Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44743—Introducing samples
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N2035/1027—General features of the devices
  • G01N2035/1034—Transferring microquantities of liquid
  • G01N2035/1037—Using surface tension, e.g. pins or wires

Definitions

• the invention relates to a method for feeding sample material by means of a sample feed part from a sample addition point to a sample receiving point of a sample processing device, preferably a sample analysis device.
• the sample material is removed from a sample vessel unit with the aid of a pipetting device and fed to the sample processing device in the form of a vertically oriented electrophoresis device (DE 38 05 808 AI, FIGS. 10 and 11). It is also possible to take several samples at the same time with the aid of a corresponding number of pipettes and to feed corresponding sample receiving pockets on the upper edge of the gel.
• the mechanical outlay for this known type of sample supply is great, especially when particularly thin gel layers are used and a large number of samples are to be analyzed simultaneously.
• the gap between the glass plates on both sides of the gel which corresponds to the gel thickness, is in the range of less than one millimeter in the case of the small gel planks sought today; the distance between successive sample pockets is in the range of a few millimeters.
• up to 100 sample recesses can then be arranged side by side.
• the effort for the corresponding number of pipettes is correspondingly high, as are the requirements for the positioning accuracy. Only a vertical orientation of the gel is practically possible, otherwise the sample recesses could leak.
• a method of the type mentioned at the outset is known from WO 94/11529.
• the sample material is specifically chemically bonded to the teeth of the comb-like sample feed part.
• the comb-like sample feeder has only eight teeth, so that accordingly only eight samples can be analyzed at the same time. This may be due to the fact that sufficient sample material can only be provided with relatively wide teeth.
• the invention has for its object to provide a method of the type mentioned, which allows an effective sample material supply in a simple manner.
• the sample supply part has at least one material section made of porous material with such a pore size of the porous material that the sample material is in the liquid phase due to the sample processing device at least when the sample is added to the sample supply part and when the sample is processed by the sample processing device is held by capillary forces.
• the sample material is therefore kept in the liquid phase, at least when adding or removing samples, only due to capillary forces in the sample material.
• the sample material may, but need not, be present in the pores of the porous material in a dried form. In any case, complicated mechanics, such as multi-pipetting devices, are spared.
• the porous material only has to be brought into contact with the sample material in the liquid phase; Reactions to chemically bind the sample material to the sample feed part are in no way necessary, just as chemical reactions during sample delivery to release the chemical bond between sample material from the sample feed part are not necessary. Since the capillary forces hold the sample material in the liquid phase during sample delivery until it is transferred to the sample processing device, regardless of the direction of gravity, there is no longer any restriction Regarding the orientation of the sample processing device.
• the porous material can have a sheet shape so that it can be easily pushed between the glass plates of a gel electrophoresis device. At least the material section of the sample supply part preferably consists of porous material throughout, so that the capacity of the material section for the sample material is comparatively high. Small-sized material sections are therefore sufficient, so that a corresponding number of material sections can be used in smaller spaces. The number of samples to be analyzed at a given width of the electrophoresis gel can therefore be increased significantly, for example to 192 to 384 samples.
• the sample material be dried after the sample has been added to the sample supply part and that the sample supply part be supplied to a liquid phase before the sample is released.
• the sample supply part which has been dry in the meantime, is particularly easy to handle; the risk of interim contamination is significantly reduced.
• the sample supply part can, for example, be mechanically processed, e.g. B. are mechanically compressed or blown by compressed gas.
• an electrical field passing through the porous material section is generated in the area of the sample acceptance point in order to cause a flow of electrically charged molecules, macromolecules or particles of the sample material from the porous material section into the sample processing device.
• the strength of the electric field is chosen depending on the electric charge so that the capillary forces are overcome.
• the use of this method step in electrophoresis is particularly advantageous since the means for generating the electric field are provided there from the outset.
• the sample supply part loaded with the sample material is easy to handle, there is the advantageous possibility that after the sample has been added the sample supply part is transported from the sample addition point to the sample acceptance point.
• the addition of the sample can therefore take place at a location which is arbitrarily distant from the sample processing device.
• the transport of the sample from the sample addition point to the sample acceptance point can also take place in that when the sample addition point is physically connected to the sample acceptance point, the capillary forces ensure the sample transport through the porous material section of the sample supply part.
• This type of sample supply will be used above all if the sample receiving point is particularly inaccessible or restricted to a very small space, as can be the case with microchip sensors, in particular DNA sensors.
