WO2004052528A1

WO2004052528A1 - Capacitive detection of bound molecules

Info

Publication number: WO2004052528A1
Application number: PCT/EP2003/013913
Authority: WO
Inventors: Ralph Müller
Original assignee: Axaron Bioscience Ag
Priority date: 2002-12-09
Filing date: 2003-12-09
Publication date: 2004-06-24
Also published as: DE10257604A1; AU2003289985A1

Abstract

A biochip (20) is selected for the detection of molecules in a solution, to which the molecules for detection can bind. An electrically-conducting substrate (24) is selected for the biochip. An electrically-conducting array (32) of electrodes (36) is also necessary. The electrode array is in a spatial arrangement complementary to the spots of the biochips. A microcapacitor can be formed from each electrode and a spot. The electrode array is applied to the biochip after the binding reaction, whereby microcapacitors with a minimal separation of electrodes and counter-electrodes are formed. By means of said microcapacitors it can be determined if molecules have bound to the spot concerned, by means of a change in the dielectric constant of the space in the relevant capacitor.

Description

Applicant in:

Axaron Bioscience AG

In Neuenheimer Feld 515

69120 Heidelberg

Capacitive detection of bound molecules

description

Field of the Invention:

Biochips are increasingly used for analysis in the field of medicine, genome research or food analysis. The biochips usually consist of a slide on which different types of DNA or RNA oligonucleotides, peptides, proteins, enzymes, etc. are immobilized in separate spots. The spots are arranged in the form of a grid or array, which is why the biochips are also generally referred to as microarrays. With the help of this

Biochips are, for example, hybridization assays, e.g. B. transcription analyzes, usually carried out with the help of appropriately labeled, such as fluorescence-labeled samples. Microarrays are generally suitable for studying affinity reactions.

State of the art:

In addition to the fluorescence spectroscopic readout methods for biochips, other readout methods are known, among others readout methods that rely on principles of electrical engineering, that is to say measure an impedance, Determine electrochemical parameters or are based on voltammetry or polarometry. Detection methods based on the measurement of a capacitance are also described in a number of documents (see, for example, US 6,440,662, WO 97/34140, WO 00/62047, US 4,072,576, US 5,114,674, WO 87/03095). However, most of these solutions have proven to be less practical.

Task:

The object of the invention is to simplify the parallel detection of a large number of different biomolecules.

Solution;

This object is achieved by the inventions with the features of the independent claims. Advantageous developments of the inventions are characterized in the subclaims.

The molecules to be detected can be any chemical substances, e.g. B. from combinatorial chemistry or from a substance library for drug screening. As a rule, they are biomolecules of the type mentioned at the beginning, that is to say DNA, RNA, PNA, proteins, enzymes, peptides, tumor markers, etc. These molecules are usually in solution. However, the detection of other molecules is also possible. B. from the gas phase. A fluid is therefore generally understood to mean a flowable substance, in particular a gas or a liquid.

According to the invention, molecules are fixed on the top of a substrate on predetermined spots, to which the molecules to be detected can bind. Such a system is usually called a biochip. This can be done on a flat substrate or on a substrate with a plurality of wells. A well is typically a closed well, a small pot in an array, as found in micro or nanotiter plates.

An electrically conductive material is selected for the substrate so that the capacitors described below can be formed in a suitable manner. For example, the substrate can be formed from a suitably doped semiconductor material, which also offers the possibility of microstructuring with the usual means.

In addition, an electrically conductive array of electrodes is selected, the electrodes of which are designed such that they are then arranged essentially above the spots of the biochip when the electrode array is brought into a suitable position (complementary position) to the top of the biochip, whereby by an electrode and a spot of the biochip as a counter electrode

Capacitor is formed, the capacitor has a capacitance and the space between the electrode and the spot has a dielectric.

As a rule, the electrodes are individually raised on a substrate by microstructuring. In this case, the substrate is formed in integrated circuit technology in such a way that voltages can be applied to the electrodes individually.

