EP2142911A2 - Biochip for carrying out a fluorescence analysis of individual transporters - Google Patents

Biochip for carrying out a fluorescence analysis of individual transporters

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Publication number
EP2142911A2
EP2142911A2 EP08748708A EP08748708A EP2142911A2 EP 2142911 A2 EP2142911 A2 EP 2142911A2 EP 08748708 A EP08748708 A EP 08748708A EP 08748708 A EP08748708 A EP 08748708A EP 2142911 A2 EP2142911 A2 EP 2142911A2
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EP
European Patent Office
Prior art keywords
biochip
membrane
recesses
carrier
transport
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Withdrawn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
EP08748708A
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German (de)
French (fr)
Inventor
Stefan Hummel
Matthias Pirsch
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.)
Synentec GmbH
Original Assignee
Synentec GmbH
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Filing date
Publication date
Application filed by Synentec GmbH filed Critical Synentec GmbH
Publication of EP2142911A2 publication Critical patent/EP2142911A2/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • the invention relates to a biochip for the optical measurement of the properties of individual transport systems.
  • Biological membranes separate cells from the outer medium and the individual cell compartments of the cells. Transport systems such as transport proteins and channels selectively control the mass transfer through these membranes. Dysfunctions of these transporters and channels are responsible for many common diseases.
  • membrane transporters were the most abundant target group. There are at least 1,302 transporter pharmaceuticals, both imported and under development, in the portfolios of 326 companies worldwide. Overall, more than 100 transporter targets are currently being researched by the pharmaceutical companies, which shows what immense economic importance they have.
  • the object of the invention is therefore to propose a device by means of which the properties of transporter molecules can be measured with high measurement accuracy and high throughput.
  • a biochip for the optical measurement of the properties of individual transport systems, which consists essentially of a transparent support and a plurality of recesses open at the top, wherein the Biochip is formed such that its openings are covered by a membrane, and so closed measuring chambers are formed and the transport of substrate molecules via the membrane in the wells is detectable.
  • the membrane is clamped over the recesses in the biochip, so that they are closed.
  • Evaluation yields parameters such as the transport rate, the conclusions about the transport protein / channel or e.g. allow an influence of a drug candidate. Both the method and the evaluation can be automated and used in high throughput.
  • a conductive layer preferably made of metal, is applied to the biochip, then it can additionally be used as an electrode, preferably for measurements of the impedance spectroscopy or else for the application of an electric field.
  • a second electrode in the solution over the membrane, e.g. statements about the electrical tightness of an applied lipid layer or a cell layer are taken by means of impedance spectroscopy. This can be used as a quality control for the quality of the lipid layer or even evaluation of the viability of the cells.
  • An applied electric field can be used for the control of voltage dependent channel proteins, e.g. To switch an ion channel to the open state and then perform as described a transport measurement by means of fluorescence measurement of an ion-dependent fluorescence indicator.
  • Proteo liposomes so artificial, hollow membrane vesicles containing inserted into the membrane transport proteins to use. These can either be coupled directly to the activated surface of the biochip or applied by fusion with a preformed lipid membrane.
  • the vesicle is reshaped to a membrane containing the transporter, which closes the measuring chambers formed in the wells in the biochip and thus enables a fluorescence measurement for characterizing the transporters and determining the transport rates.
  • the carrier is made of a material with a high
  • Refractive index such as glass, silicon or silicon dioxide.
  • refractive index is higher than the refractive index of the measuring solution used, total reflection and thus an evanescent field at the phase boundary of the material and the measuring solution can be generated by irradiating the excitation light at an angle and used for fluorescence detection.
  • the carrier (10) may have one or more layers (20) connected to its top.
  • the upwardly opened depressions (30) are provided in the layer or layers (20).
  • the diameter of the recesses is smaller than the wavelength of the excitation light, so that the recesses are formed as zero-mode waveguides.
  • the intensity of the excitation light then decreases exponentially within the measuring chamber, whereby a highly selective excitation is possible.
  • the biochip on the upper side is substantially opaque. As a result, the excitation light is shielded from the membrane. Fluorescent substrate molecules that are located in the membrane or above the membrane, ie outside the
  • a particularly suitable metal is gold because it is chemically inert, can be securely bonded to the substrate, and also has suitable light-reflecting properties. Titanium is also suitable.
  • the metal layer is firmly connected to the carrier by means of a bonding agent. It has been found that a metal, in particular chromium or titanium, is very well suited as adhesion promoter.
  • An improvement in the measurement accuracy can be achieved in that the metal layer is designed to be reflective on its underside in order to mirror the excitation light and thus excite the substrate molecules several times.
  • the opening of the recess may be partially covered by the overlying metal layer by having the opening in the metal layer selected to be smaller than the recess opening.
  • the metal layer lying over the openings can be used as an electrode for electrical measurements of the membrane or can also be used to generate an electric field. If the layer consists of silicon dioxide, fluorescence detection of the transport substrates in the recesses of the layer is possible.
  • the layer bearing the transparent support is made of a fluoropolymer, such as Teflon or Cytop, then this allows the detection of the fluorescence in the measuring chambers, e.g. using confocal laser scanning microscopy.
  • a fluoropolymer such as Teflon or Cytop
  • a further improvement can be achieved in that the diameter of the recesses decreases continuously from the bottom to the top, so that the recesses have approximately a conical shape.
  • the larger diameter of the chambers towards the carrier then make it possible to detect the fluorescence in the measuring spaces thus formed with greater accuracy.
  • the coupling that is to say the fixation of the biological membranes or artificial vesicles to the biochip, can take place in such a way that its surface has linker molecules which are in particular amino-reactive and / or lipid derivatives and which bind covalently or noncovalently to suitable constituents of the membrane.
  • the membrane has one or more proteins, in particular pore, channel or carrier proteins, whose transport activity is detected via the vesicle membrane.
  • Another application of the biochip is the characterization of production cell lines for recombinant proteins and antibodies.
  • cells or cell components are measured for the production of recombinant proteins or antibodies.
  • the cells are bound to the biochip, so that they close with their membrane, the wells of the chip. It is also possible to grow cells on the biochips.
  • Upon secretion of the produced proteins into the measuring chambers becomes generates a fluorescence signal via a reporter system. This fluorescence signal provides information on the amount of recombinant protein or antibody generated and thus allows the discovery of many producing cells that can be used for the biotechnological production of these proteins and antibodies.
  • the membranes used in the measurement may be biological or artificial lipid membranes. If biological membranes are used, particularly natural measuring conditions result.
  • the measurement is with a vesicle membrane containing reconstituted transporter molecules therein.
  • a vesicle membrane containing reconstituted transporter molecules therein. This allows fast, reproducible measurements. By embedding in the vesicle membrane, the transporter protein also regains its functional conformation.
  • the membrane stretched over a depression contains as few as possible, preferably one to three, transporter molecules.
  • the detection of the substrate transported by the transporter molecules is made possible by the fact that the substrate molecules fluoresce, preferably by being bound to a fluorescent dye, but also by binding to a substrate-dependent fluorescence indicator, e.g. for the measurement of ion currents.
  • the fluorescent substrate molecules are transported by the transporter molecule across the membrane into the wells of the biochip. There they are detected by means of a suitable fluorescence detection device. A particularly accurate measurement is made by the detection device measuring the fluorescence in a confocal plane within the well.
  • a further improvement in accuracy is achieved in that the diameter of the recesses, taking into account the wavelength of the excitation light, is selected such that an evanescent field is generated, which is used for fluorescence detection.
  • an evanescent field is generated by irradiating the excitation light at a total reflecting angle and thus used for fluorescence detection.
  • a layer is electrically conductive and designed as an electrode so as to electrically measure or excite the membrane.
  • a suitable layer may be, for example, the metal layer of gold arranged above the carrier.
  • the layer can thus additionally be used as an electrode for characterizing the electrical properties of membranes, cell layers or the transport systems present in the membrane.
  • the biochip can be used in such a way that the impedance of the membrane or epithelial cell layer stretched across the biochip is measured with transport systems, for example transport proteins. As a result, the tightness of the membrane can be determined.
  • the biochip can also be used by means of the electrode in addition to generate an electric field, in particular for the control of voltage-sensitive transport systems.
  • These are, for example, voltage-dependent ion channels, ie ion channels which open at a certain limit value of the membrane voltage or shut down.
  • By changing the applied electric field so functional switching processes can be triggered, which have a change in the transport of substrate through the membrane result.
  • the transport substrate can then be detected in the wells by means of fluorescence indicators.
  • an exemplary application of the biochip is that the top metal layer of the biochip is covered with a lipid membrane containing ion channels.
