WO2002070733A1

WO2002070733A1 - Method for detecting macromolecular biopolymers by using at least one nanoparticle provided with a scavenger molecule

Info

Publication number: WO2002070733A1
Application number: PCT/DE2002/000759
Authority: WO
Inventors: Johannes R. Luyken; Franz Hofmann; Erhard Landgraf; Thomas Schulz
Original assignee: Infineon Technologies Ag
Priority date: 2001-03-01
Filing date: 2002-03-01
Publication date: 2002-09-12
Also published as: DE10109777A1

Abstract

A unit for immobilizing macromolecular biopolymers is a nanoparticle. The unit used for immobilizing is provided with scavenger molecules that can bind macromolecular biopolymers. A sample to be examined is brought into contact with the unit used for immobilizing, whereby the sample to be examined can contain the macromolecular biopolymers to be detected. Macromolecular biopolymers contained in the sample to be examined are bound to the scavenger molecules, and the macromolecular biopolymers are detected by using a signal emitted by a marking.

Description

METHOD FOR DETECTING MACROMOLECULAR BIOPOLYMERS BY MEANS OF AT LEAST ONE NANOPARTICLE PROVIDED WITH A COLLECTING MOLECULE

The invention relates to a method for detecting macromolecular biopolymers by means of at least one unit for immobilizing macromolecular biopolymers.

Methods for detecting DNA molecules are known from [1] to [4], in which biosensors are used for the detection, which are based on electrode arrangements.

2a and 2b show such a sensor as described in [1] and [4]. The sensor 200 has two electrodes 201, 202 made of gold, which are embedded in an insulator layer 203 made of insulator material. Electrode connections 204, 205 are connected to the electrodes 201, 202, to which the electrical potential applied to the electrode 201, 202 can be supplied. The electrodes 201, 202 are arranged as planar electrodes. DNA probe molecules 206 are immobilized on each electrode 201, 202 (cf. FIG. 2a). The immobilization takes place according to the so-called gold-sulfur coupling. The analyte to be examined, for example an electrolyte 207, is applied to the electrodes 201, 202.

If the electrolyte 207 contains DNA strands 208 with a sequence that is complementary to the sequence of the DNA probe molecules 206, these DNA strands 208 hybridize with the DNA probe molecules 206 (cf. FIG. 2b).

Hybridization of a DNA probe molecule 206 and a DNA strand 208 only takes place if the sequences of the respective DNA probe molecule 206 and the corresponding DNA strand 208 are complementary to one another. If not ff M s α

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This procedure is therefore expensive, complicated and very sensitive to interference, which means that the measurement result can easily be falsified.

Furthermore, it is known from affinity chromatography (cf. [6]) to use immobilized small molecules, in particular ligands of high specificity and affinity, to generate peptides and proteins, e.g. Enzymes to bind specifically in the analyte.

Furthermore, it is also known that in the case of detection methods for antigens or antibodies, such as the so-called ELISA tests, which are based on solid phase systems, one of the two reactants is bound to a solid phase (e.g. microtiter plates). The antibody-antigen reaction carried out is verified by a labeled reaction partner (cf. [7]). More specifically, in such an antibody capture test, the antigen is first bound to a solid support. A labeled antibody in solution then reacts with the antigen. After the unbound antibody has been washed out, a qualitative or quantitative statement is obtained by measuring the label on the bound antibody (cf. [8] and [9]).

A reduction / oxidation-recycling method for detecting macromolecular biopolymers from [2] and [3] is also known.

The reduction / oxidation recycling process, hereinafter also referred to as the redox recycling process, is explained in more detail below with reference to FIGS. 4 a to 4 c.

4a shows a biosensor 400 with a first electrode 401 and a second electrode 402, which are applied to a substrate 403 as an insulator layer. A holding area, designed as a holding layer 404, is applied to the first electrode 401 made of gold. The holding area is used to immobilize DNA probe molecules

405 on the first electrode 401.

No such holding area is provided on the second electrode.

If 400 DNA strands with a sequence that is complementary to the sequence of the DNA probe molecules 405 are to be detected by means of the biosensor 400, the sensor 400 is treated with a solution 406 to be examined, e.g. an electrolyte, brought into contact in such a way that any DNA strands contained in the solution 406 to be examined can hybridize with the complementary sequence to the sequence of the DNA probe molecules 405.

Fig.4b shows the case that in the solution to be examined

406 the DNA strands 407 to be detected are contained and hybridized to the DNA probe molecules 405.

The DNA strands 407 in the solution to be examined are marked with an enzyme 408, with which it is possible to cleave the molecules described below into partial molecules.

Usually, a considerably larger number of DNA probe molecules 405 is provided than the DNA strands 407 to be determined are contained in the solution 406 to be examined.

After the DNA strands 407 possibly contained in the solution 406 to be examined have hybridized with the enzyme 408 with the immobilized DNA probe molecules, the biosensor 400 is rinsed, as a result of which the non-hybridized DNA strands are removed and the biosensor 400 from it investigating solution 406 is cleaned. An electrically uncharged substance is added to this rinsing solution used for rinsing or another solution 412, which is supplied in a separate phase, and which contains molecules which, by means of the enzyme on the hybridized DNA strands 407, into a first submolecule of a negative first electrical charge and into a second Partial molecule of a positive second electrical charge can be split.

As shown in FIG. 4c, the negatively charged partial molecules are drawn to the positively charged anode, as indicated by arrow 411 in FIG. 4c.

The negatively charged first partial molecules 410 are oxidized on the first electrode 401, which has a positive electrical potential as the anode, and are oxidized as the oxidized partial molecules 413 on the negatively charged cathode, i.e. pulled the second electrode 402 where they are reduced again.

The reduced sub-molecules 414 in turn migrate to the first electrode 401, i.e. to the anode.

In this way, an electrical circuit current is generated which is proportional to the number of charge carriers generated in each case by the enzymes 408.

The electrical parameter that is evaluated with this method is the change in the electrical current - as dt

Function of time t, as shown in diagram 500 in FIG. 5.

Furthermore, methods for the nanostructuring of substrates such as glass or silicon wafers are known, ie methods with which nano-particles are produced from metal on these substrates (cf. [12-14]. From [13] is furthermore known that streptavidin molecules can be bound to such gold nanoparticles by means of biotin derivatives and can be made visible by scanning force microscopy.

