IMPEDIMETRIC DETECTION SYSTEM AND METHOD OF PRODUCTION THEREOF

Abstract: A sensor for identifying molecular structures within a sample solution is disclosed. The sensor comprises an insulating layer with a plurality of interspaced channels therein having essentially the same direction. Said channels have a bottom and at least two opposite side-walls along said direction. The channels furthermore have submicron dimensions. A metal coating is applied on one of said two opposite side-walls of essentially each channel and on top of the dielectric layer inbetween said channels thereby forming an impedimetric device with said sample solution within and between adjacent channels. Probes for specifically binding to said molecular structures can be applied, said probes being applied to either the insulating part of the channels (said bottom and the other side-wall of said channels), or to the surface of the electrodes or both, the insulating part of the channels and the surface of the electrodes. Furthermore, means are provided for applying a voltage on the metal coatings; and means for measuring the impedance inbetween the electrodes. Furthermore, a method of fabricating a sensor for identifying molecular structures within a sample solution is disclosed. This method comprises the steps of forming a plurality of interspaced channels in an insulating layer, said channels having essentially the same direction, said channels having a bottom and at least two opposite side-walls along said direction; depositing a metal layer on said insulating layer while aligning said insulating layer with respect to the metal deposition source such that the bottom of said canals and the side-walls of said canals along the deposition direction are shadowed and not covered by metal to thereby form an impedimetric device with said sample solution within and between adjacent channels; and applying probes for binding to said molecular structures, said probes being applied to either the insulating part of the channels (said bottom and the other side-wall of said channels), or to the surface of the electrodes or both, the insulating part of the channels and the surface of the electrodes, facing the deposition direction.

Description: IMPEDIMETRIC DETECTION SYSTEM AND METHOD OF PRODUCTION THEREOF The present invention relates to an improved sensor for electronically detecting a binding reaction between molecular structures or a pair of chemical substances, such as oligonucleotides, antigens, enzymes, peptides, antibodies. DNA and RNA fragments. The present invention further provides a new production method for this improved sensor. Techniques and sensors for detecting molecular structures and specific substances such as enzymes, peptides, oligonucleotides, antigens, antibodies, DNA and RNA fragments in a solution sample are known in the art. ln a specific class of sensors, use is made of the principle of measuring the impedance between two electrodes. The absence or presence of DNA-molecules or antibodies or antigens between the electrodes affects the permittivity and/or the conductivity between the electrodes. Various techniques were proposed to measure the presence and/or concentration of a given analyte in a sample solution by using a binding substance element having specific affinity for the analyte. Such specific binding reactions occur e.g. between enzymes and their substrates, antibodies and antigens, between DNA-DNΛ. between RNA-DNA, or other molecular structures. Stoner et al. in "Adsorption of blood proteins on metals using capacitance techniques", J. Phys. Chem., 74, Mar. 5,1970, describe a differential capacity measurement for evaluation of protein adsoiption on metalic electrodes. Arwin et al. in U.S.Pat. No. 4,072,576, use an adsorbed polypeptide substrate and establish a capacitive method for the measurement of enzymatic activity and the immunological interaction assay. Giaever in U.S.Pat. No. 4.054,646, teaches an electrical method that measures the presence of antibodies in a solution, by coating a metallic substrate with an antigen. After the incubation ofthe electrodes with the sample solution, he measures capacitively the thickness ofthe molecular sheet, i.e. he distinguishes between mono- and bimolecular layer, by using a mercury drop as a second electrode Newman in Patent application WO87/03095 discloses a capacitive sensoi foi chemical analysis and measuiement Said sensor can be used to detect a bioad lange of analytes including bactena, vn uses, antibodies, antigens, enzyme substiates and hormones A thin insulating layer is coated on the surface of conductois and a substrate to form an open capacitoi A biospecific binding agent is immobilized on the surface ofthe insulating layer between the conductoi s The dielectric constant ofthe biospecific binding agent is altered by binding ofthe analyte being detected with the biospecific binding agent A similar sensing principle is disclosed in U S Pat 5, 1 14,674 Battailard et al , 1988, Anal Chem , 60, 