  • an electric field is applied to the electrically conductive layer, ie the electrode.
  • the applied voltage leads to the activation of the ion channels. This creates an ion current across the membrane into the wells, which is then detected quantitatively by fluorescence.
  • the proposed biochip thus surprisingly has the additional advantage that it can switch biological transport systems electrically functional and at the same time be able to measure the transport generated thereby via the membrane optically by means of fluorescence.
  • FIG. 1 shows a vertical section of the biochip according to the invention
  • FIG. 2 shows a vertical section as in FIG. 1 with a vesicle
  • FIG. 3 shows a vertical section as in FIG. 2 with a resting biological cell
  • Figure 4 is a plan view of an array of the biochip
  • FIG. 5a shows a detail view of the biochip with a depression in vertical section
  • Figure 5b is a detail view of the biochip with a cone-shaped recess of the biochip in vertical section and
  • Figure 6 is a detail view of a preferred embodiment of the biochip with a recess in vertical section.
  • FIG. 1 shows a vertical section through the biochip according to the invention.
  • the biochip 1 consists of a carrier 10, which is transparent to the excitation light or the fluorescent light. At his
  • the biochip 1 which serve as measuring chambers for the detection of a substrate 60.
  • the biochip 1 consists of a composite of different materials.
  • the basis is the optically transparent carrier 10 made of cover glass.
  • a layer of silicon dioxide 20 is arranged on the top of the carrier.
  • a layer of titanium is applied, which serves both as a reflector for the excitation light 80 and as a bonding agent for a further layer of gold.
  • the gold layer can be contacted and used as an electrode.
  • the three layers 20 contain through recesses 30 through which an upwardly open measuring chamber is formed in each case.
  • a membrane 40 is applied for the measurement, so that the measurement spaces 30 are closed.
  • the membrane 40 can be made from artificial proteo-liposomes 5 which contain transport proteins or pore proteins as a transport system.
  • the membrane 40 the Cell membrane of production cell lines for recombinant proteins or antibodies.
  • the membrane 40 contains transport systems 50, such as transport proteins or pore proteins.
  • transport systems 50 such as transport proteins or pore proteins.
  • transporters of the ABC transporter group which are relevant for many diseases, such as e.g. adrenoleukodystrophy ABCD 1 transporter with fatty acids as substrate or e.g. the glutamate transporter with the substrate glutamate, whose metabolism is disturbed in mental illness.
  • Fluorescence method detectable transport substrates 60 added. This is made possible, for example, by covalently marking the substrate with a fluorescent dye.
  • the transport 70 of the transport substrates through the transport systems 50 contained in the membrane 40 into the recesses 30 of the biochip is specific to the transport system 50 contained and can be quantified by fluorescence measurements in the measurement spaces 30. This makes it possible to draw conclusions about parameters specific to the transport system 50, such as transport rates and permeability, and thus the evaluation of drug candidates or the production rates of production cell lines.
  • the biochip may consist of a fluoropolymer 20 such as Teflon or Cytop, which contains the measurement spaces 30 and is applied to a light-transmissive carrier 10. This allows the detection of fluorescence in the measurement spaces, e.g. using confocal laser scanning microscopy.
  • a fluoropolymer 20 such as Teflon or Cytop
  • the biochip can also consist of a metal layer 20, into which the holes 30 are introduced, and which are applied to a light-transmitting carrier 10. If the diameter of the Holes 30 a certain size in the nanometer range, so the incident light can no longer penetrate completely into the measuring chambers, instead forms an evanescent field at the transition of the carrier and filled with measuring solution measuring space. The pits then represent "Zero Mode Waveguides" and thus allow the detection of the fluorescence in the measuring chambers formed.
  • Another way to make the biochip is to anisotropically etch conical holes 30 in silicon dioxide 20 and then apply this to a permeable support 10. The larger diameter of the holes towards the carrier 10 then permits detection of the fluorescence in these depressions.
  • the biochip may be formed by forming recesses 30 in a high refractive index material, such as a glass sheet. Glass 10 + 20, refractive index 1.53 can be produced. This refractive index is significantly higher than that of the measuring solution located in the measuring chambers 30 with a refractive index of 1.33. If the excitation light is irradiated obliquely from below, an evanescent field is generated at a transition from the carrier to the measurement solution at total reflection of the light, which can be used to detect the fluorescence in the measuring chambers 30.
  • a high refractive index material such as a glass sheet. Glass 10 + 20, refractive index 1.53 can be produced. This refractive index is significantly higher than that of the measuring solution located in the measuring chambers 30 with a refractive index of 1.33. If the excitation light is irradiated obliquely from below, an evanescent field is generated at a transition from the carrier to the measurement solution at total reflection of the light, which can be used to detect the flu
  • FIG. 2 shows a vertical section as in FIG. 1 with a vesicle (5). Pore-forming proteins (50) are reconstituted in the vesicle membrane.
  • FIG. 3 shows a vertical section as in FIG. 2 with a resting biological cell 15. This may be a complete cell 15 or just a part thereof. The cell extends over several recesses 30 and covers them. This makes it possible to measure under natural biological conditions.
  • FIG. 4 shows a plan view of an array 36 of the biochip 1. This is formed by virtue of the fact that four depressions 30, which are square in plan view, are arranged close to one another and thus form a group 35. The group 35 has a length c or width d of about 100 microns. Sixteen recessed groups 35 or sixty-four recesses 30 are arranged in each case to form an array 36 which has a length a or width b of about 500 ⁇ m in each case.
  • FIG. 5a shows a detail view of an embodiment of the biochip 1 with a depression 30 in vertical section.
  • a metal layer of gold is applied to a carrier 10 made of cover glass by means of a bonding agent made of chromium or titanium (not shown).
  • the measuring chamber 30 is thus formed in this embodiment only through the opening 31 in the metal, while the glass carrier 10 itself has no recess.
  • FIG. 5 b shows a similar embodiment as in FIG. 5 a, but the metal layer 20 has a conical or conical depression 30.
  • the opening 31 also has a diameter of 60 to 120 nm on its upper side, but widens downwards. This increases the measurement accuracy because the measurement chamber 30 contains more substrate 60 (not shown) and thus the signal / noise ratio is improved.
  • FIG. 6 shows a detailed view of a preferred embodiment of the biochip 1 with a depression 30 in vertical section.
  • a further metal layer of gold is applied by means of an adhesion promoter made of chromium or titanium (not shown) to a carrier 10 made of cover glass and a layer of silicon dioxide 20 connected thereto.
  • the two metal layers together have a thickness of about 100 nm.
  • the silicon dioxide and metal layers are provided with a layer opening 31 and a continuous recess 30 having a diameter of 200 nm.
  • the pitch is 500 nm. For the measurement of cellular membranes, the pitch is 1 to 2.5 ⁇ m.
  • the measuring chamber is formed by the depression 30 within the layer
  • the recess has a length e of about 1 .mu.m and an opening diameter 31 of about 200 nm.
  • the thickness f of the metal layer is preferably about 100 nm, the diameter of the layer opening 21 about 200 nm.
  • an advantage of this embodiment is that the measuring space formed in the glass carrier 10 by the depression 30 has a greater extent in the vertical direction.
  • the substrate molecules 50 (not shown) transported via the membrane are further removed on the average from the membrane and thus from the non-transported substrate molecules 50.
  • only the substrate molecules 50 below the lipid membrane (not shown) should be excited to fluoresce, facilitated by the greater spatial distance. This increases the signal / noise ratio.
  • the signal-to-noise ratio can be further improved by covering the upper recess opening 31 in part by the metal layer 20.
  • the excitation light is thus effectively shielded from the non-transported substrate molecules 50 (not shown) above the membrane.
  • FIG. 6 shows a schematic representation of the beams 80 of the excitation light.
  • a parallel light beam 80 is obliquely angled into the bottom of the glass carrier 10.
  • the beam path is arranged as in a commercially available TIRF microscope.
  • the beams 80 are reflected by the metal layer 20 and repeatedly irradiate the measuring volume 30 with the sample 60 (not shown).
  • the signal excitation is amplified many times, which further improves the measurement accuracy considerably.
  • the evanescent wave forming next to the excitation light is not shown in FIG. Due to the oblique incidence and thickness of the metal layer 20, the diameter is insufficient for zero mode excitation, which is desirable for signal suppression.

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  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • General Physics & Mathematics (AREA)
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  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention relates to a biochip (1) for optically measuring the properties of individual transporter systems (50). The aim of the invention is to increase the measuring accuracy and the throughput with which the properties of transporter molecules (50) can be measured. To achieve this aim, a biochip (1) for optically measuring the properties of individual transporter systems (50) substantially consists of a transparent support (10) and a plurality of recesses (30) that are open to the top. The biochip (1) is configured in such a manner that its openings (31) can be covered by a membrane (40), thereby forming closed measuring compartments (30), and that the transport of the substrate molecules (60) via the membrane (40) into the recesses (30) can be detected.

