EP0958300A1 - Preparation electrocinetique d'echantillons - Google Patents

Preparation electrocinetique d'echantillons

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Publication number
EP0958300A1
EP0958300A1 EP97954757A EP97954757A EP0958300A1 EP 0958300 A1 EP0958300 A1 EP 0958300A1 EP 97954757 A EP97954757 A EP 97954757A EP 97954757 A EP97954757 A EP 97954757A EP 0958300 A1 EP0958300 A1 EP 0958300A1
Authority
EP
European Patent Office
Prior art keywords
membrane
nucleic acid
microchannel
analysis
macromolecules
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP97954757A
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German (de)
English (en)
Inventor
Hansjörg Dürr
Ulf Brüggemeier
Karsten Dierksen
Hans-Robert Hehnen
Rainer Neumann
Eberhard Kuckert
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Bayer AG
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Bayer AG
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Publication date
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Publication of EP0958300A1 publication Critical patent/EP0958300A1/fr
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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

Definitions

  • Biomacromolecules such as Proteins and nucleic acids, but also small particles such as viruses and bacteria, are of great importance both diagnostically and for medical research.
  • the established methods for characterizing these substances from biological matrices are partly. very complex.
  • the target nucleic acid is isolated for nucleic acid analyzes, then amplified and evaluated using a suitable analysis method. Isolation is time consuming and difficult to automate.
  • the nucleic acid In order to obtain sufficient amounts of nucleic acid for the established analytical methods, the nucleic acid must be amplified in a further step. So far, only the polymerase chain reaction (PCR) has found wider application.
  • Isolation from a pure bacterial culture is a simple case for obtaining nucleic acid: in the case of E. coli, alkaline lysis releases the nucleic acid; After centrifugation and neutralization, this crude product can be used directly for PCR batches (Rolfs, A. et al. PCR: Clinical Diagnosis and Research,
  • sample material for medical diagnostics is more heterogeneous; Blood, urine, cerebrospinal fluid, swab material, sputum, tissue samples, faeces, e.g. call for specific digestion methods, which in turn depend on the question
  • Silicon microparticles which can also be embedded in membranes, can also be used for nucleic acid purification (WO 95/34569).
  • Ion exchange membranes US 832284
  • chemically modified silica phases EP
  • an isolation step usually follows the isolation of the nucleic acids (EP 229701).
  • Viruses are usually isolated and concentrated using the following methods: ultracentrifuge, electroextraction, size exclusion separation, affinity chromatography and precipitation (Polson, Alfred, Prep. Biochem., 1993; 23, Dekker, New York,
  • Bacteria are usually separated and raised by spreading on nutrient media.
  • immunological methods eg by fluorescent labeling -
  • nucleic acid determination methods after cell lysis - are available.
  • Capillary electrophoretic analysis methods have been used for several years to analyze biological macromolecules such as proteins (WO 93/22665), nucleic acids (Heller, C, J. Chromatogr. A, 1995, 698, 19-31) and more recently also viruses (DE 4438833) Detection is carried out either directly with UV or by means of fluorescence detection after marking the macromolecules (Pentoney, SL, Jr, et al Handbook of Capdlary Electrophoresis, S 147, Landers, JP (Ed) Boca Raton, CRC
  • a fundamental disadvantage of the CE is the low injection volume, which is only a few nanoliters. There are numerous attempts to compensate for this disadvantage (St. Claire R.L., Anal. Chem. 1996, 68, 569R-586R). This includes isotachophoretic concentration and stacking, both of which focus the sample components in the injection volume.
  • a patent was filed by Guzmann in 1993 for a specific solid phase adsorption in the capillary for sample concentration (WO 93/05390). Specific compounds in a sample are held or allowed to pass through specific molecular interactions. A further development of these methods was developed by Tomlinson et al. made with the membrane preconcentration capillary electrophoresis (Tomlinson, A.J., et al. J. High
  • Capillary array electrophoresis was developed by various working groups, mainly for nucleic acid sequencing, and a patent application was also filed in part (WO 96/04547).
  • fluorescent molecules were controlled and analyzed depending on the voltage.
  • Micromachines were also patented on the basis of chip technology, which enables process control of solutions for synthetic or analytical purposes through a network of channels and electrical switches (WO 96/15450).
  • Amplification-free nucleic acid analyzes
  • Fluorescence correlation spectroscopy was also used for biological screening methods using single molecule detection (EP 731173). High throughput nucleic acid sequencing is also processed based on this technology (Harding, J.D., et al. Trends in Biotechnology, 1992, 10, 55-57). These methods are based on the detection of a single fluorescent molecule in a very small volume element.
