WO1998030571A1 - Electrokinetic sample preparation - Google Patents

Abstract

The invention concerns a method which enables macromolecules (nucleic acids, proteins, viruses and bacteria) to be isolated from biological materials, such as blood, serum, fluid, urine, plants, cells, supernatant liquor from cells, etc. and preparations thereof, concentrated and rendered accessible to analysis. The macromolecules are first electrokinetically concentrated in a flat duct on a membrane. The concentrated macromolecules can be transferred electrokinetically via a transfer duct into an analytical micro-duct for further processing.

Description

Electrokinetic sample preparation

Biological macromolecules 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. For example, 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. 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.

With "electrokinetic sample preparation", the entire nucleic acid analysis from isolation to evaluation can be carried out with unprecedented speed and automation, even while avoiding additional amplification.

Purification and diagnosis of proteins

Since different proteins have individual physical properties, there are no universally applicable purification processes. Chromatographic and electrophoretic processes, precipitation, ultrafiltration, ultracentrifugation and size exclusion chromatography are predominantly used (Doonan, S., Methods Mol. Biol, 1996, 59, Totowa, NJ, Humana, 1996, 405ff). For diagnostic applications, immunological methods have become established that detect the target protein via specific antibody recognition. Purification and diagnosis of nucleic acids

In order to isolate nucleic acids from biological material for diagnostic applications, depending on the nature of the material, different digestion processes and subsequent purification processes are necessary. It must again be ensured that the nucleic acid to be isolated is not destroyed. In particular, R A can easily be degraded by ubiquitous RNAses, so that the addition of inhibitors for these interfering enzymes is necessary (Walker, J.M., Methods Mol. Biol., 1984, 2, Clifton, N.J., Humana, 1984, 113fϊ). The currently customary procedures are outlined below:

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,

Berlin, Springer, 1992).

As a rule, however, the 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

(Detection of bacteria, fungi, viruses or genomic nucleic acid from the carrier organism) must be modified.

The following is a brief summary of the most common purification procedures for these samples:

Phenol extraction of nucleic acid with Proteinase K treatment.

Retardation of the nucleic acid on membrane filters; is only used for pre-cleaned samples or slightly contaminated samples - such as urine - due to the problem of constipation. Solid phases to which nucleic acids can be bound enable separations of interfering and accompanying substances by washing steps. Examples of this are: a) Absorption on glass milk in sodium iodide buffer (Maiwald, M. et al., BlOforum 1994, 17, 232-237.). b) addition of the negatively charged nucleic acid to weakly basic polymers (EP 0707077 and US 5434270). c) Cellulose matrices for the direct absorption of blood, whereupon all treatment steps including nucleic acid release and purification take place. (Del Rio, SA et al. Biotechniques, 1996, 20, 970-974). d) 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) or chemically modified silica phases (EP

0648777) also belong to this group.

There are also electroelution devices on the market (e.g. from Biometra) for macroscopically extracting proteins, nucleic acids or viruses from gels (EP 380357).

In order to obtain sufficient amounts of nucleic acid, an isolation step usually follows the isolation of the nucleic acids (EP 229701).

There are approaches to automating nucleic acid analysis in the form of anion exchange chromatography and nucleic acid adsorption with pipetting robots (BioRobot from Quiagen). In a Japanese patent, the nucleic acid is immobilized in a capillary in order to be able to carry out the amplification therein (JP 7107962).

Purification and diagnosis of viruses

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,

NY, 1993). For diagnostic purposes, either immunological procedures are used used on the protein envelope or nucleic acid analysis methods after the release of the viral nucleic acids

Purification and characterization of bacteria

Bacteria are usually separated and raised by spreading on nutrient media. For the isolation and characterization, immunological methods - eg by fluorescent labeling -, nucleic acid determination methods - after cell lysis - are available.

