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
  • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
  • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
  • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
  • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength

Definitions

• the invention relates to biosondes which change their electrical and / or dielectric properties through specific binding to biological material and thereby enable detection and / or purification of the material modified in this way by means of electrophoresis and / or dielectrophoresis.
• bioanalytics and diagnostics the task is often to selectively separate one or more biological species from a template of biological material or from a background that consists of very similar biological species.
• the biological material can be tissue, cells, viruses, cell organelles, proteins, protein complexes, carbohydrates, lipids or other organic compounds or a mixture of the groups listed.
• the different species accordingly come from at least one of these groups.
• the differences in the properties of the different biological species on which the separation or detection principle is based can be very small, so that separation and / or detection is often very difficult or impossible to achieve.
• Methods in which the biological material is selectively labeled in a specific way and only possible because of the labeling or because of the properties changed by the labeling separation or detection of the different species are all methods which are based on an antibody-antigen interaction. Examples of this are ELISA procedures (enzyme-linked immunosorbent assay) and certain variants of FACS (fluorescence-activated cell sorting) and MACS (magnetic-activated cell sorting). In these cases, using specific binding antibodies, an enzyme, a fluorescent dye or a magnetic bead is selectively bound to certain species of the biological material in order to enable detection or purification of the species thus labeled.
• Free flow electrophoresis (Bauer, J. (1999) J. Chrom. B 722: 55) is an example of a method in which the separation of biological material is very difficult. In this process, a laminar flow of liquid is passed between two closely spaced glass plates. An electrical field applied perpendicular to the direction of flow enables the different species of the submitted biological material to be separated on the basis of their different charges. For example, if the FFE is used to separate cells, they will be attracted electrically by the cathode due to their negative charge. The lateral rate of migration of the cells depends on the surface charge density of the cells. Cells with different surface charge densities have different lateral ones
• the decisive disadvantage of the FFE is that biological material often has very similar charging properties. Cells, for example, all have a negative and moreover, an almost identical surface charge density and can therefore only be separated from one another very poorly using FFE. A sufficient degree of selectivity can often only be achieved with the FFE if the surface charge density of the cells has been sufficiently modified by a pathogenic change. For this reason, the use of the FFE has declined very sharply today and the method has been replaced by other, often more expensive and more expensive equipment.
• Dielectrophoresis is a process that takes advantage of the dielectric properties of biological material to separate it. Dielectrophoresis takes place when biological material is exposed to inhomogeneous alternating electrical fields. The biological material moves in the inhomogeneous electrical field due to its dielectric properties (permanent or inducible electrical dipole), which makes separation possible.
• dielectrophoresis In cells, for example, there are different areas that can be polarized and can thus be used for dielectrophoresis. These include the electrical charge double layer that surrounds a cell, the cell membranes that bound the cell or the cell organelles, and the polar cytoplasm in the interior of the cell. Dielectrophoresis can be used, for example, to separate cells, bacteria and other microorganisms. The use of dielectrophoresis is also conceivable for the separation of other biological material such as nucleic acids, proteins, lipids or other organic compounds. Dielectrophoresis can be used, for example, to examine drinking water, food and biological fluids for pathogenic microorganisms or to enrich stem cells from the bone marrow or peripheral blood. The disadvantage of dielectrophoresis is that the dielectric properties of biological materials are often very similar to 'make it difficult to perform separation of different species.
• the object of the invention is therefore to increase the selectivity of electrophoretic and / or dielectrophoretic methods and thus to provide an improved detection and / or cleaning method
• the object is achieved by the specific binding of bio-probes to a specific species or to a group of specific species from a template of biological material which contains at least one species, which selectively and specifically changes the electrical and / or dielectric properties of the species thus labeled and thereby the cleaning and / or detection of the species so marked or of the complex formed by binding the biosonde and biosonde and biological species is possible.
