Patent Publication Number: US-2003226768-A1

Title: Method for detecting macromolecular biopolymers by means of an electrode arrangement

Description:
[0001] The invention relates to a method for detecting macromolecular biopolymers by means of an electrode arrangement.  
       [0002] Such a method is known from [1].  
       [0003]FIG. 2 a  and FIG. 2 b  show such a sensor, as described in [1]. The sensor  200  has two electrodes  201 ,  202  made of gold, which are embedded in an insulator layer  203  made of insulator material. Electrode terminals  204 ,  205 , to which the electrical potential applied to the electrodes  201 ,  202  can be delivered, are connected to the electrodes  201 ,  202 . The electrodes  201 ,  202  are arranged as planar electrodes. DNA probe molecules  206  are immobilized on each electrode  201 ,  202  (cf. FIG. 2 a ). The immobilization is carried out according to the so-called gold-sulfur coupling. The analyte to be studied, for example an electrolyte  207 , is applied to the electrodes  201 ,  202 .  
       [0004] If DNA strands  208  with a sequence which is complementary to the sequence of the DNA probe molecules are contained in the electrolyte  207 , then these DNA strands  208  hybridize with the DNA probe molecules  206  (cf. FIG. 2 b )  
       [0005] Hybridization of a DNA probe molecule  206  and a DNA strand  208  takes place only if the sequences of the respective DNA probe molecule  206  and of the corresponding DNA strand  208  are complementary to one another. If this is not the case, then no hybridization takes place. A DNA probe molecule with a predetermined sequence is hence respectively able to bind, i.e. hybridize, only a particular DNA strand, namely the one with the complementary sequence in each case.  
       [0006] If hybridization takes place, then as can be seen from FIG. 2 b , the value of the impedance between the electrode  201 ,  202  becomes modified. This modified impedance is determined by applying an AC voltage with an amplitude of approximately 50 mV to the electrode terminals  204 ,  205  and by determining the resulting current by means of connected measuring instrument (not shown).  
       [0007] In the event of hybridization, the capacitive component of the impedance between the electrodes  201 ,  202  is reduced. This is attributable to the fact that both the DNA probe molecules  206  and the DNA strands  208 , which may hybridize with the DNA probe molecules  206  if appropriate, are non-conductive and therefore clearly shield the respective electrode  201 ,  202  electrically to a certain extent.  
       [0008] In order to improve the measurement accuracy, it is also known from [4] to use a plurality of electrode pairs  201 ,  202  and to connect them in parallel, they being clearly arranged interdigitated with one another, so that a so-called interdigital electrode  300  is obtained. The dimensioning of the electrodes and the distances between the electrodes are of the order of the length of the molecules to be detected, i.e. the DNA strand  208  or less, for example in the region of 200 nm and less.  
       [0009] A further procedure for studying the electrolyte with respect to the existence of a DNA strand with a predetermined sequence is known from [2]. In this procedure, the DNA strands with the desired sequence are marked and, on the basis of the reflection properties of the marked molecules, the existence thereof is determined. To that end, light in the visible wavelength range is shone onto the electrolyte, and the light reflected by the electrolyte, in particular by the DNA strand to be registered, is detected. On the basis of the reflection response, i.e. in particular on the basis of the detected, reflected light beams, the question of whether or not the DNA strand to be registered, with the correspondingly predetermined sequence, is or is not contained in the electrolyte is determined.  
       [0010] This procedure is highly elaborate, since very accurate knowledge about the reflection response of the correspondingly marked DNA strands is required and, furthermore, it is necessary to mark the DNA strands before the start of the method.  
       [0011] Furthermore, very accurate adjustment of the detection means for detecting the reflected light beams is required, in order to be able to detect the reflected light beams at all.  
       [0012] This procedure is therefore expensive, complicated and highly sensitive to perturbing effects, so that it is very easy for the measurement result to be vitiated.  
       [0013] It is furthermore known from affinity chromatography ([3]) to use immobilized low molecular weight molecules, in particular ligands with high specificity and affinity, in order to specifically bind peptides and proteins, e.g. enzymes, in the analyte.  
       [0014] The electrical parameter which is evaluated in the method known from [1] is the capacitance between the electrodes, or the impedance of the two electrodes.  
       [0015] In order to achieve the greatest possible sensitivity when detecting macromolecular biopolymers, it is desirable to arrange the greatest possible number of field lines between the two electrodes in the volume in which the hybridization of the DNA strands having predetermined sequences with the DNA probe molecules, in general the binding of macromolecular molecules to immobilized DNA probe molecules, takes place.  
       [0016] In the known method, the impedance, or the capacitance, is detected in terms of its change, when only DNA probe molecules are present, in relation to the case in which DNA probe molecules are hybridized with the DNA strands to be detected.  
       [0017] It is an object of the invention to provide a method for detecting macromolecular biopolymers, with which a more robust measurement signal is achieved, i.e. a larger change in the impedance signal between the state in which no holding molecules, or exclusively holding molecules, are applied to the electrodes and that in which binding with the macromolecular biopolymers to be registered has at least partially taken place.  
       [0018] The object is achieved by the method having the features according to the independent patent claim.  
       [0019] In the scope of the method, an electrode arrangement which has a first electrode and a second electrode is used. The first electrode may be provided physically (i.e. by adsorption) or chemically (i.e. by means of covalent bonds) with first molecules which can bind macromolecular biopolymers of a first type. The second electrode may be provided physically or chemically with second molecules which can bind macromolecular biopolymers of a second type.  
       [0020] The term “macromolecular biopolymers” is intended to mean, for example, proteins or peptides or even DNA strands with a respectively predetermined sequence.  
       [0021] If proteins or peptides are intended to be detected as the macromolecular biopolymers, then the first molecules and the second molecules are ligands, for example active substances with a possible binding activity, which bind the proteins or peptides to be detected to the respective electrode on which the corresponding ligands are arranged.  
       [0022] Suitable ligands include enzyme agonists or enzyme antagonists, pharmaceuticals, sugars or antibodies or any molecule which has the ability to specifically bind proteins or peptides.  
       [0023] In the scope of this description, the term “probe molecule” is intended to mean both a ligand and a DNA probe molecule.  
       [0024] The electrode arrangement may be a plate electrode arrangement or an interdigitated electrode arrangement, as is known from [1 ].  