In addition, a method for the determination of an analyte such as a protein is known from [15], in which a labeled reagent is used which is able to bind specifically to the analyte and which comprises a brine with gold particles in which at least 75 percent by weight of the gold particles have an average diameter of less than 5 nanometers. In the method from [15], this reagent, which is present in an aqueous solution, is brought into contact with a solution containing the analyte and the complex formed in this way is immobilized for the determination.

[16] also discloses a self-addressable microelectronic device which is designed in such a way that it can actively carry out molecular-biological multi-step reactions and multiplex reactions in microscopic formats. Another self-addressable microelectronic system for carrying out molecular biological reactions is known from [17].

[18] finally describes a method for screening target-ligand interactions using a chemical library of ligands, in which at least one fluorescence property of a chemical library of ligands immobilized on a solid phase, a molecular fluorescence sensor being bound to each ligand, is measured before and after adding the target.

The abovementioned methods for detecting macromolecular biopolymers have in common that the exact number of capture molecules immobilized on the substrate or surface is not precisely determined. This is particularly disadvantageous in the case of quantitative detection of macromolecular biopolymers.

The problem underlying the invention is to provide an alternative method for the detection of macromolecular biopolymers.

The problem is solved by the method with the features according to the independent claims.

Such a method for detecting macromolecular biopolymers uses at least one unit for immobilizing macromolecular biopolymers. The at least one unit for immobilizing biopolymers is a nanoparticle.

In a first method, the at least one unit for immobilizing macromolecular biopolymers is provided with capture molecules, the capture molecules being able to bind macromolecular biopolymers. A sample to be examined is then brought into contact with the at least one unit for immobilizing macromolecular biopolymers, wherein the sample to be examined can contain the macromolecular biopolymers to be detected. The macromolecular biopolymers contained in the sample to be examined are bound to the capture molecules and the macromolecular biopolymers are detected by means of a signal emitted by a label. In simple terms, the present methods are distinguished from the known methods for detecting macromolecular biopolymer in that they use nanoparticles as a unit or units for immobilizing macromolecular biopolymers (by means of capture molecules).

For the purposes of the invention, a nanoparticle is understood to mean a particle which is characterized by so-called Nanostructuring processes can be obtained. Nanostructuring methods that can be used to produce such nanoparticles on suitable substrates are, for example, the use of block copolymer microemulsions described in [12] and [13] or the use of colloid particles as structuring masks described in [14]. The method described in [14] is in principle analogous to a lithography method usually used in the field of substrate structuring. It should therefore be emphasized here that a nanoparticle in the sense of the invention is not restricted to those particles which are obtained by one of the methods mentioned here by way of example. Rather, such a nanoparticle is any particle whose diameter is in the nanometer range, ie generally between 2 and 50 nm, preferably in the range from 5 to 20 nm, particularly preferably in the range from 5 to 10 nm.

A "unit for immobilization, which is a nanoparticle", which is also referred to below as a nanoparticle-shaped unit, is consequently a nanoparticle described above, which has a surface on which the capture molecules can be immobilized, ie the surface is such that you can bind the capture molecules through physical or chemical interactions. These interactions include hydrophobic or ionic (electrostatic) interactions and covalent bonds. Examples of suitable surface materials that can be used for the at least one nanoparticle-shaped unit for immobilization are metals such as gold or silver, semiconducting materials such as silicon, plastics such as polyethylene or polypropylene or silicon dioxide, for example in the form of glass. Nanoparticulate units made of plastics and silicon dioxide are asken by using the colloid described in [14]. Process available Nanoparticulate units made of semiconducting materials such as silicon can also be formed, for example, by the Stranski-Krastanov process

By oxidizing such nanoparticles from silicon, nanoparticle-shaped units made from silicon dioxide are still available.

If one of the m [12] to [14] mentioned or a corresponding manufacturing process is used for the production of the nanoparticle-shaped units for immobilization, these units take on the substrate surfaces used, for example metal or oxide surfaces of photodiodes or electrodes a regular order. The individual units usually have distances from one another in the range of a few 10 nanometers, for example from approximately 10 to 30 nm, on these surfaces. The type of arrangement and the distance of the nanoparticles from one another, as well as their size, depend on the particular method for forming the nanoparticles.

An advantage of using one or more units for immobilizing macromolecular biopolymers that are present as nanoparticles is that a precisely defined number of capture molecules can be immobilized on these nanoparticles. In the optimal case, a single capture molecule can be bound per nanoparticle-shaped unit.Another advantage of using nanoparticles as immobilization units is that the spacing of the nanoparticles from one another, i.e. the spatial separation of the capture molecules, means that the capture olekules are more accessible for the binding macromolecular biopolymers and thus the probability an interaction between the capture molecule and the molecule to be detected is increased. Training as

Nanoparticles also increase the effective surface.

All of these advantages are particularly useful in the quantitative detection of macromolecular biopolymers, so that in a preferred embodiment the present method is used for the quantitative detection of macromolecular biopolymers.

For the purposes of the invention, detection means both the qualitative and quantitative detection of macromolecular biopolymers in an analyte to be examined. This means that the term "capture" also includes determining the absence of macromolecular biopolymers in the analyte.

In general, any substrate material can be used to form the nanoparticle-shaped unit or units thereon. So they can e.g. on a glass surface, a silicon wafer, a metal surface, a plastic surface such as a microtiter plate are formed.

In a preferred embodiment of the method, the at least one immobilization unit is applied to an electrode or a photodiode. The multiple units for immobilization are preferably applied in a regular arrangement on the electrode or the photodiode, or on the substrate in general.

In a first method disclosed here, the label, which is used to detect the macromolecular biopolymer, is attached to the capture molecule and not as in the methods described in the prior art the macromolecular biopolymer to be detected. The fact that, instead of the biopolymers to be detected, the capture molecules are provided with a label before their immobilization has the advantage that the sample to be examined which may contain the biopolymer is no longer subjected to a labeling reaction, in which part of the sample or the entire sample may be lost or may not run completely when marked.

However, it is also possible that the marking is attached to the macromolecular biopolymer to be detected.

In the first method described here, the marking generates a signal. In one embodiment, such a signal is an electrical current. In a further embodiment, the signal is visible light or UV light. The signal can also consist of radioactive radiation or X-rays.

From this it can be seen that different types of marking, which is also referred to below as a reporter group, can be used in the method.