2374-2379 and more iecently, Klein et al , 1995, Sensors and Actuators , B 26-27, pp 474-476, show that a metal-semiconductoi-insulatoi device can be used in a similai way as a MIS (metal-insulatoi-semiconductoi ) capacitoi The device is immersed in a solution togethei with a second, refeience electrode By measuring the ac capacity between the metallic layei ofthe device and the leference electiode, when dc biasing voltages aie simultaneously applied, the flat band voltage ofthe system is in fact measured in a similai way as in the case of a MIS capacitoi It was proven that the flat band voltage can be modulated by species adsoibed at the insulation-liquid interface On this principle woik the ISFET, ion- sensitive-field-effect-transistor and GENFET, gene-sensitive-field-effect-transistor U S Pat 4,219,335, issued to Richard Ebersole discusses the use of immime ieagents labeled with l eactance tags These tags can be detected ellectrically since they alter the dielectric, conductive oi magnetic properties ofthe test surface Similaily, EP 0 241 771, issued to S J.Mioczkowski, teaches the detection of metal labeled antibodies by conductometric measurements When antigens aie immobilised inbetween two electiodes, the specific intei action with a metal-labeled antibody is measuied by means of lesistance deciease ofthe int ei electrode medium M Malmros in U S Pat 4,334,880 describes conductivity variation of a semiconductive polymeric layei inbetween two planai electrodes The said polymei incorpoiates, in a way oi anothei. certain molecules able to lecognize specific analytes The lecognition piocess induces conductivity changes ofthe polymeric layei Furthei variations on this central idea ofimpedimetπc sensing appeal in the art, EP 0 543 550, EP 0241 771, U.S. Pat. 4,453,126, GB 2,137,361, US 3,999,122. Essentially in an impedimetric sensor, certain molecules are immobilised on the top, in between or both on the top and in between a pah of electrodes. Said molecules 'recognise' a specific analyte when exposed to a sample solution. This recognition process eventually ends up, directly or indirectly, in conductivity and/or permitrvity alteration of the space in the neighbourhood of the electrodes. Finally, by measuring the impedance between the two electrodes, a measure ofthe recognition process can be established. The problem associated with the so called sensors as referred to above is that to have a good resolution, the immoblised layer should be perfectly homogenous and should not contain holes, which is hard to achieve. With the advent ofthe microelectronic technology, there is a continuous effort to use it in order to develop micro- sensors. Sensors realised with microelectronic technology offer advantages such as low-cost production, increased reproducibility of the production process, uniformity, accurateness of detection, and flexibihty in development. Such microelectronic sensors can comprise a multitude of individual test sites with reproducible, uniform electrical properties, whereby enhancing the detection sensitivity of the sensor. The test sites can be made with dimensions ofthe order ofthe dimensions ofthe molecules that have to be detected. The spatial limitations are the fabrication technology resolution and the sensitivity ofthe device which is dictated by the state ofthe art in instrumentation and the density of probes. A configuration can also be realised wherein the individual test sites can each yield a di ferent type of signal according to the particular molecule which is to be detected in said test site. Another important characteristic of the microelectronic technology is its planarity: the microelectrodes patterned this way aie essentially flat elements. This feature is not a strong point in the impedimetric devices. In a planar impedimetric structure the electric field lines expand more above the device surface and out ofthe region of intrest in comparison to real 3-D structures. This is a major drawback especially when the region of interest is limited in space, i.e. it is an enzymatic or polymeric membrane or an adsorbed molecular layer at the surface ofthe structure. Any field line depassing this region of interest, introduces in the impedimetric response a shunting impedance which can be considered as noise for the measurement. Still, depending on the electrodes geometry, i.e. dimensions and interspacing, a big majority of tlie total signal is enclosed in a certain region above the surface of tlie device as shown in figure 1. From the same figure one can deduce that miniaturisation, i.e. L decrease, is crucial in obtaining impedimetric planar structures that probe the space in the very close neighbourhood ofthe device. An illustration ofthe dimension down scaling was given by DeSilva et al. DeSilva et al. in 1995, Biosensors & Bioelectronws ,10, pp. 675-682 report a new biosensing structure that combines a covalent antibody immobilization technique with a simple impedance response method. The biosensor was fabricated by