       [0025] Furthermore, various arrangements of the parallel connection of electrodes may be provided in the electrode arrangement; for example, the electrodes may be configured as cylindrical elements which are respectively arranged concentrically around one another and are electrically insulated from one another, for example, by means of a suitable dielectric so that an electric field is set up between the electrodes.  
       [0026] If DNA strands with a predetermined sequence, which are intended to be detected by means of the electrode arrangement, are used as the macromolecular biopolymers then, by means of the electrode arrangement, DNA strands with a predetermined first sequence can be hybridized with DNA probe molecules, having a sequence complementary to the first sequence, as the first molecules on the first electrode. In order to detect a DNA strand with a predetermined second sequence by means of second molecules on the second electrode, DNA probe molecules which have a sequence that is complementary to the second sequence of the DNA strand are used as the second molecules.  
       [0027] In a first method stage, a first electrical measurement is carried out on the electrodes; the first molecules and/or the second molecules may or may not already be arranged on the electrodes during the first electrical measurement.  
       [0028] A medium, for example an electrolyte, is brought into contact with the electrodes. This is done in such a way that, in the event that macromolecular biopolymers of the first type are contained in the medium, they can bind to the first molecules. In the event that macromolecular biopolymers of the second type are contained in the medium, they can bind to the second molecules.  
       [0029] It should be noted that the macromolecular biopolymers of the first type bind only to the first molecules on the first electrode, and that the macromolecular biopolymers of the second type bind only to the second molecules on the second electrode.  
       [0030] After having waited for a sufficient length of time, so that the macromolecular biopolymers can bind to the first molecules, or to the second molecules, unbound first molecules or second molecules are removed from the respective electrode on which they are located.  
       [0031] In the event that the probe molecules are DNA strands, this is done, for example, enzymatically by means of an enzyme which selectively degrades single-stranded DNA. In this case, the selectivity of the degrading enzyme for single-stranded DNA needs to be taken into account. If the enzyme selected for the degradation of unhybridized DNA single strands does not have this selectivity, then the hybridized double-stranded DNA to be detected may possibly be undesirably degraded as well.  
       [0032] In particular, in order to remove the unbound first or second DNA probe molecules from the respective electrode, it is possible to use DNA nucleases, for example a mung bean nuclease, nuclease P 1  or nuclease S 1 . The use of DNA polymerases which are capable of degrading single-stranded DNA owing to their 5′→3′ or their 3′→5′ exonuclease activity, may likewise be used.  
       [0033] In the event that the probe molecules are low molecular weight ligands, they may also be removed enzymatically if they are unbound.  
       [0034] To that end, the ligands are bonded covalently to the electrode via an enzymatically cleavable bond, for example via an ester bond.  
       [0035] In this case, for example, it is possible to use a carboxyl ester hydrolase (esterase) in order to remove unbound ligand molecules. This enzyme hydrolyzes that ester bond between the electrode and the respective ligand molecule which has not been bound by a peptide or protein. Conversely, owing to their reduced stearic accessibility due to the molecular mass of the bound peptide or protein, the ester bonds between the electrode and those molecules which have participated in a binding interaction with peptides or proteins remain intact.  
       [0036] After the removal of the unbound first molecules or second molecules has taken place, a second electrical measurement is carried out on the electrodes.  
       [0037] The values determined from the first electrical measurement and the second electrical measurement are compared with one another, and if the capacitance values differ in such a way that the difference in the determined values is greater than a predetermined threshold value, then it is assumed that macromolecular biopolymers have bound to probe molecules, or in general to the first or second molecules, and this has caused the change in the electrical signal at the electrodes.  
       [0038] If the difference between the values of the first electrical measurement and the second electrical measurement is greater than a predetermined threshold, then the result delivered is that the corresponding macromolecular biopolymers, which specifically bind the first molecules or second molecules have been bound, and therefore that the corresponding macromolecular biopolymers were contained in the medium.  
       [0039] In this way, the macromolecular biopolymers have been detected.  
       [0040] The first electrical measurement and the second electrical measurement may be carried out by measuring the capacitance between the electrodes.  
       [0041] Alternatively, the electrical resistance of the individual electrodes may also be determined.  
       [0042] In general, an impedance measurement, in the scope of which both the capacitance between the electrodes and the electrical resistances are measured, may be carried out as the first electrical measurement and as the second electrical measurement.  
       [0043] Clearly, the invention may be regarded as consisting in the following: by removing unbound first molecules or second molecules from the respective electrode, the difference between the determined values of the electrical signals between the first electrical measurement and the second electrical measurement, during the bonding of macromolecular biopolymers, is further increased by the fact that the unbound molecules, which vitiate the measurement result, no longer have any perturbing effect on the measurement result. 
     
    
    
     [0044] An exemplary embodiment of the invention is represented in the figures and will be explained in more detail below.  
     [0045]FIGS. 1 a  to  1   c  show an electrode arrangement in different method states, with the aid of which the method according to an exemplary embodiment of the invention will be explained;  
     [0046]FIGS. 2 a  and  2   b  show a sketch of two planar electrodes, by means of which the existence of DNA strands to be detected in an electrolyte (FIG. 2 a ) or their non-existence (FIG. 2 b ) can be registered;  
     [0047]FIG. 3 shows interdigitated electrodes according to the prior art;  
     [0048]FIG. 4 shows a sketch of an electrode arrangement which is used in the scope of a second exemplary embodiment.  
     [0049]FIG. 5 shows a biosensor according to an exemplary embodiment of the invention;  
     [0050]FIG. 6 shows a cross section of a biosensor with two electrodes, which are arranged as an interdigitated electrode arrangement;  
     [0051]FIGS. 7 a  to  7   d  show cross-sectional views of an interdigitated electrode in four method states in a method for producing a biosensor according to an exemplary embodiment of the invention;  
     [0052]FIGS. 8 a  to  8   c  show cross-sectional views of a biosensor during individual method stages of the method for producing an electrode of the biosensor according to a further exemplary embodiment of the invention;  
     [0053]FIGS. 9 a  to  9   c  show cross-sectional views of a biosensor during individual method stages of the method for producing an electrode of the biosensor according to a further exemplary embodiment of the invention;  
     [0054]FIGS. 10 a  to  10   c  respectively show a cross section of a biosensor at various times during the production method according to a further exemplary embodiment of the invention;  
     [0055]FIG. 11 shows a plan view of a biosensor array according to an exemplary embodiment of the invention with cylindrical electrodes;  
     [0056]FIG. 12 shows a plan view of a biosensor array according to an exemplary embodiment of the invention with cuboid electrodes;  
     [0057]FIG. 13 shows a cross-sectional view of a biosensor according to a further exemplary embodiment of the invention;  
     [0058]FIG. 14 shows a cross-sectional view of a biosensor according to a further exemplary embodiment of the invention; and  
     [0059]FIGS. 15 a  to  15   g  show cross-sectional views of a biosensor during individual method stages of a production method according to a further exemplary embodiment of the invention; 
    
    
     [0060]FIG. 1 a  shows an electrode arrangement  100  with a first electrode  101  and a second electrode  102 , which are arranged in an insulator layer  103  made of insulator material.  