  • an automated method should be developed that allows macromolecules (nucleic acids, proteins, viruses and bacteria) from biological materials, such as e.g. Blood, blood plasma, serum, cerebrospinal fluid, urine, tissue samples, plants, cells, cell supernatants etc. and preparations from them to be isolated, concentrated and made available for analysis.
  • biological materials such as e.g. Blood, blood plasma, serum, cerebrospinal fluid, urine, tissue samples, plants, cells, cell supernatants etc. and preparations from them to be isolated, concentrated and made available for analysis.
  • the central component of the sample preparation module is a thermostattable microchannel with an inserted membrane.
  • This channel filled with a conductive liquid, is in contact with replaceable or fixed on both sides Installed vessels
  • a potential difference between the sample vessels charged molecules can be mobilized electrophoretically.
  • the possibility of applying a pressure difference can also generate a laminar flow in the microchannel.
  • a membrane (2) is introduced into a microchannel (1) and is suitable for retaining the desired macromolecule.
  • a pressure difference (6) and / or a voltage (5) to the ends of the microchannel (3,4), part of the sample becomes injected into the channel (Fig. 1) The injection is continued until there is a sufficient amount of the desired macromolecule in the microchannel.
  • the injection can be carried out be matched to the desired target molecule so that either only the desired macromolecule gets into the channel or is held by the membrane
  • the microchannel has an inner diameter of 10 - 100 ⁇ m and a total length of 3 - 50 cm.
  • the microchannel is made of an electrically non-conductive material such as polymer, ceramic, glass or quartz. In principle, all are synthetic
  • the polymer must be inside the buffer solutions used and is ideally transparent for optical detection methods (e.g. polycarbonate, polyester acrylate, polymethacrylate, polyurethane, polyacrylamide) but also PTFE is suitable.
  • the channel can be coated on the inside with a polymer in order to obtain favorable surface properties (e.g. polyacrylamide, silanol or polyvinyl alcohol)
  • membranes can be used that work on the principle of large size exclusion (ultrafiltration membrane).
  • the large size exclusion range must be adapted to the molecular size of the macromolecule. Range, for large nucleic acids and viruses, down to 0.45 mm, for bacteria and cells the membranes are microstructured polymers, preferably polyether sulfone (PES), polyester, fleece-based acrylic polymer, polytetrafluoroethylene (PTFE), polysulfone, polypropylene (PP), glass fiber, nylon or polycarbonate.
  • PES polyether sulfone
  • PTFE polytetrafluoroethylene
  • PP polypropylene
  • glass fiber nylon or polycarbonate
  • ion exchange membranes and adsorption phases can be used. The choice of these membranes depends on the type of
  • the injected macromolecule is concentrated in front of or in the membrane (2) by applying a pressure difference (6) and / or a voltage (5) (Fig. 2).
  • the desired macromolecule is then in a volume of a few nanoliters.
  • Pressure difference (6) and / or a voltage (5) are brought into the microchannel (1) (Fig. 3).
  • the conditions are chosen so that the target molecule remains concentrated. In this way, the target molecule can be modified enzymatically or chemically and / or specifically recognized by hybridization or immunological recognition.
  • the ability of the microchannel (1) to be thermostatted and the ability to change the reagent tubes several times allows complex implementations and cyclical processes. In this modification step, any derivatization reactions that may be necessary for fluorescence, chemiluminescence or laser-induced fluorescence detection are carried out.
  • the required reaction temperatures are achieved by thermostatting the microchannel (1).
  • either appropriately tempered air or liquid is directed past the microchannel.
  • the wall thickness of the microchannel is chosen so that sufficient heat dissipation is ensured.
  • the target molecule After optimally changing the reagent vessels (8,9) against the buffer vessels (10,1 1), the target molecule is mobilized by applying a pressure difference (6) and / or a voltage (5) (Fig. 4).
  • optical detection methods (12) such as Absorption or fluorescence, the molecule can be determined analytically directly in the microchannel (1) (13)
  • the analog detection methods are available as in the CE (St.Claire RL, Anal. Chem. 1996, 68, 569R-586R)
  • the microchannel is either made complete or transparent at one point for optical radiation.
  • the transmission of the excitation radiation and the fluorescent radiation must be ensured. These are preferably the non-conductive materials explained at the beginning.
  • the fluorescent radiation is perpendicular or at a defined angle of 0-180 ° measured in reflection to the irradiation wavelength.