• the biological material in particular comprises at least one species from at least one group selected from tissue, cells, cell organelles, viruses, proteins, peptides, nucleic acids, carbohydrates, lipids or other organic compounds, it being possible for the species to also be modified.
• the main advantage of the invention described here compared to the conventional methods lies in the possibility of simple and rapid separation and / or detection of the complex of biological species and biosonde which is formed by binding the biosonde to the biological species, which on the one hand can be used to to enable the detection or purification of the species so selectively labeled, but conversely it can also be used to selectively separate one or more biosondes from a template of biosondes with different binding specificities.
• the biological material can be different before performing the separation process, in particular with a library of biosondes according to the invention Binding specificity can be contacted so that selectively binding biosondes can be identified and isolated from the library.
• the decisive advantage of the method according to the invention compared to methods such as FACS and MACS is that the biosonde does not have to be modified in a complex manner with a fluorescent dye or a magnetic bead in order to enable detection and / or purification of the labeled biological material, and also that the outlay on equipment is also high , which is inevitable with processes such as FACS or MACS, is no longer required.
• the method according to the invention for the detection or purification of biological material can in particular comprise the following steps: a) Presentation of biological material which contains different species. b) adding a biosonde containing a part A which binds specifically to at least one of the species and a part B which changes the electrical and / or dielectric properties of the species (s) marked by specific binding of the biosonde so that the detection and / or the purification of these labeled species (s) is made possible or improved by electrophoresis and / or dielectrophoresis, c) by establishing an electric field for the detection or purification of the complex or complexes from biological species (s) and specifically bound biosonde Electrophoresis or dielectrophoresis.
• the method according to the invention can, in order to identify and isolate bio-probes that bind specifically to submitted biological material, in particular comprise the following steps: a) presentation of biological material which contains at least one species, b) addition of a bio-probe or different bio-probes, each containing part A, can bind specifically to at least one of the species, and a part B, which is the electrical and / or dielectric Properties of the species (s) marked by binding of a biosonde are changed, so that detection and / or purification of a complex of biological species (s) and biological material which is formed in this way is possible, the different biosondes having different binding specificities and at least one of the added ones
• Biosonde binds to the biological material, c) Establishment of an electric field for the detection or purification of the complex or complexes of biological species (s) and specifically bound biosonde by electrophoresis or dielectrophoresis.
• the various bio-probes according to point (b) can in particular be a library of bio-probes of different substrate specificity, which comprises more than 10 9 , in particular more than 10 12 different bio-probes.
• biosondes according to the invention comprise part A, which is the specific one
• Part B which changes the properties of the biological material in the desired manner, i.e. with regard to electrophoresis, the charge, with respect to dielectrophoresis, the dielectric constant or the specific conductivity or Polarizability changed.
• Parts A and B do not have to be structurally separated.
• Part A of the biosonde is or comprises, for example, a peptide that specifically binds to biological material.
• a peptide receptor that is located on the biological material.
• An example of such an interaction is that of the factor receptor Ste2p from Saccharomyces cerevisiae with the associated peptide ligand ⁇ factor.
• Further examples are the TSH receptor (Schuppert et al. (1996) Thyroid 6: 575), the FSH receptor (Tilly et al. (1992) Endocrinology 131: 799), the EGF receptor (Christensen et al. (1998 ) Dan. Med. Bull. 45: 121), the TNF receptor (Murphy et al.
• Thymus 23: 177 the transferrin receptor (Ponka and Lok (1999) Int. J. Biochem. Cell Biol. 31: 1111), the insulin receptor (Milazzo et al. (1992) Cancer Res. 52: 3924), the FGF receptor (Kiefer et al. (1991) Growth Factors 5: 1 15), the TGFß receptor (Derynck et al. (1994) Princess Takamatsu Symp. 24: 264), the IGF receptor (Peyrat and Bonneterre (1992) Breast Cancer Res. Treat. 22: 59) , the angiotensin II receptor (Smith and Timmermans (1994) Curr. Opin. Nephrol. Hypertens.