     [0061] The first electrode  101  is provided with a first electrical terminal  104 , and the second electrode  102  is provided with a second electrical terminal  105 .  
     [0062] The first electrode  101  and the second electrode  102  are made of gold.  
     [0063] Alternatively, the electrodes  101  and  102  may also be made of silicon oxide. They may be coated with a material which is suitable for immobilizing the probe molecules thereon.  
     [0064] For example, known alkoxysilane derivatives may be used, such as  
     [0065] 3-glycidoxypropylmethyloxysilane,  
     [0066] 3-acetoxypropyltrimethoxysilane,  
     [0067] 3-aminopropyltriethoxysilane,  
     [0068] 4-(hydroxybutyramido)propyltriethoxysilane,  
     [0069] 3-N,N-bis(2-hydroxyethyl)aminopropyltriethoxysilane, or other related materials which are capable, with one of their ends, of forming a covalent bond with the surface of the silicon oxide and, with their other end, of offering a chemically reactive group, such as an epoxy, acetoxy, amine or hydroxyl radical, for reaction to the probe molecules to be immobilized.  
     [0070] If a probe molecule to be immobilized reacts with such an activated group, then it will be immobilized on the surface of the coating on the electrode via the selected material as a kind of covalent linker.  
     [0071] DNA probe molecules  106 ,  107  are applied to the immobilized regions of the electrodes  101 ,  102 .  
     [0072] On the first electrode  101 , first DNA probe molecules  106  with a sequence complementary to a predetermined first DNA sequence are applied.  
     [0073] On the second electrode  102 , second DNA probe molecules  107  with a sequence which is complementary to a predetermined second DNA sequence are applied.  
     [0074] Sequences of DNA strands that are respectively complementary to the sequences of the probe molecules can hybridize onto the pyrimidine bases adenine (A), guanine (G), thymine (T) or cytosine (C) in the usual way, i.e. base pairing via hydrogen bridge bonds between A and T or between C and G.  
     [0075]FIG. 1 a  furthermore shows an electrolyte  108 , which is brought into contact with the electrodes  101 ,  102  and the DNA probe molecules  106 ,  107 .  
     [0076]FIG. 1 b  shows the electrode arrangement  100  for the case in which DNA strands  109  with the predetermined first sequence, which is complementary to the sequence of the first DNA probe molecules  106 , are contained in the electrolyte  108 .  
     [0077] In this case, the DNA strands  109  complementary to the first DNA probe molecules hybridize with the first DNA probe molecules  106 , which are applied on the first electrode  101 .  
     [0078] Since the sequences of DNA strands hybridize only 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  107 .  
     [0079] As can be seen from FIG. 1 b , the result after hybridization has taken place is that hybridized molecules, i.e. double-stranded DNA molecules, are applied on the first electrode  101 . On the first electrode, only the second DNA probe molecules  107  are still present as single-stranded molecules.  
     [0080] In a further stage, hydrolysis of the single-stranded DNA probe molecules  107  of the second electrode  102  is brought about by means of a biochemical method, for example by means of DNA nucleases in the electrolyte  108 .  
     [0081] In this case, the selectivity of the degrading enzyme for single-stranded DNA needs to be taken into account. If the enzyme selected for the degradation of unhybridized DNA single strands does not have this selectivity, then the hybridized double-stranded DNA to be detected may possibly be undesirably degraded as well, which would lead to vitiation of the measurement result.  
     [0082] After having removed the single-strand DNA probe molecules, i.e. the second DNA probe molecules  107  on the second electrode  102 , only the double-strand molecules of the hybridized DNA strands with the sequence complementary to the first sequence of the first DNA probe molecules  106  are present (cf. FIG. 1 c ).  
     [0083] For example, in order to remove the single-strand DNA probe molecules  107  on the second electrode, one of the following substances may be added:  
     [0084] mung bean nuclease,  
     [0085] nuclease P 1 , or  
     [0086] nuclease S 1 .  
     [0087] DNA polymerases which, owing to their 5′→3′ exonuclease activity or their 3′→5′ exonuclease activity, are capable of degrading single-stranded DNA may likewise be used for this purpose.  
     [0088] If a sample substance, which contains DNA strands with a sequence that is complementary to the sequence of the second DNA probe molecules  107  on the second electrode  102 , is added to the electrolyte, then hybridization of the added DNA strands having the sequence complementary to the second DNA probe molecules  107  takes place with the second DNA probe molecules  107 , and the first DNA probe molecules  106  remain as single-strand probe molecules on the first electrode.  
     [0089] According to the method represented in FIG. 1 b , these are removed from the first electrode  101  in a similar way by means of the biochemical method described above.  
     [0090] By means of a measuring instrument (not shown) connected to one of the electrode terminals  104 ,  105 , according to this first exemplary embodiment a capacitance measurement is carried out between the electrodes  101 ,  102  in the state represented in FIG. 1 a , i.e. in the unhybridized state.  
     [0091] In the scope of the first capacitance measurement, a reference capacitance value is determined and stored in a memory (not shown).  
     [0092] A second capacitance measurement is carried out after the single-stranded DNA probe molecules  107  have been removed from the respective electrode.  
     [0093] This is again carried out by means of the measuring instrument, which is not represented. By means of the second capacitance measurement, a capacitance value is determined which is compared with the reference capacitance value.  
     [0094] If the difference between these capacitance values is greater than a predetermined threshold value, then this means that DNA strands that have hybridized either with the first DNA probe molecules or the second DNA probe molecules were contained in the electrolyte  108 .  
     [0095] In this case, a corresponding output signal is delivered by the measuring instrument to the user of the measuring instrument.  
     [0096] In this context, it should be pointed out that, depending on the substance which is used for removing the single-strand DNA probe molecules  107  on the second electrode, the single-stranded component of the hybridized DNA strands  109  may remain behind or may also be removed as well.  