  • the irradiation is preferably at 45 ° or 90 °.
  • the highly concentrated analysis target is, however, also available for further analysis (FIG. 5).
  • the target molecule can thus be fractionated into the analysis vessel (14) or into or onto any other analysis target
  • the analytical vessel (14) contains 1-1000 ml of a buffer suitable for further analysis.
  • a buffer suitable for further analysis is PBS buffer, Tris / borate or a tris-glycine buffer.
  • the analysis vessel (14) can also be a planar analysis target, for example a mass spectrometric sample holder. The electrical contact is either made directly via the conductive one
  • Analysis target reached or by wetting the surface between the electrode and microchannel with an electrically conductive liquid.
  • the concentrated target molecule can be brought into further channels for further analysis or conversion by switching over pressure or voltage and is therefore directly compatible with the CE chip technology (WO 96/04547)
  • the highly concentrated macromolecule is eluted in less than a microliter and can be applied directly to suitable liquid matrices or to solid sample carriers
  • a schematic representation of the parallel structure is shown in FIG. 6.
  • the microchannels (1) are produced from non-conductive materials, such as polymer, ceramic, glass, quartz or ceramic (15), and are coated if necessary.
  • the channel blocks are joined with an intermediate membrane layer (2) , so that the channels meet on the membrane The arrangement of the channels depends on the
  • thermostatic elements may be introduced to dissipate the Joule heat.
  • the channels are tapered at the ends so that they can be inserted into the respective vessels or are tightly connected to permanently installed vessels.
  • electrodes and supplied with a high voltage source are used for electrical contact, either on attached to the channel ends or in the vessels.
  • channels are placed in the analysis block that are perpendicular to the direction and between the levels of the microchannels. These channels allow air or liquid to be pumped at a suitable temperature
  • the height of the channel corresponds to the dimension of the microchannel (10-100 ⁇ m), so that the Joule heat can still be dissipated well.
  • the width of the channel (100 ⁇ m to 10 mm) allows rivers up to 10 3 higher than in the microchannel (1)
  • the membrane is clamped between the module blocks (15).
  • the entire module is 3 to 10 cm long, 1 to 50 mm wide and 0.1 to 50 mm thick
  • the channel ends are in turn connected either to exchangeable or permanently installed sample vessels.
  • a parallel arrangement can also be used to achieve an analog structure as in FIG. 6.
  • the macromolecules are concentrated in the channel (16) and then transferred via the transfer channel (17) to the microchannel and analogously the method from FIGS. 1-6 further processed.
  • the schematic enrichment of macromolecules with this rapid concentration is described below using the example of nucleic acids Nucleic acid from saline solution is enriched by applying a voltage to the flat channel (16) (Fig. 8a). In addition to the excess of small anions (small black balls), the nucleic acid is also injected from the sample. The anionic molecules migrate through the membrane (2 ) and are thus removed from the nucleic acid.
  • modification reactions can also be carried out simultaneously on the concentrated macromolecule
  • Nucleic acid is released from the sample to be examined using a suitable, established method (lysis, hydrolysis, ultrasound, etc.) and mixed with a suitable acidic extraction buffer.
  • the buffer must be designed in such a way that non-nucleotide components of the solution do not carry an anionic excess charge below one pH
  • a value of approx. 5 shows that proteins no longer have a negative excess charge.
  • Inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid as well as organic phosphoric acid derivatives or sulfonic acids can be used.
  • a polymer-bound, acidic ion exchanger eg polystyrene sulfonic acid
  • the acidic pH value is reached without additional anions being introduced into the solution. The electrokinetic nucleic acid extraction is thereby promoted
  • the nucleic acid is extracted electrophoretically from this acid buffered solution.
  • an electrode is introduced into the solution and brought to cationic potential.
  • the electrode in the buffer vessel (4) (FIG. 1) is brought to anodic potential and the Microchannel (1) is filled with an electrically conductive liquid.
  • the composition of the electrolyte depends on the type of immobilization technique. It is preferably an aqueous buffer based on borate, phosphate or citrate. Glycine is also a suitable buffer ion.
  • the buffer concentration is between 10 and 100 mmol / 1.
  • the pH is between 2.5 and 8.5.
  • a modifier, preferably a chaotropic agent, such as urea is optionally added in molar concentration.
  • the task can be carried out using UV or fluorescence detection - according to the previous one
  • Derivatization - can be followed in the microchannel (1).
  • the extracted nucleic acid is immobilized and concentrated in the channel with the aid of the membrane introduced while maintaining the tension (FIG. 2).