On the one hand, the label can be a (chemical) compound or group that is directly capable of generating a signal that can be used to detect the macromolecular biopolymers. The generation of this signal can be stimulated externally, but the marking can also emit the signal without external excitation. In the former case, the label is, for example, a fluorescent dye (fluorophore) or a chemiluminescent dye, in the latter case, for example, a radioisotope.

On the other hand, the label can be a substance that only indirectly generates a signal for the detection of the macromolecular biopolymers, ie a substance that generates it of the signal. For example, such a reporter group can be an enzyme that catalyzes a chemical reaction and the chemical reaction is used to detect the biopolymers. Examples of such enzymes are alkaline phosphatase, glutathione-S-transferase, superoxide dismutase, marine right peroxidase, alpha-galactosidase and beta-galactosidase. These enzymes are capable of cleaving suitable substrates which give colored end products or, for example, compounds which can be used in the above-described reduction / oxide recycling process. The group of labels that only indirectly generate a signal that can be used to detect macromolecular biopolymers also includes ligands for binding proteins and substrates for enzymes. This label is commonly referred to here as enzyme ligands. Examples of such enzyme ligands that can be used as labels are biotm, digoxigenm or substrates for the above-mentioned enzymes.

A second method for detecting macromolecular biopolymers disclosed here uses one

Electrode arrangement which has a first electrode and a second electrode.

In this second method, the first electrode is provided with at least one unit for immobilizing macromolecular biopolymers. The immobilization unit, like the first method, is a nanoparticle. In this method, the at least one unit for immobilizing macromolecular biopolymers on the first electrode is provided with capture molecules, the capture molecules being able to bind macromolecular biopolymers. Then, in the method, a medium is brought into contact with the electrodes, and the medium to be examined can contain the macromolecular biopolymers to be detected. The macromolecular biopolymers contained in the sample to be examined are bound to the capture molecules. The macromolecular biopolymers are then recorded using an electrical measurement.

In a further development of the second method, the second electrode is also provided with at least one unit for immobilizing macromolecular biopolymers, this at least one unit for immobilizing also being a nanoparticle. In this embodiment of the method, the at least one unit for immobilizing macromolecular biopolymers on the second electrode is provided with capture molecules, the capture molecules being able to bind macromolecular biopolymers.

In a further embodiment of the second method, a first electrical measurement is carried out on the electrodes before step a) or step b), i.e. either at a time when the capture molecules are present on the electrodes (before step c) or not (before step a). In these cases, a second electrical measurement is carried out on the electrodes after step c). Then, depending on a comparison of the results of the two electrical measurements on the electrodes, the macromolecular biopolymers are recorded.

In this embodiment, the capacitance between the electrodes, the electrical resistance of the electrodes or an impedance measurement are preferably measured in the first electrical measurement and the second electrical measurement.

In a further embodiment of the second method, first and second capture molecules are used. The at least one unit for immobilizing macromolecular biopolymers with the is located on the first electrode provide first capture molecules that can bind macromolecular biopolymers of a first type. The at least one unit for immobilizing macromolecular biopolymers located on the second electrode is provided with second capture molecules which can bind macromolecular biopolymers of a second type.

In this embodiment, a medium is brought into contact with the electrodes in such a way that if there are macromolecular biopolymers of the first type in the medium, they can bind to the first capture molecules and if there are macromolecular biopolymers of the second type in the medium , can bind them to the second capture molecules. Subsequently, unbound first catcher molecules or second catcher molecules are removed from the respective electrode and the second electrical measurement is then carried out on the electrodes.

A plate electrode arrangement or an electrode arrangement can be used as the electrode arrangement which has the one electrode which is provided with at least one nanoparticle-shaped unit for immobilization

Interdigital electrode arrangement, as known from [1], can be used. In the case of an interdigital electrode arrangement, several or all “fingers” of the arrangement can of course be nanostructured.

Furthermore, different arrangements of the parallel connection of electrodes can be provided in the electrode arrangement, for example the electrodes can be configured as cylindrical elements, which are each arranged concentrically around one another and are electrically isolated from one another, for example by means of a suitable dielectric, so that an electric field between the electrodes Forms electrodes. It should be noted that it is of course also possible with the first method not to record just one type of biopolymer in a single series of measurements. Rather, several macromolecular biopolymers can be recorded simultaneously or one after the other. For this purpose, several types of capture molecules, each of which has a (specific) binding affinity for a specific biopolymer to be detected, can be bound on the immobilization unit and / or several immobilization units can be used, each of these units only a kind of capture molecule is bound. In these multiple determinations, a mark that can be distinguished from the other markings is preferably used for each macromolecular biopolymer to be detected. For example, two or more fluorophores can be used as labels, each of these fluorophores preferably having a specific excitation and emission wavelength.

Macromolecular biopolymers that can be detected with the aid of the methods disclosed here include nucleic acids such as DNA and RNA molecules or shorter nucleic acids such as oligonucleotides with 10 to 20 base pairs

(bp) understood. The nucleic acids can be double-stranded, but also have at least single-stranded regions or, for example by preceding thermal denaturation (strand separation) for their detection, can be present as single strands. The sequence of the nucleic acids to be detected can be at least partially or completely predetermined, i.e. be known. Other macromolecular biopolymers are proteins or peptides. These can be made up of the 20 amino acids usually found in proteins, but can also contain naturally occurring amino acids or e.g. due to sugar residues

(Oligosaccharides) may be modified or post-translational Modifications included. Furthermore, complexes from several different macromolecular biopolymers can also be detected, for example complexes from nucleic acids and

Proteins.

If proteins or peptides are to be detected as macromolecular biopolymers, ligands which can specifically bind the proteins or peptides to be detected are preferably used as capture molecules. The capture molecules / ligands are preferably linked to the immobilization agent by covalent bonds.

Low-molecular enzyme agonists or enzyme antagonists, pharmaceuticals, sugars or antibodies or any molecule which has the ability to specifically bind proteins or peptides can be considered as ligands for proteins and peptides.

If DNA molecules (nucleic acids or oligonucleotides) of a given nucleotide sequence are recorded using the methods described here, they are preferably recorded in single-stranded form, ie, if necessary, they are converted into single strands by denaturation as explained above. In this case, DNA probe molecules with a sequence complementary to the single-stranded region are then preferably used as capture molecules. The DNA probe molecules can in turn have oligonucleotides or longer nucleotide sequences as long as they do not form any of the intermolecular structures that prevent hybridization of the probe molecule with the nucleic acid to be detected. However, it is also possible to use DNA-binding proteins or agents as capture molecules. In a first process step of the processes described here, the at least one immobilization unit is provided with the capture molecules. In the first method, these capture molecules can have a label that can generate a detectable signal.