All methods, regardless of the macromolecule, are time-consuming, involve numerous dilution steps and often do not guarantee the separation of interfering factors. The technical state of microchannel technology and amplification-free nucleic acid analysis will be briefly outlined below

Microchannels

Capillary electrophoresis is a relatively new analytical separation technique (St Claire

R L, anal. Chem. 1996, 68, 569R-586R) The principle is based on the separation of analytes in an electrolyte-filled capillary by applying a high-voltage field between the capillary ends

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

Press, 1994) Almost all device manufacturers offer analysis kits for nucleic acid analysis with the CE. Since 1995 there has also been a fully automated nucleic acid Analyzer based on the CE, the ABI Prism 310 from Perkin-Elmer (Applied Biosystems). The nucleic acid is usually injected into the capillary electrokinetically by applying a voltage. However, the electrokinetic application quantity is limited, since otherwise peak broadening and sample discrimination occur (Butler, JM, et al. J. Chromatogr. B, 1994, 658, 271-280).

The introduction of laser-induced fluorescence detection in capillary electrophoresis (St.Claire R.L., Anal. Chem. 1996, 68, 569R-586R), which Beckman has already commercialized, has resulted in a significant increase in sensitivity. This means that intact viruses can also be analyzed using CE (DE 4438833). Proteins can only be detected after specific modification with fluorescence.

Concentration in the microchannel

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

Res. Chromatogr. 1995, 18, 381-383). By inserting a reverse phase membrane into the capillary, lipophilic sample components are retained in the membrane in a solid phase extraction, then eluted through the membrane with an organic solvent and separated by capillary electrophoresis. CE chips

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). In photolithographic microcapillary systems, 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

Based on the high sensitivity of fluorescence detection, a patent was filed for flow cytometry for single molecule detection (WO 90/14589).

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.

With the present invention, 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.

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 By applying 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 simplified schematic representation can be found in Fig. 1-5

A membrane (2) is introduced into a microchannel (1) and is suitable for retaining the desired macromolecule. By applying 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. By combining the parameters pH value, membrane properties, nature of the microchannel, polarity of the voltage and direction of the pressure gradient, 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 μm 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

Suitable for polymers 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)

Regardless of the type of macromolecule examined, 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. In addition, ion exchange membranes and adsorption phases can be used. The choice of these membranes depends on the type of

Macromolecule and is therefore treated individually.

After optimally changing the sample vessel (3) to a concentration buffer vessel (7), 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.

After optimally changing the tubes (4,7) against the reagent tubes (8,9), the solutions contained therein, or components thereof, can be created by applying a

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). For this purpose, 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.

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). By 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)

For this purpose, 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. For example, it 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.

If the channel is additionally branched, 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)

Fractionated macromolecules can be further analyzed using all conceivable methods. 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

Sample format The following additional devices are not shown. Additional 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. For electrical contact, either on attached to the channel ends or in the vessels, electrodes and supplied with a high voltage source

For thermostatting, for example, 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

A parallel capillary arrangement was developed for a much faster concentration of the samples. The schematic representation of this module is shown in Fig. 7 Next to the microchannel (1) is the much wider channel

(16) The height of the channel corresponds to the dimension of the microchannel (10-100 μm), so that the Joule heat can still be dissipated well.The width of the channel (100 μm 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. The voltage is maintained until all nucleic acids are immobilized on the membrane surface (FIG. 8b). If a voltage is now applied between the flat channel (16) and the microchannel (1), as indicated, then the purified and concentrated nucleic acid from the large membrane surface of the flat channel (16) to the membrane of the microchannel (FIG. 8c) and is then available for further treatment and analysis (FIG. 3-6)

In this transfer step, modification reactions can also be carried out simultaneously on the concentrated macromolecule

Description of nucleic acid concentration

1 1 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. However, a polymer-bound, acidic ion exchanger (eg polystyrene sulfonic acid) is preferably used The acidic pH value is reached without additional anions being introduced into the solution. The electrokinetic nucleic acid extraction is thereby promoted

1 2 The nucleic acid is extracted electrophoretically from this acid buffered solution. For this purpose, 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. For example sodium citrate (20 mmol / 1, pH 5.0) or tris / borate (100 mmol / 1, pH 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).