covalently binding anti-SEB antibodies onto an ultra-thin, island-like, electrically continuous, Pt film deposited onto a sihcon chip. They register an impedance decrease when the specific interaction with SEB takes place. However, the reproducibility is low due to the somewhat random behavior ofthe fabrication process, i.e. the Pt deposition and the immobilisation procedure. A true electrode patterning process is likely to insure a good reproducibility ofthe structures and to improve the control upon sensor behaviour. Devices with patterned features, said features having dimensions of hundreds of nanometers are expected to be highly sensitive to DNA fragments of 300 bases, i.e. exhibiting a total molecular lenth of about 180 nm. or to other large molecules like enzymes or antibodies (tens of nanometers diameter). This dimension range is usually achieved in two ways: 1. deep UV or X-ray lithography, techniques where about 100 nm features can be achieved with a fully optimised process. 2. electron-beam patterning, a tedious and very expensive technique where features of tens of nm can be obtained. It is an aim of the present invention to provide an electrochemical sensor suitable for measuring the presence or absence of molecular structures. It is another aim ofthe present invention to provide a method for fabricating as defined above. It is yet another aim ofthe present invention to provide a method for detecting the presence of molecular structures in a sample. The present invention relates to a new electrochemical sensor, based on the interference of an electrical field between electrodes with the analyte. The analyte to be tested is brought in the close neighbourhood ofthe structure by means of probes. Tlie present invention relates more particularly to a sensor for identifying molecular structures within a sample solution is disclosed. The sensor comprises an insulating layer with a plurality of interspaced channels therein having essentially the same direction. Said channels have a bottom and at least two opposite side-walls along said direction. The channels furthermore have submicron dimensions. A metal coating is applied on one of said two opposite side-walls of essentailly each channel and on top of the insulating layer in between said channels thereby formmg an impedimetric device with said sample solution within and between the channels. Optionally probes for binding to said molecular structures are already applied on said sensor. Said probes can be applied to either the insulating part ofthe channels (said bottom and the other side- wall of said channels), or to the surface of the electrodes or to both, the insulating part ofthe channels and the surface ofthe electrodes. Furthermore means are provided for applying a voltage on the metal coatings; as well as means for measuring the impedance in between the electrodes. The term electrochemical sensor or shortly sensor according to the present invention refers to a device which transforms a (bio)chemical information into an electrical signal. The present invention overcomes the problem of sensitivity compared to prior art sensors and methods. One important feature of this novel design is the high degree of miniaturisation. This is likely to reduce the noise ofthe structure and subsequently to increase its sensitivity. Another remarkable feature of the proposed sensor is its tridimensional geometry. This improves the electric field penetration in the area of interest with an eventual sensitivity increase. Said sensor has an interdigitated electrode structure which can be fabricated in a cheap way. even for large active areas. The probes ofthe present invention are functionally defined as molecules able to react with another molecule to form a complex an/or induce a secondary reaction. It is by the way of example and not by way of limitation that probes can be enzymes, antibodies, antigens, peptides, DNA fragments, RNA fragments or oligonucleotides. Preferred probes according to the present invention aie described in the following patents and patent applications held by one ofthe present applicants: EP 0 337 896; EP 0 345 375; EP 0 657 532; EP 0 419 355; EP 0 525 095; EP 0 494 317: EP 0489968; EP 0 644 202; WO 92/10514; EP 0 499 003; WO 92/11366; WO 92/16628; WO 92/19770; WO 93/08302; WO 93/18054; EP 0 561 087; WO 93/22437; WO 94/01554; EP 0 637 342; WO 94/13795; WO 94/18325; WO 94/21818; WO 94/25601 ; WO 95/12666; WO 95/17429; WO 95/33851 ; WO 96/00298; WO 96/04309; EP 0 721 505; WO 96/13590; WO 96/13608; WO 96/17065; and PCT apphcations filed under number 96/03091, 96/04146; as well as EP apphcations filed under No. 95870136.9, 96870006.2, 96870081.5, 96870053.4, 96870122.8 or 96870131.8. The contents of these patent (apphcations) and any other document refened to in this text are to be considered as incorporated by reference. The probes as well as the methods for making these probes are further discussed in the above-mentioned documents. It should be clear that these probes may