A sample to be examined, preferably a liquid medium such as an electrolyte, is then brought into contact with the immobilization unit. This is done in such a way that the macromolecular biopolymers can bind to the capture molecules. In the event that there are several macromolecular biopolymers to be detected in the medium, the conditions are selected such that they can bind to their corresponding capture molecule at the same time or one after the other.

After waiting a sufficient period of time for the macromolecular biopolymers to bind to the corresponding capture molecule or molecules, unbound capture molecules are removed from the immobilization unit or units on which they are located.

If proteins or peptides are to be detected as macromolecular biopolymers, the unbound ligands used as capture molecules are removed from the at least one immobilization unit by bringing a material into contact with the at least one immobilization unit, the material being able to do so to hydrolyze the chemical bond between the ligand and the immobilization unit. In the event that the capture molecules are low molecular weight

If they are ligands, they can, if unbound, also be removed enzymatically

For this purpose, the ligands are covalently linked to the immobilization unit via an enzymatically cleavable compound, for example via an ester compound.

In this case, for example, a carboxyl ester hydrolase (esterase) can be used to remove unbound ligand molecules. This enzyme hydrolyzes the ester bond between the immobilization unit and the respective ligand molecule that was not bound by a peptide or protein. In contrast, the ester compounds between the immobilization unit and those molecules that have entered into a binding interaction with peptides or proteins remain intact due to the reduced steric accessibility that occurs due to the space filling of the bound peptide or protein.

In the event that the capture molecules are DNA strands, the unbound probe molecules are removed enzymatically, for example with the aid of an enzyme with nuclease activity. An enzyme which selectively degrades single-stranded DNA is preferably used as the enzyme with nuclease activity. The selectivity of the degrading enzyme for single-stranded DNA must be taken into account here. If the enzyme selected for the degradation of non-hybridized DNA single strands does not have this selectivity, the DNA to be detected, which is in the form of a double-stranded hybrid with the olekul probe, may also be undesirably degraded In particular, DNA nucleases, for example a nuclease from mung beans, the nuclease P1 or the nuclease S1 can be used to remove the unbound DNA probe molecule from the respective electrode. Likewise, DNA polymerase, which due to their 5 '->3' exonuclease activity or their

3 '-> 5' exonuclease activity are able to degrade single-stranded DNA.

After the unbound capture molecules have been removed, the macromolecular biopolymers are recorded. In the first method, this is done by means of the marking. For example, either a signal spontaneously emitted by the marking, such as radioactive radiation, or a signal caused by external excitation, such as emitted fluorescent radiation, is measured. In the second method, the detection takes place by means of an electrical measurement.

Embodiments of the invention are shown in the figures and are explained in more detail below.

Show it

Figures la to ld the formation of a biosensor with which a method described here for the detection of macromolecular biopolymers can be carried out;

FIGS. 2a and 2b show a sketch of two planar electrodes, by means of which the existence of DNA strands to be detected in an electrolyte (FIG. 2a) or their nonexistence (FIG. 2b) can be verified;

Figures 3a to 3e a biosensor to different

Process states on the basis of which a process according to an embodiment of the invention is explained;

Figures 4a to 4c sketches of a biosensor according to the prior art, on the basis of which individual conditions are explained in the context of the redox recycling process;

FIG. 5 shows a functional curve of a circulating current according to the prior art in the context of a redox recycling process;

FIGS. 6a and 6b show a biosensor with which a redox

Recycling process can be carried out as a further embodiment of a method described here.

FIGS. 7a to 7d an electrode arrangement for different process states, on the basis of which a further method according to an embodiment of the invention is explained;

1 shows a biosensor 100 which is designed with at least one immobilization unit in the form of nanoparticles and with which the method described here can be carried out.

The biosensor 100 has a layer 101 made of a suitable substrate material such as e.g. Silicon or gold and another layer 102 thereon on (Fig.la). The second layer 102 consists of a metal that is not suitable for the immobilization of macromolecular biopolymers. Such a metal is e.g. Platinum.

The units for immobilizing macromolecular biopolymers in the form of nanoparticles are formed on layer 102 by the following method. A solution of 0.5% by weight block copolymer polystyrene (PS) - block-poly (2-vinylpyridine) (P2VP) of the general formula PS (x) -b-P2VP (y) is, as in [12] and [13], with 0.5 equivalents of HAuCl ₄ -H ₂ 0 per pyridine unit to form monodisperse (micellar dissolved) gold particles, x and y in the formula give the number of basic units according to the ratio between monomer and initiator.

After the formation of homogeneous micelles, a monolayer of gold nanoparticles is deposited on the layer 102 from this solution by reduction with hydrazine, as described in [12] and [13]. The organic components of the separated micelles, i.e. the block copolymer, removed from the layer 102 by plasma etching using an oxygen plasma (cf. [13]). The gold particles 103, which serve as the units for immobilizing macromolecular biopolymers, remain intact during this treatment with plasma and, as illustrated in the sectional view and the top view of FIG. 1b, form a regular arrangement on the layer 102 (cf. [ 12]). The distances between the gold nanoparticles 103 are usually a few 10 nm, e.g. approx. 20 to 30 nm. The nanoparticles preferably have a size in the range from approx. 5 to 10 nm.

In addition to the block polymer mentioned above, other block polymers can of course also be used to form the nanoparticles.

Alternatively, the units 103 for immobilization in nanoparticle form can be generated on the biosensor, as described in [14], by first forming a mask for the nanostructuring from colloid particles on the layer 102 and then depositing gold particles, for example by means of vacuum deposition. After the application of the nanoparticles 103 made of gold, the sensor 100 is structured in such a way that the layer 102 made of platinum together with the units 103 for immobilization only remains on selected areas, as is shown in the sectional view and the top view of FIG. 1c. This structuring is possible, for example, using any suitable common chemical etching process.

With the help of the biosensor 100 designed in this way, one of the methods described here for detecting macromolecular biopolymers can be carried out. FIG. 1d shows a DNA capture molecule 104 immobilized on a gold nanoparticle 103 by means of the gold-sulfur coupling.