1.3. The extracted nucleic acid is immobilized and concentrated in the channel with the aid of the membrane introduced while maintaining the tension (FIG. 2). In addition to the size exclusion membranes, 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. For example, the membranes contain nanoparticles, preferably silica-based or metal oxide pigments. With immobilized oligonucleotides, specific nucleic acid strands are immobilized by solid-phase hybridization.

1.4. 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. For example, the nucleic acid can be labeled with intercalating dyes. They are preferably 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

Background fluorescence occurs because these dyes only fluoresce in the intercalated state. The positively charged YOYO can be introduced electrokinetically, for example, from the reaction vessel (9) with the connection of the focusing voltage

Significantly higher specificity can be achieved, for example, using a fluorescence-labeled gene probe. 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

Applying pressure and / or voltage can be eluted in a few nanoliters and is available for further analysis

1 5 For this purpose, 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. As the subsequent analyzes, PCR reactions, CE separations, DNA sequencing, hybridization reactions, mass spectrometric analyzes or molecular biological methods can be carried out.

1.6. 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.

For this purpose, the buffer vessels (10, 11) and the microchannel (1) are filled with a polymer-containing buffer solution. These 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.

1.7. By using fluorescence-labeled probes, fluorescence-labeled terminators in Sanger sequencing or intercalating dyes, direct fluorescence detection of the nucleic acid in the microsystem is possible.

Viruses

2.1. 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

Pressure difference between the buffer vessels (3 and 4)

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

Voltage Combined with a suitable temperature control, all derivatization methods for proteins and nucleic acids are possible in the microsystem (cf. 1 4 and 3 4) the membrane is lysed and then the nucleic acids and / or the proteins are analyzed (cf. 1 4-1 7 and 3 4-3.7)

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

For this purpose, the voltage is reversed so that the anode is now in the analysis vessel (14) (FIG. 5). The microchannel (1) and the buffer vessel (11) are preferably filled beforehand with an aqueous buffer of the abovementioned composition. After fractionation into a buffer-filled analysis vessel (14) 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).

The 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

3.1. 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.

3.2. The positively charged proteins are extracted electrophoretically from this buffered solution. For this purpose, 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. Optionally, 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.

3.3. The extracted proteins are introduced into the channel using the

Membrane immobilized and concentrated (Fig. 2). In addition to the size exclusion membrane, 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. In the case of the cationic proteins discussed here, mainly carboxylic acid phases are suitable as soft exchangers and sulfonic acid phases are suitable as strong exchangers. For special proteins, the membranes can be coated with appropriate antibodies and thus enriched for affinity.

3.4. 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

Maintain focus. Combined with a suitable temperature control, enzymatic cleavage, complexation, chemical derivatization or antibody binding are possible in the microsystem.

For example, the protein can be reacted with reactive dyes. It is preferably amine-specific dyes such as e.g. Fluorescein isothiocyanate (FITC). 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.

Significantly higher specificity can be achieved, for example, with fluorescence-labeled antibodies. In the subsequent separation, the protein-antibody complex is then determined by means of fluorescence.

3.5. 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.

3.6. The proteins can be analyzed electrophoretically within the microsystem (FIG. 4).

3.7. By using fluorescent labeled antibodies, fluorescent

Enzyme substrates, fluorescent binding partners or derivatizing reagents, the proteins can also be identified by fluorescence spectroscopy.

4. Bacteria

4.1. 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.

4.3. 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 μm. However, the same anion exchangers as for the proteins can also be used (cf. 3.3.)

4.4. 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. Combined with a suitable temperature control, all derivatization methods for proteins and nucleic acids are possible in the microsystem (cf. 1.4 - 1.7 and 3.4 -

3.7).