Description

Biochip für die Fluoreszenzanalyse von einzelnen Transportern Biochip for the fluorescence analysis of individual transporters
Die Erfindung betrifft einen Biochip zur optischen Messung der Eigenschaften von einzelnen Transport-Systemen.The invention relates to a biochip for the optical measurement of the properties of individual transport systems.
Biologische Membranen trennen Zellen vom äußeren Medium und die einzelnen Zellkompartimente der Zellen voneinander ab. Transport- Systeme wie Transportproteine und Kanäle steuern selektiv den Stoffdurchlass durch diese Membranen. Funktionsstörungen dieser Transporter und Kanäle sind für zahlreiche verbreitete Krankheiten verantwortlich. Unter den 100 am meisten verkauften Arzneimitteln in den USA im Jahre 2004 waren die Membrantransporter die am häufigsten vorkommende Targetgruppe. Es sind mindestens 1.302 Transporter- Pharmaka, sowohl eingeführte als auch noch in der Entwicklung sich befindende Arzneimittel, in den Portfolios von 326 Firmen weltweit vorhanden. Insgesamt werden zurzeit mehr als 100 Transporter-Targets bei den Pharmafirmen erforscht, was zeigt, welche immense wirtschaftliche Bedeutung diese haben.Biological membranes separate cells from the outer medium and the individual cell compartments of the cells. Transport systems such as transport proteins and channels selectively control the mass transfer through these membranes. Dysfunctions of these transporters and channels are responsible for many common diseases. Among the top 100 drugs sold in the US in 2004, membrane transporters were the most abundant target group. There are at least 1,302 transporter pharmaceuticals, both imported and under development, in the portfolios of 326 companies worldwide. Overall, more than 100 transporter targets are currently being researched by the pharmaceutical companies, which shows what immense economic importance they have.
Für die Entwicklung solcher Wirkstoffe werden Messmethoden benötigt, mit denen Eigenschaften wie die Transportraten von spezifischen Substraten durch das Transporter-Target und der Einfluss von Wirkstoffkandidaten evaluiert werden kann. Hierbei werden insbesondere Methoden benötigt, die einzelne Targetmoleküle sogar automatisiert im Hochdurchsatz charakterisieren können.For the development of such drugs, measurement methods are needed to evaluate properties such as the transport rates of specific substrates through the transporter target and the influence of drug candidates. In particular, methods are needed that can characterize individual target molecules even in high-throughput automated fashion.
Für die Analyse von Transportraten von Ionen und geladenen Teilchen können elektrische Messungen eingesetzt werden. Dieses Verfahren findet bereits eine Anwendung im Hochdurchsatz in der biotechnologischen und pharmazeutischen Forschung. Es ist jedoch auf geladene Transportsubstrate beschränkt und wird daher in der Regel für die Gruppe der lonenkanäle eingesetzt. Der Transport von ungeladenen Molekülen wie Aminosäuren, Peptiden, Zuckerverbindungen und Fettsäuren, aber auch biologischen Makromolekülen wie RNA, DNA und Proteinen kann nur indirekt mit elektrischen Verfahren gemessen werden.For the analysis of transport rates of ions and charged particles electrical measurements can be used. This process is already being used in high throughput biotechnological and pharmaceutical research. However, it is limited to loaded transport substrates and is therefore usually for the group of ion channels used. The transport of uncharged molecules such as amino acids, peptides, sugar compounds and fatty acids, but also biological macromolecules such as RNA, DNA and proteins can only be measured indirectly by electrical methods.
Die Fluoreszenzanalyse kann dagegen den Transport dieser Moleküle sichtbar machen. Erste Vorarbeiten dazu wurden von einer akademischen Gruppe für den Transport von Biomolekülen durch den Kernporenkomplex in Kernhüllen aus Xenopus Laevis durchgeführt. Es wurde auch für die Messung des Transports von Calcium-Ionen durch die α-Hämolysin-Pore angewendet, welche direkt in vorgefertigte, künstliche Lipidmembranen insertiert wurde und sich dabei aus einer denaturierten Struktur in eine funktionale Form rückfaltet.In contrast, fluorescence analysis can visualize the transport of these molecules. Preliminary work was carried out by an academic group on the transport of biomolecules through the nuclear pore complex in Xenopus Laevis core casings. It has also been used to measure the transport of calcium ions through the α-hemolysin pore, which has been inserted directly into prefabricated, artificial lipid membranes, refolding from a denatured structure into a functional form.
In den Veröffentlichungen wurden dazu Polycarbonatfilter oder Polycarbonatstrukturen eingesetzt, deren Vertiefungen für die Fluoreszenzmessung von Transportraten mittels konfokaler Laser-In the publications, polycarbonate filters or polycarbonate structures were used, the wells of which were used for the fluorescence measurement of transport rates by means of confocal laser beams.
Scanning-Mikroskopie genutzt wurden. Dies bedingt schlechte optische Eigenschaften, u.a. auf Grund von Divergenzen in den Brechungsindices von Polycarbonat und Messpuffer. Weitergehende Experimente, die über die Grundlagenforschung hin zu einer biotechnologischen oder pharmazeutischen Anwendung des Verfahrens im Hochdurchsatz führen oder hierfür geeignete Chips verwenden, sind nicht publiziert worden.Scanning microscopy were used. This causes poor optical properties, i.a. due to divergences in refractive indices of polycarbonate and measuring buffer. Further experiments that lead beyond basic research towards a biotechnological or pharmaceutical application of the method in high throughput or use suitable chips for this have not been published.
Aufgabe der Erfindung ist es daher, eine Vorrichtung vorzuschlagen, durch die die Eigenschaften von Transportermolekülen mit hoher Messgenauigkeit und hohem Durchsatz gemessen werden können.The object of the invention is therefore to propose a device by means of which the properties of transporter molecules can be measured with high measurement accuracy and high throughput.
Diese Aufgabe wird dadurch gelöst, dass ein Biochip zur optischen Messung der Eigenschaften von einzelnen Transport-Systemen vorgeschlagen wird, der im Wesentlichen aus einem transparentem Träger sowie mehreren nach oben geöffneten Vertiefungen besteht, wobei der Biochip derart ausgebildet ist, dass seine Öffnungen durch eine Membran abdeckbar sind, und so geschlossene Messkammern gebildet werden und der Transport von Substratmolekülen über die Membran in die Vertiefungen nachweisbar ist. Dazu wird die Membran über die Vertiefungen im Biochip aufgespannt, so dass diese verschlossen sind. Über der Membran zugegebene und mit Fluoreszenzverfahren detektierbare Transportsubstrate gelangen somit nur mittels der in der Membran enthaltenen Transportproteine oder Kanäle in die Messräume des Biochips. Durch Fluoreszenzmessungen können diese Substrate in den Vertiefungen nachgewiesen und quantifiziert werden. EineThis object is achieved by proposing a biochip for the optical measurement of the properties of individual transport systems, which consists essentially of a transparent support and a plurality of recesses open at the top, wherein the Biochip is formed such that its openings are covered by a membrane, and so closed measuring chambers are formed and the transport of substrate molecules via the membrane in the wells is detectable. For this purpose, the membrane is clamped over the recesses in the biochip, so that they are closed. Thus, transport substrates added via the membrane and detectable by fluorescence are only able to enter the measurement spaces of the biochip by means of the transport proteins or channels contained in the membrane. Fluorescence measurements can be used to detect and quantify these substrates in the wells. A
Auswertung ergibt Parameter wie die Transportrate, die Rückschlüsse auf das Transportprotein/ den Kanal oder z.B. einen Einfluss eines Wirkstoffkandidaten erlauben. Sowohl das Verfahren als auch die Auswertung kann automatisiert und im Hochdurchsatz eingesetzt werden.Evaluation yields parameters such as the transport rate, the conclusions about the transport protein / channel or e.g. allow an influence of a drug candidate. Both the method and the evaluation can be automated and used in high throughput.