3 shows a section of a biosensor 300 with which an embodiment of a method described here can be carried out.

The biosensor 300 has a first photodiode 301 and a second photodiode 302, which are arranged in an insulator layer 303 made of insulator material such as silicon. The biosensor 300 furthermore has an oxide layer 304 and a second layer 305 thereon. The second layer 305 consists of a metal such as platinum, which is not suitable for the immobilization of macromolecular biopolymers.

The nanoparticle-shaped units 306 for immobilizing macromolecular biopolymers are located on the layer 305, the units 306 being able to be formed by the method explained with reference to FIG. 1 (cf. the sectional view of FIG. 3a and the top view of FIG. 3a). The immobilization units 306 are made of gold.

The first photodiode 301 and the second photodiode 302 are via first electrical connections 307 and second electrical connections, respectively Connections 308 connected to an evaluation unit (not shown).

Alternatively, immobilization units 306 may be made of silicon oxide, e.g. by forming silicon particles through the masks and vacuum deposition described in [14] or by the Stranski-Krastanov method and then oxidizing them to silicon oxide. These silicon dioxide units 306 thus obtainable can be coated with a material which is suitable for immobilizing capture molecules.

For example, known alkoxysilane derivatives can be used, such as

3-glycidoxypropylmethyloxysilane,

3-acetoxypropyltrimethoxysilane,

3-aminopropyltriethoxysilane,

4- (hydroxybutyra ido) propyltriethoxysilane,

• 3-N, N-bis (2-hydroxyethyl) aminopropyltriethoxysilane, or other related materials that are able to form a covalent bond with the surface of the silicon oxide at one end and a chemically reactive group at the other end to the probe molecule to be immobilized such as offering an epoxy, acetoxy, amine or hydroxyl radical for the reaction.

If a capture molecule to be immobilized reacts with such an activated group, it is bound via the selected material as a kind of covalent linker on the surface of the coating on the immobilization unit.

DNA probe molecules 309, 310 are applied as capture molecules to the nanoparticulate units for immobilization 306 (FIG. 3c, the units 306 are not shown for the sake of simplicity). First DNA probe molecules 309 with a sequence complementary to a predetermined first DNA sequence are applied to the first photodiode 301 by means of the units 306. The DNA probe molecules 309 are each labeled with a first fluorophore 311.

For example, fluorescein can be used as the fluorophore. The capture molecules 309 can be labeled by enzymatically labeling a correspondingly labeled nucleotide such as ChromaTide Fluorescein-12-dUTP (Molecular Probes, Inc., Eugene, Oregon, USA, Product No. C-7604). by means of suitable polymerases such as DNA polymerase or Klenow polymerase, into which oligonucleotides (capture molecules) 309 are incorporated (cf. [10]).

Second DNA probe molecules 310 with a sequence that is complementary to a predetermined second DNA sequence are applied to the second photodiode 302. The DNA probe molecules 310 are each with a second fluorophore

Marked 312. As a marker 312, e.g. the

TM fluorophore "Oregon Green 488" serve that also at a

Nucleotide coupled like dUTP (Molecular Probes, Inc., Eugene,

Oregon, USA, Product No. C-7630) is enzymatically incorporated into the DNA molecules 310.

To the pyrimidine bases adenine (A), guanine (G), thymine (T) or uracil (U) in the case of a label described above, or cytosine (C), sequences of DNA strands complementary to the sequences of the probe molecules can be in each case the usual way, ie hybridize by base pairing via hydrogen bonds between A and T or U or between C and G.

3c also shows an electrolyte 313 which is brought into contact with the photodiodes 301, 302 and the DNA probe molecules 308, 309. 3d shows the biosensor 300 in the event that the electrolyte 313 contains DNA strands 314 which have a predetermined first nucleotide sequence which is complementary to the sequence of the first DNA probe molecules 309.

In this case, the DNA strands 314 complementary to the first DNA probe molecules 309 hybridize with the first DNA probe molecules 309, which are applied to the first photodiode 301.

Since the sequences of DNA strands only hybridize with the specific complementary sequence in each case, the DNA strands complementary to the first DNA probe molecules do not hybridize with the second DNA probe molecules 310.

As can be seen from FIG. 3d, the result after hybridization has occurred is that on the first photodiode

301 hybridized molecules, i.e. double-stranded DNA molecules are immobilized. On the second photodiode

302, only the second DNA probe molecules 310 are present as single-stranded molecules.

In a further step, the single-stranded DNA probe molecules 310 on the second photodiode 302 are hydrolysed by means of a biochemical method, for example by adding DNA nucleases to the electrolyte 313.

The selectivity of the degrading enzyme for single-stranded DNA must be taken into account. If the enzyme selected for the degradation of the non-hybridized single DNA strands does not have this selectivity, the nucleic acid to be detected as double-stranded DNA may also be degraded (undesirably), which would lead to a falsification of the measurement result. After removal of the single-stranded DNA probe molecules, ie the second DNA probe molecules 310 on the second photodiode 302, only the hybrids of the DNA molecules 314 to be detected and the first DNA probe molecules 309 complementary thereto (cf. FIG. 3e) are present.

For example, to remove the unbound single-stranded DNA probe molecules 310 on the second photodiode 302, i.e. the second immobilization unit, one of the following substances is added:

Mung bean nuclease,

• Nuclease Pl, or

Nuclease Sl.

For this purpose it is also possible to use DNA polymerases which, owing to their 5 '- 3' exonuclease activity or their 3 '~> 5' exonuclease activity, are able to break down single-stranded DNA.

After the single-stranded probe molecules have been broken down, the electrolyte can optionally be removed from the photodiodes 301 and 302. This increases the contrast, i.e. reduces the background during the subsequent fluorescence measurement.

Using a laser, not shown, light symbolized by arrows 315 is then irradiated with a wavelength which is suitable for exciting the first fluorophore 311 and the second fluorophore 312 for fluorescence. Depending on the type of fluorophores, different wavelengths can also be used, either simultaneously or in succession.

The incident light only excites the fluorophore 311 located on the first DNA probe molecules 309, since the unbound second DNA probe molecules 310 together with the fluorophore 312 have been removed from the second photodiode 302 by the nuclease treatment (see FIG. 3e ). The fluorescence radiation symbolized by the arrow 316, which is emitted by the fluorophore 311, is detected by the first photodiode 301. In contrast, no fluorescence radiation is detected at the second photodiode 302.