• a separate section of material is generally used for each sample, which is independent of the material sections of the other samples in order to avoid "crosstalk", i.e. H. a mixing of sample material to prevent from the outset, which would otherwise be possible due to the comparatively large amounts of sample material to be used.
• the possibility of mixing can also be specifically exploited by wetting the porous material with the substances to be mixed at different points or in succession at the same point.
• proteins eg antigens
• proteins can be mixed with antibodies by adding them in the porous material, or DNA with complementary hybridization DNA or DNA with labeling agents.
• porous material sections Due to the ease of handling mentioned and the relatively high sample material capacity of the porous material sections, there is the preferred possibility that a corresponding number of porous material sections are used in a sample processing device with a large number of sample receiving points.
• the sample supply part comprises, according to the invention, a material section carrier which carries the material sections in an arrangement corresponding to the geometric arrangement of the sample receiving points.
• the sample supply part can be shaped essentially like a comb if the sample receiving points are arranged in a linear or arcuate manner.
• the sample supply part can be produced particularly easily, for example by stamping, if it comprises a sheet of porous material having all porous material sections.
• the material section carrier can also carry a corresponding plurality of separate material sections. Such an arrangement is recommended when the risk of cross-talk cannot otherwise be excluded.
• the material section carrier can be formed by a base film or can also take another form, such as that of a clamping device.
• a porous material with an average pore size below 100 ⁇ m, preferably below 10 ⁇ m, preferably below 1 ⁇ m, or with an average pore size between 1.5 ⁇ m and 0.2 ⁇ m, preferably around 0.5 ⁇ m, is preferably used .
• Such porous material has sufficiently high capillary forces for the sample materials usually used, particularly in the biochemical field.
• Porous material made of porous cellulose acetate or porous polyethylene or porous glass or agarose gel or other wide-meshed gel matrices has proven to be particularly suitable.
• the sample acceptance of the sample material by the sample processing device under the influence of the named electric field requires an electrical charge of the substances to be moved. This charge can be obtained in the desired manner in the case of biological macromolecules by adjusting the pH of the liquid phase in a corresponding manner.
• a sample vessel unit be used with a large number of sample receptacles in a geometric arrangement corresponding to the geometric arrangement of the material sections of the sample supply part. It is therefore sufficient if the comb-like sample feed part with all comb teeth is immersed simultaneously in the large number of sample receptacles.
• the described method can be used for a large number of sample processing devices, in particular for the detection and / or for the production of a biochemical reaction product.
• Another application example is a mass spectrometer.
• the use for supplying sample liquid for an electrophoresis device or a chromotography device is particularly preferred.
• the separating agent, preferably separating gel, of the electrophoresis device can not only be oriented vertically as before, but also horizontally or in any spatial orientation.
• a liquid phase preferably a buffer solution
• the liquid phase serves to dissolve the sample material reversibly adsorbed in the pores within the pores.
• the capillary forces that then occur keep the sample material in the pores as long as no external forces attack as long as the electrical field forces of the electrophoresis device.
• the sample liquid addition points in the separating agent form sample liquid recesses for receiving corresponding porous material sections of the sample feed part.
• the sample liquid recesses can be made independently of the sample supply part and before the sample supply part is supplied or, alternatively, by inserting the comb-shaped sample supply part already provided with sample material into the separating gel material prior to its polymerization.
• the method can also be used to supply sample liquid to a microchip arrangement, in which case the sample supply part or several sample supply parts in the form of separate material sections can also be stationary in order to cover inaccessible sensor areas with dimensions of, for example, 2 mm ⁇ 2 mm to be permanently connected with more accessible addition points.
• the invention also relates to a sample supply part for carrying out the described method with at least one material section made of a correspondingly porous material.
• the invention also relates to an electrophoresis device with a sample feed part for carrying out the aforementioned method, wherein, as also mentioned above, in addition to the previously vertical arrangement, a horizontal arrangement or any arrangement of the separating gel is also possible.
• Another aspect of the invention relates to a method for carrying out a reaction between at least a first and at least a second reaction partner, preferably for the detection or / and for the production of a biochemical reaction product in a sample liquid, which is characterized in that at least one first reaction partner, which is reversibly adsorbed on a porous material, is brought into contact with at least one second reaction partner in a liquid phase, the Pore size of the porous material is such that the liquid phase is at least partially held in the porous material due to capillary forces.