In the case of bacterial lysis, the size exclusion membrane must be dimensioned such 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, urea or sodium dodecyl sulfate (SDS). 4.5. 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.

For this purpose, the voltage is reversed so that the anode is now in the analysis vessel (14) (FIG. 5). The microchannel (1) and the buffer vessel (11) are preferably filled beforehand with an aqueous buffer of the above-mentioned composition. After fractionation into a buffer-filled analysis vessel (14), pathogenicity assays or electrophysiological experiments can be carried out with the bacteria, for example. After fractionation on a planar analysis target (14), 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.

4.6. The bacteria can be analyzed electrophoretically within the microsystem (FIG. 4).

4.7. The bacteria can also be identified by fluorescence spectroscopy using fluorescent-labeled antibodies or fluorescent binding partners.

All common methods of macromolecule isolation require the processing of the entire sample volume through the extraction medium. The handling of large-volume samples prevents miniaturization, which in turn is absolutely necessary in order to increase the analysis speed and sensitivity. The combination of direct electrophoretic extraction with a micro-scale immobilization membrane makes it possible for the first time to combine large sample volumes directly with a nanoanalysis technique.

Compared to the established processes, 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

By concentrating nucleic acids, additional amplification steps can be avoided in many cases. The method can thus replace PCR, for example.

The method can also be used as a digestion method for viruses, bacteria and other cells. For example, 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. In the case of human DNA, 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. Here, the analysis of tumor suppressor genes and oncogenes is of particular interest

Another use is the combination with 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 As a quality assurance method, the invention also enables the control of genetically engineered products in which freedom from nucleic acids must be guaranteed.

The investigation of intact viruses, bacteria or their nucleic acid or their

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.

For the first time, 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. B. transferrin, fibrinogen, ß-2 microglobulin, hCG or tumor marker (AFP, CEA, CA 15-3, CA 19-9) high diagnostic relevance.

0 Examples

Electrokinetic injection of nucleic acid

To check the electrokinetic injection of nucleic acid, model

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.

There was a dilution series of pBr-DNA (Boehringer Mannheim) from 250 to

1.25 mg / 1 prepared and placed in the sample vessel. After the injection, electrophoretic separation was carried out. The measurement conditions are in Table 1.

If the peak areas are plotted against the DNA concentration, the peak area increase becomes saturated for high DNA concentrations. The injected

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.

Recovery rate from size exclusion membrane

To check the retardation of nucleic acid on a size exclusion membrane, 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.

Detector (see Fig. 4). The pBR-DNA (250 mg / 1) was electrophoresed past the detector and the electrophoresis was continued so long that the DNA would have left the channel without a membrane. Then the voltage was reversed and the retarded DNA moved past the detector again. The measurement parameters are in Table 2. Table 2: Measurement parameters for the electrophoretic nucleic acid recovery from a size exclusion membrane.

The electrokinetically injected DNA migrated through the UV detector after 1.7 min. Without the size exclusion membrane, the DNA would leave the channel after 7 min. However, the electrophoresis was continued for a total of 10 minutes and the voltage was then reversed directly. The retarded DNA in the capillary migrated back through the UV detector. After 12.05 minutes, a signal could be detected that corresponded exactly to the peak area of the injected DNA. The analogous experiment with 3-nitrobenzenesulfonic acid showed, as expected, only the injection peak and therefore no retardation on the size exclusion membrane.

In a further experiment, three successive injections of nucleic acid were carried out and analyzed under identical conditions. The electropherogram clearly showed the concentration of the three nucleic acid injections in one signal. The peak areas of the three individual injections corresponded exactly to the peak area of the retarded DNA. This showed that macromolecules on the Membrane in the microchannel can be electrophoretically immobilized and quantitatively remobilized

Nucleic acid extraction

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

In order to keep the amount of DNA as low as possible, 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 μl TBE buffer) was placed in front of it and mixed with 1-4 μl 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

Table 3: Measurement parameters for electrophoretic nucleic acid extraction

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.