The presence of the DNA molecules 314 is determined in this way. The use of the biosensor 300 described here permits locally resolved detection and offers a significant simplification of the entire measuring arrangement, since no external unit for detecting the fluorescence radiation is necessary.

FIG. 6 shows a biosensor 600 with which a redox recycling process can be carried out as a further exemplary embodiment of the invention. In this exemplary embodiment, a macromolecular biopolymer is detected both with the aid of a signal emitted by a marker and with the aid of an electrode arrangement which has a first electrode which is provided with at least one nanoparticle-shaped unit for immobilizing macromolecular biopolymers.

The biosensor 600 has three electrodes, a first electrode 601, a second electrode 602 and a third electrode 603.

The electrodes 601, 602, 603 are electrically insulated from one another by means of an insulator material as the insulator layer 604.

A holding area 605 is provided on the first electrode 601 for holding probe molecules which can bind macromolecular biopolymers. This holding area 605 has units for immobilizing 606 in the form of nanoparticles, the units 606 being made of gold. The probe molecules (capture molecules) 607 according to this exemplary embodiment, which are immobilized on the holding area, are DNA probe molecules with which DNA strands can hybridize with a sequence complementary to the sequence of the DNA probe molecules. At its 5 'terminus, the probe molecules 607 carry a Biotm group as the marker 608, which is used, for example, by using the “FluoReporter Biotm-X-C5 Oligonucleotide Labelmg Kit” (product no. F-6095) from Molecular Probes, Eugene, Oregon, USA can be added there (see [11]).

The DNA probe molecules 607 are immobilized on the units 606 for immobilization on the first electrode 601 by means of the known gold-sulfur coupling. If another material is used to bind the probe molecules, the material is provided with the corresponding coating material on which the probe molecules can be immobilized.

During the immobilization of the DNA probe molecules on the first electrode 601, different electrical potentials are applied to the electrodes, so that an electrical field arises between the electrodes in such a way that immobilization of the DNA probe molecules is only possible on the first electrode 601 and on the second electrode 602 and / or on the third electrode 603 is prevented.

In a manner analogous to the method according to the prior art as described above (cf. FIG. 4), in a further step a solution 610 to be examined, for example an electrolyte, with the macromolecular biopolymer to be detected, ie the DNA strands, which can hybridize with the DNA probe molecules, are brought into contact with the biosensor 600, ie in particular with the first electrode 601 and the labeled DNA probe molecules 607 m thereon. This is done in such a way that possible m of the solution to be examined contained DNA strands 609 can hybridize with the DNA probe molecules 606.

Then capture molecules 607 to which no DNA strands to be detected have hybridized are removed. This can be done by adding the above-mentioned DNA nucleases to the electrolyte 610. In this case too, one of the following substances can be used for the hydrolysis of the single-stranded DNA probe molecules 607:

Mung bean nuclease,

* Nuclease Pl, β Nuclease Sl, or

«DNA polymerases, which because of their

5 '- ^ 3' exonuclease activity or its

3 '-7> 5' exonuclease activity is able to degrade single-stranded DNA.

After the nuclease treatment, only the hybrids of labeled capture molecules 607 and DNA molecules 609 to be detected are located on the biosensor. This state is shown in FIG.

In this state, the biosensor 600 is rinsed using a rinsing solution, not shown, i.e. the fragments of the non-hybridized DNA strands and the solution to be examined are removed.

In a next step, a further solution (not shown) is brought into contact with the biosensor 600, in particular with the first electrode 601.

This further solution contains an enzyme 611 which binds to the label 608 of the hybridized DNA probe molecules 607 and which can cleave the molecules explained below, which are added in a further solution 612. According to this exemplary embodiment, enzyme 611 can be, for example

Alpha-galactosidase,

Beta-galactosidase,

Beta-glucosidase,

Alpha-mannosidase,

• Alkaline phosphatase,

Acid phosphatase,

Oligosaccharide dehydrogenase,

Glucose dehydrogenase, β laccase,

Tyrosinase,

• or related enzymes can be used.

The enzyme 611 is used here in the form of an avidin conjugate. The reason for this is that avidin forms a specific bond with the biotin label 608 used here as an example (FIG. 6b).

It should be noted here that low molecular weight enzymes can ensure the highest conversion efficiency and therefore also the highest sensitivity when used as an enzyme which effects redox recycling.

The further solution 612 contains molecules 613 which can be split by the enzyme 611 into a first sub-molecule 614 with a negative electrical charge and into a second sub-molecule with a positive electrical charge (cf. FIG. 6b).

Above all, for example, as the cleavable molecule 613

P-aminophenylhexopyranoside,

P-aminophenyl phosphates,

P-nitrophenyl hexopyranoside,

P-nitrophenyl phosphate, or

• suitable derivatives of e) diamines, f) catecholamines, g) Fe (CN -, h) ferrocene, i) dicarboxylic acid, j) ferrocene lysine, k) osmium bipyridyl-NH, or 1) PEG-ferrocene ² .

The electrodes 601, 602, 603 are used for this

Embodiment now each applied an electrical potential.

A first electrical potential V (E1) is applied to the first electrode 601, a second electrical potential V (E2) to the second electrode 602 and a third electrical potential V (E3) to the third electrode 603.

During the actual measurement phase, which is basically analogous to the procedure from the prior art, as described above, depending on the sign of the charge, the following potential gradient of the electrical potentials is applied to the electrodes 601, 602, 603 in such a way that applies:

V (E3)> V (E1)> V (E2).

For example, if the third electrode 603 has a positive electrical potential V (E3), then the third electrode 603 has the greatest electrical potential of the electrodes 601, 602, 603 of the biosensor 600.

This causes the generated first partial molecules 614 with negative charge to be drawn to the positively charged third electrode 603 due to the greatest electrical potential V (E3) which is present at the third electrode 603, and no longer, as in the prior art, to the first electrode 601.

Consequently, in this exemplary embodiment, the first electrode 601 is no longer used both as a holding electrode for holding the probe molecules and as a measuring electrode for oxidizing or reducing the respective partial molecules. Rather, the electrode 601 only serves to immobilize the probe molecules or the complexes of probe molecules and macromolecular biopolymers to be detected.

The third electrode 603 now takes over the function of the electrode on which the oxidation or reduction of the partial molecules generated takes place.