• the reaction in the process just described is preferably a biochemical reaction, i. H. a reaction in which biological macromolecules such as proteins, glycoproteins and / or nucleic acids or complexes of such macromolecules, e.g. B. immune complexes between antibodies and antigens, protein-nucleic acid complexes or nucleic acid hybridization complexes are involved as reaction partners or arise as reaction products.
• the method is particularly suitable for a reaction to nucleic acids, e.g. B. an in vitro transcription, nucleic acid sequencing or nucleic acid amplification.
• a first reaction partner is used, which is reversibly adsorbed on a porous material with a suitable pore size, i. H. adsorption on the porous material does not take place via chemical covalent bonds or high-affinity interactions (eg biotin-streptavidin), so that elution of the first reaction partner and / or the reaction products from the porous material is essentially completely successful under suitable conditions .
• a suitable pore size i. H. adsorption on the porous material does not take place via chemical covalent bonds or high-affinity interactions (eg biotin-streptavidin), so that elution of the first reaction partner and / or the reaction products from the porous material is essentially completely successful under suitable conditions .
• first reaction partners can also be adsorbed on the porous material. This can be done, for example, by soaking the material with a liquid containing several reactants at the same time, or by successive or at different points in the porous material. soak the material with several liquids.
• the porous material can be used so that the first reactant is adsorbed onto the porous material either dry or wet. Dry adsorption is preferred in order to improve the shelf life of the adsorbed reaction partner or to reduce the reaction volume.
• the drying of the porous material can be done in the usual way, e.g. B. by freeze drying or vacuum drying.
• the reaction carried out is a detection reaction for determining an analyte in a sample of biological origin, eg. B. a sample that comprises a body fluid, such as serum, blood, plasma, urine, saliva, etc.
• a sample of biological origin eg. B. a sample that comprises a body fluid, such as serum, blood, plasma, urine, saliva, etc.
• body fluid such as serum, blood, plasma, urine, saliva, etc.
• other biological samples z. B. tissue samples can be used.
• the detection reaction for determining an analyte is the first reaction partner which is reversibly adsorbed on the porous material, preferably a substance which is specifically capable of binding with the analyte to be determined or a substance which competes with the analyte for a further binding partner.
• the analyte is, for example, an immunochemically detectable antigen
• the first reaction partner can be an antibody which is capable of binding to the analyte or an analyte analogue which can compete with the analyte for binding to an antibody.
• the first reaction partner can be a nucleic acid which is complementary to the analyte or a corresponding nucleic acid analog (for example a peptidic nucleic acid).
• the reaction it is expedient for the reaction to have a labeling group which indicates the occurrence and / or the strength of the reaction and thus enables a qualitative or quantitative determination of the analyte.
• the labeling group is preferably a non-radioactive labeling group, e.g. B. a fluorescent or luminescent group, an enzyme, a metal particle, an NMR active group or another labeling group familiar to those skilled in the field of biochemical detection methods.
• the reaction is carried out in such a way that the reaction products are kept essentially completely in the pores. It is further preferred that the reaction products are essentially completely removable from the porous material. In this way, a quantitative determination of the reaction is possible.
• the sample liquid containing the reaction product can - as described above - be fed to a sample processing device, preferably a sample analysis device.
• Another advantage in carrying out a reaction according to the invention in the pores of a porous material is that after the desired reaction time has elapsed, no non-aqueous stop or denaturing reagent, e.g. B. formamide, must be added.
• a non-aqueous stop or denaturing reagent e.g. B. formamide
• the addition of such a formamide reagent is necessary in order to achieve sufficient denaturation of the nucleic acids present in the sample.
• this formamide addition can be dispensed with in the process according to the invention.
• Another object of the invention is a reagent for carrying out a reaction, in particular for detecting and / or for producing a biochemical reaction product, which comprises a part with at least one portion of material made of porous material, wherein at least one reactive substance is reversibly adsorbed on the porous material and wherein the pore size of the porous material is such that the reactive substance at least partially in contact with liquid in the porous material due to capillary forces is held.
• This reagent according to the invention can be used in addition to other test components as part of a reagent kit.
• the invention also relates to an electrophoresis device with a porous, preferably comb-shaped sample feed part, comprising a separating agent, preferably separating gel, and an electric field arrangement for applying an electric field to the separating agent.
• the object of the invention is to provide an electrophoresis device of the type mentioned, which provides improved measurement results, in particular with higher resolution.