     [0097]FIG. 4 shows a sensor arrangement  400  in which, instead of the capacitance measurement, an impedance measurement is carried out in a second exemplary embodiment.  
     [0098] The sensor arrangement  400  represented in FIG. 4 is represented in the state in which hybridization of the DNA strands complementary to the first DNA probe molecules  106  has already taken place with the first DNA probe molecules  106 , and after the second DNA probe molecules  107 , which are not hybridized, have been removed from the second electrode  102 .  
     [0099] For each electrode  101 ,  102 , a reference electrode  401 ,  402  is respectively provided, and these are designed in such a way that the DNA probe molecules  106 ,  107  do not adhere to this reference electrode  401 ,  402 .  
     [0100] This may be guaranteed by selecting a material for the reference electrode  401 ,  402  which does not permit any sulfur bonding.  
     [0101] Alternatively, undesired adhesion of the DNA probe molecules on the reference electrode may be prevented if the coating material suitable for immobilizing the DNA probe molecules (see above) is not applied in advance to the reference electrode. Therefore, there will not be any chemically reactive groups on the reference electrode, which would otherwise bond covalently with the DNA probe molecules so as to immobilize them there.  
     [0102] Alternatively, this may be guaranteed by applying a sufficiently large negative electric field, which ensures that the negatively charged DNA probe molecules  106 ,  107  do not adhere to the reference electrodes  401 ,  402 .  
     [0103] Each reference electrode  401 ,  402  is coupled to an electrical reference terminal  403 ,  404 .  
     [0104] In the scope of the second exemplary embodiment, a first impedance measurement is carried out in the uncoated state, i.e. for example in a state without probe molecules  106 ,  107  on the electrodes  101 ,  102  or with unhybridized DNA probe molecules  106 ,  107 .  
     [0105] After the single-stranded DNA probe molecules have been removed, following possible hybridization of the appropriate DNA probe molecules  106 ,  107  with DNA strands having the predetermined sequence complementary to the respective DNA probe molecule  106 ,  107 , a second impedance measurement is carried out in the known way and, on the basis of the possibly changed impedance values, the question of whether or not hybridization of probe molecules  106 ,  107  and DNA strands  109  with a respectively complementary sequence has taken place is determined.  
     [0106] The intention is not restricted to an electrode arrangement with only two electrodes, and in particular not to the plate electrode arrangement explained according to the exemplary embodiment.  
     [0107] Without modifying the method, it is possible to carry it out in the scope of an interdigitated electrode arrangement or else with an arbitrary number of different electrodes, to which different DNA probe molecules with different sequences are applied, so that it is possible to detect a plurality of different DNA strands with different sequences in an array fashion.  
     [0108] It should furthermore be pointed out that the invention is not restricted to use in the case of a planar electrode arrangement.  
     [0109] Rather, the invention can also be used in the case of an electrode arrangement in which the first electrode and the second electrode are arranged, relative to one another, in such a way that essentially uncurved field lines of an electric field produced between the first electrode and the second electrode can be formed between the first holding region and the second holding region.  
     [0110] A few such electrode arrangements, and methods for their production, will be explained in more detail below.  
     [0111]FIG. 5 shows a biosensor chip  500  with a further electrode configuration.  
     [0112] The biosensor chip  500  has a first electrode  501  and a second electrode  502 , which are arranged on an insulator layer  503  in such a way that the first electrode  501  and the second electrode  502  are electrically insulated from one another.  
     [0113] The first electrode  501  is coupled to a first electrical terminal  504 , and the second electrode  502  is coupled to a second electrical terminal  505 .  
     [0114] The electrodes  501 ,  502  have a cuboid structure, with a first electrode face  506  of the first electrode  501  and a first electrode face  507  of the second electrode  502  facing one another while being aligned essentially parallel.  
     [0115] This is achieved, according to this exemplary embodiment, by the fact that the electrodes  501 ,  502  have side walls  506 ,  507  which are essentially perpendicular with respect to the surface  508  of the insulator layer  503 , and which respectively form the first electrode face  506  of the first electrode  501  and the first electrode face  507  of the second electrode  502 .  
     [0116] If an electric field is applied between the first electrode  501  and the second electrode  502 , then owing to the electrode faces  506 ,  507  which are aligned essentially parallel with one another, a field line profile is produced with field lines  509  which are essentially uncurved between the surfaces  506 ,  507 .  
     [0117] Curved field lines  510  occur only between a second electrode face  511  of the first electrode  501  and a second electrode face  512  of the second electrode  502 , which respectively form the upper surface for the electrodes  501 ,  502 , as well as in an edge region  513  between the electrodes  501 ,  502 .  
     [0118] The first electrode faces  506 ,  507  of the electrodes  501 ,  502  are formed as holding regions for holding probe molecules, which can bind macromolecular biopolymers that are to be detected by means of the biosensor  500 .  
     [0119] The electrodes  501 ,  502  are made of gold according to this exemplary embodiment.  
     [0120] Covalent bonds are produced between the electrodes and the probe molecules, the sulfur for forming gold-sulfur coupling being present in the form of a sulfide or a thiol.  
     [0121] For the case in which DNA probe molecules are used as the probe molecules, such sulfur functionalities are part of a modified nucleotide which is incorporated by means of phosphoramidite chemistry during an automated DNA synthesis method at the  3 ′ end or at the  5 ′ end of the DNA strand to be immobilized. The DNA probe molecule is therefore immobilized at its  3 ′ end or at its  5 ′ end.  
     [0122] For the case in which ligands are used as the probe molecules, the sulfur functionalities are formed by one end of an alkyl linker or of an alkylene linker, the other end of which has a chemical functionality suitable for the covalent bonding of the ligand, for example a hydroxyl radical, an acetoxy radical or a succinimidyl ester radical.  
     [0123] The electrodes, i.e. in particular the holding regions, are covered during measurement use with an electrolyte  514 , in general with a solution to be studied.  
     [0124] If the solution  514  to be studied contains the macromolecular biopolymers to be detected, for example DNA strands to be detected which have a predetermined sequence and which can hybridize with the immobilized DNA probe molecules on the electrodes, then the DNA strands hybridize with the DNA probe molecules.  
     [0125] If the solution  514  to be studied does not contain any DNA strands with the sequence complementary to the sequence of the DNA probe molecules, then no DNA strands from the solution  514  to be studied can hybridize with the DNA probe molecules on the electrodes  501 ,  502 .  