be purified from a living source or may be made by any method of synthesis known in the art. The targets to be detected in the sample or analyste can be any molecule present in a sample which binds or reacts with said probes. The targets can thus also include enzymes, antibodies, antigens, peptides, DNA fragments, RNA fragments, oligonucleotides or even whole cells. Depending on the type of targets and type of apphcation, a specific type of recognition circuitry for processing the information related to target detection may be provided with or separately fiom the sensor. The sample can be any biological sample (tissue or fluid) containing target molecules to be detected taken directly or after culturing (enrichment) from a healthy or an infected human being or animal More specifically these samples can include expectorations of any kind, blood, plasma, respiratoiy tract samples such as sputum, broncheolavages, skin tissue, biopsies, lymphoyte blood culture material, colonies, cerebrospinal fluid, brain tissue, urine, gastrointestinal tract, food, feed or environmental samples. Said samples may be prepared or ecxtracted by any method known in the art. The sample may also be any preparation as described below (such as urea) or any other industrial product. Alternatively, the sample to be tested may contain partially or fully purified target or analyte molecules, such as for instance amplified nucleotides, which have been solubilized in a solution. These solutions can be chosen from any type of solution known in the art which is suited for esatblisbing a binding reaction between the specific probe and its target. In the case of nucleotide detection, the sample material will include either genomic DNA or precursor RNA or amplified versions thereof. The solution will be what is refened to a as hybridization solution. Upon hybridisation under what is refened to as "desired hybridisation characteristics according to the present invention", the probe (in this case an oligonucloetide) will only hybridize to the DNA or RNA from the specific organisms or molecules for which it was designed and not to the DNA or RNA from other organisms or molecules such as closely related organisms or variant or mutated molecules which may also be present in a particular sample. In practice, this often implies that the intensity ofthe hybridization signal is at least two, three, four, five or even ten times stronger with the target DNA or RNA from the organisms fiom which the probes were designed, as compared to non-target sequences. Often it is desirable and achievable to detect nucleotide which perfectly match the probe nucleotide (implying that hybridization conditions are used in which one mismatch is detectable). The hybridization conditions can be monitored relying upon several parameters, such as the nature and concentration ofthe components ofthe media or solutions, and the temperatures under which the hybrids are formed and washed. When modifications are introduced, be it either in the probes or the media, the temperatures at which the nucleotide probes can be used to obtain the required specificity should be changed according to known relationships, such as those described in Hames and Higgins (eds ). Nucleic acid hybridization. A practical approach, IRL Press, Oxford, UK.. 1985. The probes may be applied to the sensor ofthe present invention in any manner known in the art, for instance immobilized by means of high resolution probe dispensing systems or even synthesized on the spot. In another set-up also comprised within the scope ofthe present invention, the sample may be applied to the sensor and the probes may be added in solution to the ative test site area ofthe sensor to bring about a recognition which may be detected. It should be stressed that the ability to simultaneously generate recognition results with a number of probes is an outstanding benefit ofthe sensors ofthe present invention. In case of detection of antibodies present in a sample, the probe will be an antigen (e.g. a peptide or a polypeptide) or an anti-idiotype antibody known in the art. In case of detection antigens or polypeptides or peptides possibly present in a sample, the probe will be an antibody or a derivative thereof specifically binding to certain antigens, an antisense peptide specifically binding to certain peptides or polypeptides, a receptor or chemical molecule specifically binding to said polypeptide or peptide. The solution in which the possibly prepared or purified target material present in the sample may be dissolved, will be any solution which allows the binding between said binding molecules to occur. The conditions under which this foimation may occur are well known in the art and are for instance further described in the above-mentioned patents and apphcations ofthe one ofthe applicants.. The present invention also relates to method of fabricating a sensor for identifying molecular structures within a sample substance. This method comprises the steps of forming