EP2593772A1 - Puce de mesure microstructurée pour la mesure optique de propriétés de membranes biologiques ou artificielles et procédé de fabrication de cette puce - Google Patents

Puce de mesure microstructurée pour la mesure optique de propriétés de membranes biologiques ou artificielles et procédé de fabrication de cette puce

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Publication number
EP2593772A1
EP2593772A1 EP11719758.2A EP11719758A EP2593772A1 EP 2593772 A1 EP2593772 A1 EP 2593772A1 EP 11719758 A EP11719758 A EP 11719758A EP 2593772 A1 EP2593772 A1 EP 2593772A1
Authority
EP
European Patent Office
Prior art keywords
measuring
layer
microstructured
silicon
measuring chambers
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.)
Withdrawn
Application number
EP11719758.2A
Other languages
German (de)
English (en)
Inventor
Guido BÖSE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanospot GmbH
Original Assignee
Nanospot GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Nanospot GmbH filed Critical Nanospot GmbH
Publication of EP2593772A1 publication Critical patent/EP2593772A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/168—Specific optical properties, e.g. reflective coatings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T156/00—Adhesive bonding and miscellaneous chemical manufacture
    • Y10T156/10—Methods of surface bonding and/or assembly therefor
    • Y10T156/1052—Methods of surface bonding and/or assembly therefor with cutting, punching, tearing or severing
    • Y10T156/1062—Prior to assembly

Definitions

  • the invention relates to a microstructured measuring chip for the optical measurement of properties of artificial or biological membranes with a lower, transparent carrier layer and at least one opaque main layer arranged thereon, which are designed as measuring chambers
  • the invention further relates to a method for producing the measuring chip and a holder for these measuring chips.
  • Membrane transport systems such as e.g. Transport proteins, channel proteins, secretory systems and
  • Membrane permeability selectively the passage through these membranes.
  • receptors mediate signals, e.g. an extracellular signal
  • Drug candidates can be evaluated.
  • you have to Membrane transport systems can be automatically characterized in high throughput, thus finding a drug candidate by statistically significant evidence of a change in the transport rate of a given
  • Membrane permeability is crucial for the availability of drugs in cells, as well as in the brain, as it requires the blood-brain barrier to be penetrated. In drug development, availability at the target site is thus a crucial property of potential drugs.
  • Effective cellular secretion is critical to the production of biopharmaceutical products such as antibodies, proteins and the like by cultures of producing eukaryotic and prokaryotic cells, e.g. Mammalian cells, ciliates, yeasts and bacteria. Since divergences in the production rates of the individual cells occur in such crops in spite of desired monoclonality, the finding and selection of high-producing cells are decisive for the production rate of the culture.
  • the global market for biopharmaceutical products is estimated at $ 70 billion in 2010.
  • Membrane receptors play a central role in the development of many economically important diseases, such as e.g. Allergies, neurological disorders, depression, pain, inflammation, diabetes, epilepsy, high blood pressure or asthma.
  • Allergies neurological disorders, depression, pain, inflammation, diabetes, epilepsy, high blood pressure or asthma.
  • the subgroup of protein kinases had a market share of $ 12.7 billion and a projected increase to $ 58.6 billion in 2010 (Biophoenix Consulting).
  • Receptor proteins such as G-protein coupled receptors (GPCR for short) possess extracellular regions for ligand binding, transmembrane
  • Receptor activation is a detection of the signal, so the
  • electrical measurements can be used to analyze transport rates of ions and charged particles. This process already has an application in higher throughput in the biotechnological and
  • RNA, DNA and proteins can not or only indirectly be measured by electrical methods.
  • Nuclear pore complex in core casings made from Xenopus Laevis has also been used to measure the transport of calcium ions through the a-hemolysin pore, which has been inserted directly into prefabricated, artificial lipid membranes, refolding from a denatured structure into a functional form.
  • a-hemolysin pore which has been inserted directly into prefabricated, artificial lipid membranes, refolding from a denatured structure into a functional form.
  • Polycarbonate filter or polycarbonate structures were used, the wells were used for the fluorescence measurement of transport rates using confocal laser scanning microscopy. This causes poor optical properties, i.a. due to divergences in refractive indices of polycarbonate and measuring buffer.
  • measuring chips with measuring chambers in translucent material known whose wells can be covered by an upper membrane or cells or tissue and thus closed measuring chambers are formed and the transport of substrate molecules through the membrane or the secretion of cells can be measured in the measuring chambers.