• the porous sample feed part inserted into the electrophoresis device is arranged at a non-zero distance from the separating means and that the electric field arrangement is designed to generate an electric field in the distance area for transferring sample material from the sample feed part into the Release agent.
• the electric field arrangement is designed to generate an electric field in the distance area for transferring sample material from the sample feed part into the Release agent.
• the said distance must in any case be so large that it precludes direct mechanical contact between the sample feed part and the release agent. In order to avoid crosstalk to neighboring samples, the distance should not be too large.
• a range between 0.2 mm and 2 mm has proven to be particularly favorable.
• a volume area between the sample feed part and the release agent have an electrically insulating and / or higher density than liquid. speed, preferably Ficoll ® solution or dextran solution.
• the precision of the electrophoresis measurement, in particular the selectivity of the bands, is in turn improved by this measure. This may be due to the fact that the increased density of the liquid makes it difficult for the biomolecules to migrate out of the porous sample supply part, so that the biomolecules only penetrate into the liquid when the electric field is applied, ie at a well-defined point in time.
• the use of the electrically insulating liquid may have the effect that, apart from the migration of the biomolecules, no ion migration takes place, which could otherwise impair the homogeneity of the electric field, at least in the region of the bevel, and thus also lead to a deterioration in the resolution.
• the invention also relates to a method for providing the smallest sample volumes of sample material containing biological macromolecules, preferably for the subsequent capillary-force-receiving by a porous, preferably comb-shaped sample feed part.
• the object of the invention is to provide a method which leads in a simple manner to the smallest sample volumes, for example in the range of 0.5 ⁇ l. This process is characterized by the successive steps: A) providing an initial sample comprising sample material and a first volume of a first solution liquid (preferably water),
• Formamide has proven to be a particularly suitable second solvent, also because it has other properties which are advantageous in connection with DNA electrophoresis, namely, it denatures DNA and can also be used as a stop solution. Dextran is also suitable as a second solvent.
• a comparatively small amount of second solution liquid is therefore added - the sample material can be dissolved in both solution liquids.
• the first solution liquid has evaporated, so that only the second solution liquid with the sample material contained therein remains.
• the residual volume (corresponding approximately to the second volume) is therefore independent of the first volume of the first liquid.
• the remaining volume can also be pressed below the second volume if only a partial volume of the intermediate sample is used in step C, for example half.
• the remaining volume is then only half of the second volume. For example, assuming a volume of the initial sample of 4 ⁇ l and adding a stop solution of 2 ⁇ l consisting of 1 ⁇ l formamide and 1 ⁇ l buffer solution, a total of 6 ⁇ l is obtained.
• the resulting formamide-containing sample can then be taken up by direct contact with the porous sample feed part, since it is sucked into the sample feed part due to corresponding capillary forces. It has been found that the sample supply part prepared in this way can be temporarily stored without further measures before it is supplied to the electrophoresis device.
• the sample feed part Before being inserted into the electrophoresis device, the sample feed part is moistened according to a further development of the invention, and preferably with the same liquid that is also used in the space between the sample feed part used and the release agent. This pre-moistening of the sample feed part prevents blistering of the sample feed part used. Such bubbles can interfere with the biomolecule movement.
• FIG. 1 is a top view, partially cut along the section line II in FIG. 2, of a first embodiment of an electrophoresis device according to the invention with a comb-like sample feed part
• FIG. 2 shows a side view of the electrophoresis device according to FIG. 1 cut along the section line II-II in FIG. 1,
• FIG. 3 is a top view, similar to FIG. 1, of a second embodiment with a smooth-contoured gel molded body
• FIG. 4 shows a top view of the electrophoresis device, partly or partially cut along the section line IV-IV in FIG. 5, but with a modified sample supply part,
• FIG. 5 shows a side view of the embodiment according to FIG. 4, cut along the section line V-V in FIG. 4
• FIG. 5A shows a detail VA of FIG. 5,
• FIG. 6 is a top view, sectioned along the line VI-VI, of a third embodiment of the electrophoresis device with a plurality of separation capillaries, with a corresponding sample supply part,
• FIG. 7 is a sectional view of the arrangement according to FIG. 6 cut along the line VII-VII,
• FIG. 8 is a sectional front view of a comb-like sample feed part inserted into the sample receptacles of a sample vessel unit
• FIG. 11 shows a simplified isometric view of a further modified electrophoresis device with a sample feed part.