a plurality of interspaced channels in a insulating layer, said channels having essentially the same direction, said channels having a bottom and at least two opposite side-walls along said direction; depositing a metal layer on said insulating layer while aligning said dielectric layer with respect to the metal deposition source such that the bottom of said channels and the sidewalls of said canals along the deposition duection are shadowed and not covered by metal to thereby form an impedimetric device with said sample substance within and between the channels and eventually immobilising probes for binding to said molecular structures, said probes being applied to either the insulating part ofthe channels (said bottom and the other side-wall of said channels), or to the surface of the electrodes or to both, the insulating part ofthe channels and the surface ofthe electrodes. The present invention represents an important tool in a wide field of apphcations and it is by the way of example and not by way of limitation suited for measuring specific interactions like the reaction between an enzyme and its substrate or the recognition reaction between an antibody and an antigen, between DNA-DNA between RNA-DNA or other molecular structures; in the study ofthe reaction kinetics of said specific interactions; for sequencing molecules such as peptides, enzymes, nucleotides, DNA RNA and so on; for detecting genes mutations; for epidemiology and geno- or sero typing or for instance HLA and HCV; for drag susceptibility testing like the resistance against beta-lactamase and tetracycline in Neisseria gonorrhoeae. the detection of rifampicin resistant Mycobacterium tuberculosis strains or the detection of AZT-resistance in HIV: in screening and diagnosis:viral diagnosis: like in the case of HIV, HCV, HBV, herpes and relatives, CMV, HPV or HTLV; bacterial diagnosis like in the case of sexually transmitted diseases, cerebral spinal fluid analysis, detection of different mycobacterial species, evaluation of anaerobic infections, otitis, respiratory tract, gastro-intestinal tract, periodontal pathogens, pathogenic fungi; genetic diseases, like cystic fibrosis, Alzheimer, detection of mitochondrial mutations, platelet antigens, drug receptors, risk factors for atherioscrerosis and coronary heart dieases, cancer. APOE, AChE, APOB, LDL and so on; in chnical analysis: hke in the case of conductometric urea or creatinine quantitation. The high sensor miniaturisation also allows the constmction of integrated microdiagnostic devices capable of simultaneous detection of a multitude of parameters, i.e. multiparametei testing, and ultimately screening assays. ABBREVIATIONS BCB Benzocyclobuteen LPCVD Low Pressure Chemical Vapour Deposition PECVD Plasma Enhanced Chemical Vapour Deposition PMMA polymethylmetacrylaat PEEK Po ly( eth er ether )ket on PC Polycarbonaat PVE Polyvinylethyleen PEI Polyethyleneimine CMV Cytomegalovirus HPV Human papilloma virus HTLV Human T-cell leukemia virus APOE Apolipoprotein E APOB Apolipoprotein B AchE Acetylcholinesterase LDL Low density lipoprotein HLA Human leukocyte antigen HCV Hepatitis C virus HTV Human immunodeficiency vims HBV Hepatitis B virus LIGA Lithographie, Galvanik Abfoimung BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an illustration ofthe dependence ofthe electrical field penetration depth in the case of a planar structure with the electrodes geometry (different ratios of (electrode width )/( electrode interspacing)) and dimension, L. Figure 2 is a schematic drawing of the active sensor test site area of an embodiment of a bioelectronic sensor according to the present invention. Numbering used in Figures 2-7 is as follows: ( 1 ) direction of evaporation or deposition of metal; (2) first bonding pad of one sensor; (3) second bonding pad of one sensor; (4) 'even' planes of electrode fingers; (5) 'odd' planes of electrode figures; (6) hills, blocking planes (4) from (5); (7) channels; (8) mask for separation of different sensors; (9) shaded arrays (shaded fiom metal deposition); (10) probes; ( 11 ) field lines of a sensor on which a voltage is applied; (12) to be detected molecules; (13) sacrificed electrodes in the separation step; (14) width of all interdigitated electrodes together of one sensor; ( 15 ) left side of devide with separate functions: electrodes; ( 16) right side of said device: area with probes. Figure 3a is a schematic drawing, representing a base plate for making an anay of 2x2 sensors (by way of example and not by way of limitation) according to an embodiment of the present invention; Figure 3b, 3c and 3d give a cross-sectional detail of one sensor according to the present invention. (Tlie numbering used is as described for Figure 2) Figure 4a, 4b and 4c show schematically a metallization process ofthe sensor according to the present invention. (The numbering used is as described for Figure 2) Figure 5a illustrates how a shadow mask is applied for achieving a final stmcture. Figure 5b shows