     [0126]FIG. 6 shows a biosensor  600  with a further electrode configuration according to a further exemplary embodiment of the invention.  
     [0127] In the biosensor  600 , in the same way as in the biosensor  500  according to the exemplary embodiment shown in FIG. 5, two electrodes  501 ,  502  are provided which are applied on the insulator layer  503 .  
     [0128] In contrast to the biosensor  500  with only two cuboid electrodes, the two electrodes according to the biosensor  600  represented in FIG. 6 are arranged as a plurality of respectively alternately arranged, parallel-connected electrodes in the form of the known interdigitated electrode arrangement.  
     [0129] For further illustration, FIG. 6 also shows a schematic electrical equivalent circuit diagram, which is indicated in the representation of the biosensor  600 .  
     [0130] Since essentially uncurved field lines occur with respect to the surface  508  of the insulator layer  503  between the electrode faces  506 ,  507  of the electrodes  501 ,  502 , which face one another while being essentially parallel, as was represented in FIG. 7, the component of the first capacitance  602  and of the first admittance  603  produced by the uncurved field lines predominates compared with the second capacitance  604  and the second admittance  605 , which are produced by the curved field lines  510 .  
     [0131] This significantly greater component of the first capacitance  602  and of the first admittance  603  in relation to the total admittance, which is obtained from the sum of the first capacitance  602  and the second capacitance  604  as well as the first admittance  603  and the second admittance  605 , has the effect of significantly increasing the sensitivity of the biosensor  600  when the state of the biosensor  600  changes, i.e. when DNA strands in the solution  514  to be studied hybridize with DNA probe molecules  601  immobilized on the holding regions on the electrode faces  506 ,  507 .  
     [0132] Clearly, with the same lateral dimensions of the electrodes  501 ,  502  and the same dimensions of the previously introduced active region, i.e. with the same area of the holding regions on the electrode faces, a substantially greater component of field lines of an applied electric field between the electrodes  501 ,  502  is therefore contained in the volume in which the hybridization takes place when DNA strands to be detected are contained in the solution  514  to be studied, than in the case of a planar electrode arrangement.  
     [0133] In other words, this means that the capacitance of the arrangement according to the invention per unit chip area is significantly greater than the capacitance per unit chip area in the case of a planar electrode arrangement.  
     [0134] A few alternative possibilities for producing a cuboid sensor electrode with essentially vertical side walls will be explained below.  
     [0135] First Method for Producing Metal Electrodes With Essentially Vertical Side Walls, Which can Immobilize Probe Molecules  
     [0136]FIG. 7 a  shows a silicon substrate  700 , as is produced for known CMOS processes.  
     [0137] On the silicon substrate  700 , which already contains integrated circuits and/or electrical terminals for the electrodes to be formed, an insulator layer  701  which is also used as a passivation layer is applied with a sufficient thickness, with a thickness of 500 nm according to the exemplary embodiment, by means of a CVD method.  
     [0138] The insulator layer  701  may be made of silicon oxide SiO 2  or silicon nitride Si 3 N 4 .  
     [0139] The interdigitated arrangement of the biosensor  600  according to the exemplary embodiment described above is defined by means of photolithography on the insulator layer  701 .  
     [0140] By means of a dry etching method, e.g. reactive ion etching (RIE), steps  702  are subsequently produced, i.e. etched, in the insulator layer  701  with a minimum height  703  of approximately 100 nm according to the exemplary embodiment.  
     [0141] The height  703  of the steps  702  must be large enough for a subsequent self-aligning process to form the metal electrode.  
     [0142] It should be pointed out that an evaporation coating method or a sputtering method may alternatively also be used for applying the insulator layer  701 .  
     [0143] During the structuring of the steps  702 , care should be taken that the flanks of the steps  702  are steep enough so that they form sufficiently sharp edges  705 . An angle  706  of the step flanks, measured with respect to the surface of the insulator layer  701 , should be at least 50° according to the exemplary embodiment.  
     [0144] In a further stage, an auxiliary layer  704  (cf. FIG. 7 b ) made of titanium with a thickness of approximately 10 nm is applied to the stepped insulator layer  701 .  
     [0145] The auxiliary layer  704  may comprise tungsten and/or nickel-chromium and/or molybdenum.  
     [0146] It is necessary to guarantee that a metal layer applied in a further stage, according to the exemplary embodiment a metal layer  707  made of gold, grows porously at the edges  705  of the steps  702  so that, in a further method stage, it is possible to respectively etch a gap  708  at the step junctions, into the gold layer  707  which is applied surface-wide.  
     [0147] The gold layer  707  for the biosensor  600  is applied in a further method stage.  
     [0148] According to the exemplary embodiment, the gold layer  707  has a thickness of from approximately 500 nm to approximately 2000 nm.  
     [0149] In terms of the thickness of the gold layer  707 , it is merely necessary to guarantee that the thickness of the gold layer  707  is sufficient for the gold layer  707  to grow porously in columns.  
     [0150] In a further stage, openings  708  are etched into the gold layer  7  so that gaps are formed (cf. FIG. 7 c ).  
     [0151] For wet etching of the openings, an etchant solution made up of 7.5 g Super Strip 100™ (trademark of Lea Ronal GmbH, Deutschland) and 20 g KCN in 1000 ml water H 2 O is used.  
     [0152] Owing to the columnar growth of the gold, in general of the metal, during the evaporation coating onto the adhesion layer  704 , anisotropic etching attack is achieved so that the surface erosion of the gold takes place approximately in the ratio 1:3.  
     [0153] The gaps  708  are formed as a function of the duration of the etching process by the etching of the gold layer  707 .  
     [0154] This means that the duration of the etching process dictates the basic width, i.e. the distance  709  between the gold electrodes  710 ,  711  which are being formed.  
     [0155] After the metal electrodes have a sufficient width and the distance  709  between the gold electrodes  710 ,  711  which are being formed is achieved, the wet etching is ended (cf. FIG. 7 d ).  
     [0156] It should be noted that, because of the porous evaporation coating, etching in a direction parallel to the surface of the insulator layer  701  takes place substantially faster than in a direction perpendicular to the surface of the insulator layer  701 .  
     [0157] It should be pointed out that alternatively to a gold layer, it is possible to use another noble metal, for example platinum, titanium or silver, since these materials can likewise have holding regions or can be coated with a suitable material for holding immobilized DNA probe molecules, or in general for holding probe molecules, and they exhibit columnar growth during evaporation coating.  