  • the membrane or the cells are clamped over the measuring chambers in the measuring chip, so that these
  • the measuring chip is suitable for analysis of permeability through artificial or biological membranes or cells.
  • optical measurements such as fluorescence measurements substrate molecules can be detected and quantified in the measuring chambers. It should as few substrate molecules outside the measuring chambers are excited to fluorescence, so as not to falsify the measurement result.
  • the metal layer contains a measuring chamber with a much smaller diameter than the wavelength of the radiated from below excitation light and thereby acts as a so-called zero-mode waveguide.
  • the excitation light thus does not penetrate into the depressions, but some of the light energy reaches the measuring chambers. Above the measuring chambers no substrate molecules are excited.
  • a measuring chip has numerous disadvantages. Due to their small dimensions, the volume of the measuring chamber with few zeptoliters is very small, so that they can not be used for transport operations. In addition, only the lower area of the measuring chamber can be used, since only there excitation occurs. As a result, the signal-to-noise ratio of the measurements is very poor and it requires a complex measuring device.
  • the object of the invention is to provide a measuring chip with which the properties of membranes or transport systems can be measured by means of commercially available, conventional measuring devices with higher measuring accuracy and higher throughput.
  • a microstructured measuring chip having a lower, transparent carrier layer and at least one substantially opaque main layer, which has recesses formed as measuring chambers with an upper opening and an inner side wall or a plurality of inner side walls, in that the
  • Measurements can be made of conventional fluorescence microscopes with the noncoherent light of a fluorescent lamp, but also laser scanning microscopes.
  • the measurement is made from below through the translucent carrier layer of the measuring chip. Therefore, it is permeable to the excitation light of a fluorescent lamp.
  • the excitation light for example, suitable
  • the measuring accuracy achievable with the measuring chip now depends to a considerable degree on the extent to which emission light is measured which is generated exclusively by fluorescence excitation within the measuring chamber. If, in addition, emissions are measured that are generated outside the measuring chamber, these are emissions that are the result of emissions
  • Measuring chambers are stimulated.
  • the side wall or the side walls of the measuring chambers are not smooth, but according to the invention have depressions and / or elevations. Smooth reflecting side surfaces would reduce the emissions from above the
  • Chambers continue to reflect to the camera, while the pits and bumps scatter the spurious emissions, thus minimizing propagation within the measuring chambers. In addition, a spread of the
  • Membrane or measuring fluid reduced. Overall, an undesirable optical detection of emissions of substrate molecules within and above minimized membrane and achieved a predominant detection of emissions of substrate molecules within the measuring chambers. This is also possible with conventional fluorescence microscopy.
  • Recesses and elevations alternate, that are formed like a groove and the grooves in the direction of the longitudinal axis of the measuring chambers a corrugated
  • the alternating recesses or elevations or grooves each have a periodic spacing of 0.1 to 0.6 ⁇ on.
  • the distance can also be a few nanometers to several micrometers.
  • the distance between the highest point of the elevations and the lowest point of the depressions is 20 to 1 10 nm, but can also be up to a few micrometers.
  • the measuring chambers each have the basic shape of a circular cylinder or a truncated cone. Because the
  • Measuring chambers are formed by depressions in the main layer, they themselves have no external shape.
  • the basic shape of the measuring chambers is therefore to be understood as meaning their hollow volume that is delimited by the main layer. In this sense, the sidewall of a
  • the grooves and bumps forming the grooves circulate the sidewall, i. both the
  • Measuring chambers and the grooves are approximately circular in section perpendicular to the longitudinal axis of the measuring chambers.
  • Such a corrugated surface structure can be achieved by the reactive silicon ion deep etching (DRIE, Bosch process) for the deep etching of silicon. This includes an alternating sequence of etching process and passivation step and thereby generates such a wave or comb structure of the side walls perpendicular to the etching direction.
  • Shape and Gap spacing varies with process settings and etch depth.
  • the grooves in the sidewalls of opaque, rather reflective material produce scattering of both the incident excitation light and the emission light in the measurement chambers, while smooth sidewalls would allow reflection of light longitudinally through the measurement chambers and thus propagation as in an optical fiber.
  • excitation light irradiated from the underside of the measuring chip is better shielded from an exit from the upper opening of the measuring chamber.
  • interference emissions above the measuring chambers are better shielded from passage from the bottom of the measuring chambers to the camera, and measurement accuracy is further improved overall.