  • soft anion exchangers for example based on amines, are also suitable for nucleic acids. They are preferably alkylamine, imidazole or pyrollidone-substituted polymers.
  • the nucleic acids can also be retarded by adsorption on membranes.
  • the membranes contain nanoparticles, preferably silica-based or metal oxide pigments.
  • the nucleic acid concentrated to a few nanoliters can be modified and analyzed in many ways (FIG. 3).
  • the open channel system allows reagents to be added and removed using pressure or tension. The polarity of the voltage is maintained as in the extraction and focusing. Combined with a suitable temperature control, enzymatic cleavages, Sanger sequencing, gene probe hybridization, but also PCR reactions are possible in the microsystem.
  • the nucleic acid can be labeled with intercalating dyes.
  • fluorescent derivatives such as ethidium bromide, acridine orange, or their dimers, such as 1,1 '- (4,4,7,7-tetramethyl-4,7diazaundecamethylene) -bis-4- [3-methyl-2 , 3-dihydromethyl- (benzo-l, 3-oxazole) -2methylidene] quinolinium
  • Tetraiodide (YOYO)
  • the choice of the dye depends primarily on the selected detection unit YOYO is ideal, for example, for fluorescence detection after excitation with an argon laser, while the corresponding YOPRO dimer is ideally suited to the infrared laser. These dyes are also characterized by this from that hardly
  • the positively charged YOYO can be introduced electrokinetically, for example, from the reaction vessel (9) with the connection of the focusing voltage
  • the gene probe consists of a nucleotide sequence that is complementary to the target nucleic acid and carries one or more fluorescent dyes.
  • the choice of the dye is primarily based on the selected detection unit used after excitation with an argon laser
  • Enzyme-catalyzed reactions such as restriction enzyme digestion, PCR
  • Reaction and Sanger sequencing are carried out by transporting the necessary enzymes and required substrates to the nucleic acid in the microchannel
  • the concentrated nucleic acid can be in a suitable buffer
  • the voltage is reversed so that the anode is now in the analysis vessel (14) (FIG. 5).
  • the microchannel and the buffer vessel (11) are preferably filled beforehand with an aqueous buffer of the above-mentioned composition.
  • PCR reactions, CE separations, DNA sequencing, hybridization reactions, mass spectrometric analyzes or molecular biological methods can be carried out.
  • the nucleic acid can be analyzed electrophoretically within the microsystem (FIG. 4). As with the elution, the buffer vessel (10) is brought to anodic potential. In a suitable sieving medium, nucleic acid fragments can be separated depending on their size.
  • the buffer vessels (10, 11) and the microchannel (1) are filled with a polymer-containing buffer solution.
  • a polymer-containing buffer solution are preferably linear soluble polymers, for example acrylamide, polyvinyl alcohol, cellulose (modified and unmodified), dextran or agarose.
  • the other buffer composition corresponds to the general composition.
  • the virus-containing sample is brought to a pH such that the virus to be examined, or the viruses to be examined, carries a negative excess charge. If necessary, nuclei digestion or virus modifications can be carried out beforehand.
  • the suitable buffers have already been described in the general procedure. The pH of the buffer must be well above the pK of the virus. Acid buffers such as sodium citrate are therefore not used, and modification reagents are also not used. Nuclease digestion is preferably carried out by adding RNAses or DNAses
  • the benzonase modification reactions for example, are extremely suitable and can be carried out in the form of staining reactions with intercalating dyes (cf. 14) or by incubation with fluorescence-labeled antibodies. In both cases, the choice of dye depends on the type of detection chosen
  • the negatively charged viruses are electrophoretically extracted from this buffered solution.
  • An electrode is placed in the solution and brought to cationic potential
  • the buffer vessel (4) (Fig. 1) is brought to anodic potential and the microchannel (4) is filled with an electrically conductive liquid.
  • the composition of the electrolyte corresponds to the general buffer composition.
  • the task can be monitored by means of UV or fluorescence a
  • the extracted viruses are introduced into the channel using the
  • Membrane immobilized and concentrated (Fig. 2) The membrane works according to the principle of large exclusion, the pore size being between 10 and 200 nm depending on the virus
  • the viruses concentrated on a few nanoliters can be modified and analyzed in a variety of ways (FIG. 3).