However, it is also possible to design the electrodes in a flat substrate merely as spatial areas that can be subjected to voltages individually. The electrodes can also be produced by influence when the counter electrode is energized. It is sufficient to drive one side of a capacitor with electrical voltage; on the opposite side, a counterelectrode forms due to the influence, provided that the opposite side is electrically conductive. This then works as a ground electrode. Whether the coated side is controlled or the uncoated side - which will be the rule for practical reasons - or both sides is irrelevant.

Ultimately, it does not matter whether the molecules to be detected are bound on the electrode or the counterelectrode, on the electrically individually controllable element or not, on raised or recessed microstructured elements, in wells or on a flat substrate. Conceptually, but not structurally, the part on which molecules are bound is referred to as a biochip, the other as an electrode array. A system is also conceivable in which the electrode array simultaneously functions as a biochip, for example when such a system is only opposed to a conductive plate.

If the system is prepared in this way, the fluid with the molecules to be detected is brought into contact with the top of the biochip. As a result, the molecules to be detected are bound, provided they are present in the fluid and the physico-chemical boundary conditions for the binding reaction have been met.

The actual capacitive detection of the molecules follows. For this purpose, the electrode array and the top of the biochip are brought together in a complementary position. Typically, the biochip will have wells and the electrodes will protrude from the electrode array as small columns. In this In the usual case, the electrodes are lowered into the wells in a complementary position until they almost touch the bottom of the wells. As a result, there is only a minimal distance between the electrode and the counter electrode of the capacitor. This distance can be less than 1 μm, for example 50 nm.

The capacitance C of a capacitor is calculated in the textbook case of the plate capacitor according to the formula

A

C - ε ε _n

where ε is the dielectric constant of the substance between the capacitor plates, ε _{Q is} the dielectric constant of the

Vacuum, A is the area of the two opposing plates and d is the distance between the plates.

Since the capacitance of a capacitor is inversely proportional to the distance between the two electrodes, an extremely high capacitance results for the detection according to the invention. This in turn leads to an extremely high sensitivity when

Detection of changes in the dielectric constant of the space between the two electrodes. The dielectric constant changes when molecules are bound in the space between the electrodes. The very close merging of the two electrodes thus results in a high sensitivity in the capacitive detection of bound molecules. The sensitivity is sufficient for the detection of a few less bound molecules.

For capacitive detection, a change in the capacitance and / or the dielectric of the interspace of the individual capacitors compared to at least one comparison standard is determined. Determining the change in The capacitance and / or the dielectric can be done in different ways.

For this purpose, for example, the capacitance can be determined at a predetermined distance. The dielectric can then be concluded from the capacitance or its change. It is also possible to track the capacitance as a function of the distance between the electrode and the spot. There are then different ways of evaluating the function obtained. In the extreme case that the electrode touches the bound molecules and mechanically compresses it, for example, there will be a deviation from the 1 / d dependence of the capacitance on the distance.

As a rule, the difference or quotient to a reference is considered during the measurement. As a reference or comparison standard z. B. measurements before the supply of the fluid or measurements on spots that are not coated and therefore do not specifically bind molecules from the fluid. A well that is closed and does not come into contact with the sample can also serve as a reference. It therefore always offers constant conditions, in particular with regard to the dielectric of the substance filling it.

It can also be useful to train references for specific distances. This can be done by suitable elevations or depressions on the substrate of the biochip or the electrode array.

In order to be able to detect a large number of different molecules, a different type of molecule is usually fixed on each spot, which specifically binds a certain type of molecule from the fluid. For differentiated detection of all these different types of molecules, each capacitor formed from the electrode and spot must be individual be read out. For this purpose, the electrode array must be suitably designed electronically so that each electrode can be controlled individually for a sequential or parallel reading. It is not necessary that the conductive substrate of the biochip also have an individual address.

A localized counter electrode forms in the conductive substrate opposite to the electrode to which voltage is applied. The voltage between the electrode and this counterelectrode created by the influence can be measured as a mass between the electrode and the substrate. The capacitance or the dielectric of the intermediate space can be concluded from this.

