Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/5432—Liposomes or microcapsules

Definitions

• tetraetheripids with their natural head group structure are not suitable for covalent bonding to material surfaces, but lipidation of material surfaces under specific conditions is only possible by targeted activation of their head groups.
• DE 102 28 857 A1 describes tetraetheripids with small head groups which are said to be suitable for covalent bonding to certain surfaces.
• the tetraether lipids in this document are chemically derivatives of caldarchaeol. Caldarchaeol is the hydrolysis product of the main phospholipid from the archaebacterium Thermoplasma acidophilum.
• lipidation using organic solvents is not an option for broad industrial application. It has been shown that no vesicles could be produced in the aqueous medium with caldarchaeol and also with dieducationated Caldarchaeol and so Caldarchaeol is not suitable for lipidation in aqueous medium. It is the object of the present invention to provide easy-to-handle tetraether lipid-based biosensor chips for chip-based analytical methods.
• the underlying immobilization matrix should be producible by means of lipidation of material surfaces in aqueous medium by self-assembly.
• the prepared lipid layers of the matrix should be stable and allow efficient coupling of ligands, preferably proteins.
• the biochips produced in this way are said to have high sensitivities and low proportions of non-specific bonds.
• the object of the present invention is achieved according to independent claims 1, 5, 11 and 14.
• the subclaims represent preferred embodiments. Glasses, z. Borosilicate glasses (eg Borofloat®33, Schott Jena AG, Germany) or glasses with a layered structure of Borofloat® 33 glass, tantalum (V) oxide and SiOx (BIAffinity chips, LOS Jenoptik, Jena, Germany) used.
• the immobilization matrix is formed by contacting the solid substrate which is at least partially aminated
• aqueous suspension of unilameilar lipid vesicles of tetraether of general formula I is preferably carried out for 2 to 15 hours, more preferably 9 to 15 hours, at ambient temperature.
• ambient temperature is understood to mean 15-29 ° C., preferably 18-25 ° C.
• concentration of the aqueous suspension of unilameilar vesicles is preferably from 1 to 10 mg / ml, more preferably from 2 to 4 mg / ml.
• the thus-immobilized matrix is subsequently tempered.
• the annealing may be carried out, for example, at 80 to 80 ° C for one to three hours, preferably at 70 ° C for two hours.
• the aqueous suspension of the unilamellar vesicles of the tetraether lipid of the general formula I is prepared by repeated extrusion, preferably 1 to 21 times, extrusion of an aqueous suspension of multilamellar lipid vesicles of this tetraether lipid through a polycarbonate membrane of desired and defined pore size.
• the pore size is preferably between 100 and 1000 nm. More preferably, a polycarbonate membrane of 100 nm is used so that lipid vesicles having a defined hydrodynamic diameter of 100 nm can be produced.
• Tetraether lipid of the general formula 1 is carried out by hydration of an evaporated lipid film and subsequent treatment in an ultrasonic bath.
• the treatment in the ultrasonic bath may preferably be carried out at a temperature between 50 and 70 ° C, in particular at about 60 ° C and preferably for a period between 10 and 20 minutes, preferably for 15 minutes.
• the final lipid concentration of the aqueous suspension is preferably 1 to 10 mg / ml, more preferably 2 to 4 mg / ml.
• the surface of the solid substrate used in addition to the gold, silver or copper surface, the surface of the solid substrate used must be at least partially aminated to ensure the reliable Anfeindung the Cyanurchloridrestes on the amino group. Methods for the amination of surfaces are known.
• the amination of the surface can be achieved for example by a plasma activation or by incubation with an amino-functional silane.
• an aminoalkylsilane can be used, with particular preference being given to 3-aminopropyldimethylethoxysilane, 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane.
• the immobilization matrix of claim 1 and its preferred embodiments for producing a biosensor chip is also an object of the present invention.
• biosensor chips are referred to as carrier materials which comprise the immobilization matrix according to the invention to which ligands, for example proteins, are bound.
• the ligands are able to bind to specific target molecules (targets) and the target molecules can be detected and / or influenced in this way, so that biochemical evidence or label-free optical biomolecular interaction analyzes (RlfS, SPR) are possible.
• the biosensor chips of the present invention are suitable for reflectometric interference spectroscopy (RIfS).
• the biochips of the present invention have the advantage that they are storage and acid stable, do not swell and thus do not require equilibration time.
