with a plurality of interface areas 66, each interface area 66 having three connection ports for connection to the first and second columns and the waste tubing.

The interface area is shown enlarged on the right side of FIG. At the top, this area 66 is provided with an inlet 68 for buffers or reagents from one of the reservoirs 40, 42 and 44. A connection port 72 is provided for connection to the first capillary column 12 and a second connection port 74 is provided for connection to the second column 14. A central port 76 is provided for connection to a waste outlet.

That is, each interface area 66 can fulfill the same function as the interface 22 in the first embodiment. But in this case,
Multiple capillary detectors can be used to analyze the signal from the capillary array.
As many as 96, or as many as 96, capillary systems (ie, pairs of capillaries 12, 14) can be operated in parallel.

The operation of the interface multicapillary system is similar to that of the single-capillary type described in the detailed description of the first embodiment, except for differences depending on the particular system. Briefly, an analyte is introduced into the isoelectric focusing capillary, an acidic buffer is introduced into the interface area, and an electric field is applied across the capillary. As the components are concentrated to their respective isoelectric points, the electric field is extinguished.

Filling the interface area with a basic buffer mixed with a labeling reagent fluidizes the fraction of the contents of the isoelectric focusing capillary. An electric field is briefly applied across the isoelectric focusing capillary to flow the fraction to the interface.

The temperature of the interface can be raised to a level sufficient to increase the rate of the fluorescent labeling reaction. Upon completion of the reaction, labeled molecules are provided to the SDS-gel capillary by creating an electric field across the SDS-gel capillary.

Once the sample has been applied to the SDS-gel capillary, the interface chamber is flushed with a suitable buffer. An electric field is applied across the SDS-gel capillary to separate the components contained in the labeled fraction.

A second aspect of the preferred embodiment of the multiple capillary version of the present invention is a multiple capillary fluorescence detector. According to this embodiment of the present invention, the detector is disclosed in US Pat.
These detectors can be detectors similar to those described in US Pat. Nos. 39,578, 5,584,982, 5,741,412 or, preferably, 5,567,294. Is incorporated herein by reference.

For multi-capillary operation, an array of antibodies is prepared on a microscope slide.
The immobilization technique will be based on classical microphotolithography. A clean microscope slide is coated with a thin layer of photoresist. This resist
It is irradiated with UV light through a mask having 96 spots of 5 μm diameter in an 8 × 12 array with a center spacing of 1 mm. Opaque spots cast shadows on the photoresist and prevent photopolymerization in the shadowed areas. The light-irradiated resist will polymerize and unpolymerized resist will be washed away from the 96 spots. The slide is then treated with an aminopropyl-silane reagent that is covalently attached to the glass surface. The photoresist is removed with an organic solvent, leaving an 8 × 12 array of 5 μm diameter amino-propyl based pads. The use of classical diisothiocyanate chemistry will attach the antibody to the amino group. It should be noted that other and possibly more effective reagent selections can be made. For example, a gold pad can be formed and the antibody immobilized with a thiol-containing reagent.

The cell suspension is flushed onto the treated slide surface to capture 96 cells. The 5 μm diameter pads are small enough to ensure that only one eukaryotic cell will be trapped in each pad. Once the cells have been captured, the adsorption of the second cell at that location will be blocked.

[0098] Many different antibodies can be used to select surface markers that will isolate and enrich a particular cell. This method can be used to immobilize antibodies against human blood group A. This antibody expresses HT29, which expresses the antigen on the cell surface.
Will catch the cell. Other capture reagents, such as lectins and aptamers, can also be used.

The microscope slide is supported on a machined holder, which will place the slide in a precise position on the microscope stage. Each pad will be sequentially centered in the field of view and a photograph will be taken using a CCD camera. The images will be saved for later examination by a pathologist. At the same time, the UV fluorescent signal from each cell will be recorded to determine ploidy. The inventors,
Each cell is imaged, each ploidy determined, 15 times to move to the next cell.
It requires only seconds and expects that a preliminary examination of whole cells will take less than 30 minutes.

A capillary array holder will be fabricated to support a bundle of 96 capillaries in precise alignment with the antibody pads formed on the microscope slide. The capillary array would be placed above the microscope slide and lowered to capture one cell in each capillary. A three second vacuum pulse is applied to the distal end of each capillary to draw the cells into the capillary and lyse the cells, similar to a single capillary device.

The inventors have developed several sensitive multi-capillary fluorescence detectors for DNA sequencing, as disclosed in the above-mentioned US patents. The newest form of detector is based on a two-dimensional capillary array in a sheath flow cuvette.

Referring to FIG. 5, a two-dimensional sheath flow cuvette 70 is shown. Laser 72 provides a laser beam that passes through cylindrical lenses 74 and 76 to create an elliptical beam. This laser is an argon ion laser.