Since each system of spot and electrode forms a small capacitor, an array of microcapacitors is obtained overall. The capacities obtained can be output as an array in the form of a capacitive image if appropriately displayed. The chip is then read out by reading out the capacitive image.

From the change determined in the respective measurement, it is determined whether molecules to be detected are bound to individual spots on the biochip.

If a substrate is selected which has an array of elevations and depressions, and additionally an electrode array which has an array of projecting electrodes which is complementary to the depressions and which can be inserted in a complementary position in the depressions of the substrate, this results in each of the individual capacitors to a strong concentration of the electric field that is building up on this capacitor alone, without there being any significant crosstalk or stray fields towards other capacitors. On a flat biochip, spaces of the dimensions of the spots exist between the spots on which binding molecules are immobilized. Typical values for the size of these spots are, for example, 100 µm. Since the binding reactions with the molecules to be detected are diffusion-limited, the typical lengths beyond which there is no longer any binding are in the range of 10 μm for the time usually available for the detection. This means that large parts of the spaces between the spots remain unused. It is therefore advisable to partially use these rooms with elevations or small columns. This has several advantages:

On the one hand, the gaps between the spots, which are anyway not to be used for the detection reaction, are included

Material filled that displaces the fluid used for the detection. Less fluid is then required for the detection.

On the other hand, this creates a system of increases and

Wells, similar to the wells of a conventional microtiter plate. In contrast to the usual wells, however, the depressions formed communicate with one another in such a way that a fluid can flow freely between them. It is therefore not a question of wells in the actual sense, but rather of so-called pseudowells.

Through such a biochip, a fluid can finally flow completely without the fluid having to be pipetted into each well individually. Due to the small spatial dimensions that the columns and depressions usually have in such a biochip, a liquid is distributed evenly over the biochip due to capillary forces and due to the grid-screen-like structure. A cover glass, but preferably an electrically insulating film, can be arranged between the biochip and the electrode array. A material with a dielectric constant close to 1 should be selected as the material for the film, so that the measurement of the capacitance or dielectric is not influenced by the film if possible. The film should also be as thin as possible so that the distance between the electrode and the counterelectrode of the capacitor is not unnecessarily increased by the film, which would reduce the sensitivity of the detection. The film should have a thickness in the range of 50 to 500 nm.

The arrangement of the film between the biochip and the electrode array has a number of advantages.

If a biochip with pseudowells is covered by such a film, a liquid is more easily distributed over the entire biochip when it is applied laterally by capillary forces. Such a chip can also be easily rinsed and dried completely.

In addition, such a film electrically isolates the biochip and the electrode array from one another, so that short circuits cannot occur in one of the capacitors.

The film also ensures that the electrode array is not contaminated during the measurement. So that in such a system i. d. R. reused the electrode array formed in semiconductor technology and the biochip as

Consumables designed.

The biochip and / or the electrode array can also be coated with an electrically insulating film. Nitrocellulose, for example, is suitable for such a coating on. Coating the biochip and / or the electrode array with nitrocellulose offers a number of advantages.

Nitrocellulose is electrically insulating. It thus prevents short circuits between the biochip and the electrode array.

Furthermore, coating the biochip with nitrocellulose allows DNA molecules to bind to the biochip in a very simple manner. DNA can be bound to nitrocellulose by irradiation with UV light (so-called UV crosslinking). Nitrocellulose itself is a network. UV radiation leads to chemical bonds between the backbone of the DNA and the nitrocellulose. For this purpose, the DNA to be bound can be applied to the nitrocellulose dissolved in a buffer. The DNA bound in this way lies flat on the nitrocellulose coating. At. As a result, the hybridization of a complementary DNA strand leads to an ideal alignment of the dipole moments of the base pairings, namely perpendicular to the surface of the spot, parallel to the electrical field lines between the electrodes of the capacitance measurement. This results in a maximum dielectric constant of the bound molecules.