  • the open channel system allows the supply and removal of reagents by means of pressure or
  • the size exclusion membrane In the case of virus lysis, the size exclusion membrane must be dimensioned so that the target proteins or nucleic acids are also retarded. In principle, any lysis protocol is suitable. Denaturing conditions such as extreme pH values, chaotropic reagents or detergents are preferably used. Examples are dilute sodium hydroxide solution, guanidinium hydrochloride or sodium dodecyl sulfate (SDS). Non-ionic detergents (eg NP-40) can be used to remove the lipoprotein membrane from enveloped viruses
  • the concentrated viruses can be eluted in a suitable buffer by applying pressure and / or voltage in a few nanoliters and are available for further analyzes
  • the microchannel (1) and the buffer vessel (11) are preferably filled beforehand with an aqueous buffer of the abovementioned composition.
  • the viruses can be used, for example, to carry out pathogenicity assays or CE separations. After fractionation on a planar analysis target (14), the viruses can be examined directly using electron microscopy, for example
  • the viruses can be analyzed electrophoretically within the microsystem. As with the elution, the buffer vessel (10) is brought to anodic potential (FIG. 4).
  • viruses can also be identified by fluorescence spectroscopy using fluorescent labeling methods for proteins or nucleic acids (cf. 1 4 and 3 4) 3. Proteins
  • the protein-containing sample is brought to a pH value which is at least one log level next to the pK value of the protein. If the solubility properties of the protein allow, the pH is set below the pK of the protein so that the protein is positively charged. In the following, this case will be discussed. For negatively charged proteins, the voltage relationships are reversed accordingly.
  • Suitable buffers meet the general conditions. Alkali-buffered phosphate and citrate buffers are preferably used, e.g. Sodium citrate, 20 mmol / 1, pH 2.5.
  • the positively charged proteins are extracted electrophoretically from this buffered solution.
  • an electrode is placed in the solution and brought to anionic potential.
  • the electrode in the buffer vessel (4) (Fig. 1) is brought to cathodic potential and the microchannel (4) is filled with the buffer.
  • the composition of the electrolyte depends on the type of immobilization technique and corresponds to the general conditions.
  • a modifier preferably an organic solvent, such as e.g. Methanol between 5 and 30% added.
  • the task can be followed in the microchannel (1) by means of UV or fluorescence detection - after prior derivatization.
  • ion exchange membranes are also suitable for proteins.
  • DEAE phases are preferably used as the soft anion exchanger for negatively charged proteins.
  • Quaternary ammonium phases are mainly found as strong anion exchangers Use.
  • carboxylic acid phases are suitable as soft exchangers and sulfonic acid phases are suitable as strong exchangers.
  • the membranes can be coated with appropriate antibodies and thus enriched for affinity.
  • the proteins concentrated on a few nanoliters can be modified and analyzed in many ways (FIG. 3).
  • the open channel system allows reagents to be added and removed using pressure or tension.
  • the polarity of the voltage becomes the same as for the extraction and
  • the protein can be reacted with reactive dyes. It is preferably amine-specific dyes such as e.g. Fluorescein isothiocyanate (FITC).
  • FITC Fluorescein isothiocyanate
  • the choice of the dye depends primarily on the selected detection unit. For example, FITC is ideal for fluorescence detection after excitation with an argon laser.
  • the concentrated proteins can be eluted in a suitable buffer by applying pressure and / or voltage in a few nanoliters and are available for further analyzes.
  • the voltage is reversed so that the cathode is now in the analysis vessel (14) (Fig. 5).
  • the microchannel and the buffer vessel (11) previously filled with an aqueous buffer of the above composition.
  • CE analyzes, mass spectrometric analyzes, enzyme assays, binding studies or immunological processes can be carried out as subsequent analyzes.
  • the proteins can be analyzed electrophoretically within the microsystem (FIG. 4).
  • Enzyme substrates, fluorescent binding partners or derivatizing reagents, the proteins can also be identified by fluorescence spectroscopy.
  • the sample containing bacteria is brought to a pH such that the bacterium to be examined has a negative excess charge. If necessary, nuclease digestion or protein modifications can be carried out beforehand.
  • the appropriate buffers have already been described in the general procedure.
  • the pH of the buffer must be well above the pK of the bacterium. Acid buffers such as sodium citrate are therefore not used, and modification reagents are also not used.
  • Nuclease digests are preferably carried out by adding RNAses or DNAses. Benzonase, for example, is extremely suitable. Modification reactions can be carried out in the form of staining reactions with intercalating dyes (cf. 1.4) or by incubation with fluorescence-labeled antibodies. In both cases, the choice of dye depends on the type of detection chosen. 4.2.