• the first embodiment shown in FIGS. 1 and 2 of the sample processing device according to the invention in the form of an electrophoresis device is generally designated 10. It comprises a first glass plate 12 and a second glass plate 14 arranged parallel to the latter, which is held at a defined distance from the first glass plate 125 by two spacers arranged laterally, hereinafter also called spacers 16.
• the resulting cavity between the two glass plates 12 and 14 and the spacers 16 is filled with a layer 18 of polymerized gel.
• Different gels can be used, e.g. B. polyacrylamide gels, agarose gels or hydroxyethyl cellulose gels.
• the gel layer 18 On one o of the two end faces, the gel layer 18 has a comb-like structure, which is composed of a multiplicity of depressions, so-called sample receiving points 20, and gel walls 22 lying between the depressions.
• This structure is known to be produced by (DE-C2- 3 024 288) that a comb-like molded body made of gel-repellent material between the two glass plates 12 and 14 of the gel layer 18 and between the two spacers 16 at the appropriate place in the not yet polymerized gel is pressed in 0 and remains there until the gel has polymerized.
• a comb-like structure is understood here to mean that the contour of the face of the gel in the top view according to FIG. 15 forms a linear succession of rectangular teeth.
• Protruding sections 24 of a comb-like sample feed part 26 are inserted into the sample receiving points 20 of the gel layer 18.
• the first glass plate 12 projects significantly beyond the second glass plate 14. This facilitates the insertion of the protruding sections 24 of the sample supply part 26 into the sample receiving points 24 provided for this purpose in the gel.
• the sample supply part 26 has a thickness D which is smaller than that of the gel layer 18 (in the range between 0.1 mm and 0.3 mm).
• the length L of the sections ("teeth") 24 is approximately 3 mm to 8 mm and their width B is approximately 1 mm (essentially corresponding to the depth and width of the sampling points 20).
• the clear distance A between successive sections 29 is in generally 1 mm to 3 mm corresponding to the thickness of the gel walls 22.
• the sample receiving points 20 can also be produced in the form of the multiplicity of depressions with gel walls 22 lying between the depressions in that instead of the comb-like molded body made of gel-repellent material, the sample supply part 26 itself is inserted between the glass plates 12 and 14 during gel manufacture.
• the sample feed part 26 which has already been impregnated with sample material or a sample feed part which has been dried after the impregnation with the various samples.
• a sample feed part free of sample material can also be inserted into the gel material, sample material then being finished by polymerization before the actual measurement is brought into the area of the individual material sections 24, for example, by feeding by means of a pipette.
• the sample material is transported into the area of the gel solely by the capillary forces.
• FIG. 3 shows an electrophoresis device which has essentially the same structure as that according to FIG. 1.
• the end face of the gel layer 28 has a linear contour instead of a comb-like structure.
• This linear contour is produced in that a suitable shaped body 30 with flat or also curved end faces extending along a straight line surfaces 32 between the two glass plates 12 and 14 is pressed into the not yet polymerized gel and remains there until the gel is polymerized.
• the comb-like sample feed part 26 5 can then be fed, the shape of which can correspond to FIGS. 1 and 2.
• the clear distance A between successive sections 24 can be reduced, since it is no longer necessary to take into account the sufficient stability of the corresponding gel walls 22, as in the exemplary embodiment according to FIGS. 1 and 2.
• a larger number of tracks 19 can thus be realized, so that a larger number of samples can be measured simultaneously. Nevertheless, after inserting the sample supply part 26, mixing of sample liquid of adjacent comb sections
• the addition of electrolyte liquid in the area of the introduced sample supply part 26 can wait until immediately before the build-up of the electric field, so that a previous lateral migration of sample liquid
• FIGS. 4 and 5 Another embodiment, designated 26 ', is shown in FIGS. 4 and 5.
• a large number of separate sample feed material sections 34 are covered by a material
• This material section carrier 36 is composed of two plates 38 and 40 arranged parallel to one another, which are connected by two connecting elements 52, e.g. B. cap screws, are pressed together.
• the material sections 34 are between these two plates 38 and 40
• FIGS. 4 and 5 corresponds to the embodiment according to FIG. 3 except for a bevel of the second glass plate 14 'in the region between the gel face 43 and the glass plate face 45 on the inside of the glass plate facing the first glass plate 12.