This clearly means that the third electrode 603 shields the first electrode 601 from the split partial molecules.

That way, another benefit to this

Embodiment covering the first electrode with the

DNA probe molecules 607 can be increased significantly.

The negatively charged partial molecules 614 are oxidized at the third electrode 603 and the oxidized first partial molecules 615 are drawn to the second electrode 602, since this has the smallest electrical potential V (E2) of all electrodes 601, 602, 603 in the biosensor 600.

The oxidized sub-molecules are reduced at the second electrode 602 and the reduced sub-molecules 616 are again drawn to the third electrode 603, where oxidation is again carried out.

In this way, in the present case, analogously to the manner known in the prior art, a circulating current results which is also detected in a known manner. Hence it follows here too a course of the circulating current over time as a signal. The number of hybridized DNA strands 607 and thus of the DNA molecules 609 to be detected can in turn be determined from this (by means of the enzyme 611 bound via the marker 608) on the basis of the proportionality of the circulating current to the number of charge carriers generated by the enzyme 611.

However, it should be noted here that a redox recycling process in the sense of the invention can also be carried out with the known “2-electrode arrangement” according to FIG. 4. However, then the advantage of the biosensor 600 described here cannot be used through the design of the electrode 601 as

Immobilization electrode, a higher coverage density than the arrangement known from the prior art allows.

FIG. 7 a shows an electrode arrangement 700 with a first electrode 701 and a second electrode 702, which are arranged in an insulator layer 703 made of insulator material.

The first electrode 701 is provided with a first electrical connection 704 and the second electrode 702 is provided with a second electrical connection 705.

The first electrode 701 and the second electrode 702 both have a regular arrangement of immobilization units 706, the units 706 being designed as gold nanoparticles.

Alternatively, as described above, the units 706 can also be made of silicon oxide. These can also be coated with one of the materials listed above, which is suitable for immobilizing the probe molecules thereon. DNA probe molecules 707, 708 are applied to the units 706 for immobilizing the electrodes 701, 702.

First DNA probe molecules 707 with a sequence complementary to a predetermined first DNA sequence are applied to the first electrode 701 (FIG. 7b).

Second DNA probe molecules 708 with a sequence that is complementary to a predetermined second DNA sequence are applied to the second electrode 702 (FIG. 7b).

7b also shows an electrolyte 709 which is brought into contact with the electrodes 701, 702 and the DNA probe molecules 707, 708.

7c shows the electrode arrangement 700 in the event that the electrolyte 709 contains DNA strands 710 with the predetermined first sequence, which is complementary to the sequence of the first DNA probe molecules 707.

In this case, the DNA strands 710 complementary to the first DNA probe molecules hybridize with the first DNA probe molecules 707, which are applied to the first electrode 701.

Since the sequences of DNA strands only hybridize with the respectively specific complementary sequence, the DNA strands complementary to the first DNA probe molecules do not hybridize with the second DNA probe molecules 708.

As can be seen from FIG. 7c, the result after hybridization has taken place is that hybridized molecules are applied to the first electrode 701, ie double-stranded DNA molecules. Only the second DNA probe molecules 708 are still present on the first electrode as single-stranded molecules. In a further step, the single-stranded DNA probe molecules 708 of the second electrode 702 are hydrolysed by means of a biochemical method, for example by adding DNA nucleases to the electrolyte 709 as described above.

After removal of the single-stranded DNA probe molecules, i.e. of the second DNA probe molecules 708 on the second electrode 702 are only the double-stranded molecules from the DNA strands 710 with the first DNA probe molecules 707 (cf. FIG. 7d).

By means of a measuring device (not shown) connected to the electrode connections 704, 705, according to this first exemplary embodiment, a capacitance measurement is carried out between the electrodes 701, 702 in the state shown in FIG. m non-hybridized state.

As part of the first capacitance measurement, a reference capacitance value is determined and stored in a memory (not shown).

A second capacitance measurement takes place after the single-stranded DNA probe molecules 708 have been removed from the respective electrode.

This again takes place by means of the measuring device, not shown. A capacitance value is determined by means of the second capacitance measurement, which is compared with the reference capacitance value

If the difference between these capacitance values is greater than a predetermined threshold value, this means that the electrolyte contained 710 DNA strands which are either hybridized with the first DNA probe molecules or with the second DNA probe molecules In this case, a corresponding output signal is output by the measuring device to the user of the measuring device.

In this context, it should be noted that, depending on the substance used to remove the single-stranded DNA probe molecules 708 on the second electrode, the single-stranded portion of the hybridized DNA strands 710 can be retained or can also be removed.

The method according to this exemplary embodiment has the advantage over the method known from [1] or [4] that a more robust measurement signal is achieved. This is because there is a major change in the impedance signal between the state in which no catcher molecules or only catcher molecules are attached to the electrodes and the state that there has at least partially been a bond with the macromolecular biopolymers to be detected.

However, in the sense of the invention it is of course also possible to carry out the method described in [1] and [4] with the aid of electrodes which have the nanoparticulate units for immobilization disclosed here.

The following publications are cited in this document:

[1] R. Hintsche et al., Microbiosensors Using Electrodes Made in Si-Technology, Frontiers in Biosensorics, Fundamental Aspects, edited by F. W. Scheller et al. , Dirk Häuser Verlag, Basel, pp. 267-283, 1997

[2] R. Hintsche et al, Microbiosensors using electrodes made in Si-technology, Frontiers in Biosensorics, Fundamental Aspects, edited by F. W. Scheller et al, Birkhauser Verlag, Basel, Switzerland, 1997

[3] M. Paeschke et al, Voltammetric Multichannel

Measurements Using Silicon Fabricated Microelectrode Arrays, Electroanalysis, Vol. 7, No. 1, pp. 1-8, 1996

[4] P. van Gerwen, Nanoscaled Interdigitated Electrode Arrays for Biochemical Sensors, IEEE, International Conference on Solid-State Sensors and Actuators, Chicago, pp. 907-910, June 16-19, 1997

[5] N.L. Thompson, B.C. Lagerholm, Total Internal Reflection Fluorescence: Applications in Cellular Biophysics, Current Opinion in Biotechnology, Vol. 8, pp. 58-64, 1997P.