Wird auf den Biochip eine leitende Schicht vorzugsweise aus Metall aufgebracht, so kann diese zusätzlich als Elektrode vorzugsweise für Messungen der Impedanzspektroskopie oder auch für das Anlegen eines elektrischen Feldes genutzt werden. Mit einer zweiten Elektrode in der Lösung über der Membran kann z.B. mittels Impedanzspektroskopie Aussagen über die elektrische Dichtigkeit einer aufgebrachten Lipidschicht oder einer Zellschicht getroffen werden. Dies kann als Qualitätskontrolle für die Güte der Lipidschicht oder auch Bewertung der Lebensfähigkeit der Zellen genutzt werden. Ein angelegtes elektrisches Feld kann für die Steuerung von spannungsabhängigen Kanalproteinen eingesetzt werden, um z.B. einen lonenkanal auf den offenen Zustand zu schalten und dann wie beschrieben eine Transportmessung mittels Fluoreszenzmessung eines ionenabhängigen Fluoreszenzindikators durchzuführen.If a conductive layer, preferably made of metal, is applied to the biochip, then it can additionally be used as an electrode, preferably for measurements of the impedance spectroscopy or else for the application of an electric field. With a second electrode in the solution over the membrane, e.g. statements about the electrical tightness of an applied lipid layer or a cell layer are taken by means of impedance spectroscopy. This can be used as a quality control for the quality of the lipid layer or even evaluation of the viability of the cells. An applied electric field can be used for the control of voltage dependent channel proteins, e.g. To switch an ion channel to the open state and then perform as described a transport measurement by means of fluorescence measurement of an ion-dependent fluorescence indicator.
Für die biotechnologische und pharmazeutische Anwendung dieses Verfahrens ist es notwendig, mittels Standardverfahren hergestellte Proteo-Liposomen, also künstliche, hohle Membranvesikel, die in die Membran insertierte Transportproteine enthalten, einzusetzen. Diese können entweder direkt an die aktivierte Oberfläche des Biochips gekoppelt werden oder durch Fusion mit einer vorgeformten Lipidmembran aufgebracht werden. Dabei wird der Vesikel zu einer den Transporter enthaltenden Membran umgeformt, die die aus den Vertiefungen gebildeten Messkammern im Biochip verschließt und somit eine Fluoreszenzmessung zur Charakterisierung der Transporter und Bestimmung der Transportraten ermöglicht.For the biotechnological and pharmaceutical application of this method, it is necessary, produced by standard methods Proteo liposomes, so artificial, hollow membrane vesicles containing inserted into the membrane transport proteins to use. These can either be coupled directly to the activated surface of the biochip or applied by fusion with a preformed lipid membrane. In this case, the vesicle is reshaped to a membrane containing the transporter, which closes the measuring chambers formed in the wells in the biochip and thus enables a fluorescence measurement for characterizing the transporters and determining the transport rates.
Mit Vorteil besteht der Träger aus einem Material mit hohemAdvantageously, the carrier is made of a material with a high
Brechungsindex, wie Glas, Silizium oder Siliziumdioxid. Hierdurch werden optische Artefakte vermindert und die Fluoreszenzdetektion in den Vertiefungen mit Abmessungen im Nanometerbereich möglich. Ist der Brechungsindex höher als der Brechungsindex der verwendeten Messlösung, kann durch Einstrahlen des Anregungslichtes unter einem Winkel eine Totalreflektion und somit ein evaneszentes Feld an der Phasengrenze von Material und Messlösung erzeugt und für die Fluoreszenzdetektion genutzt werden.Refractive index, such as glass, silicon or silicon dioxide. As a result, optical artifacts are reduced and the fluorescence detection in the wells with dimensions in the nanometer range possible. If the refractive index is higher than the refractive index of the measuring solution used, total reflection and thus an evanescent field at the phase boundary of the material and the measuring solution can be generated by irradiating the excitation light at an angle and used for fluorescence detection.
Der Träger (10) kann eine oder mehrere mit seiner Oberseite verbundene Schichten (20) aufweisen. Die nach oben geöffneten Vertiefungen (30) sind in der Schicht oder den Schichten (20) vorgesehen. Hierdurch kann unterschiedliches Material für den Träger und Messkammern verwendet werden, was weitere vorteilhafte Eigenschaften ermöglicht.The carrier (10) may have one or more layers (20) connected to its top. The upwardly opened depressions (30) are provided in the layer or layers (20). As a result, different material for the carrier and measuring chambers can be used, which allows further advantageous properties.
In einer bevorzugten Ausführungsform ist der Durchmesser der Vertiefungen kleiner als die Wellenlänge des Anregungslichts, so dass die Vertiefungen als Zero-Mode-Waveguides ausgebildet sind. Die Intensität des Anregungslichts nimmt dann exponentiell innerhalb der Messkammer ab, wodurch eine hochselektive Anregung möglich ist. Wenn mindestens eine der Schichten aus lichtundurchlässigem Material, insbesondere Metall besteht, ist der Biochip an der Oberseite im Wesentlichen lichtundurchlässig. Hierdurch wird das Anregungslicht von der Membran abgeschirmt. Fluoreszierende Substratmoleküle, die sich in der Membran oder oberhalb der Membran, also außerhalb derIn a preferred embodiment, the diameter of the recesses is smaller than the wavelength of the excitation light, so that the recesses are formed as zero-mode waveguides. The intensity of the excitation light then decreases exponentially within the measuring chamber, whereby a highly selective excitation is possible. If at least one of the layers is made of opaque material, in particular metal, the biochip on the upper side is substantially opaque. As a result, the excitation light is shielded from the membrane. Fluorescent substrate molecules that are located in the membrane or above the membrane, ie outside the
Messkammer befinden, können so nicht angeregt werden. Hierdurch wird ein störendes Hintergrundsignal vermindert oder vermieden.Can not be stimulated so. As a result, a disturbing background signal is reduced or avoided.
Ein besonders geeignetes Metall ist Gold, da es chemisch inert ist, sicher mit dem Trägermaterial verbunden werden kann und außerdem geeignete Lichtreflektionseigenschaften hat. Titan ist ebenfalls geeignet.A particularly suitable metal is gold because it is chemically inert, can be securely bonded to the substrate, and also has suitable light-reflecting properties. Titanium is also suitable.
Die Metallschicht wird mit dem Träger mittels eines Haftvermittlers fest verbunden. Es hat sich herausgestellt, dass als Haftvermittler ein Metall, insbesondere Chrom oder Titan, sehr gut geeignet ist.The metal layer is firmly connected to the carrier by means of a bonding agent. It has been found that a metal, in particular chromium or titanium, is very well suited as adhesion promoter.
Eine Verbesserung der Messgenauigkeit lässt sich dadurch erreichen, dass die Metallschicht an ihrer Unterseite reflektierend ausgebildet ist, um das Anregungslicht zu spiegeln und so die Substratmoleküle mehrfach anzuregen.An improvement in the measurement accuracy can be achieved in that the metal layer is designed to be reflective on its underside in order to mirror the excitation light and thus excite the substrate molecules several times.
Die Öffnung der Vertiefung kann zum Teil von der darüber angeordneten Metallschicht abgedeckt, indem die Öffnung in der Metallschicht so gewählt ist, dass sie kleiner ist als die Vertiefungsöffnung. Hierdurch wird das Anregungslicht von den Substratmolekülen noch mehr abgeschirmt, die sich nicht in der Messkammer befinden und so die Messgenauigkeit verbessert.The opening of the recess may be partially covered by the overlying metal layer by having the opening in the metal layer selected to be smaller than the recess opening. As a result, the excitation light from the substrate molecules is shielded even more, which are not in the measuring chamber and thus improves the accuracy of measurement.
Zusätzlich kann die über den Öffnungen liegende Metallschicht als Elektrode für elektrische Messungen der Membran eingesetzt werden oder auch zur Erzeugung eines elektrischen Feldes genutzt werden. Sofern die Schicht aus Siliziumdioxid besteht, ist die Fluoreszenzdetektion der Transportsubstrate in den Vertiefungen der Schicht möglich.In addition, the metal layer lying over the openings can be used as an electrode for electrical measurements of the membrane or can also be used to generate an electric field. If the layer consists of silicon dioxide, fluorescence detection of the transport substrates in the recesses of the layer is possible.
Besteht die dem transparenten Träger aufliegende Schicht aus einem Fluoropolymer, wie Teflon oder Cytop, dann erlaubt dies die Detektion der Fluoreszenz in den Messkammern, z.B. mittels konfokaler Laser Scanning Mikroskopie.If the layer bearing the transparent support is made of a fluoropolymer, such as Teflon or Cytop, then this allows the detection of the fluorescence in the measuring chambers, e.g. using confocal laser scanning microscopy.