  • the negatively charged bacteria are extracted electrophoretically from this buffered solution. For this purpose, an electrode is placed in the solution and brought to cationic potential. The electrode in the
  • Buffer vessel (4) (Fig. 1) is brought to anodic potential and the microchannel (4) is filled with an electrically conductive liquid.
  • the composition of the electrolyte depends on the type of immobilization technique.
  • the task can be followed using UV or fluorescence.
  • the extracted bacteria are immobilized and concentrated in the channel with the help of the membrane introduced (FIG. 2).
  • the membrane works according to the size exclusion principle (ultrafiltration membrane) or the ion exchanger principle (anion exchanger).
  • Membranes are preferably used for sterile filtration with an exclusion size of 0.1-1.45 ⁇ m.
  • anion exchangers as for the proteins can also be used (cf. 3.3.)
  • the bacteria concentrated on a few nanoliters can, for example, be lyophilized on the membrane and then the proteins or
  • Nucleic acids are modified and analyzed in many ways (Fig. 3).
  • the open channel system allows reagents to be added and removed using pressure or tension.
  • all derivatization methods for proteins and nucleic acids are possible in the microsystem (cf. 1.4 - 1.7 and 3.4 -
  • the size exclusion membrane In the case of bacterial lysis, the size exclusion membrane must be dimensioned such that the target proteins or nucleic acids are also retarded.
  • any lysis protocol is suitable. Denaturing conditions such as extreme pH values, chaotropic reagents or detergents are preferably used. Examples are dilute sodium hydroxide solution, guanidinium hydrochloride, urea or sodium dodecyl sulfate (SDS). 4.5.
  • SDS sodium dodecyl sulfate
  • the concentrated bacteria can be eluted in a suitable buffer by applying pressure and / or voltage in a few nanoliters and are available for further analyzes.
  • the microchannel (1) and the buffer vessel (11) are preferably filled beforehand with an aqueous buffer of the above-mentioned composition.
  • pathogenicity assays or electrophysiological experiments can be carried out with the bacteria, for example.
  • the bacteria can, for example, be examined directly by light or electron microscopy, or e.g. can be identified microbiologically on an agar plate via plaques.
  • the bacteria can be analyzed electrophoretically within the microsystem (FIG. 4).
  • the bacteria can also be identified by fluorescence spectroscopy using fluorescent-labeled antibodies or fluorescent binding partners.
  • the process is characterized above all by the simplified isolation and extreme enrichment rates. If the sample is eluted electrophoretically or with pressure from the microchannel, further nanoanalysis methods can then be carried out. After dilution, the isolated sample is also available for conventional macroscopic analysis methods. The method then represents a very efficient sample preparation module for these techniques
  • the method can thus replace PCR, for example.
  • the method can also be used as a digestion method for viruses, bacteria and other cells.
  • bacterial material is isolated, then the bacterium is digested in the microchannel and the released nucleic acid is derivatized and analyzed
  • the method can advantageously be used for direct nucleic acid sequencing for diagnostics and research.
  • an analysis for hereditary genetic defects, which are caused by deletions, mutations or translocations is possible here.
  • Possible areas of use are cystic fibrosis, Down 's Syndrome, sickle cell amia, Huntington 's chorea, hamophilia A and B
  • a further application of this nucleic acid analysis is in tumor diagnosis and general recognition for genetic predispositions to certain diseases.
  • the analysis of tumor suppressor genes and oncogenes is of particular interest
  • nucleic acid amplification methods such as PCR
  • the method can also be used for direct gene probe analysis of drug-resistant germs or for subclassification
  • the invention also enables the control of genetically engineered products in which freedom from nucleic acids must be guaranteed.
  • Proteins can be used for infection diagnosis. Nucleic acids or proteins from fungi or parasites can also be analyzed for these purposes.
  • the most important viral representatives in this case are HIV lu. 2, HTLV, HSV, CMV, HPV, hepatitis A, B, C, D, E, F, G, VZV, rotaviruses, EBV and adenoviruses.
  • the most important bacterial representatives include chlamydia, mycobacteria, shigella, campylobacter, salmonella, neisserie, staphylococci, streptococci, pneumococci.
  • the most important pathogens in fungi are Candida, Aspergillus and Cryptococcus.
  • Another area of application is the security monitoring of biological samples.
  • the meaning lies here e.g. when checking blood donations and all products made from blood.
  • the areas of application largely correspond to those of infection diagnostics.
  • this method allows the direct, highly sensitive detection of intact
  • Viruses Any, even unknown viruses can be measured directly. This is of enormous importance both for infection diagnosis and for the safety of products made from biological materials, since only special viruses can be detected individually with all other methods.