• a ligand preferably a protein having at least one free NH 2 group, covalently, optionally via spacers, to the cyanuric chloride, thiol or 1, 2-dithiolan-3-yl -Rest bound to the monomolecular tetraether lipid layer of Immobilmaschinesmatrix.
• the immobilization matrix is incubated with a buffer solution of the ligand. This can be performed, for example so that a continuous liquid flow is preferably particularly preferably passed for 20 to 40 minutes, "25 minutes on the lipid matrix.
• the flow rate should be in the range of 5 ⁇ / min to 10 ⁇ / min.
• a BIAffinity® flux (Analytik Jena AG, Germany) may be used for this purpose.
• proteins can be attached. Streptavidin, C-reactive protein, protein G, protein A, His-tag or GFP binding protein (GBP) are particularly preferably bound according to the invention as ligands, and the corresponding protein chips are thus produced.
• GFP GFP binding protein
• the non-specific binding sites can be blocked very well with BSA.
• a BSA solution with a concentration of 80-100 pg / ml BSA is used in the particular buffer used to bind the ligands (in particular PBS or acetate buffer).
• biosensor chips of the present invention are highly sensitive and show low levels of non-specific binding events.
• the invention will be explained in more detail below with reference to embodiments, without limiting it thereto.
• Example 1 Isolation of the Main Phospholipid (MPL) from the Archea Dry Mass of Sulfolobus acidocaldarius and Activation of the Lipid Head Groups with Cyanuric Chloride
• the terminal hydroxyl groups of the MPL were activated with cyanuric chloride.
• the lipid was dissolved in chloroform and NaHCO 3 (1.5 g per 100 mg lipid) was added. The mixture was stirred at reflux for 1 hour. Subsequently, 5 eq. Add cyanuric chloride and heat at reflux for one week. The course of the reaction was monitored by thin-layer chromatography, R f values (eluent: petroiether / diethyl ether / glacial acetic acid 5/5 / 0.1) of 0.67 ⁇ 0.0173 for the pure MPL and 0.75 ⁇ 0.0824 were found for the activated MPL.
• Hydrodynamic diameters of diaactivated sulfolobus MPL liposomes before (right curve) and after (left curve) of extrusion In the figure above, the hydrodynamic diameters of the dicotivated sulfolobus MPLs are shown and it becomes clear that with this lipid vesicles with defined diameters of 100 nm.
• Example 3 Amination of glass surfaces and Liposomenspreitung for the amination, the glass substrates (Borofloat ® 33 or BIAffinity chips) were placed in a specially made PTFE chamber.
• a chloroform solution was prepared with 1% by volume of aminopropyldimethyfethoxysilane (APDMES).
• APDMES aminopropyldimethyfethoxysilane
• the chamber was completely filled with the silane solution and sealed with a lid.
• the chamber was incubated overnight at 60 ° C in a drying oven. Thereafter, the chamber was taken out of the oven, and after cooling, the substrates were removed from the chamber and rinsed successively in chloroform, methanol, and water for 10 minutes each in an ultrasonic bath.
• the glass substrates were incubated in a freshly extruded Sulfolobus MPL liposome suspension (see Example 2) overnight at room temperature. Subsequently, the substrates were again stored for two hours at 70 ° C before they Ultrasonic bath in chloroform were cleaned. The lipid substrates produced in this way had water contact angles of ⁇ 75 °.
• Example 4 Chip Production and Functionality Testing by means of RlfS All RifS analyzes were carried out using a BIAffinity System (Analytik Jena AG, Jena, Germany). In the functionality test, the detection of the complementary analytes was carried out by means of RrfS, whereby the ratio nonspecificity / specificity was quantified. In addition, the chip modifications were tested for regenerability and the cycle stability of the bond-active surface was tested. Finally, the kinetic parameters such as the dissociation constant K D were determined and compared with the literature values.
• the ligands (catcher molecules) streptavidin (Biomol, Hamburg, Germany), GFP binding protein (Chromotek, Kunststoff, Germany), protein G and C-reactive protein (CRP) were in the flow cell (dimension 6 mm x 1 mm x 125 ⁇ ) immobilized within the BIAffinity system on the lipidated glass transducers of Example 3. All buffers for immobilizations and measurements were degassed and filtered.
• anti-streptavidin antibodies and a biotinylated HRP were detected as complementary analytes by means of RlfS.
• the unspecificity was less than 20%, the chip could be regenerated into antibodies for the streptavidin / anti-streptavid system and the dissociation constant KD was consistent with the literature values.
• GFP could be detected as complementary analyte by means of RifS.
• the chip was regenerable.

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  • 2012-09-14 DE DE201210216378 patent/DE102012216378B4/de active Active
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