Eine weitere Verbesserung lässt sich dadurch erzielen, dass sich der Durchmesser der Vertiefungen von unten zur Oberseite hin kontinuierlich verringert, so dass die Vertiefungen annähernd eine Kegelform aufweisen. Die zum Träger hin größeren Durchmesser der Kammern ermöglichen dann eine Detektion der Fluoreszenz in den so gebildeten Messräumen mit höherer Genauigkeit.A further improvement can be achieved in that the diameter of the recesses decreases continuously from the bottom to the top, so that the recesses have approximately a conical shape. The larger diameter of the chambers towards the carrier then make it possible to detect the fluorescence in the measuring spaces thus formed with greater accuracy.
Die Kopplung, das heißt die Fixierung der biologischen Membranen oder künstlichen Vesikeln an den Biochip kann so erfolgen, dass dessen Oberfläche Linkermoleküle, welche insbesondere aminoreaktiv sind, und/oder Lipidderivate aufweist und die an geeignete Bestandteile der Membran kovalent oder nichtkovalent binden.The coupling, that is to say the fixation of the biological membranes or artificial vesicles to the biochip, can take place in such a way that its surface has linker molecules which are in particular amino-reactive and / or lipid derivatives and which bind covalently or noncovalently to suitable constituents of the membrane.
Die Membran weist als Transportermolekül ein oder mehrere Proteine, insbesondere Poren-, Kanal- oder Carrierproteine, auf, deren Transport- Aktivität über die Vesikelmembran nachgewiesen wird.As a transporter molecule, the membrane has one or more proteins, in particular pore, channel or carrier proteins, whose transport activity is detected via the vesicle membrane.
Eine weitere Anwendung des Biochips ist die Charakterisierung von Produktionszelllinien für rekombinante Proteine und Antikörper. Hierzu werden Zellen oder Zellbestandteile für die Produktion von rekombinanten Proteinen oder Antikörpern gemessen. Dabei werden die Zellen an den Biochip gebunden, so dass sie mit ihrer Membran die Vertiefungen des Chips verschließen. Es ist auch möglich, Zellen auf den Biochips wachsen zu lassen. Bei Sekretion der hergestellten Proteine in die Messräume wird über ein Reportersystem ein Fluoreszenzsignal erzeugt. Dieses Fluoreszenzsignal gibt Aufschluss über die erzeugte Menge an rekombinantem Protein oder Antikörper und erlaubt somit das Auffinden von viel produzierenden Zellen, die für die biotechnologische Herstellung dieser Proteine und Antikörper eingesetzt werden können.Another application of the biochip is the characterization of production cell lines for recombinant proteins and antibodies. For this purpose, cells or cell components are measured for the production of recombinant proteins or antibodies. The cells are bound to the biochip, so that they close with their membrane, the wells of the chip. It is also possible to grow cells on the biochips. Upon secretion of the produced proteins into the measuring chambers becomes generates a fluorescence signal via a reporter system. This fluorescence signal provides information on the amount of recombinant protein or antibody generated and thus allows the discovery of many producing cells that can be used for the biotechnological production of these proteins and antibodies.
Die bei der Messung verwendeten Membranen können biologische oder künstliche Lipidmembranen sein. Sofern biologische Membranen verwendet werden, ergeben sich besonders natürliche Messbedingungen.The membranes used in the measurement may be biological or artificial lipid membranes. If biological membranes are used, particularly natural measuring conditions result.
Vorzugsweise erfolgt die Messung mit einer Vesikelmembran, die darin rekonstituierte Transportermoleküle enthält. Dies erlaubt schnelle, reproduzierbare Messungen. Durch die Einbettung in die Vesikelmembran nimmt das Transporterprotein außerdem wieder seine funktionelle Konformation ein.Preferably, the measurement is with a vesicle membrane containing reconstituted transporter molecules therein. This allows fast, reproducible measurements. By embedding in the vesicle membrane, the transporter protein also regains its functional conformation.
Eine genaue Messung ist dann möglich, wenn die über eine Vertiefung gespannte Membran möglichst wenig, vorzugsweise ein bis drei, Transportermoleküle enthält.Accurate measurement is possible if the membrane stretched over a depression contains as few as possible, preferably one to three, transporter molecules.
Der Nachweis des durch die Transportermoleküle transportierten Substrates wird dadurch ermöglicht, dass die Substratmoleküle fluoreszieren, vorzugsweise indem sie an einen Fluoreszenzfarbstoff gebunden sind, aber auch durch Bindung an einen substratabhängigen Fluoreszenzindikator z.B. zur Messung von lonenströmen.The detection of the substrate transported by the transporter molecules is made possible by the fact that the substrate molecules fluoresce, preferably by being bound to a fluorescent dye, but also by binding to a substrate-dependent fluorescence indicator, e.g. for the measurement of ion currents.
Die fluoreszierenden Substratmoleküle werden von dem Transportermolekül über die Membran in die Vertiefungen des Biochips transportiert. Dort werden sie mittels einer geeigneten Fluoreszenzdetektionsvorrichtung nachgewiesen. Eine besonders genaue Messung erfolgt dadurch, dass die Detektionsvorrichtung die Fluoreszenz in einer konfokalen Ebene innerhalb der Vertiefung misst.The fluorescent substrate molecules are transported by the transporter molecule across the membrane into the wells of the biochip. There they are detected by means of a suitable fluorescence detection device. A particularly accurate measurement is made by the detection device measuring the fluorescence in a confocal plane within the well.
Eine weitere Verbesserung der Genauigkeit wird dadurch erzielt, dass der Durchmesser der Vertiefungen unter Berücksichtigung der Wellenlänge des Anregungslichtes so gewählt ist, dass ein evaneszentes Feld erzeugt wird, welches zur Fluoreszenzdetektion verwendet wird.A further improvement in accuracy is achieved in that the diameter of the recesses, taking into account the wavelength of the excitation light, is selected such that an evanescent field is generated, which is used for fluorescence detection.
In einer weiteren Ausführungsform wird ein evaneszentes Feld erzeugt, indem das Anregungslicht unter einem totalreflektierenden Winkel eingestrahlt und so zur Fluoreszenzdetektion verwendet wird.In another embodiment, an evanescent field is generated by irradiating the excitation light at a total reflecting angle and thus used for fluorescence detection.
In einer weiteren bevorzugten Ausführungsform ist eine Schicht elektrisch leitend und als Elektrode ausgebildet, um so die Membran elektrisch zu vermessen oder anzuregen. Eine geeignete Schicht kann beispielsweise die oberhalb des Trägers angeordnete Metallschicht aus Gold sein.In a further preferred embodiment, a layer is electrically conductive and designed as an electrode so as to electrically measure or excite the membrane. A suitable layer may be, for example, the metal layer of gold arranged above the carrier.
Überraschenderweise kann die Schicht damit zusätzlich als Elektrode für eine Charakterisierung der elektrischen Eigenschaften von Membranen, Zellschichten oder der in der Membran befindlichen Transport-Systeme verwendet werden.Surprisingly, the layer can thus additionally be used as an electrode for characterizing the electrical properties of membranes, cell layers or the transport systems present in the membrane.
Der Biochip kann dabei so eingesetzt werden, dass die Impedanz der über den Biochip gespannten Membran oder Epithelzellschicht mit Transport- Systemen, beispielsweise Transportproteinen, gemessen wird. Hierdurch kann die Dichtigkeit der Membran bestimmt werden.The biochip can be used in such a way that the impedance of the membrane or epithelial cell layer stretched across the biochip is measured with transport systems, for example transport proteins. As a result, the tightness of the membrane can be determined.
Der Biochip kann mittels der Elektrode zusätzlich aber auch zur Erzeugung eines elektrischen Feldes verwendet werden, insbesondere zur Steuerung von spannungssensitiven Transport-Systemen. Dies sind beispielsweise spannungsabhängige lonenkanäle, d.h. lonenkanäle die sich bei einem bestimmten Grenzwert der Membranspannung öffnen oder schließen. Durch Veränderung des angelegten elektrischen Feldes können so funktionelle Schaltvorgänge ausgelöst werden, die eine Veränderung des Transports von Substrat über die Membran zur Folge haben. Das Transportsubstrat kann dann in den Vertiefungen mittels Fluoreszenzindikatoren detektiert werden.The biochip can also be used by means of the electrode in addition to generate an electric field, in particular for the control of voltage-sensitive transport systems. These are, for example, voltage-dependent ion channels, ie ion channels which open at a certain limit value of the membrane voltage or shut down. By changing the applied electric field so functional switching processes can be triggered, which have a change in the transport of substrate through the membrane result. The transport substrate can then be detected in the wells by means of fluorescence indicators.