• the bevel angle a is shown in FIG. 5; the lower transverse edge 47 of the beveled surface in FIGS. 4 and 5 is indicated in FIGS. 4 and 5.
• This beveling of the second glass plate 14 ' is independent of the design of the sample feed part 26 or 26' and can therefore also be used, for example, in the arrangement according to FIGS. 1 and 2 or FIG. 3.
• FIG. 5A clarifies that the beveled area designated there with 49 facilitates the introduction of the sample feed part 26, which is shown here as a sheet element according to FIG. 3 for the sake of simplicity.
• the bevel angle a need only be chosen accordingly large.
• FIG. 5A further clarifies, in a particularly preferred embodiment of the invention there is a clear distance a between the preferably linearly running edge 51 of the separating gel and the outer ends 53 of the projecting comb sections 24.
• the distance range, as well as that which adjoins upwards, almost up to the top end of the plate 14 thus reaching and adjacent to the Abschrägungsflache 49 Volumenbe- is rich filled by a liquid which contains 1% Ficoll ® and is accordingly denoted in Figure 5A.
• this liquid is electrically insulating (di-electrical tric liquid) and on the other hand it also has a density exceeding the density of water.
• Ficoll ® is understood to mean hydrophilic copolymers made from sucrose and epichlorohydrin and having a molecular weight between 70,000 and 400,000.
• the Ficoll ® solution ensures a homogeneous electric field despite the wedge shape of the space above the separating gel due to the bevel surface 49.
• FIG. 5A this is also shown as a possible variant in FIG. 4; where the upper dash-dotted edge of the separating gel is also designated 51. It can be seen that the edge 51 (in contrast to the embodiment according to FIGS. 1 and 2) runs linearly, which simplifies production and allows a larger number of samples (ie tooth-shaped tooth sections 24).
• sample feed part 26, 26 is also conceivable, such as the use of a material section carrier in the form of an underlay film, e.g. B. in comb form, which in turn carries a corresponding number of individual material sections 34 or a comb-like sheet made of porous material.
• a material section carrier in the form of an underlay film, e.g. B. in comb form, which in turn carries a corresponding number of individual material sections 34 or a comb-like sheet made of porous material.
• FIGS. 6 and 7 show a further embodiment of the invention, in which the electrophoresis device 50 has no has a flat gel, but a multiplicity of gel-filled separating capillaries 54 within a base body 52.
• the protruding sections 62 of a comb-like sample supply part 56 are introduced into the openings of the separating capillaries 54, which, as shown, is either formed as a single sheet of porous material or, according to FIGS. 4, 5, is formed by a material section carrier with a multiplicity of separate porous material sections.
• the comb-like sample supply part 56 made of porous material also facilitates the sample supply to a large number of closely adjacent sample acceptance points (here openings of the separating capillaries 54), even if the risk of mixing sample liquid from adjacent sample acceptance points ("crosstalk") is low from the outset.
• the insertion of the free ends of the sections 62 into the openings of the separating capillaries 54 can be facilitated by tapering or rounding off the sections 62 or (in a manner not shown analogously to FIG. 5) by correspondingly tapering the separating capillary openings.
• all sections 62 deliver their sample liquid to the respective sample receiving point practically simultaneously, so that the risk that the sample liquid diffuses prematurely into the gel in some samples, as is the case with sequential sample supply, for example using a pipette, which is the case here does not exist.
• the capillary forces ensure that sample liquid only escapes from the porous material if a sufficiently large electric field is applied.
• the start of diffusion can also be postponed accordingly by delaying the addition of electrolyte.
• the comb-like sample supply part described above in its various embodiments made of continuously porous material or porous material sections also enables simple and rapid supply of sample liquid to the sample supply part.
• the geometrical arrangement of these corresponds to the geometrical arrangement of the protruding sections (teeth) 62 of the sample feed part 60. If, as shown, the teeth are arranged linearly one after the other, the sample receptacles 64 are accordingly linear Row arranged.
• all sections 62 of the sample supply part can be simultaneously soaked with sample liquid by appropriately immersing them in the receptacles 64.
• the sample supply part 60 is then fed to the respective electrophoresis device.
• the loading of the sample vessel unit 66 can be carried out in advance, e.g. B. by means of a corresponding manual or automatic multiple pipetting device, that is to say independently of the actual sample loading of the electrophoresis device by means of sample supply part 60.