  • the lower, translucent carrier layer consists for example of plastic or glass. It has been found that, in particular, borosilicate glass is suitable, which is produced by the float process or as a polished wafer.
  • the opaque, microstructured main layer with the wells serving as measuring chambers comprises metal, plastic or silicon.
  • silicon also includes silicon compounds. Silicon has the advantage that in the processing, ie the production of the wells, some known methods in the field of electronic microchips can be used.
  • a cover layer, preferably of silicon dioxide and / or metal, may be arranged on the main layer. The cover layer then has openings which are arranged above the openings of the measuring chambers. Preferably, in each case an opening in the cover layer is arranged above the opening of a measuring chamber.
  • the aperture of the openings of the cover layer is smaller than the aperture of the openings of the measuring chambers.
  • diaphragms are formed by the cover layer, which can partially shield the excitation light irradiated from the underside of the measuring chip when exiting the upper opening of the measuring chamber. Likewise, spurious emissions are shielded above the cover layer. This further improves the measurement accuracy.
  • the embodiment of the biological membrane measuring chip is suitable with a biologically predetermined transporter density. As the number of If transport proteins per area in the case of biological membranes can not be readily changed, the aperture of the cover layer opening can be selected and optimized with an unchanged measuring chamber volume such that preferably only one or only a few transport proteins lie above the cover layer opening. This allows more accurate measurements with a longer measurement duration.
  • the surface of the measuring chip may contain one or more chemically reactive and / or polar coatings, in particular poly-L-lysine and / or
  • Membrane direct or indirect, covalent or noncovalent to bind to the measuring chip.
  • Measuring chambers facing bottom of the cover layer and / or the top of the main layer may additionally each have an opaque coating, preferably of metal, in particular of gold or titanium. This is preferably done by means of known PVD methods (abbreviation for "physical vapor deposition") .
  • the coating has several advantages
  • Residual light transmission of the main layer is, this is through the
  • silicon is substantially opaque to wavelengths of visible light up to 600 nm.
  • silicon becomes increasingly permeable. This would be annoying if that
  • standardized thiol compounds such as ß-mercaptoethanol or
  • Mercaptopropionic acid but also components of a detection system of the Couple receptor activation.
  • the layer of silicon or silicon oxide does not bind these and can therefore be selectively modified by silanization. This allows a different modification of the measuring chambers and the top of the measuring chip, which is advantageous for certain measuring tasks.
  • the ratio of depth to diameter of the measuring chambers is greater than one, preferably greater than five, and particularly preferably ten to fifty, only the small portion of the excitation light irradiated parallel to the side wall of the measuring chamber can propagate through the upper opening of the measuring chamber.
  • the invention further comprises a holder for the above-described microstructured measuring chip.
  • the holder comprises a plate having a top, a bottom, and one or more top-fillable reservoirs.
  • the holder has, for example, a similar cuboid shape as commercially available microtiter plates and can also be used in a similar manner. In a preferred embodiment, it has standardized dimensions in terms of width, length and / or height that the ANSI standards for
  • Microplates or coverslips meet.
  • the reservoirs are continuous channels, i. they initially have no bottom but a lower opening.
  • reservoirs are first formed by a measuring chip attached to the underside of the plate. All measuring chips of the holder can also be first glued to a thin glass carrier in holder size and then sunk from below into the reservoirs, so that the glass carrier is glued under the holder.
  • the attachment can be made by a waterproof and -êtn adhesive in such a way that the measuring chambers point in the direction of the reservoir.
  • the chip is adhered with a UV-curing adhesive or adhesive film under the lower opening of a reservoir, the adhesive after adjustment by irradiation with UV light
  • Glass carrier a silicone adhesive, preferably Sylguard 184, proved to be suitable for sticking the measuring chips on the glass carrier.
  • the reservoirs form in this way for the microstructured measuring chips a container for a suitable measuring liquid.
  • the optical measurement is carried out from the underside of the holder through the translucent carrier layer of the measuring chips or additionally by glass carrier and adhesive as described above.
  • the invention also includes a method for producing microstructured measuring chips, in particular having the features described above.
  • a silicon wafer is used as the starting material or as a substrate, as it is used in microelectronics for the production of integrated circuits.
  • known methods of microstructuring such as
  • Photolithography and etching can be applied.