After preparation of the surfaces, the nitrocellulose can be sprayed on with high pressure and in small quantities using chemicals from silane chemistry. A small amount of nitrocellulose, less than 1 μl, is added to a dry nitrogen jet. This results in layer thicknesses of less than 10 nm, typically between 1 and 100 nm. The layers formed in this way are very well fixed on the substrate. The invention is explained in more detail below on the basis of exemplary embodiments which are illustrated schematically in the figures. The same reference numbers in the individual figures denote the same elements. In detail shows:

1 shows a schematic representation of a biochip suitable for carrying out the invention; 2 shows a schematic illustration of a silicon wafer with a column array;

3 shows a schematic representation of a biochip with a film lying thereon; and FIG. 4 shows a schematic illustration of the capacitive detection.

1 shows in the upper right part an array 10 of spots 12 on which molecules are immobilized which can selectively bind the molecules to be detected from a solution.

An array 14 of rectangular columns 16 is shown in the central region of FIG. 1. Between the pillars 16 are the above-described pseudo-wells 18 which communicate with one another such that the solution can flow to the ^'molecules to be detected or a rinsing liquid freely between them.

The finished biochip 20 is shown in the lower right area. It arises from the fact that the spots 12 are placed in the pseudowells 18 of the column array 14. The spotting can be done in different ways, for example photolithographically. The spotting for all spots 12 is preferably carried out simultaneously using a stamp which has a multiplicity of projecting capillaries. Each of these projecting capillaries is connected to one Reservoir for the molecules to be applied in the respective spot 12. Such a stamp can be produced using microstructuring methods, among other things.

2 shows a silicon wafer 22 with a conductively doped silicon substrate 24. The thickness of the silicon wafer 22 is 675 μm including the columns 16. The columns 16 have a height of 10 μm. The columns 16 have a distance from the center of the column to the center of the column of preferably 300 μm to the nearest neighbor, measured parallel to the edges of the entire array 14. The column diagonal is in any case smaller than the distance, that is to say smaller than 300 μm, since otherwise the free flow of solutions between the pseudowells 18 would not be possible. Typical edge lengths of the columns 16 are 10 μm, 100 μm, 230 μm or 280 μm. The columns are prepared on the silicon wafer 22 using microstructuring processes.

3 shows the biochip 20 with the columns 16 prepared thereon. A film 26 lies on the biochip 20. The film is electrically insulating and preferably has a thickness between 50 and 500 nm. A thickness of one or a few micrometers is also conceivable. In order to have the required elasticity which becomes clearer below in connection with FIG. 4, a film made of PET (polyethylene terephthalate), PC (polycarbonate), PEN (polyethylene naphthalene), silicone film or PP (polypropylene) is preferably used. To the left of the biochip 20, an arrow 28 pointing to the right indicates that the solution to be examined is on one side of the solution covered by the film 26

Biochips 20 can be applied laterally. Due to capillary forces, the solution is distributed independently over the entire biochip 20. The checkerboard-like pattern of the columns 16 or the column array 14 (as can be seen in the middle part of FIG. 1) helps to further close the solution mix thoroughly. The solution can - as indicated on the right of the biochip 20 by the arrow 30 pointing to the right - be easily sucked out of the biochip 20 again or flushed through the biochip 20 with pressure and dried by nitrogen or dried air introduced.

4 illustrates the actual capacitive detection. 4A, the electrode array 32 is shown in the upper area. It consists of an electrically conductive semiconductor substrate 34, on which truncated pyramid-shaped electrodes 36 are formed. The electrodes 36 are formed from the substrate 34 using microstructuring methods. The electrodes 36 are preferably designed as truncated pyramids in order not to tear the foil 26 which may be present in the event of detection.

The electrode array 32 has a total height of preferably 675 μm. The electrodes 36 themselves have a height of 9 or 10 μm. The substrate 34 contains corresponding dopants

Conductor tracks and the formation of circuits an integrated circuit, which makes it possible to drive each electrode 36 individually.