the sensor array after etching through this mask. (The numbering used is as described for Figure 2) Figure 6 illustrates the working principle of one of the possible embodiment of the sensor according to the present invention. Figure 6a shows a sensor without recognized' molecules and Figure 6b with 'recognized' molecules. (The numbering used is as described for Figure 2) Figure 7a and 7b show a specific embodiment of the bioelectronic sensor of te present invention. (The numbering used is as described for Figure 2) EXAMPLES AND DETAILED DESCRIPTION OF THE INVENTION Preferred exemplary embodiments of the present invention will hereafter be described in conjunction with the appended drawings. It is to be understood that the examples given are only for the purpose of teaching ofthe invention, the spirit and scope of this patent application being limited only by the terms ofthe appended claims. The sensor ofthe present invention comprises an insulating layer with metallic electrodes on the top. A submicron pattern is made in the insulating layer. The metal top layers are in a specific geometiy thereby enhancing the detection sensitivity of the sensor. Said sensor can further comprise a base layer. Figure 2 shows a detailed view of a preferred embodiment of an electronic sensor according to the present invention. This figure shows the active test site area ofthe sensor in a schematic drawing. This sensor or test site may be fabricated in a sequence of steps as detailed hereunder. A substrate is to be provided. Said substrate, designating said base layer, can be a cristaline wafer (quartz, silicon, geimanium), an amorphaus material (glass wafer), a polymer (PMMA, PC, PEEK, PVE, PEI ) or thick film substrate, such as Al 2 O An insulating layer is formed on said substrate. The insulating layer can be a polymer layer such as polyimide or BCB. The dielectric or insulating layer can as well be Si 3 N 4 being deposited by LPCVD or PECVD techniques. It can also be a layer of SiO 2 that is deposited or thermally grown on said sihcon wafer. A specific geometiy can then be patterned using known hthography techniques, e.g. photohthography, preferably UV litography, even more preferably deep UV hthography, followed by a selective etching in the SiO 2 layer. Another way to produce such insulating structure with a specific geometiy is using a moulding process. The reproduction is then done by injection moulding or any other way of making replicates. The mould can then be made with LIGA using X-ray or photohthogiaphy, preferably UV litography, more preferably deep UV lithography, which allows to achieve veiy small dimensions. Plastics such as PMMA, PEEK, PVE and PEI can be used as a substrate. The use of these plastics for making microstructures is known in the art. The moulds fabricated by means ofthe above-defined methods can be used again as a tool for further replication processes, e.g. as mould inserts for micromoulding or reaction injection moulding. Materials to be used for the replication processes are usually melted polymers and casting resins. After hardening in the metaUic form, the mould materials have reached a sufficient strength and the separation of mould and mould insert can take place. For the realization of micromoulding and micro-reaction injection moulding the extremely low roughness ofthe walls of LIGA fabricated mould inserts is most important. Materials which have been used for microreplication include low viscosity thermoplastic polymers like polymethyl methacrylate (PMMA), polyoxymethylene (POM), polyamide (PA), or polycarbonate, as well as reaction resins based on methacrylates. silicones and caprolactames. However, many more materials could be used. Except for filled moulding materials, almost any material suitable for macroscopic moulding can be used for micromoulding. Ceramic microstructures can be fabricated by slurry casting, by using sol-gel processes or by means of electrophoretic and other processes. It is e.g. possible to fill the gaps of a LIGA fabricated polymer stmcture with a slurry of microcrystalline ceramic powder. After drying and filing, the polymer degrades, evaporates or is oxidized, which results in a ceramic microstructure ("method ofthe lost form"). The characteristic dimensions ofthe ceramic structures are smaller than the polymer foim, due to shrinkage during the filing process. Mechanically very stable and temperature persistent materials, piezoelectric materials and ionic conductors can thus be microstructured by means ofthe LIGA process. Figure 3 shows a plate with 2x2 sensors. The chemical composition of this plate can be an insulating on its own or the plate can be composed of a substrate (e.g. a sihcon wafer) with an insulating layer (e.g. a SiO 2 layer) thereon. The topography ofthe plate shows pits (7) and hills (6) . Figure 3b gives a detail of one sensor. Figure 3c and 3d show cross-sectional views. In a preferred embodiment, the pits (7) are channels (7) having dimensions of the same order of