  • SOI wafer silicon on insulator wafer
  • SOI wafers are known from the prior art and consist of three layers: a lower silicon layer, an upper silicon layer and a so-called buried layer arranged therebetween, which has electrically insulating properties
  • electrical components made from an SOI wafer, such as integrated circuits have shorter switching times and lower power consumption because the buried layer reduces leakage currents.
  • the buried layer stops the etching process when an etchant is used that selectively attacks and dissolves only silicon.
  • an etchant an acid or a gas, preferably in the method of Deep Reactive Ion Etching (DRIE), can be used.
  • Coating such as titanium and / or gold, applied to the upper silicon layer.
  • the upper silicon layer of the silicon wafer with the etched measuring chambers is then connected to the carrier layer, preferably by anodic bonding, wherein the previous opening of the measuring chamber with the carrier layer as a bottom to its underside.
  • the lower silicon layer of the silicon wafer is removed, preferably by etching.
  • the buried layer stops the etching process when an etchant is used that selectively attacks and dissolves only silicon.
  • the buried layer is completely or partially removed, preferably by etching, using a special etchant that attacks and dissolves the buried layer, for example hydrofluoric acid.
  • a special etchant that attacks and dissolves the buried layer, for example hydrofluoric acid.
  • the buried layer is selectively photolithographically patterned at the locations where it covers the measuring chamber openings. The buried layer then forms the cover layer with the cover layer openings of the measuring chip.
  • Sawed silicon wafer in particular in sizes of 2 times 2 mm to 10 times 10 mm.
  • a particularly preferred size is 2.5 by 2.5 mm.
  • the measuring chips can be used individually or in particular glued to the holder described above. The invention will be exemplified with reference to a drawing
  • Figure 2 is a detail view of a measuring chamber of another
  • FIG. 3 shows the measuring chip from FIG. 1 with a lipid membrane
  • Figure 4 is a vertical section through another embodiment of the
  • Figure 5 is a detail view of a measuring chamber with cover layer and a
  • FIG. 6 shows a vertical section through a further embodiment of the invention
  • Figure 7 is a plan view of the measuring chip in a partial view
  • FIG. 8a shows a vertical section through a holder
  • Figure 8b is a plan view of the holder of Figure 8a; 9 shows a vertical section through another embodiment of the
  • Partial view a vertical section through an SOI wafer as in Figure 10 with etched measuring chambers; a vertical section through an SOI wafer as in Figure 1 1 with an upper, bonded support layer; a vertical section through an SOI wafer as in Figure 12 after removal of the lower silicon layer; a vertical section through an SOI wafer as in Figure 13 after turning over; and a vertical section through an SOI wafer as in FIG. 14 after the partial removal of the hidden layer.
  • FIG. 1 shows a partial view of a measuring chip 1 according to the invention
  • the measuring chip 1 consists of interconnected layers or materials. As a base, it has a lower, translucent carrier layer 10 made of floated or polished borosilicate glass. Proven have "Borofloat 30" or "Pyrex". The thickness of the carrier layer 10 is about 140-200 ⁇ , although it may also be thicker or thinner. The carrier layer 10 is permeable to excitation light 80 or emitted fluorescent light 81. On the carrier layer 10, a substantially opaque main layer 20 of silicon is arranged, which forms the top 17 of the measuring chip 1. It should be noted for the sake of completeness that the main layer 20 of silicon oxidizes externally in the air and so a superficial, but only nanometer thick
  • Silicon dioxide layer forms.
  • the main layer 20 is firmly connected to the carrier layer 10 by anodic bonding.
  • the main layer 20 has circular cylindrical, continuous
  • the depressions thus form measuring chambers 30 with a circular cylindrical hollow volume.
  • the one, inner side wall 26 of Messkannnnern 30 is so to speak, through the lateral surface of the
  • Circular cylinder formed and the circular upper opening 25 through his
  • each metering chamber 30 is formed by the top surface of the translucent support layer 10.
  • the measuring chambers 30 have a depth 33 of 10 to 30 ⁇ , but there are also depths of a few nanometers to millimeters possible.
  • the diameter 31 of the measuring chambers 30 is about 1 ⁇ , but there are also diameter 31 of a few nanometers to a millimeter possible.
  • the distance 32 between the longitudinal axes of the individual measuring chambers 30 is 2.5 ⁇ to 4 ⁇ ; but it is also distances 32 from a few nanometers to a millimeter possible.