  • the proteins obtained by electrokinetic sample preparation are more easily accessible for subsequent immunodiagnostic analysis due to their enrichment and purification.
  • Here have different proteins in human diagnostics such.
  • DNA is injected into a microchannel by applying voltage and measured using UV.
  • the experiment should show that it is possible to extract nucleic acid electrokinetically.
  • the amount of DNA is limited to over 100 mg / 1 by the electric current and not by the DNA concentration. This showed that nucleic acid can be electrokinetically concentrated from a solution over a wide concentration range.
  • Table 1 Measurement parameters for electrokinetic nucleic acid injection.
  • pBr-DNA was electrokinetically injected into the microchannel and then electrophoretically mobilized in the channel to the anode. There was a UV between the injection end of the channel and the size exclusion membrane in the channel.
  • nucleic acid For the concentration of nucleic acid it is necessary to transfer the amount of nucleic acid from a particular solution into the microchannel as quantitatively as possible. If the entire nucleic acid is injected, only a very small amount of nucleic acid should be extractable from the same sample vessel after a 2 injection
  • the DNA was stained with the intercalating dye YOYO (Molecular Probes) and detected with laser-induced fluorescence detection (LIF).
  • YOYO (0.4 mmol / 1, in 76 ⁇ l TBE buffer) was placed in front of it and mixed with 1-4 ⁇ l of the pBr-DNA solution (1 mg / 1) and incubated at RT for at least 30 min before the measurement.
  • the measurement parameters are in Table 3
  • the detection profiles were such that after about 10 minutes there was a significant increase in fluorescence, which quickly reached a plateau. After about 30 minutes the fluorescence decreased again, combined with a gradual increase in the current in the channel. The height of the plateau correlated with the amount of DNA. In the second injection, no fluorescence was injected from any of the samples. The data demonstrate the complete extraction of the pBr DNA on the 1st injection. We conclude from the fluorescence course that the entire nucleic acid was completely injected after 30 min.
  • Nucleic acid was injected into the microchannel with the membrane installed using the measurement conditions in Table 2.
  • pBr-DNA 2.5 mg / 1, 50 ⁇ l
  • the separating buffer FIG. 2
  • the DNA was not directly detectable.
  • the voltage was reversed and the concentrated DNA migrated back through the detector as an intense peak after 12 min.
  • Virtually the entire DNA of the 50 ⁇ l sample (0.1 mg) was concentrated in less than 50 nl (1 cm in the capillary). The enrichment factor was therefore a factor of 1000.
  • pBr-DNA was electrokinetically injected as described above and immobilized on the membrane (Tab. 2). After immobilization, the tension was not immediately reversed, but that of DNA on the other side of the membrane.
  • Bran buffer vessel was replaced with a buffer solution containing cationic intercalating dye (YOYO, 0.4 mmol / 1, Molecular Probes). The tension was maintained for an additional 20 minutes with the dye migrating through the microchannel and through the membrane. The DNA was thus incubated on the membrane with YOYO. The mixture was then electrophoresed for another 10 min without dye in order to remove any remaining YOYO from the capillary. Then the voltage was reversed and the retarded and stained DNA was moved past the detector again.
  • YOYO cationic intercalating dye
  • the electrokinetically injected DNA migrated through the UV detector after 2 min.
  • the UV-VIS spectrum of the injected DNA generated by means of diode array detection (DAD) in the microchannel showed the typical UV spectrum with 2 absorption maxima at 200 and 260 nm. After incubation with YOYO on the membrane, the DNA also showed an absorption maximum at 490 nm. This corresponds exactly to the absorption maximum of the DNA intercalated with YOYO. This proved that macromolecules can be derivatized in the microchannel.
  • DAD diode array detection
  • Herpes simplex viruses (type 2) were stained with YOYO (Molecular Probes) and detected with laser-induced fluorescence detection (LIF).
  • YOYO (0.4 mmol / 1, in 76 ⁇ l TBE buffer) was introduced and 4 ⁇ l of the HSV-2 solution (5 ⁇ 10 5 viruses / ml) were added and incubated at RT for at least 30 min before the measurement.
  • the measurement parameters are in Table 3.
  • the few injected, intercalated viruses (10-20) were detected as individual signals. To identify the signals, the same sample was compared under The electrophoresis conditions on a Prince-CE (Lauerlabs) were analyzed with a nanofraction collector (Probot, BAI-Instruments). The end of the microchannel was fractionated in a time-controlled manner on different electron microscopy carriers and examined in the EM after negative contrasting with uranyl acetate.