Eine beispielhafte Anwendung des Biochips besteht darin, dass die obere Metallschicht des Biochips mit einer Lipidmembran bedeckt ist, die lonenkanäle enthält. Für eine Messung wird an die elektrisch leitende Schicht, also die Elektrode, ein elektrisches Feld angelegt wird. Die angelegte Spannung führt zur Aktivierung der lonenkanäle. Hierdurch entsteht ein lonenstrom über die Membran in die Vertiefungen, der dann mittels Fluoreszenz quantitativ nachgewiesen wird.An exemplary application of the biochip is that the top metal layer of the biochip is covered with a lipid membrane containing ion channels. For a measurement, an electric field is applied to the electrically conductive layer, ie the electrode. The applied voltage leads to the activation of the ion channels. This creates an ion current across the membrane into the wells, which is then detected quantitatively by fluorescence.
Der vorgeschlagene Biochip hat damit den überraschenderweise den zusätzlichen Vorteil, dass er biologische Transportsysteme elektrisch funktionell schalten und gleichzeitig den dadurch erzeugten Transport über die Membran optisch mittels Fluoreszenz messen kann.The proposed biochip thus surprisingly has the additional advantage that it can switch biological transport systems electrically functional and at the same time be able to measure the transport generated thereby via the membrane optically by means of fluorescence.
Die Erfindung wird in einer bevorzugten Ausführungsform unter Bezugnahme auf eine Zeichnung beispielhaft beschrieben, wobei weitere vorteilhafte Einzelheiten den Figuren der Zeichnung zu entnehmen sind.The invention will be described by way of example in a preferred embodiment with reference to a drawing, wherein further advantageous details are shown in the figures of the drawing.
Funktionsmäßig gleiche Teile sind dabei mit denselben Bezugszeichen versehen.Functionally identical parts are provided with the same reference numerals.
Die Figuren der Zeichnung zeigen im Einzelnen:The figures of the drawing show in detail:
Figur 1 einen Vertikalschnitt des erfindungsgemäßen Biochips;FIG. 1 shows a vertical section of the biochip according to the invention;
Figur 2 einen Vertikalschnitt wie in Fig. 1 mit einem Vesikel;FIG. 2 shows a vertical section as in FIG. 1 with a vesicle;
Figur 3 einen Vertikalschnitt wie in Fig. 2 mit aufliegender biologischer Zelle; Figur 4 eine Draufsicht auf ein Array des Biochips;FIG. 3 shows a vertical section as in FIG. 2 with a resting biological cell; Figure 4 is a plan view of an array of the biochip;
Figur 5a eine Detailansicht des Biochips mit einer Vertiefung im Vertikalschnitt;FIG. 5a shows a detail view of the biochip with a depression in vertical section;
Figur 5b eine Detailansicht des Biochips mit einer konusförmigen Vertiefung des Biochips im Vertikalschnitt undFigure 5b is a detail view of the biochip with a cone-shaped recess of the biochip in vertical section and
Figur 6 eine Detailansicht einer bevorzugten Ausführungsform des Biochips mit einer Vertiefung im Vertikalschnitt.Figure 6 is a detail view of a preferred embodiment of the biochip with a recess in vertical section.
Figur 1 zeigt einen Vertikalschnitt durch den erfindungsgemäßen Biochip.FIG. 1 shows a vertical section through the biochip according to the invention.
Der Biochip 1 besteht aus einem Träger 10, der für das Anregungslicht beziehungsweise das Fluoreszenzlicht transparent ist. An seinerThe biochip 1 consists of a carrier 10, which is transparent to the excitation light or the fluorescent light. At his
Oberseite weist der Chip Vertiefungen 30 auf, die als Messkammern zum Nachweis eines Substrates 60 dienen. In der dargestellten Ausführungsform besteht der Biochip 1 aus einem Verbund unterschiedlicher Materialien. Die Basis bildet der optisch durchlässige Träger 10 aus Deckglas. Auf der Oberseite des Trägers ist eine Schicht aus Siliziumdioxid 20 angeordnet. Auf der Siliziumdioxidschicht 20 ist eine Schicht aus Titan aufgebracht, die sowohl als Reflektor für das Anregungslicht 80 als auch als Haftvermittler für eine weitere Schicht aus Gold dient. Die Goldschicht kann kontaktiert und als Elektrode genutzt werden. Die drei Schichten 20 enthalten durchgehende Vertiefungen 30, durch die jeweils eine nach oben geöffnete Messkammer gebildet wird.Top side, the chip recesses 30, which serve as measuring chambers for the detection of a substrate 60. In the illustrated embodiment, the biochip 1 consists of a composite of different materials. The basis is the optically transparent carrier 10 made of cover glass. On the top of the carrier, a layer of silicon dioxide 20 is arranged. On the silicon dioxide layer 20, a layer of titanium is applied, which serves both as a reflector for the excitation light 80 and as a bonding agent for a further layer of gold. The gold layer can be contacted and used as an electrode. The three layers 20 contain through recesses 30 through which an upwardly open measuring chamber is formed in each case.
Auf die Oberfläche des Biochips 1 wird zur Messung eine Membran 40 aufgebracht, so dass die Messräume 30 verschlossen werden. Die Membran 40 kann aus künstlichen Proteo-Liposomen 5 hergestellt werden, welche als Transport-System Transport-Proteine oder Poren- Proteine enthalten. Andererseits kann die Membran 40 auch die Zellmembran von Produktionszelllinien für rekombinante Proteine oder Antikörper sein.On the surface of the biochip 1, a membrane 40 is applied for the measurement, so that the measurement spaces 30 are closed. The membrane 40 can be made from artificial proteo-liposomes 5 which contain transport proteins or pore proteins as a transport system. On the other hand, the membrane 40, the Cell membrane of production cell lines for recombinant proteins or antibodies.
Die Membran 40 enthält Transport-Systeme 50, wie Transport-Proteine oder Poren-Proteine. Exemplarisch können hierbei Transporter der ABC- Transportergruppe genannt werden, die für viele Krankheiten relevant sind, wie z.B. der Adrenoleukodystrophie ABCD 1 -Transporter mit Fettsäuren als Substrat oder z.B. der Glutamat-Transporter mit dem Substrat Glutamat, dessen Stoffwechsel bei psychischen Erkrankungen gestört ist.The membrane 40 contains transport systems 50, such as transport proteins or pore proteins. By way of example, transporters of the ABC transporter group which are relevant for many diseases, such as e.g. adrenoleukodystrophy ABCD 1 transporter with fatty acids as substrate or e.g. the glutamate transporter with the substrate glutamate, whose metabolism is disturbed in mental illness.
Oberhalb der Membran werden ein oder mehrere mitAbove the membrane are one or more with
Fluoreszenzverfahren detektierbare Transport-Substrate 60 zugegeben. Dies wird beispielsweise dadurch ermöglicht, indem das Substrat mit einem Fluoreszenzfarbstoff kovalent markiert ist. Der Transport 70 der Transportsubstrate durch die in der Membran 40 enthaltenen Transport- Systeme 50 in die Vertiefungen 30 des Biochips ist spezifisch für das enthaltene Transport-System 50 und kann durch Fluoreszenzmessungen in den Messräumen 30 quantifiziert werden. Dies ermöglicht Rückschlüsse auf für das Transport-System 50 spezifische Parameter wie Transportraten und Durchlässigkeit und somit die Evaluation von Wirkstoffkandidaten oder die Produktionsraten von Produktionszelllinien.Fluorescence method detectable transport substrates 60 added. This is made possible, for example, by covalently marking the substrate with a fluorescent dye. The transport 70 of the transport substrates through the transport systems 50 contained in the membrane 40 into the recesses 30 of the biochip is specific to the transport system 50 contained and can be quantified by fluorescence measurements in the measurement spaces 30. This makes it possible to draw conclusions about parameters specific to the transport system 50, such as transport rates and permeability, and thus the evaluation of drug candidates or the production rates of production cell lines.
Der Biochip kann aus einem Fluoropolymer 20 wie Teflon oder Cytop bestehen, welches die Messräume 30 enthält und auf einen lichtdurchlässigen Träger 10 aufgebracht wird. Dies erlaubt die Detektion der Fluoreszenz in den Messräumen z.B. mittels konfokaler Laser Scanning Mikroskopie.The biochip may consist of a fluoropolymer 20 such as Teflon or Cytop, which contains the measurement spaces 30 and is applied to a light-transmissive carrier 10. This allows the detection of fluorescence in the measurement spaces, e.g. using confocal laser scanning microscopy.