• the specifically binding part A can also be an antibody, in particular, for example, an antibody that recognizes marker structures on cell surfaces.
• An example of such a marker structure is the
• Part A can also consist of a low-molecular structure that specifically binds to biological material.
• a low molecular structure is acetylcholine, which is specifically bound by the acetylcholine receptor.
• Part A of the biosonde can, for example, also be a carbohydrate, a lipid, an inorganic compound, a nucleic acid or another ligand that specifically binds to biological material, or can comprise one of these groups.
• Part B of the biosonde which is linked to Part A, is or comprises a structure which, when bound to the biological material, changes its electrical and / or dielectric properties so that the behavior of the biological material thus modified changes in an electrical and / or dielectric field changed. This can be done, for example, by introducing electrical charge, by changing the dielectric constant and / or by changing the specific conductivity of the biological material.
• Examples of carriers of electrical charge in this sense are acidic and basic amino acids (aspartate, glutamate, lysine, arginine), nucleic acids (single-stranded and double-stranded DNA and RNA, DNA / RNA heteroduplex), organic acids and bases (tartrate, citrate, amines) , polyquaternary amines and inorganic charge carriers.
• Structures that change the dielectric behavior of the biological material include, in addition to the structures mentioned above, hydrophobic, electrically neutral structures such as lipids, fatty acids, waxes, oils and sterols.
• part B of the biosonde is or comprises a nucleic acid which is covalently linked to part A of the biosonde directly or via an intermediate member.
• the nucleic acid here preferably comprises at least 5 or 10, particularly preferably at least 15 or 20, nucleotides.
• part B comprises the
• Biosonde is a nucleic acid comprising the genetic information for Part A of the biosonde, wherein Part A of the biosonde comprises a protein or polypeptide and Part B of the biosonde comprises a single or double stranded RNA or DNA or an RNA / DNA heteroduplex which is covalent is linked to part A of the biosonde.
• part A of the biosonde also comprises a ligand for the Ste2p receptor, the TSH receptor, the FSH receptor, the EGF receptor, the TNF receptor, the transferrin receptor, the insulin receptor, the FGF - Receptor, the TGFß receptor, the IGF receptor, the angiotensin II receptor or the somatostatin receptor and part B of the biosonde each have a nucleic acid which comprises the sequences coding for the ligands mentioned.
• part A and part B of the biosonde are preferably covalently linked to one another via a unit which is able to take over the growing peptide chain in a ribosomally catalyzed translation reaction with the formation of a covalent bond.
• this unit comprises, for example, a puromycin- or puromycin-analogous molecule and / or an amino acid or an amino acid-analogous molecule.
• a puromycin- or puromycin-analogous molecule and / or an amino acid or an amino acid-analogous molecule.
• Roberts et al. (1997) Proc. Natl. Acad. Be. USA 94: 12297, WO98 / 16636 and Krayevsky et al. (1979) Progress in Nucleic Acids Research and Molecular Biology 23: 1.
• a labeling of these species with a fluorescent dye, a dye (such as propidium iodide, calcofluor), a correspondingly labeled specifically binding antibody (e.g. FITC), a radioactive label (eg 35 S-methionine) or another compound that enables simple detection of the biological material.
• a fluorescent dye such as propidium iodide, calcofluor
• a correspondingly labeled specifically binding antibody e.g. FITC
• a radioactive label eg 35 S-methionine
• Biochips as described by Cheng et al. (Cheng et al. (1998) Anal. Chem. 70: 2321). Biochips allow separation and / or detection to be carried out in miniaturized form. Due to the miniaturization, the experimental effort e.g. compared to FACS can also be reduced.
• the dielectric separation of cells on a chip has already been reported (Cheng et al. (1998) Anal. Chem. 70: 2321), in which case the differences in the dielectric properties of the species to be separated were large enough to to enable a separation.