  • FIG. 1 Schematic representation of the purification apparatus
  • a microchannel (1) with a built-in membrane (2) connects 2 buffer vessels (3,4). These buffer vessels can be brought to different voltage potentials by means of electrodes (5) )
  • the sample to be examined is in (3)
  • Fig. 2 Schematic representation of the concentration.
  • the macromolecule to be investigated was electrokinetically injected into the microchannel (1) with the membrane (2) installed.
  • the sample vessel was already replaced by the concentration buffer (7).
  • the macromolecules migrate to the membrane and are held there
  • Fig. 3 Schematic representation of the sample modification In the reaction vessels (8,9) are the derivatization reagents, which are brought either electrokinetically and / or with the help of pressure to the concentrated macromolecules
  • Fig. 4 Schematic representation of an on-line analysis method.
  • the concentrated and modified macromolecule becomes electrokinetic mobilized in the microchannel (1) past a detector window (12) so that the spectroscopic properties can be analyzed and evaluated (13).
  • Fig. 5 Schematic representation of the fractionation of the purified
  • the concentrated sample is collected in the sample vessel or on the analysis target (14) and is available for further analysis.
  • Fig. 6 Schematic representation of the high-throughput purification apparatus.
  • a variety of microchannels are arranged to be compatible with the sample format (e.g. microtiter plate).
  • the membrane (2) is introduced over the entire format and connected to a second microchannel array (15). The procedure of these multiple arrangements corresponds to Fig. 1-5.
  • Fig. 7 Schematic representation of the enrichment apparatus for fast
  • Fig. 8 Schematic representation of the rapid enrichment from saline

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  • Electrochemistry (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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  • Saccharide Compounds (AREA)
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Abstract

L'invention concerne un procédé permettant d'isoler des macromolécules (acides nucléiques, protéines, virus et bactéries) hors de matériaux biologiques tels que du sang, du sérum, de l'urine, des végétaux, des cellules, des surnageants cellulaires etc. et hors de préparations, de les concentrer et de les rendre accessibles à l'analyse. Les macromolécules sont d'abord concentrées de manière électrocinétique dans un canal plat, sur une membrane. Les macromolécules ainsi concentrées peuvent être transférées de manière électrocinétique par l'intermédiaire d'un canal de transfert dans un microcanal analytique en vue d'un autre traitement.
EP97954757A 1997-01-08 1997-12-24 Preparation electrocinetique d'echantillons Withdrawn EP0958300A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19700364 1997-01-08
DE1997100364 DE19700364A1 (de) 1997-01-08 1997-01-08 Elektrokinetische Probenvorbereitung
PCT/EP1997/007306 WO1998030571A1 (fr) 1997-01-08 1997-12-24 Preparation electrocinetique d'echantillons

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EP0958300A1 true EP0958300A1 (fr) 1999-11-24

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US6699986B2 (en) * 1998-08-04 2004-03-02 Ortho-Clinical Diagnostics, Inc. Electrophoretic separation of nucleic acids from proteins at low ph
GB2372464B (en) * 2001-02-22 2003-05-14 Vivascience Ltd Method of isolating a charged compound
DE10149875A1 (de) * 2001-10-10 2003-07-03 Alpha Technology Ges Fuer Ange Vorrichtung zur Extraktion elektrisch geladener Moleküle
WO2004081530A2 (fr) * 2003-03-10 2004-09-23 The Johns Hopkins University Procede et appareil pour la surveillance et la bioprospection de l'environnement
GB2416030B (en) * 2004-01-28 2008-07-23 Norchip As A diagnostic system for carrying out a nucleic acid sequence amplification and detection process
DE102006002258B4 (de) 2006-01-17 2008-08-21 Siemens Ag Modul zur Aufbereitung einer biologischen Probe, Biochip-Satz und Verwendung des Moduls
US8999636B2 (en) 2007-01-08 2015-04-07 Toxic Report Llc Reaction chamber
US8361716B2 (en) 2008-10-03 2013-01-29 Pathogenetix, Inc. Focusing chamber
US8685708B2 (en) 2012-04-18 2014-04-01 Pathogenetix, Inc. Device for preparing a sample

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US5202010A (en) * 1987-11-25 1993-04-13 Princeton Biochemicals, Inc. Automated capillary electrophoresis apparatus

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AU5985798A (en) 1998-08-03
DE19700364A1 (de) 1998-07-09
WO1998030571A1 (fr) 1998-07-16

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