     [0158] For the case in which the adhesion layer  704  needs to be removed in the opened columns  712  between the metal electrodes  710 ,  711 , this is likewise carried out in a self-aligning fashion by using the gold electrodes  710 ,  711  as an etching mask.  
     [0159] Compared with the known interdigitated electrodes, the structure according to this exemplary embodiment has the advantage, in particular, that owing to the self-aligning opening of the gold layer  707  over the edges  705 , the distance between the electrodes  710 ,  711  is not tied to a minimum resolution of the production process, i.e. the distance  709  between the electrodes  710 ,  711  can be kept very narrow.  
     [0160] According to this method, the biosensor  600  according to the exemplary embodiment represented in FIG. 6 with the corresponding metal electrodes is therefore obtained.  
     [0161] Second Method for Producing Metal Electrodes With Essentially Vertical Side Walls, Which can Immobilize Probe Molecules  
     [0162] The production method represented in FIG. 8 a  to FIG. 8 c  starts with a substrate  801 , for example a silicon substrate wafer (cf. FIG. 8 a ), on which metallization  802  is already provided as an electrical terminal, an etch stop layer  803  of silicon nitride Si 3 N 4  already having been applied on the substrate  801 .  
     [0163] A metal layer  804 , according to the exemplary embodiment a gold layer  804 , is applied on the substrate by means of an evaporation coating method.  
     [0164] Alternatively, a sputtering method or a CVD method may also be used to apply the gold layer  804  to the etch stop layer  803 .  
     [0165] In general, the metal layer  804  comprises the metal on which the electrode to be formed is intended to be formed.  
     [0166] An electrically insulating auxiliary layer  805  of silicon oxide SiO 2  is applied on the gold layer  804  by means of a CVD method (alternatively by means of an evaporation coating method or a sputtering method).  
     [0167] By using photolithographic technology, a resist structure, for example a cuboid structure, is formed from a resist layer  806 , which resist structure corresponds to the shape of the electrode to be formed.  
     [0168] If a biosensor array, described below, with a plurality of electrodes is to be produced, a resist structure whose shape corresponds to the electrodes to be formed, which form the biosensor array, is produced by means of photolithography.  
     [0169] Put another way, this means that the lateral dimensions of the resist structure which is formed correspond to the dimensions of the sensor electrode to be produced.  
     [0170] The thickness of the resist structure, i.e. the thickness of the resist layer  806 , corresponds essentially to the height of the electrodes to be produced.  
     [0171] After application of the resist layer  806  and the corresponding illumination, which defines the corresponding resist structures, the resist layer is removed in the “undeveloped”, i.e. unilluminated regions, for example by means of ashing or wet chemically.  
     [0172] The auxiliary layer  805  is also removed by means of a wet etching method in the regions not protected by the photoresist layer  806 .  
     [0173] In a further stage, after removal of the resist layer  806 , a further metal layer  807  is applied conformally as an electrode layer over the remaining auxiliary layer  805 , in such a way that the side faces  808 ,  809  of the residual auxiliary layer  805  are covered with the electrode material, according to the exemplary embodiment with gold (cf. FIG. 8 b ).  
     [0174] The application may be carried out by means of a CVD method or a sputtering method or by using an ion metal plasma method.  
     [0175] In a last stage (cf. FIG. 8 c ), spacer etching is carried out, during which the desired structure of the electrode  810  is formed by deliberate over-etching of the metal layers  804 ,  807 .  
     [0176] The electrode  810  therefore has the spacers  811 ,  812 , which have not been etched away in the etching stage of etching the metal layers  804 ,  807 , as well as the part of the first metal layer  804 , arranged immediately below the residual auxiliary layer  805 , which has not been etched away by means of the etching method.  
     [0177] The electrode  810  is electrically coupled to an electrical terminal, i.e. the metallization  802 .  
     [0178] The auxiliary layer  805  of silicon oxide may if necessary be removed by further etching, for example in a plasma or wet chemically, by means of a method in which selectivity with respect to the etch stop layer  803  is provided.  
     [0179] This is guaranteed, for example, if the auxiliary layer  805  consists of silicon oxide and the etch stop layer  803  comprises silicon nitride.  
     [0180] The steepness of the walls of the electrode in the biosensor chip  500 ,  600 , represented by the angle  813  between the spacers  811 ,  812  and the surface  814  of the etch stop layer  803 , is therefore determined by the steepness of the flanks of the residual auxiliary layer  805 , i.e. in particular the steepness of the resist flanks  815 ,  816  of the structured resist layer  806 .  
     [0181] Third Method for Producing Metal Electrodes With Essentially Vertical Side Walls, Which can Immobilize Probe Molecules  
     [0182]FIG. 9 a  to FIG. 9 c  represent a further possibility for producing an electrode with essentially vertical walls.  
     [0183] This also, as represented in the first example of producing an electrode, starts with a substrate  901  on which a metallization  902  is already provided for the electrical terminal of the biosensor electrode to be formed.  
     [0184] A metal layer  903  is evaporation coated as an electrode layer on the silicon substrate  901 , the metal layer  903  comprising the material to be used for the electrode, according to this exemplary embodiment gold.  
     [0185] Alternatively to evaporation coating of the metal layer  903 , the metal layer  903  may also be applied on the substrate  901  by means of a sputtering method or by means of a CVD method.  
     [0186] A photoresist layer  904  is applied on the metal layer  903  and is structured by means of photolithographic technology so as to produce a resist structure which, after development and removal of the developed regions, corresponds to the lateral dimensions of the electrode to be formed, or in general of the biosensor array to be formed.  
     [0187] The thickness of the photoresist layer  904  corresponds essentially to the height of the electrodes to be produced.  
     [0188] During structuring in a plasma with process gases which cannot lead to any reaction of the electrode material, in particular in an inert gas plasma, for example with argon as the process gas, the erosion of the material according to this exemplary embodiment is carried out by means of physical sputter erosion.  
     [0189] In this case, the electrode material is sputtered from the layer  903  in a redeposition process onto the essentially vertical side walls  905 ,  906  of the structured resist elements that are not removed after ashing the developed resist structure, where it is no longer exposed to any sputter attack.  
     [0190] Redeposition of electrode material onto the resist structure protects the resist structure from further erosion.  