Der Biochip kann aber auch aus einer Metallschicht 20 bestehen, in die die Löcher 30 eingebracht werden, und die auf einem lichtdurchlässigen Träger 10 aufgebracht werden. Unterschreitet der Durchmesser der Löcher 30 eine bestimmte Größe im Nanometer-Bereich, so kann das eingestrahlte Licht nicht mehr vollständig in die Messräume eindringen, stattdessen bildet sich ein evaneszentes Feld am Übergang von Träger und mit Messlösung gefülltem Messraum aus. Die Vertiefungen stellen dann „Zero Mode Waveguides" dar und erlauben so die Detektion der Fluoreszenz in den gebildeten Messräumen.However, the biochip can also consist of a metal layer 20, into which the holes 30 are introduced, and which are applied to a light-transmitting carrier 10. If the diameter of the Holes 30 a certain size in the nanometer range, so the incident light can no longer penetrate completely into the measuring chambers, instead forms an evanescent field at the transition of the carrier and filled with measuring solution measuring space. The pits then represent "Zero Mode Waveguides" and thus allow the detection of the fluorescence in the measuring chambers formed.
Eine weitere Möglichkeit, den Biochip herzustellen besteht darin, kegelförmige Löcher 30 anisotrop in Siliziumdioxid 20 zu ätzen und dieses dann auf einen durchlässigen Träger 10 aufzubringen. Der zum Träger 10 hin größere Durchmesser der Löcher erlaubt dann eine Detektion der Fluoreszenz in diesen Vertiefungen.Another way to make the biochip is to anisotropically etch conical holes 30 in silicon dioxide 20 and then apply this to a permeable support 10. The larger diameter of the holes towards the carrier 10 then permits detection of the fluorescence in these depressions.
Außerdem kann der Biochip durch Erzeugen von Vertiefungen 30 in ein Material mit einem hohen Brechungsindex, wie z.B. Glas 10+20, Brechungsindex 1,53 hergestellt werden. Dieser Brechungsindex ist deutlich höher als der der in den Messräumen 30 befindlichen Messlösung mit einem Brechungsindex von 1,33. Wird das Anregungslicht schräg von unten eingestrahlt, so wird ab einem bestimmten Winkel am Übergang vom Träger zur Messlösung bei Totalreflektion des Lichtes ein evaneszentes Feld erzeugt, welches zur Detektion der Fluoreszenz in den Messkammern 30 genutzt werden kann.In addition, the biochip may be formed by forming recesses 30 in a high refractive index material, such as a glass sheet. Glass 10 + 20, refractive index 1.53 can be produced. This refractive index is significantly higher than that of the measuring solution located in the measuring chambers 30 with a refractive index of 1.33. If the excitation light is irradiated obliquely from below, an evanescent field is generated at a transition from the carrier to the measurement solution at total reflection of the light, which can be used to detect the fluorescence in the measuring chambers 30.
Figur 2 zeigt einen Vertikalschnitt wie in Fig. 1 mit einem Vesikel (5). In der Vesikelmembran sind porenbildende Proteine (50) rekonstituiert.FIG. 2 shows a vertical section as in FIG. 1 with a vesicle (5). Pore-forming proteins (50) are reconstituted in the vesicle membrane.
Figur 3 zeigt einen Vertikalschnitt wie in Fig. 2 mit einer aufliegenden biologischen Zelle 15. Dies kann eine vollständige Zelle 15 sein oder auch nur ein Teil davon. Die Zelle erstreckt sich über mehrere Vertiefungen 30 und bedeckt diese. Hierdurch ist eine Messung unter natürlichen biologischen Bedingungen möglich. Figur 4 zeigt eine Draufsicht auf ein Array 36 des Biochips 1. Dieses wird dadurch gebildet, dass jeweils vier in der Draufsicht quadratische Vertiefungen 30 dicht beieinander angeordnet sind und so eine Gruppe 35 bilden. Die Gruppe 35 hat dabei eine Länge c bzw. Breite d von jeweils etwa 100 μm. Jeweils sechzehn Vertiefungsgruppen 35 bzw. vierundsechzig Vertiefungen 30 sind zu einem Array 36 angeordnet, welches eine Länge a bzw. Breite b von jeweils etwa 500 μm aufweist.FIG. 3 shows a vertical section as in FIG. 2 with a resting biological cell 15. This may be a complete cell 15 or just a part thereof. The cell extends over several recesses 30 and covers them. This makes it possible to measure under natural biological conditions. FIG. 4 shows a plan view of an array 36 of the biochip 1. This is formed by virtue of the fact that four depressions 30, which are square in plan view, are arranged close to one another and thus form a group 35. The group 35 has a length c or width d of about 100 microns. Sixteen recessed groups 35 or sixty-four recesses 30 are arranged in each case to form an array 36 which has a length a or width b of about 500 μm in each case.
Figur 5a zeigt eine Detailansicht einer Ausführungsform des Biochips 1 mit einer Vertiefung 30 im Vertikalschnitt. Hierbei ist auf einen Träger 10 aus Deckglas mittels eines Haftvermittlers aus Chrom oder Titan (nicht gezeigt) eine Metallschicht aus Gold aufgebracht. Die Messkammer 30 wird bei dieser Ausführungsform also ausschließlich durch die Öffnung 31 im Metall gebildet, während der Glasträger 10 selbst keine Vertiefung aufweist.FIG. 5a shows a detail view of an embodiment of the biochip 1 with a depression 30 in vertical section. In this case, a metal layer of gold is applied to a carrier 10 made of cover glass by means of a bonding agent made of chromium or titanium (not shown). The measuring chamber 30 is thus formed in this embodiment only through the opening 31 in the metal, while the glass carrier 10 itself has no recess.
Figur 5b zeigt eine ähnliche Ausführungsform wie in Figur 5a, allerdings weist die Metallschicht 20 eine konus-, bzw. kegelförmige Vertiefung 30 auf. Die Öffnung 31 hat an ihrer Oberseite ebenfalls einen Durchmesser von 60 bis 120 nm, erweitert sich aber nach unten hin. Hierdurch erhöht sich die Messgenauigkeit, weil die Messkammer 30 mehr Substrat 60 (nicht gezeigt) enthält und so das Signal/Rauschverhältnis verbessert wird.FIG. 5 b shows a similar embodiment as in FIG. 5 a, but the metal layer 20 has a conical or conical depression 30. The opening 31 also has a diameter of 60 to 120 nm on its upper side, but widens downwards. This increases the measurement accuracy because the measurement chamber 30 contains more substrate 60 (not shown) and thus the signal / noise ratio is improved.
Figur 6 zeigt eine Detailansicht einer bevorzugten Ausführungsform des Biochips 1 mit einer Vertiefung 30 im Vertikalschnitt. Hierbei ist auf einen Träger 10 aus Deckglas und einer darauf verbundenen Schicht aus Siliziumdioxid 20 eine weitere Metallschicht aus Gold mittels eines Haftvermittlers aus Chrom oder Titan (nicht gezeigt) aufgebracht. Die beiden Metallschichten haben zusammen eine Dicke von etwa 100 nm. Die Siliziumdioxid- und Metallschichten sind mit einer Schichtöffnung 31 und einer durchgehenden Vertiefung 30 versehen, die einen Durchmesser von 200 nm hat. Der Pitch beträgt 500 nm. Für die Vermessung von zellulären Membranen beträgt der Pitch 1 bis 2,5 μm.FIG. 6 shows a detailed view of a preferred embodiment of the biochip 1 with a depression 30 in vertical section. In this case, a further metal layer of gold is applied by means of an adhesion promoter made of chromium or titanium (not shown) to a carrier 10 made of cover glass and a layer of silicon dioxide 20 connected thereto. The two metal layers together have a thickness of about 100 nm. The silicon dioxide and metal layers are provided with a layer opening 31 and a continuous recess 30 having a diameter of 200 nm. The pitch is 500 nm. For the measurement of cellular membranes, the pitch is 1 to 2.5 μm.
Im Unterschied zu den oben gezeigten Ausführungsformen wird die Messkammer durch die Vertiefung 30 innerhalb der Schicht ausIn contrast to the embodiments shown above, the measuring chamber is formed by the depression 30 within the layer
Siliziumdioxid 20 und den beiden Metallschichten gebildet. Die Vertiefung hat dabei eine Länge e von etwa 1 μm und einen Öffnungsdurchmesser 31 von etwa 200 nm. Die Dicke f der Metallschicht beträgt vorzugsweise etwa 100 nm, der Durchmesser der Schichtöffnung 21 etwa 200 nm.Silicon dioxide 20 and the two metal layers formed. The recess has a length e of about 1 .mu.m and an opening diameter 31 of about 200 nm. The thickness f of the metal layer is preferably about 100 nm, the diameter of the layer opening 21 about 200 nm.