[6] Cuatrecasas, Affinity Chromatography of Macromolecules, Advances in Enzy ology, Vol. 36, pp. 29-89, 1972

[7] Römpp-Lexikon Biotechnologie, Gentechnik, p. 280, 1999 Thieme Verlag, Stuttgart, Germany, 2nd edition.

[8] Römpp-Lexikon Biotechnologie, Gentechnik, p. 397, 1999 Thieme Verlag, Stuttgart, Germany, 2nd edition.

[9] J. Dodt et al. , Script for the biochemical internship Ild (immunochemistry), ELISA, p. 1, Institute of Biochemistry, Technical University of Darmstadt, description of the experiment ELISA, February 10-28, 1992.

[10] Handbook of Fluorescent Probes and Research Chemicals, pp. 157-160, Molecular Probes, Ine, 1996

[11] Handbook of Fluorescent Probes and Research Chemicals, pp. 83-84, Molecular Probes, Ine, 1996

[12] J.P. Spatz et al. , Mineralization of Gold Nanoparticles in a Block Gold Copolymer Microemulsion, Chem. Eur. J., Vol. 2, pp. 1552-1555, 1996

[13] J.P. Spatz et al. , Ordered Deposition of Inorganic

Cluster from Micellar Block Copolymer Films, Langmuir, Vol. 16, pp. 407-415, 2000

[14] F. Burmeister et al., With capillary forces too

Nanostructures, Physikalische Blätter, Vol. 36, pp. 49 - 51, 2000

[15] US 5,556,756

[16] US 6,017,696

[17] WO 96/07917

[18] DE 199 25 402 AI

Claims

claims

1. A method for detecting macromolecular biopolymers by means of at least one unit for immobilizing macromolecular biopolymers, the at least one unit for immobilizing biopolymers being a nanoparticle, a) in which the at least one unit for immobilizing macromolecular biopolymers is provided with capture molecules, wherein the capture molecules can bind macromolecular biopolymers, b) in which a sample to be examined is brought into contact with the at least one unit for immobilizing macromolecular biopolymers, the sample to be examined can contain the macromolecular biopolymers to be detected, c) in which in the investigating sample containing macromolecular biopolymers are bound to the capture molecules, d) in which the macromolecular biopolymers are detected by means of a signal emitted by a label.

2. The method according to claim 1, wherein the at least one immobilization unit is applied to an electrode or a photodiode.

3. The method of claim 2, wherein a plurality of units for immobilization on the electrode or the photodiode are applied in a regular arrangement.

4. The method according to any one of claims 1 to 3, wherein the label is attached to the capture molecule.

5. The method according to any one of claims 1 to 3, in which the label is attached to the macromolecular biopolymer to be detected.

6. The method according to any one of claims 1 to 5, wherein the label is selected from the group consisting of fluorescent and chemiluminescent dyes, radioisotopes, enzymes and enzyme ligands.

7. A method for detecting macromolecular biopolymers using an electrode arrangement, which comprises:

A first electrode,

A second electrode,

Wherein the first electrode is provided with at least one unit for immobilizing macromolecular biopolymers, the unit for immobilizing being a nanoparticle, a) in which the at least one unit for immobilizing macromolecular biopolymers on the first electrode is provided with capture molecules, wherein the capture molecules can bind macromolecular biopolymers, b) in which a medium is brought into contact with the electrodes, the medium to be examined can contain the macromolecular biopolymers to be detected, c) in the macromolecular biopolymers contained in the sample to be examined on the catcher molecules are bound, d) in which the macromolecular biopolymers are detected by means of an electrical measurement.

8. The method according to claim 7,

Wherein the second electrode is provided with at least one unit for immobilizing macromolecular biopolymers, the unit for immobilizing being a nanoparticle, and

In which the at least one unit for immobilizing. Located on the second electrode Macromolecular biopolymers are provided with catcher molecules, the catcher molecules being able to bind macromolecular biopolymers.

9. The method according to claim 8,

In which a first electrical measurement is carried out on the electrodes before step a) or step b),

• in which a second electrical measurement is carried out on the electrodes after step c), and

• in which, depending on a comparison of the results of the two electrical measurements on the electrodes, the macromolecular biopolymers are recorded.

10. The method according to claim 9,

In which the at least one unit for immobilizing macromolecular biopolymers on the first electrode is provided with first capture molecules which can bind macromolecular biopolymers of a first type, and

In which the at least one unit for immobilizing macromolecular biopolymers on the second electrode is provided with second capture molecules which can bind macromolecular biopolymers of a second type,

in which a medium is brought into contact with the electrodes in such a way that, if there are macromolecular biopolymers of the first type in the medium, they can bind to the first capture molecules, in the event that there are macromolecular biopolymers in the medium of the second type, can bind them to the second capture molecules,

in which unbound first catcher molecules or second catcher molecules are removed from the respective electrode, - in which the second electrical measurement is then carried out on the electrodes.

11. The method according to any one of the preceding claims, in which nucleic acids, oligonucleotides, proteins, or complexes of nucleic acids and proteins are detected as macromolecular biopolymers.

12. The method according to claim 11,

• in which proteins or peptides are recorded as macromolecular biopolymers, and

• in which ligands are used as capture molecules that can specifically bind the proteins or peptides.

13. The method of claim 12, wherein unbound ligands are removed from the at least one immobilization unit by contacting a material with the at least one immobilization unit, the material being capable of chemical bonding between the ligand and hydrolyze the immobilization unit.

14. The method of claim 13, wherein the material that is contacted with the at least one immobilization unit is an enzyme.

15. The method of claim 14, wherein the enzyme that is contacted with the at least one immobilization unit is a carboxyl ester hydrolase (esterase).

16. The method according to claim 11, in which DNA or RNA molecules are detected as macromolecular biopolymers.

17. The method according to claim 16, In which DNA single strands are detected as macromolecular biopolymers with a predetermined nucleotide sequence, and

• in which DNA probe molecules with a nucleotide sequence complementary to the specified nucleotide sequence are used as capture molecules.

18. The method of claim 17, wherein unbound DNA probe molecules are removed from the at least one immobilization unit by contacting an enzyme with nuclease activity with the immobilization unit.

19. The method according to claim 18, in which at least one of the following substances is used as the enzyme with nuclease activity:

Mung bean nuclease,

Nuclease Pl,

• Nuclease Sl, or

DNA polymerases due to their

5 '- 3' exonuclease activity or its

3 '-> 5' exonuclease activity are capable of being single-stranded

To break down DNA.