The laser light is focused at right angles from the end of the capillary, passes through a camera lens 78, a bandpass filter 80, a prism 82 and another camera lens 84 and enters a CCD camera indicated at 86. This configuration shows that the fluorescence from each capillary is imaged on a unique area of the camera's CCD. Prisms 82 are included in the array of optics to distribute the fluorescence across the camera plane so that the complete spectrum from each capillary can be recorded. The dispersion of the system is carefully adjusted to ensure that there is no spatial overlap of the fluorescence from each capillary.

The fluorescence is distributed in the area between adjacent capillaries. Prisms are used over diffraction gratings for two reasons. First, the low dispersion of the prism simplifies the design of the optical system. Second, the prism does not generate higher order spectra that would interfere with spectra from neighboring capillaries.

The detector of FIG. 5 continuously monitors the fluorescence from all capillaries at 28 wavelengths over the fluorescence spectral band. The system provides three important advantages over other configurations. First, the elimination of the light loss associated with reduced imaging systems through the use of one-to-one imaging optics, which is seen when images of a large one-dimensional capillary array are created on a small photodiode array. Second, there is no sensitivity loss due to the low duty cycle associated with detectors scanning the array end-to-end. 96
The time during which the apparatus for scanning one capillary can observe the fluorescence from any one capillary is 1% or less of the whole. Third, there is no sensitivity loss due to sequential detection of fluorescence by a rotating filter wheel or prism array.

Rather than recording fluorescence at 28 wavelengths simultaneously, the fluorescence will be recorded in two spectral bands corresponding to the emission spectra of fluorescein and FQ labeled proteins. This sorted and recorded data will occupy little computer memory.

Although the present invention has been described with reference to embodiments presently considered to be preferred examples,
Naturally, the invention is not limited to the disclosed embodiments. On the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are to the same extent as if the individual publications, patents and patent applications were specifically and individually indicated to be incorporated by reference in their entirety.
They are hereby incorporated by reference in their entirety.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the apparatus of the present invention.

FIG. 2 is a schematic diagram of a fluid flow interface in the device of the present invention.

FIG. 3 is a raster image for generating a two-dimensional image of data from the apparatus of the present invention.

FIG. 4 is a schematic diagram of a multiple capillary version of the device of the present invention.

FIG. 5 is a perspective view of a 2D sheath flow cuvette detector.

[Explanation of symbols]

 12,14 Separation column 16,24 High voltage power supply 22 Interface 62 Manifold 70 Two-dimensional sheath flow cuvette

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Claims (21)