These data clearly show that the entire nucleic acid of a sample volume can be electrokinetically injected into a microchannel. In combination with the size exclusion membrane, it should be possible to concentrate this nucleic acid in a few nanoliters and thus make it detectable.

Nucleic acid concentration

Nucleic acid was injected into the microchannel with the membrane installed using the measurement conditions in Table 2. For this, pBr-DNA (2.5 mg / 1, 50 μl) was injected for 25 min and then focused with the separating buffer (FIG. 2). At this low concentration, the DNA was not directly detectable. After 10 min 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 μl 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.

Derivatization on the membrane

To check the derivatization of nucleic acid on a size exclusion membrane, 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.

The electrokinetically injected DNA migrated through the UV detector after 2 min.

Two minutes after the polarity reversal, a signal with a larger peak area corresponding to the stained DNA could be detected. 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.

Coupling with electron microscopy

Coupling with electron microscopy (EM) should serve as evidence of the further analysis of prepared macromolecules.

Herpes simplex viruses (type 2) were stained with YOYO (Molecular Probes) and detected with laser-induced fluorescence detection (LIF). For this purpose, YOYO (0.4 mmol / 1, in 76 μl TBE buffer) was introduced and 4 μl 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.

Intact fractions of HSV particles were clearly detected in the expected fractions. It was shown for the first time, using viruses as an example, that macromolecules are available as intact particles for further analysis after electrophoretic separation

Legends for illustrations

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

Macromolecule. 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

Concentrations. There is a next to the microchannel (1)

Flat channel (16) which is connected to the microchannel (1) via the transfer channel (17). The flat channel allows much higher flow rates and thus higher enrichment factors. Details in the text.

Fig. 8: Schematic representation of the rapid enrichment from saline

Solution in supervision using the example of nucleic acids. Enrichment takes place by applying a voltage (6) to the flat duct

(16) (Fig. 8a). The voltage is maintained until all nucleic acids are immobilized on the membrane surface (2) (FIG. 8b). If a voltage (6) is now applied between the flat channel (16) and the microchannel (1), as indicated, the purified and concentrated nucleic acid migrates from the large membrane surface of the flat channel (16) to the membrane of the microchannel ( 1) (Fig. 8c) and is then available for the analog methods (Fig. 3 -6). Details in the text.

Claims

claims

1. A process for the isolation and concentration of macromolecules, characterized in that the macromolecules are electrokinetically collected in a microchannel on a membrane and then analyzed.

2. The method according to claim 1, characterized in that the nucleic acids, viruses, proteins, bacteria or fungi are electrokinetically collected in a microchannel on a membrane and then analyzed.

3. The method according to claims 1 and 2, wherein the sample is derivatized on the membrane.

4. The method according to claims 1 to 3 as sample preparation for others

Analytical method.

5. The method according to claims 1 to 4 as sample preparation for MS, gel electrophoresis, PCR, TEM, nucleic acid sequencing, immunodiagnostics and hybridizations.

6. Device for performing the method according to claims 1 to 5 in the form of a chip module with an embedded membrane, wherein 1 -400 capillaries are arranged side by side.

7. The device according to claim 2, containing a membrane made of polyethersulfone (PES), polyester, fleece-based acrylic polymer, polytetrafluoroethylene (PTFE), polysulfone, polypropylene (PP), glass fiber, nylon or polycarbonate.

8. Device for performing the method according to claims 1 to 7 in the form of a flat channel for the analysis of saline samples.

9. Use of the device according to claims 6 to 8 for enrichment and analysis of macromolecules.

10. Use of the device according to claims 6 to 8 in the quality control of biological preparations.

11. Use of the device according to claims 6 to 8 for direct infection diagnosis.

12. Use of the device according to claims 6 to 8 for amplification-free nucleic acid analysis.

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