In FIG. 4A, the arrow 38 indicates how the electrode array 32 is brought up to the biochip 20 for the capacitance measurement.

4B illustrates the positioning of the electrode array 32 and the biochip 20 during the capacitance measurement. The electrodes 36 are in the pseudowells

18 sunk in, so that the smallest possible distance between the electrode 36 and the bottom of the pseudo well 18 results. If the biochip 20 was covered by a film 26, the distance between the electrode 36 and the bottom of the Pseudowells 18 essentially determined by the thickness of the film 26.

If the fluid was an aqueous solution, it is removed before the capacitive measurement, since the dielectric constant of water with 81 would overlap the measured values of all other substances. For this purpose, the biochip can first be dried, for example with the aid of nitrogen. Alternatively, the electrodes 36 and optionally the film 26 can be designed such that they lie directly on the bottom of the pseudowells 18 for the capacitance measurement and thereby displace all residues of the solution. The half-open structure of the pseudowells helps here.

reference numeral

Array of spots 12 Spot with immobilized molecules Array of columns Pillar Pseudowell Biochip Silicon wafer Substrate of the silicon wafer Foil Solution inlet direction Solution outlet direction Electrode array Substrate of the electrode array 32 Electrode Direction of movement of the electrode array 32 for capacitive detection

cited literature

WO 87/03095 WO 97/34140 WO 00/62047 US 4,072,576 US 5,114,674 US 6,440,662

Claims

claims

1. A method for the detection of molecules in a fluid, comprising the following steps: a) on the upper side of a substrate (24), molecules are fixed (biochip, 20) on predetermined spots (12) to which the molecules to be detected can bind; b) an electrically conductive material is selected for the substrate (24); c) it is an electrically conductive array (32) from

Electrodes selected, the electrodes (36) of which are designed such that they are arranged essentially above the spots (12) of the biochip (20) when the electrode array (32) is brought into a suitable position (complementary position) to the top of the biochip whereby a capacitor is formed by an electrode (36) and a spot (12) of the biochip as a counter electrode, the capacitor having a capacitance and the space between the electrode and the spot having a dielectric; d) the fluid with the molecules to be detected is brought into contact with the top of the biochip (20); e) the electrode array (32) and the top of the biochip (20) are brought together in a complementary position; f) a change in the capacitance and / or the dielectric of the interspace of the individual capacitors compared to at least one comparison standard is determined; and g) it is determined from the determined change whether molecules to be detected are bound to individual spots of the biochip (20).

2. The method according to the preceding claim, characterized in that a substrate (24) is selected which has an array of elevations (16) and depressions (18); and that an electrode array (32) is selected which has an array of projecting electrodes (36) which is complementary to the depressions (18) and which can be countersunk into the depressions (18) of the substrate (24) in a complementary position. 5

3. The method according to the preceding claim, characterized in that the depressions (18) are selected such that they communicate with one another and the fluid can flow freely between them 10.

4. The method according to any one of the preceding claims, characterized in that an electrically insulating film (26) is arranged between the biochip (20) and the electrode array (32) 15.

5. The method according to any one of the preceding claims, characterized in that the biochip (20) and / or the electrode array (32) is coated with 20 an electrically insulating film.

6. The method according to the preceding claim, characterized in that a film of 25 nitrocellulose is selected as the electrically insulating film.

7. Arrangement for the detection of molecules in a fluid: a) with a substrate (24), on the upper side of which molecules are fixed on predetermined spots (12) (biochip, 20),

_30th to which the molecules to be detected can bind, the substrate being essentially formed from an electrically conductive material; and b) with an electrically conductive array (32) of electrodes (36), the electrodes of which are designed such that they then

35 essentially over the spots (12) of the biochip (20) are arranged when the electrode array (32) is brought to the top of the biochip in a suitable position; c) wherein the electrode array (32) is designed such that the electrodes (36) of the electrode array can be individually subjected to an electrical voltage.