• sample liquid can be fed to the respective electrophoresis device in a simple manner, the distance between adjacent porous sections and thus also the distance between the individual samples from one another, which can be kept small Measuring effectiveness of the electrophoresis device significantly increased. Simultaneous start of the migration of the electrically charged components of the sample liquid at the start of the electrophoresis is ensured, so that the precision of the electrophoresis measurements, in particular with regard to the comparison of neighboring tracks, is high.
• porous material for taking up sample liquid also gives the possibility of treating the sample liquid within the porous material.
• the porous material thus serves as a sample vessel or reaction vessel.
• the porous material can be added one after the other or at different points in the porous material with the liquids to be reacted soak, in the latter case the liquids in the porous material are mixed as a result of the capillary forces. It is also possible to impregnate and dry the porous material beforehand with at least one of the reaction liquids, so that the porous material is "impregnated” accordingly. At any later point in time, the porous material can then be added to the further reagent liquid, e.g. B. the sample liquid and / or other reagent solutions, are immersed to allow the desired reaction to take place.
• the further reagent liquid e.g. B. the sample liquid and / or other reagent solutions
• the reaction can be any biochemical reaction, e.g. B. an enzymatic reaction, a nucleic acid hybridization reaction, an immunochemical reaction or the like.
• Reactions on nucleic acids are preferably carried out, such as in vitro transcription, an RNA strand being generated by an RNA polymerase on a nucleic acid matrix, sequencing, preferably by enzymatic reactions, eg. B. with T7 DNA polymerase, the nucleotide sequence of a nucleic acid is determined, or an amplification, the amount of nucleic acid present in the sample liquid can be increased by enzymatic reactions.
• a preferred example of an amplification is the PCR (polymerase chain reaction).
• the PCR is preferably carried out at elevated temperatures, the enzymatic reaction at 65 ° C. to 80 ° C., particularly preferably at 72 ° C. to 75 ° C. and the cleavage reaction at 80 ° C. to 100 ° C., particularly preferably at 90 ° C. expires up to 95 ° C.
• thermocycling with thermostable enzymes.
• the sensitivity of the sample analysis can be increased considerably by PCR or another amplification reaction.
• the porous material withstands the drastic conditions during thermocycling, without the decomposition of the porous material which significantly impairs the subsequent analysis or isolation of the reaction product.
• Fig. 9a shows a comb-like sample supply part 70 in the form of a sheet of porous material.
• the protruding portions 72 of the sample supply part are with the aid of a dosing device 74 with suitable reagents, such as. B. dyes, enzymes, nucleic acids or the like soaked.
• suitable reagents such as. B. dyes, enzymes, nucleic acids or the like soaked.
• These reagents are used to facilitate sample liquid analysis, e.g. B. by coloring, increasing the resolution or the like, or they are added to trigger certain chemical or biochemical reactions before analysis or to retain certain sample proportions.
• This step can also be carried out several times in succession with different reagents, in order in this way to prepare the porous material sections 72 of the sample supply part 60 for a specific sample analysis.
• FIG. 9 b shows how the recesses 76 of a sample vessel unit 78 are filled with sample liquid separately via a pipette 80. This step can also be carried out automatically with the aid of an appropriate multiple pipetting device.
• sample supply part 70 made of porous material, with its protruding sections 72, is inserted into the recesses 76 of the sample vessel unit 78.
• sample liquid sample addition
• the protruding sections 72 of the sample feed part 70 are subsequently removed from the recesses 76, the sample liquid remains adhering to the pores of the respective material sections 72 due to the capillary forces.
• sample liquid can be transferred from the sample vessel unit 78 to the sample processing device 82, e.g. B. electrophoresis or chromatography device, as shown in Fig. 9d.
• FIG. 9B symbolizes a special way of producing particularly small sample volumes, for example in the range of 0.5 ⁇ l.
• a starting sample which comprises the biological material to be examined, in particular DNA fragments, and a first volume of a solution liquid S1, preferably water.
• the volume can be 4 ⁇ l, for example.
• 0.2 ⁇ l of a stop solution is added, which in turn consists of 1 ⁇ l of a second solution liquid (formamide (formic acid amide)) and 1 ⁇ l of a buffer solution.
• the resulting intermediate sample of 6 ⁇ l is taken up by the automatic pipetting device and half of it (3 ⁇ l) is filled into the respective bowl-shaped recess 76 of the sample vessel unit 78.
• the first solution liquid (water) is then evaporated, preferably supported by a blower 81 indicated by a broken line in FIG. 9B. soft of the first solution liquid S1 is also indicated there with corresponding arrows.