     [0191] Because of the sputtering, side layers  907 ,  908  of the electrode material, according to the exemplary embodiment of gold, are formed at the side walls  905 ,  906  of the resist structure.  
     [0192] The side layers  907 ,  908  are electrically coupled to an unremoved part  909  of the metal layer  903 , which lies immediately below the residual resist structure  906 , and furthermore to the metallization  903  (cf. FIG. 9 b ).  
     [0193] In a last stage (cf. FIG. 9 c ), the resist structure  906 , i.e. the photoresist which is found in the volume formed by the side walls  907 ,  908  as well as the remaining metal layer  909 , is removed by means of ashing or wet chemically.  
     [0194] The result is the electrode structure  910  represented in FIG. 9 c , which is formed with the side walls  907 ,  908  as well as the unremoved part  909 , which forms the bottom of the electrode structure and is electrically coupled to the metallization  903 .  
     [0195] As in the production method presented above, the steepness of the side walls  907 ,  908  of the electrode that is formed in this method is determined by the steepness of the resist flanks  905 ,  906 .  
     [0196]FIG. 10 a  to FIG. 10 c  represent a further exemplary embodiment of the invention with cylindrical electrodes protruding vertically from the substrate.  
     [0197] In order to produce the biosensor  1000  with cylindrical electrodes, which are arranged essentially vertically on a substrate  1001  of silicon oxide, a metal layer  1002  is applied by means of an evaporation coating method as an electrode layer of the desired electrode material, according to the exemplary embodiment of gold.  
     [0198] A photoresist layer is applied on the metal layer  1002 , and the photoresist layer is illuminated by means of a mask so that the cylindrical structure  1003  represented in FIG. 10 a  is obtained on the metal layer  1002  after the unilluminated regions have been removed.  
     [0199] The cylindrical structure  1003  has a photoresist torus  1004  as well as a cylindrical photoresist ring  1005 , which is arranged concentrically around the photoresist torus  1004 .  
     [0200] The photoresist is removed between the photoresist torus  1004  and the photoresist ring  1005 , for example by means of ashing or wet chemically.  
     [0201] Through the use of a sputtering method, as in conjunction with the method described above for producing an electrode, a metal layer  1006  is applied around the photoresist torus  1004  by means of a redeposition process.  
     [0202] In a similar way, an inner metal layer  1007  is formed around the photoresist ring  1005  (cf. FIG. 10 b ).  
     [0203] In a further stage, the structured photoresist material is removed by means of ashing or wet chemically, so that two cylindrical electrodes  1008 ,  1009  are formed.  
     [0204] The substrate  1001  is removed in a last stage, for example by means of a plasma etching process that is selective with respect to the electrode material, to the extent that the metallizations in the substrate are exposed and electrically couple to the cylindrical electrodes.  
     [0205] The inner cylindrical electrode  1008  is therefore electrically coupled to a first electrical terminal  1010 , and the outer cylindrical electrode  1009  is electrically coupled to a second electrical terminal  1011 .  
     [0206] The residual metal layer  1002 , which has not yet been removed by the sputtering between the cylindrical electrodes  1008 ,  1009 , is removed in a last stage by means of a sputter-etching process. The metal layer  1002  is likewise removed in this way.  
     [0207] It should be mentioned in this context that, according to this exemplary embodiment as well, the metallizations for the electrical terminals  1010 ,  1011  are already provided in the substrate  1001  at the start of the method.  
     [0208]FIG. 11 shows a plan view of a biosensor array  1100 , in which cylindrical electrodes  1101 ,  1102  are contained.  
     [0209] Each first electrode  1101  has a positive electrical potential.  
     [0210] Each second electrode  1102  of the biosensor array  1100  has an electrical potential that is negative in relation to the respectively neighboring first electrode  1101 .  
     [0211] The electrodes  1101 ,  1102  are arranged in rows  1103  and columns  1104 .  
     [0212] The first electrode  1101  and the second electrode  1102  are respectively arranged alternately in each row  1103  and each column  1104 , i.e. a second electrode  1102  is respectively arranged in a row  1103  or a column  1104  immediately next to a first electrode  1101 , and a first electrode  1101  is respectively arranged in a row  1103  or a column  1104  next to a second electrode  1102 .  
     [0213] This ensures that an electric field with essentially uncurved field lines in the height direction of the cylinder electrodes  1101 ,  1102  can be produced between the individual electrodes.  
     [0214] As described above, a large number of DNA probe molecules are respectively immobilized on the electrodes.  
     [0215] If a solution to be studied (not shown) is then applied to the biosensor array  1100 , then the DNA strands hybridize with DNA probe molecules complementary thereto which are immobilized on the electrodes.  
     [0216] In this way, by means of the redox recycling process described above, the existence or non-existence of DNA strands of a predetermined sequence in a solution to be studied can in turn be detected by means of the biosensor array  1100 .  
     [0217]FIG. 12 shows a further exemplary embodiment of a biosensor array  1200  with a plurality of cuboid electrodes  1201 ,  1202 .  
     [0218] The arrangement of the cuboid electrodes  1201 ,  1202  is in accordance with the arrangement of the cylindrical electrodes  1201 ,  1202  as presented in FIG. 12 and explained above.  
     [0219]FIG. 13 shows an electrode arrangement of a biosensor chip  1300  according to a further exemplary embodiment of the invention.  
     [0220] The first electrode  501  is applied on the insulator layer  503  and is electrically coupled to the first electrical terminal  504 .  
     [0221] The second electrode  502  is likewise applied on the insulator layer  503  and is electrically coupled to the second electrical terminal  505 .  
     [0222] As shown in FIG. 13, the second electrode  502  according to this exemplary embodiment has a different shape compared with the second electrode described previously.  
     [0223] The first electrode, as can be seen from FIG. 13, is a planar electrode and the second electrode is configured with a T-shape.  
     [0224] Each T-shaped second electrode has a first branch  1301 , which is arranged essentially perpendicular to the surface  1307  of the insulator layer  703 .  
     [0225] Furthermore, the second electrode  502  has second branches  1302  which are arranged perpendicular to the first branch  1301  and are arranged at least partially over the surface  1303  of the respective first electrode  501 .  
     [0226] As can be seen in FIG. 13, several first electrodes  501  and several second electrodes  502  are connected in parallel, so that because of the T-shaped structure of the second electrode  502 , a cavity  1304  is created which is formed by two second electrodes  502  arranged next to one another, a first electrode  501  and the insulator layer  503 .  