  • each metering chamber 30 is not smooth, but has alternating recesses 27 and protrusions 28 forming a corrugated surface structure.
  • the period of the waves is on the order of 100-600 nm, but can also be a few nanometers to a few micrometers.
  • FIG. 2 shows a detailed view of a measuring chamber of another
  • the ratio of depth 22 to diameter 31 of the measuring chambers 30 is about 1 to 10. This can only substantially parallel to
  • Measuring chamber side wall 26 radiated excitation light 80 through the opening of the measuring chamber.
  • the side walls 26 of the measuring chambers 30 partially have a corrugated surface structure extending through in the direction of
  • Noise emissions 82 are reduced within the measuring chambers 30 or out of the measuring chambers 30 (this is undesirable because only substrate molecules 60 within the measuring chambers 30 are to be excited and detected). This effect is illustrated by the non-coherent bundle of excitation light 80 (not shown) fluorescent lamp which is scattered or deflected by the recesses 27 and elevations 28. This will cause a spread of
  • FIG. 3 shows a section of a measuring chip as in FIG. 1.
  • a lipid membrane 40 is shown which is used in measurements with the measuring chip 1.
  • the lipid membrane 40 is applied to the top 17 of the measuring chip 1, so that at least some of the measuring chamber openings 25 are covered and closed by the lipid membrane.
  • the membrane 40 has been produced from artificial proteol liposomes, which upon addition can spontaneously fuse with the chip surface and thus form the membrane 40.
  • the membrane 40 contains individual transport proteins 50 for transporter analyzes, for example
  • Substrate molecules 60 are added which either fluoresce intrinsically or are covalently labeled with a fluorescent dye.
  • Substrate molecules 60 through the introduced into the membrane 40 transport proteins 50 in the measuring chambers 30 of the measuring chip 1 is specific to the
  • Transport protein 50 and can be detected by detecting the fluorescence in the
  • Measuring chambers 30 are measured. This allows conclusions to be drawn about specific parameters such as transport rates and permeability and allows, for example, the evaluation of drug candidates for drugs.
  • Measurement takes place in an aqueous medium, i. Measuring chambers 30, membranes 40, proteins 50 and substrate molecules 60 are of a type (not shown).
  • a measuring liquid for example a suitable saline
  • the measuring chip 1 forms the bottom of a reservoir 203, which is filled with measuring liquid above the measuring chip 1.
  • the measurement is carried out for example by a fluorescence microscope (not shown), which provides both a fluorescent lamp or a laser for the excitation light 80 to excite the fluorescence of the substrate molecules 60 as well as a magnifying optics.
  • the excitation light 80 becomes (shown in phantom) approximately orthogonally radiated from below through the transparent carrier layer 10 in the measuring chambers 30 to excite the transported from the top of the measuring chip 1 through the membrane 40 into the measuring chambers 30 substrate molecules 60 for fluorescence.
  • Substrate molecules 60 emitted fluorescent emissions 81 radiate from the measuring chamber 30 through the translucent
  • Carrier layer 10 and are measured by a suitable camera or detector (not shown) of the fluorescence microscope.
  • the wavy surface structure reduces unwanted propagation of excitation light 80 out of the measuring chambers 30. Nevertheless, should a certain residual portion of the excitation light 80 radiate through the measuring chamber 30 and through the membrane 40, then undesirably also the substrate molecules 60 above the
  • Spurious emissions 82 from. Due to the corrugated surface structure 27, 28, the propagation of the interference emissions 82 above the measuring chip 1 through the
  • FIG. 4 shows a vertical section through another, preferred embodiment of the measuring chip 1, which essentially corresponds to that shown in FIG. 1 but has an additional covering layer 12.
  • the cover layer 12 is on the
  • Main layer 20 is arranged.
  • the cover layer has openings 14, which are arranged above the openings 25 of the measuring chambers 30.
  • an opening 14 in the cover layer 12 is centrally above the opening of a
  • Measuring chamber 30 is arranged.
  • the aperture of the openings 14 of the cover layer 12 is smaller than the aperture of the openings 25 of the measuring chambers 30.
  • Transporter density is suitable. Since the number of transport proteins 50 per area can not be changed as in artificial membranes 40 can unchanged volume of the measuring chamber 30, the aperture of the cover layer opening 14 are selected and optimized so that preferably only one or only a few transport proteins 50 are above the cover layer opening 14.