• the second solution liquid S2 (formamide) remains, which then also contains the DNA fragments.
• the sample volume is only 0.5 ⁇ l, depending on the formamide content.
• sample volumes can then be picked up by immersing the teeth of the sample feed part 70, the pretreatment of the sample feed part 70 according to FIG. 9A possibly. can also be omitted.
• the use of the second solution liquid S2 with a comparatively low diffusion rate also makes it possible to temporarily store the correspondingly impregnated sample feed part 70 (FIG. 9e) before it is fed to the electrophoresis or chromatography device according to FIG. 9d.
• sample supply part 70 is temporarily stored or not, it is advantageous if it is moistened immediately before being inserted into the electrophoresis or chromatography device, as indicated in FIG. 9f.
• the moistening prevents air bubbles from forming after inserting the sample supply part 70 into the electrophoresis or chromatography device, which can impair the measurement.
• the same liquid can be used for moistening as is also used in the spacing area (FIG. 5A). This is preferably a one-percent Ficoll ® solution.
• the sample processing device 82 can have a different structure. Thus, all of the previously described embodiments can be used.
• the sample liquid is prepared in such a way that the biomolecules contained therein, e.g. B. proteins are electrically charged.
• the capillary forces that hold the sample liquid in the sample supply part 70 are overcome and thus cause the charged molecules to move in accordance with the electrical forces acting on them.
• the sample liquid can either enter the gel layer through direct contact between the sample supply part 70 and the gel layer of the sample processing device 82 or via a suitable electrolyte solution, in which case there is a distance of up to a few millimeters between the sample supply part and the gel layer can.
• sample liquid is sufficient to impregnate the projecting sections 72 of the sample feed part 70.
• sample liquid is of course to be avoided in order to prevent sample liquid from passing from one section 72 to the next section 72 via the porous material itself.
• the porous material is also suitable for the forwarding of sample liquid to sample reception points that are otherwise difficult to access.
• sample liquid supply to a microchip arrangement, in particular a DNA sensor arrangement, as is greatly simplified, is mentioned as an example in FIG. 10.
• sample liquid can be supplied directly only with great difficulty.
• sample feed parts 94 made of porous material, however, sample feed can be carried out very easily.
• any other geometric arrangement can also be selected, such as, for example, B. parallel next to each other, preferably with varying length of feed part from sample feed part. In this way, the distance between the addition ends of the sample feed parts can be increased. With these arrangements, too, the forwarding of the sample material from the sample feed part to the respective sensor area can be forced by a corresponding electric field.
• a further electrophoresis device 100 is shown in simplified form in FIG. 11, consisting of a lower glass plate 102, an upper glass plate 104 and an intermediate one Gel layer 106, wherein the gel layer 106 here extends to the lower left end of the upper glass plate 104 in FIG. 11.
• the electrophoresis device 100 like the electrophoresis devices described above, can also be operated in any other spatial orientation, since here, as in known electrophoresis devices with sample pockets on the edge of the gel, there is no danger that the sample material will flow out.
• the porous material holds the sample material due to capillary forces; it is only the electric field that causes the sample material to migrate into the gel.
• a support strip 112 can be used, as shown, with a gap between it which essentially corresponds to the thickness of the sample feed part 108 and the end face 110.
• the ends of the material sections ("teeth") 114 of the sample supply part 108 loaded with the sample material are either held directly on the corresponding end face 116 of the gel layer 106 or at a short distance therefrom.
• the electric field is built up in the usual way by forming a cathode or anode in the region of the two end regions of the gel layer or on the distal edge of the sample feed part.

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• 1997
  • 1997-01-10 DE DE19700626A patent/DE19700626A1/de not_active Withdrawn
• 1998
  • 1998-01-12 AU AU66139/98A patent/AU6613998A/en not_active Abandoned
  • 1998-01-12 JP JP53056198A patent/JP2001508174A/ja active Pending
  • 1998-01-12 CA CA002278310A patent/CA2278310A1/en not_active Abandoned
  • 1998-01-12 EP EP98907938A patent/EP0951647A2/de not_active Withdrawn
  • 1998-01-12 WO PCT/EP1998/000130 patent/WO1998030911A2/de not_active Application Discontinuation
• 1999
  • 1999-07-09 US US09/350,090 patent/US6428668B1/en not_active Expired - Fee Related

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