• the use of the biosondes according to the invention also allows biological species to be separated if the differences in the dielectric properties of the biological species themselves are not large enough to enable separation by means of dielectrophoresis.
• the biosonde is based on a DNA sequence via an in vitro
• the peptide formed during translation is covalently linked to its mRNA via a puromycin.
• the peptide Share represents the specific binder, while the mRNA share changes due to its strong negative charge in the neutral pH range ' the physical properties of the biological material so that a selection or detection is made possible.
• the biological material is a population of yeast cells of the species Saccharomyces cerevisiae of the mating type MATa, which express the ⁇ -factor receptor (STE2) (Davis and Davey (1997) Biochem. Soc. Trans. 25: 1015).
• PCR Chain reaction
• the DNA molecule contains the following sequence regions: a T7 promoter sequence, a TMV translation initiation sequence, a coding region with a 5 'coded E-tag, a ct factor sequence and a 3' coded Strep tag.
• the implementation of the PCR is state of the art and looks as follows: The following components are placed in a PCR reaction vessel:
• 1 ⁇ l DNA (from the oligonucleotide synthesis corresponds to 1 nmol, Seq. 1) 10 ⁇ l Taq polymerase buffer 10x (Promega, Mannheim)
• the PCR program is characterized as follows: 10 cycles with 1 min 95 ° C, 2 min 55 ° C, 2 min 72 ° C.
• the DNA is quantified and about 1 nmol of the double-stranded DNA is expressed as
• Template used for in vitro transcription The transcription is carried out using a commercially available system from Ambion (Austin, USA). The standard approach is as follows: 50 ⁇ l DNA template (1 nmol)
• Chloroform extraction worked up (Davis et al. 1994) and then quantified.
• the mRNA must be modified at the 3 'end so that there is a short DNA sequence on the mRNA and a puromycin at its 3' end. This technique is described by Roberts and Szostak (1997; see above). In the exemplary embodiment, the modification is as follows:
• the mixture is heated to 70 ° C. for 3 min and cooled at RT for 15 min.
• the puromycin-bearing linker is ligated to the mRNA at room temperature.
• the entire batch is mixed with 10 nmol antisplint (Seq. 7) and opened for 5 min
• the ligation mixture is then cooled on ice and the ligated mRNA is quantified.
• Methionine (APbiotech, Freiburg), 15 ⁇ l amino acid master mix without methionine (Ambion, Austin) and 200 ⁇ l Retic Lysate IVT (Ambion, Austin) added and translated. It is made up to 300 ⁇ l with H 2 O. The mixture is incubated at 30 ° C for 30 min. Then 100 ⁇ l 2.5 M KCI and 70 ⁇ l 1 M MgCI 2 are added. The batch is stored at -20 ° C. overnight.
• the translation mixture is mixed with 10 ml binding buffer (100 mM Tris / HCl pH 8.0; 10 mM EDTA pH 8.0; 1 M NaCl; 0.25%
• the mixture is incubated at 4 ° C. for one hour.
• the cellulose with the bound bio-probes is separated by filtration and with 8 ml Binding buffer washed.
• the biosondes are eluted with 4 times 100 ⁇ l H 2 0.
• the biosondes are quantified by determining the 35 S decays in a scintillation counter and can then be used for binding to the biological material.

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• 2000
  • 2000-07-07 DE DE10033194A patent/DE10033194C2/de not_active Expired - Fee Related
• 2001
  • 2001-06-26 AU AU2001277519A patent/AU2001277519A1/en not_active Abandoned
  • 2001-06-26 JP JP2002509509A patent/JP2004502462A/ja active Pending
  • 2001-06-26 CA CA002415253A patent/CA2415253A1/en not_active Abandoned
  • 2001-06-26 WO PCT/EP2001/007259 patent/WO2002004656A2/de not_active Ceased
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