  • Measurement accuracy can be increased by the cover layer 12, because fewer substrate molecules per unit time are transported into the measuring chamber 30 and it can be time-resolved measurements perform, which is due to high
  • FIG. 5 shows, in a vertical section, a detailed view of a further embodiment of the measuring chip 1 with a measuring chamber 30 with covering layer 12 of silicon dioxide and an additional opaque coating 21 made of titanium and / or gold. If gold is used, titanium serves as a bonding agent.
  • the components of the measuring chip 1 with metal coating 21 are shown in FIG. 6 by means of thicker line thicknesses. These are the side walls 26 of the measuring chambers 30, the underside 16 of the cover layer 12 facing the measuring chamber 30 and the underside 24 of the main layer 20 resting on the carrier layer 10.
  • the metal coating 21 has several advantages. On the one hand, translucent silicon dioxide can be used as cover layer 12. This has advantages in the production of the measuring chip 1 (see below). Although the main layer 20 of silicon is substantially opaque to wavelengths of visible light up to 600 nm. For deep red and infrared light, however, silicon becomes increasingly permeable. This would be annoying if the excitation light 80 or interference emissions 82 (not shown) are in this wavelength range. However, titanium and gold are far into the infrared wavelength range
  • shutters 12 are formed by the coated cover layer, which are irradiated from the underside of the measuring chip 1
  • Metal coating 21 therefore improves the measurement accuracy.
  • the same effect can be achieved by a metal coating above the cover layer 12.
  • Another advantage is that the metal coating 21 can be contacted and used as an electrode for electrical measurements or excitations (not shown). The metal coating 21 can in this way for
  • the measuring chip 1 can be used in such a way that the impedance of a membrane 40 stretched over it or cells (not shown) can be measured. As a result, the tightness of membranes 40, cell or tissue layers can be determined.
  • the measuring chip 1 can also be used to generate an electric field by means of the gold coating 21, in particular for controlling voltage-sensitive transport systems.
  • voltage dependent ion channels i. Ion channels that open or close at a certain threshold of membrane voltage.
  • the measuring chip 1 By changing the applied electric field so functional switching processes can be triggered, which result in a change of the transport 70 of substrate molecules 60 via a membrane 40 (not shown). The substrate molecules 60 can then be detected in the measuring chambers 30 by means of fluorescence.
  • Another application of the measuring chip 1 is that the upper
  • Covering layer 12 of the measuring chip 1 is covered with a lipid membrane
  • pore proteins for example ion channels.
  • Messchipoberseite 17 of the measuring chip acting electrode applied an electric field. Another electrode in the solution above the membrane creates a membrane potential. , The applied voltage leads to the activation of the
  • the illustrated embodiment of the measuring chip 1 thus has the advantage that biological transport systems are switched electrically functional and at the same time the transport generated thereby can be measured optically by means of fluorescence via the membranes 40. Yet another advantage results in combination with a chemically reactive or polar coating (not shown). To a gold coating 21 on the side wall 26 of the measuring chambers 30 can be standardized
  • Thiol compounds such as ⁇ -mercaptoethanol or mercaptopropionic but also components of a detection system of receptor activation are bound.
  • Silicon oxide does not bind these and can therefore be selectively modified by silanization. As a result, a different modification of
  • FIG. 5 also shows the effect of the cover layer 12. Openings 14 of the cover layer 14 are arranged centrally above the openings 25 of the measuring chambers 30. If a bundle of excitation light 80 is irradiated from below, it is partially dimmed by the cover layer 12 or reflected by the gold coating 21 on the underside 16 of the cover layer 12 and only reaches the area above the measuring chip 1 with reduced intensity, which results in the measurement accuracy elevated. In the embodiment shown, interference emissions from above the measuring chip 1 penetrate the light-permeable covering layer 21
  • FIG. 6 shows a vertical section through a further embodiment of the invention
  • a frusto-conical shape is understood to mean that the lower diameter 35 of the measuring chambers 30 decreases from the measuring chamber bottom 18 of the carrier layer 10 as far as the upper measuring chamber opening 25. In this case, excitation light 80 irradiated from the underside of the measuring chip 1 becomes better against an escape from the upper opening 25 of the measuring chamber 30
  • FIG. 6 shows that, due to the corrugated surface structure of the side walls 26 in connection with the upwardly tapering measuring chamber 30, the excitation light 80 radiated from below does not leave the measuring chamber opening 25 for the most part. Also, spurious emissions 82 (not shown) above the main layer 20 through the smaller upper opening 25 better shielded. Due to the synergistic effect of both features, the measurement accuracy achievable with the measuring chip 1 is further increased considerably.