Ein Vorteil dieser Ausführungsform besteht zum einen darin, dass der im Glasträger 10 durch die Vertiefung 30 gebildete Messraum eine größere Ausdehnung in vertikaler Richtung hat. Hierdurch sind die über die Membran transportierten Substratmoleküle 50 (nicht gezeigt) im Mittel weiter von der Membran und damit von den nicht transportierten Substratmolekülen 50 entfernt. Idealerweise sollen nur die unterhalb der Lipidmembran (nicht gezeigt) befindlichen Substratmoleküle 50 zur Fluoreszenz angeregt werden, was durch den größeren räumlichen Abstand erleichtert wird. Dadurch erhöht sich das Signal/Rauschverhältnis.On the one hand, an advantage of this embodiment is that the measuring space formed in the glass carrier 10 by the depression 30 has a greater extent in the vertical direction. As a result, the substrate molecules 50 (not shown) transported via the membrane are further removed on the average from the membrane and thus from the non-transported substrate molecules 50. Ideally, only the substrate molecules 50 below the lipid membrane (not shown) should be excited to fluoresce, facilitated by the greater spatial distance. This increases the signal / noise ratio.
Das Signal/Rauschverhältnis kann dadurch weiter verbessert werden, indem die obere Vertiefungsöffnung 31 zum Teil durch die Metallschicht 20 abgedeckt wird. Das Anregungslicht wird so effektiv von den nicht transportierten Substratmolekülen 50 (nicht dargestellt) oberhalb der Membran abgeschirmt.The signal-to-noise ratio can be further improved by covering the upper recess opening 31 in part by the metal layer 20. The excitation light is thus effectively shielded from the non-transported substrate molecules 50 (not shown) above the membrane.
Ein weiterer und überraschender Vorteil besteht darin, dass die Metallschicht 20 das Anregungslicht reflektiert. Um dieses zu verdeutlichen, zeigt die Figur 6 eine schematische Darstellung der Strahlen 80 des Anregungslichts. Ein paralleles Lichtbündel 80 wird schrägwinklig in die Unterseite des Glasträgers 10 eingestrahlt. Der Strahlengang ist dabei wie bei einem handelsüblichen TIRF-Mikroskop angeordnet. Die Strahlen 80 werden von der Metallschicht 20 reflektiert und durchstrahlen mehrfach das Messvolumen 30 mit der Probe 60 (nicht gezeigt).Another and surprising advantage is that the metal layer 20 reflects the excitation light. To clarify this, FIG. 6 shows a schematic representation of the beams 80 of the excitation light. A parallel light beam 80 is obliquely angled into the bottom of the glass carrier 10. The beam path is arranged as in a commercially available TIRF microscope. The beams 80 are reflected by the metal layer 20 and repeatedly irradiate the measuring volume 30 with the sample 60 (not shown).
Hierdurch wird die Signalanregung um ein Vielfaches verstärkt, was die Messgenauigkeit weiter erheblich verbessert.As a result, the signal excitation is amplified many times, which further improves the measurement accuracy considerably.
Die sich neben dem Anregungslicht ausbildende evaneszente Welle ist in der Figur 6 nicht gezeigt. Durch den Schrägeinfall und die Dicke der Metallschicht 20 reicht der Durchmesser für eine „Zero Mode'-Anregung nicht aus, was jedoch zur Signalunterdrückung erwünscht ist. The evanescent wave forming next to the excitation light is not shown in FIG. Due to the oblique incidence and thickness of the metal layer 20, the diameter is insufficient for zero mode excitation, which is desirable for signal suppression.
BEZUGSZEICHENLISTELIST OF REFERENCE NUMBERS
1 Biochip1 biochip
5 Vesikel5 vesicles
10 Träger 15 Biologische Zelle10 carrier 15 biological cell
20 Schicht20 shift
21 Schichtöffnung21 layer opening
30 Vertiefung30 deepening
31 Vertiefungsöffnung 35 Vertiefungsgruppe31 depression opening 35 depression group
36 Array36 array
40 Membran40 membrane
50 Transportermolekül50 transporter molecule
60 Substrat 80 Anregungslicht 60 substrate 80 excitation light

Claims

PATENTANSPRÜCHE
1. Biochip (1) zur optischen Messung der Eigenschaften von einzelnen Transport-Systemen (50), bestehend im Wesentlichen aus einem transparentem Träger (10) sowie mehreren nach oben geöffneten Vertiefungen (30), wobei der Biochip (1) derart ausgebildet ist, dass seine Öffnungen (31) durch eine Membran (40) abdeckbar sind, und so geschlossene Messkammern (30) gebildet werden und der Transport von Substratmolekülen (60) über die Membran (40) in die Vertiefungen (30) nachweisbar ist.1. biochip (1) for optical measurement of the properties of individual transport systems (50), consisting essentially of a transparent carrier (10) and a plurality of upwardly open recesses (30), wherein the biochip (1) is formed in such a way that its openings (31) can be covered by a membrane (40), and thus closed measuring chambers (30) are formed and the transport of substrate molecules (60) via the membrane (40) into the recesses (30) is detectable.
2. Biochip (1) nach Anspruch 1, dadurch gekennzeichnet, dass der Träger (10) aus einem Material mit hohem Brechungsindex, insbesondere aus Glas, Silizium oder Siliziumdioxid, besteht, wobei der Brechungsindex vorzugsweise höher als der Brechungsindex der verwendeten Messlösung ist.2. biochip (1) according to claim 1, characterized in that the carrier (10) consists of a material having a high refractive index, in particular of glass, silicon or silicon dioxide, wherein the refractive index is preferably higher than the refractive index of the measuring solution used.
3. Biochip (1) nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass der Träger (10) eine oder mehrere mit seiner Oberseite verbundene Schichten (20) aufweist.3. biochip (1) according to claim 1 or 2, characterized in that the carrier (10) has one or more connected to its top layers (20).
4. Biochip (1) nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die nach oben geöffneten Vertiefungen (30) in der Schicht oder den Schichten (20) vorgesehen sind.4. biochip (1) according to any one of the preceding claims, characterized in that the upwardly open recesses (30) in the layer or layers (20) are provided.
5. Biochip (1) nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass der Durchmesser der Vertiefungen (30) kleiner als die Wellenlänge des Anregungslichts (80) ist, so dass die Vertiefungen (30) als Zero-Mode-Waveguides ausgebildet sind.5. biochip (1) according to any one of the preceding claims, characterized in that the diameter of the recesses (30) is smaller than the wavelength of the excitation light (80), so that the recesses (30) are formed as zero-mode waveguides.
6. Biochip (1) nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass mindestens eine der Schichten (20) aus lichtundurchlässigem Material, insbesondere Metall, vorzugsweise Gold oder Titan, besteht, um Anregungslicht (80) von der Membran (40) und darüber liegenden Substratmolekülen (60) abzuschirmen.6. biochip (1) according to any one of the preceding claims, characterized in that at least one of the layers (20) of opaque material, in particular metal, preferably Gold or titanium, to shield excitation light (80) from the membrane (40) and overlying substrate molecules (60).
7. Biochip (1) nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass eine oder mehrere der Schichten (20) mittels eines Haftvermittlers mit dem Träger (10) verbunden sind, wobei der7. biochip (1) according to any one of the preceding claims, characterized in that one or more of the layers (20) by means of an adhesion promoter to the carrier (10) are connected, wherein the
Haftvermittler vorzugsweise Chrom oder Titan ist.Adhesion promoter is preferably chromium or titanium.
8. Biochip (1) nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Schicht (20) an ihrer Unterseite reflektierend ausgebildet ist, um das Anregungslicht (80) zu spiegeln und die Substratmoleküle (60) mehrfach anzuregen.8. biochip (1) according to any one of the preceding claims, characterized in that the layer (20) is formed on its bottom reflective to reflect the excitation light (80) and to excite the substrate molecules (60) multiple times.
9. Biochip (1) nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass seine Oberfläche Linkermoleküle, welche insbesondere aminoreaktiv sind, und/oder Lipidderivate zur Kopplung der Membran (40) an den Chip (1) aufweist.9. biochip (1) according to any one of the preceding claims, characterized in that its surface has linker molecules, which are in particular amino-reactive, and / or lipid derivatives for coupling the membrane (40) to the chip (1).
10. Biochip (1) nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass eine Schicht (20) elektrisch leitend und als Elektrode ausgebildet ist, um die Membran (40) elektrisch zu vermessen oder elektrisch zu steuern oder zu schalten. 10. biochip (1) according to any one of the preceding claims, characterized in that a layer (20) is electrically conductive and designed as an electrode to electrically measure or electrically control or switch the membrane (40).
EP08748708A 2007-04-04 2008-04-02 Biochip for carrying out a fluorescence analysis of individual transporters Withdrawn EP2142911A2 (en)

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