[Claims] 1. An apparatus for providing separation and detection of a plurality of components in a sample, said apparatus comprising a first separation means, an interface means, and a second separation means, wherein said interface means comprises: Apparatus, characterized in that it comprises a detector for connecting a first separating means and the second separating means and for detecting a plurality of components provided to the apparatus. 2. The apparatus according to claim 1, further comprising a first high-voltage power supply connected to said first separating means and a second high-voltage power supply connected to said second separating means. apparatus. 3. The method according to claim 2, wherein said first separation means is a capillary electrophoresis system, and said second separation means is a molecular sieve electrophoresis system.
The described device. 4. An apparatus for providing sensitive detection of a plurality of components of a biological sample, said apparatus comprising: first and second separation means, each of said first and second separation means. , Isoelectric focusing system, SDS-polyacrylamide gel electrophoresis system, free solution electrophoresis system, micellar electrokinetic chromatography system, reversed phase liquid chromatography system, normal phase chromatography system, ion exchange chromatography system, and exclusion chromatography system An interface chamber in which a plurality of components separated according to said first separating means are mixed with an inducing agent before being applied to said second separating means; implementing said first separating means A first power supply and one of the first pumps and the second A device comprising one of a second pump and a second power supply for implementing the separating means; and a detector. 5. The apparatus according to claim 4, wherein said detector is a sensitive laser-induced fluorescence detector. 6. The apparatus according to claim 5, wherein said first separation means is an isoelectric focusing system, and said second separation means is a molecular sieve electrophoresis system. 7. A plurality of first separation means, a plurality of second separation means, and a manifold comprising a plurality of interface areas, wherein the plurality of first separation areas respectively corresponding to the plurality of interface areas. Apparatus according to any of the preceding claims, providing an interface between one of the means and one of the plurality of second separating means. 8. The manifold has an inlet for connection to a buffer reservoir, valve means for allowing selective connection to a desired buffer reservoir, and a channel network connecting the inlet to the plurality of interface regions. Wherein each of the plurality of interface areas corresponds to one of the plurality of first separating means.
8. The apparatus of claim 7, comprising a connection port with one, a connection port with a corresponding one of the plurality of second separating means, and a third, waste port. 9. A two-dimensional sheath flow cuvette, wherein the second separating means includes: a plurality of capillaries mounted in the two-dimensional sheath flow cuvette in a two-dimensional array; a light source; and light emitted from the light source. An optical system for illuminating the ends of the plurality of capillaries; and a condensing optical system aligned with the ends of the plurality of capillaries for condensing, wherein the condensing optical system is required. 9. Apparatus according to claim 7 or claim 8, comprising a camera lens, a bandpass filter, a prism and a camera accordingly, axially aligned with the ends of the plurality of capillaries. 10. A method for separating and detecting a plurality of components in a sample, comprising:
The method comprises providing the sample to an apparatus with high resolution separation and high sensitivity detection of a plurality of components in the sample, wherein the apparatus comprises a first separation means, an interface means, a second separation means and the apparatus. A detector for detecting a given plurality of components, wherein said interface means connects said first separation means and said second separation means, and said method achieves a first separation. Passing the biological sample through the first separation means, passing the sample from the first separation means through the interface means, dividing the sample into a plurality of fractions by the interface means, and Separately passing each of said fractions through said second separation means. 11. The method according to claim 1, further comprising the step of applying an electric field to each of said first separating means and said second separating means by a high voltage power supply.
0. The method of claim 0. 12. The method of claim 11, wherein said first separation means is a capillary electrophoresis system and said second separation means is a molecular sieve electrophoresis system. 13. A method for providing high resolution separation and sensitive detection of a plurality of components in a biological sample, the method comprising: (a) applying a isoelectric focusing system, SDS- A first separation selected from the group consisting of a polyacrylamide gel electrophoresis system, a free solution electrophoresis system, a micellar electrokinetic chromatography system, a reversed phase liquid chromatography system, a normal phase chromatography system, an ion exchange chromatography system, and an exclusion chromatography system (B) passing the sample from the first separating means through an interface means to divide the sample into a plurality of fractions; (c) applying isoelectric focusing to each of the plurality of fractions Electrophoresis system,
A second selected from the group consisting of SDS-polyacrylamide gel electrophoresis system, free solution electrophoresis system, micellar electrokinetic chromatography system, reversed phase liquid chromatography system, normal phase chromatography system, ion exchange chromatography system, and exclusion chromatography system (D) detecting a plurality of components of said sample coming from said second separation means with a detector, wherein said method comprises the steps of: Applying a voltage across the means and the second separating means. 14. The method of claim 13, including the step of mixing said sample with an inducing agent at said interface means. 15. The apparatus of claim 14, wherein the detector is a sensitive laser-induced fluorescence detector, and wherein the inducer reacts with a plurality of components of the sample to render the plurality of components fluorescent. The described method. 16. The first separating means is an isoelectric focusing system,
The method according to claim 15, wherein the second separation means is a molecular sieve electrophoresis system. 17. The method, comprising: providing a plurality of first separating means and a plurality of second separating means and a plurality of interface means, wherein each of the plurality of interface means corresponds to the plurality of the plurality of interface means. Providing a connection between one of the first separating means and one of the plurality of second separating means; the method comprising: connecting the first and second parts by a corresponding interface means. Passing, for each of the separation means, a biological sample through the first separation means to achieve a first separation, transferring the sample from the first separation means to the corresponding interface means Passing the sample, dividing the sample into a plurality of fractions by the interface means, and passing the plurality of fractions individually through the second separation means. The method according to any one of claims 10 to 16, further comprising a step. 18. The method wherein all of the plurality of interface means are provided in a common manifold, an inlet for connection to a buffer reservoir in the manifold, and a selective connection to a desired buffer reservoir. Valve means for
And providing a channel network connecting the inlet to the plurality of interface areas, and a port connected to a corresponding first separating means, a port connected to a corresponding second separating means, for each of the plurality of interface areas. Providing a plurality of buffer storage tanks connected to the inlet of the manifold, and the valve for connecting a selected buffer storage tank to the manifold. 18. The method of claim 17, including the step of manipulating the means, so that the same buffer reservoir is connected to all of the plurality of interface areas, and wherein the same processing steps are performed simultaneously in the plurality of interface areas. 19. Providing a flat surface having a plurality of immobilizer sites for capturing cells, providing a first separation means having a plurality of capillaries each having an inlet end, and on the flat surface. Arranging the plurality of inlet ends in the form of an array corresponding to the plurality of immobilizer sites, and further providing a biological sample on the planar surface, thereby providing the plurality of immobilizer sites. Capturing at least one cell by each of: aligning an inlet end of said plurality of capillaries of said first separating means with said plurality of immobilizer sites; and within each of said plurality of capillaries. 19. The method of claim 17 or 18, further comprising the step of sucking said cells into said plurality of capillaries of said first separation means to perform a first separation.
The described method. 20. The method of claim 19, wherein each of said plurality of immobilizing agent sites is sized to hold only one cell. 21. A manifold for separating and detecting a plurality of components from a sample, wherein the manifold has an inlet for connecting to a plurality of buffer storage tanks and a selective connection to a desired buffer storage tank. Valve means for enabling and a channel network connecting said inlet with a plurality of interface areas, each of said plurality of interface areas having a port for connection with a first separation means, a connection with a second separation means. A manifold comprising a connection port and a third waste liquid port.