  • FIG. 7 shows a plan view of the measuring chip 1.
  • the drawing shows in a partial view of the measuring chip 1, the measuring chambers 30, 30 'again, which are arranged in the form of an array.
  • the measuring chambers 30 shown have one
  • Diameter 31 of 1 ⁇ but are also possible embodiments with diameters from a few nanometers to several hundred
  • the distance 32 of the centers of the measuring chambers is 2.5 to 4 ⁇ , but are also possible a few nanometers.
  • the measuring chambers 30 have the basic shape of a circular cylinder. However, as FIG. 8 shows, the measuring chip 1 also has differently shaped, as shown in FIG.
  • measuring chambers 30 are each provided with an oval measuring chamber 30 ', both in the longitudinal and in the transverse direction of the array.
  • These measuring chambers 30 ' serve as visual markings recognizable by the camera and enable a simplified, unambiguous assignment of the position of the measuring chambers 30 as well as a manual or automated correction of lateral displacements of the measuring chip 1 during the measurements.
  • Figure 8a shows a vertical section through a holder 200 for the above
  • the holder 200 comprises a cuboid plate with reservoirs 203 which can be filled through upper openings 205, preferably similar to commercially available microtiter plates, but also similar chambers in length and width suitable for slide format.
  • the holder 200 preferably also has the standardized height of a microtiter plate. in the
  • the reservoirs 203 are continuous channels, i. they have no bottom but a lower opening 210.
  • the bottom of the reservoirs 203 is first secured by a to the bottom 203 of the plate, for example, with a UV-curing adhesive
  • Measuring chip 1 is formed, wherein the adhesive is cured after an adjustment of the measuring chip 1 by means of UV light.
  • the reservoirs 203 form in this way with the microstructured measuring chips 1, a chamber with a desired
  • Measuring liquid can be filled.
  • the measuring chip 1 is arranged so that its upper side 17 faces the reservoirs 203 with the measuring chamber openings 25 (not shown) so that the measuring chambers 30 can be filled by the reservoirs 203.
  • the optical measurement takes place from the lower side 202 of the holder 200 through the lower, transparent carrier layer 10 of the measuring chips 1.
  • the volume of the reservoirs 203 is increased by increasing the diameter thereof in its lower portion upwards, i. the diameter of the upper opening 205 of the reservoir 203 is greater than the diameter of its lower opening 210, which is slightly smaller than the surface of the measuring chips 1, so that they can be glued sealingly under the lower opening 210.
  • FIG. 8b shows a plan view of the upper side 201 of the holder 200 of FIG. 9.
  • the holder 200 has the length and width of a commercial slide. For example, 16 reservoirs 203 are provided with measuring chips 1 glued underneath.
  • the distance between the centers of the upper openings 205 is 9 mm and the diameter of the upper openings 205 6 mm, wherein the diameter tapers down, so that the lower opening 210 has a diameter of 2 mm.
  • Under the lower opening 210 is a
  • square measuring chip 1 side lengths of 3 x 3 mm glued.
  • FIG. 9 shows a vertical section through a further, preferred embodiment of the holder 200b.
  • the holder 200b like the one shown in FIG. 8a, comprises a parallelepiped plate having reservoirs 203 which can be filled through upper openings 205.
  • the bottom of the reservoirs 203 is formed by a cover glass 215 of about 50-200 ⁇ m thickness.
  • cover glass 215 of about 50-200 ⁇ m thickness.
  • the entire cover glass 215 is then glued under the holder 200b and seals off all the reservoirs 203, the measuring chips 1 being sunk in the reservoirs 203 become.
  • the reservoirs 203 form in this way with the cover glass 215 a chamber which can be filled with a desired measuring liquid.
  • the lower opening 210 is somewhat larger than in the case of the holder 200 shown in FIG. 8 a.
  • the cover glass 215 is translucent, so that the optical measurement can be made from below through the cover glass 215 ,
  • FIG. 10 shows a vertical section through an SOI wafer 300 known from the prior art in a partial view. It is used in the prior art as a starting material or substrate for the production of electronic components and integrated circuits. In the present invention, however, it serves as a starting material or substrate for the production of the microstructured measuring chips 1.
  • known methods for producing electronic components such as photolithography and etching are advantageously used.