     [0227] The individual first and second electrodes  501  and  502  are electrically insulated from one another by means of the insulator layer  503 .  
     [0228] An opening  1305  is provided between the individual second branches  1302  of the second electrode  502  for each cavity  1304 , which opening  1305  is large enough so that when an electrolyte  1306  is being applied to the biosensor  1300 , the electrolyte and DNA strands possibly contained in the solution  1306  to be studied, for example an electrolyte, can pass through the opening  1305  into the cavity  1304 .  
     [0229] DNA probe molecules  1309 , which can hybridize with the corresponding DNA strands of a predetermined sequence that are to be detected, are immobilized on holding regions on the first and second electrodes.  
     [0230] As can be seen in FIG. 13, because of the mutually facing surfaces, aligned essentially parallel with one another, of the second electrode  1308  and of the first electrode  1303 , on which the holding regions for holding the DNA probe molecules  1309  are provided, essentially uncurved field lines are formed when an electric field is applied between the first electrode  501  and the second electrode  502 .  
     [0231]FIG. 14 shows a biosensor  1400  according to a further exemplary embodiment of the invention.  
     [0232] The biosensor  1400  according to a further exemplary embodiment corresponds essentially to the biosensor  1300  explained above and shown in FIG. 13, with the difference that no holding regions with immobilized DNA probe molecules  1309  are provided on side walls of the first branch  1301  of the second electrode  502 , but rather the surface  1401  of the first branch  1301  of the second electrode  502  is covered with insulator material of the insulator layer  503  or a further insulating layer.  
     [0233] According to the exemplary embodiment shown in FIG. 14, holding regions on the first electrode and on the second electrode  501 ,  502  are consequently only on directly facing surfaces of the electrodes, i.e. on the surface  1402  of the second branch of the second electrode  502  and on the surface  1403  of the first electrode  501 .  
     [0234]FIG. 15 a  to FIG. 15 g  represent individual method stages for producing the first electrode  501  and the second electrode  502  in the biosensors  1300 ,  1400 .  
     [0235] In the insulator layer  503  as a substrate, according to the exemplary embodiment made of silicon oxide, a structure whose shape corresponds to the first electrode  501  to be formed is etched into the insulator layer  503  by using a mask layer, for example made of photoresist.  
     [0236] After removal of the layer by ashing or by a wet chemical method, a layer of the desired electrode material is applied surface-wide on the insulator layer  503 , in such a way that the previously etched structure  1501  (cf. FIG. 15 a ) is at least completely filled; the structure  1501  may even be overfilled (cf. FIG. 15 b ).  
     [0237] In a further stage, the electrode material  1502 , preferably gold, located outside the prefabricated structure  1501  is removed by means of a chemical mechanical polishing method (cf. FIG. 15 c ).  
     [0238] After the completion of the chemical mechanical polishing method, the first electrode  501  is therefore embedded flush in the insular layer  503 .  
     [0239] Electrode material  1502  outside, i.e. between the further second electrodes  502  or between the first electrodes  501 , is removed without leaving any residue.  
     [0240] A cover layer  1503 , for example made of silicon nitride, may furthermore be applied to the first electrode  501  by means of a suitable coating method, for example a CVD method, a sputtering method or an evaporation coating method (cf. FIG. 15 d ).  
     [0241]FIG. 15 e  shows several first electrodes  1501  made of gold, which are embedded next to one another in the insulator layer  503 , and the cover layer  1503  located on top.  
     [0242] In a further stage (cf. FIG. 15 f ), a second electrode layer  1504  is applied on the cover layer  1503 .  
     [0243] After completed structuring of a mask layer  1506  of, for example, silicon oxide, silicon nitride or photoresist, in which the desired openings  1505  between the second electrodes are taken into account, and which is intended to be formed from the second electrode layer  1504 , the desired cavities  1304  are formed according to the biosensors  1300 ,  1400  represented in FIG. 12 or FIG. 13 in the second electrode layer  1504  over the first electrode layer  1502 , by using an isotropic etching method (dry etching method, e.g. in a downstream plasma or wet etching method) (cf. FIG. 15 g ).  
     [0244] It should be noted in this context that the cover layer  1503  is not absolutely indispensable, but it is advantageous in order to protect the first electrode  501  from superficial etching during the formation of the cavity  1304 .  
     [0245] In an alternative embodiment, the T-shaped structure of the second electrode  502  may be formed as follows: after forming the first electrode  501  according to the method described above, a further insulator layer is formed by means of a CVD method or another suitable coating method on the first insulator layer or, if the cover layer  1503  exists, on the cover layer  1503 . Subsequently, corresponding trenches are formed in the cover layer  1503 , which are used to accommodate the first branch  1301  of the T-shaped structure of the second electrode  502 . These trenches are filled with the electrode material gold and, according to the damascene method, the electrode material is removed which has been formed in the trenches and above the second insulator layer by means of chemical mechanical polishing, until a predetermined height which corresponds to the height of the second branch  1302  of the T-shaped second electrode  502 .  
     [0246] The opening  1305  between the second electrodes  502  is formed by means of photolithography, and the insulator material is subsequently removed, at least partially, by means of a dry etching method in a downstream plasma from the volume which is intended to be formed as the cavity  1304 .  
     [0247] It should furthermore be pointed out that the embodiments described above are not restricted to an electrode whose holding region is produced by means of gold. Alternatively, electrodes may be coated in the holding regions with materials, for example with silicon monoxide or silicon dioxide, which can form a covalent bond with the aforementioned amine, acetoxy, isocyanate, alkysilane residues in order to immobilize probe molecules, in this variant in particular in order to immobilize ligands.  
     [0248] The following publications are cited in this document:  
     [0249] [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 Hauser Verlag, Basel, pp. 267-283, 1997  
     [0250] [2] N. L. Thompson, B. C. Lagerholm, Total Internal Reflection Fluorescence: Applications in Cellular Biophysics, Current Opinion in Biotechnology, Vol. 8, pp. 58-64, 1997  
     [0251] [3] P. Cuatrecasas, Affinity Chromatography, Annual Revision Biochem, Vol. 40, pp. 259-278, 1971  
     [0252] [4] P. van Gerwen, Nanoscaled Interdigitated Electrode Arrays for Biochemical Sensors, IEEE, International Conference on Solid-State Sensors and Actuators, Chicago, pp. 907-910, 16-19 June 1997