  • the known SOI wafer 300 is composed of a sandwich of three interconnected layers: a bottom thick opaque silicon layer 31 1, an upper thin opaque silicon layer 320, and a very thin so-called buried layer 312 disposed therebetween (The English term is "buried layer”), the electrically insulating
  • the upper, thin, opaque silicon layer 320 is first deposited by photolithography and suitable etching techniques such as DRIE (Deep Reactive Ion Etching, Bosch Process) or wet chemical etching
  • Chambers serving recesses 30 introduced.
  • alternating alternating recesses 27 and elevations 28 form in the side walls 26, which produce a substantially corrugated or corrugated surface structure, as a result of the usual alternating etching and passivation steps.
  • Etching employs etchants that dissolve only silicon but no silica. Therefore, the etching advantageously takes place only up to the buried silicon dioxide layer 312, which effectively acts as a "stop layer” and stops the etching process
  • Silicon layer 320 If desired, a metallization of, for example, titanium or gold may now be applied to the top silicon layer.
  • Borosilicate glass mounted on top silicon layer 320 by anodic bonding. Then, the lower silicon layer 31 1 is removed by etching, as shown in FIG. Also in this process step, the etching is advantageously carried out only up to the buried silicon dioxide layer 312, which can bring the etching process to a standstill.
  • FIG. 14 shows that the SOI wafer 300 processed in this way is subsequently turned over and is in an "upside down" position, whereby the carrier layer 10 becomes the lowermost layer and the upper silicon layer 320 of the SOI wafer 300 becomes the later main layer 20 of the measuring chips 1.
  • the originally buried layer 312 of the SOI wafer 300 is the uppermost layer and forms the later covering layer 12 of the measuring chips 1.
  • the buried layer 312 forming the covering layer 12 of the measuring chip 1 is photolithographically and with suitable etching methods partially structured or completely removed, so that the apertures 14 acting as apertures are formed, which are preferably arranged centrally above the measuring chambers 30. This is illustrated in FIGURE 15, which corresponds to FIGURE 4.
  • the buried Layer 312 may also be completely removed, thereby producing an embodiment of measuring chip 1, as shown in FIG.
  • measuring chips 1 are sawn from the SOI wafer.
  • the measuring chips 1 can be used singly or glued under the holder 200 described above, as shown in FIG.

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Abstract

L'invention concerne une puce de mesure (1) microstructurée pour la mesure optique de propriétés de membranes (40) biologiques ou artificielles, cette puce comportant une couche support (10) inférieure transparente et au moins une couche principale (20) opaque qui est disposée sur la couche support et qui comporte des évidements (30) formant des chambres de mesure présentant une ouverture (25) supérieure et une ou plusieurs parois latérales (26) internes. L'invention vise à améliorer une puce de mesure (1) de manière à pouvoir mesurer des systèmes biologiques avec une précision et un rendement accrus. A cet effet, la ou les parois latérales (26) des chambres de mesure (30) présentent des creux et/ou des reliefs (28). L'invention porte également sur un support (200) pour ces puces de mesure (1) et sur un procédé de fabrication de ces puces de mesure (1) à partir d'une tranche de silicium (300).
EP11719758.2A 2010-07-12 2011-03-10 Puce de mesure microstructurée pour la mesure optique de propriétés de membranes biologiques ou artificielles et procédé de fabrication de cette puce Withdrawn EP2593772A1 (fr)

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DE102010036344 2010-07-12
PCT/DE2011/075041 WO2012006995A1 (fr) 2010-07-12 2011-03-10 Puce de mesure microstructurée pour la mesure optique de propriétés de membranes biologiques ou artificielles et procédé de fabrication de cette puce

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WO (2) WO2012006995A1 (fr)

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JP2015194442A (ja) * 2014-03-31 2015-11-05 株式会社ニコン 支持装置
EP3305721B1 (fr) * 2015-06-08 2023-10-18 Japan Science And Technology Agency Réseau de micro-chambres à haute densité et procédé de mesure l'utilisant
DE102020100244A1 (de) 2020-01-08 2021-07-08 X-FAB Global Services GmbH Verfahren zur Herstellung eines Membran-Bauelements und ein Membran-Bauelement

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DE102011051723A1 (de) 2012-03-01
WO2012006995A1 (fr) 2012-01-19
WO2012025110A1 (fr) 2012-03-01
US20140134711A1 (en) 2014-05-15

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