magnitude as tlie molecules to be detected. Thus the channels are preferably about 100 nm deep and about 100 nm wide, about 100 nm spaced. The spacing in one duection defines adjacent planes (4) (5). The dimensions ofthe channels can range between about 500 nm deep and about 500 nm wide down to about 10 nm deep and about 10 nm wide, preferably less than 250 nm deep and less than 250 nm wide. This width and deepness may vary independently. The spacing between two channels is of the same order of magnitude as the width and deepness of the channels. The channels are as long as the length ofthe active test sites area ofthe bioelectronic sensor. In the sequel, and for the purpose of explaining the invention, this length is assumed to be 0.5 mm. Lenghts between 100 μm and 1 mm or smaller or larger are possible. The active area can be made in any geometiy , for production purposes by preference a square. It can as well be rectangular. Also the channels can have any shape , e.g. trapezoidal, triangular, rectangular or cylindrical. The hills (6) are elevations of a specific height. In this embodiment a height of 1 μm is assumed. The height ofthe hills can be anything above the width ofthe channels. Then purpose is to separate the (4) and (5) planes between the channels. The shape ofthe hills by preference is rectangular but does not necessarely have to be so. The hills are located at the end ofthe planes (4) (5) between the channels compulsory depassing over the edge ofthe channel. If at one side ofthe sensor, they are located at the 'even' planes (4), then they are located at the 'odd' planes at the other side (5) (Figure 2b). In this embodiment, tlie hills are about 200 nm long and about 200 nm wide. Figure 4 illustrates the next step in the processing. A metal layer is deposited on the plate under an angle, by preference by means of e-beam evaporation. The direction ofthe deposition is shown by the big arrow (1). The directionality has to be such that some places (9) on the plate are shadowed and not covered by metal. The angle of metal deposition therefore has to be smaller than 90° as measured with respect to the surface of the plate. By preference the angle of deposition is smaller than 60°, 45° and even smaller than 30°, such as 20°, 10°. 5° or 1°. Said places (9) are at the bottom ofthe channels and at the sidewalls ofthe channels and ofthe hills along the deposition direction ( 1 ). The planes (4) (5) between the channels are isolated one from another because there is no metal at the bottom of the channels and at the sidewalls along the deposition direction ( 1). Nor are they shortcutted at the edges due to the hills (6) (see Figure 2 for a 3-D impression). Any metal that does not react with the sample solution can be used. Examples are Pt, Pd, Au or less noble metals like Ag or Al provided that chemical reactions at the electrodes are expelled. The thickness ofthe metal layer or metal coating can range inbetween about 2 nm, 50 nm, 100 n , 200 nm or thicker, preferably the metal thickness is 20 nm. The metal deposition can be achieved using thermal evaporation, sputtering, e-beam deposition or any other technique known for depositing metals such as an impinging flux of metals. The different sensors in an array (in this example 2x2 sensors) are however still shortcutted. Selective etching ofthe evaporated metal separates the different sensors. Figure 5a shows the apphcation of a mask (8). This mask can be lithographically transferred to a resist pattern, or it can be a shadow mask. If the structure is etched like this, the structures under the masking layer remain and the non-covered area is etched. Figure 5b shows the result of this etching in separated sensors. These separated sensors can be a possible final stmcture. These separated sensors with probes attached thereon are also a possible final structure. Separated bonding pads (2) and (3) are achieved and thus an interdigitated electrode stmcture results. The shaded area ofthe mask (8) in figure 5a determines the active array ofthe sensor and the positive (2) and negative bonding pads (3) The open area ofthe mask is etched away (cf. above) and separates the different sensors from each other. The alignment ofthe mask (8) is not critical. An alignment accuracy of 10 μm is sufficient for this embodiment. The up and down sides in figure 5a are defining the bonding pads (2) and (3) and then final dimension is not critical. The dimensions ofthe left and right sides on figure 5a are not critical neither. The mask (8) is namely 50 μm smaller than the width (14) of all the channels. The final active area is thus determined by mask (8). Some fingers (13) are sacrificed and etched away. This means that a misalignment of half this width of 50 μm (i.e. 25 μm) does not have any influence, because the active area is still fully covered with channels (7) and the resulting finger electrodes (4) and (5), being the metallized planes. This procedure prevents from needing structuring methods with a sub-micron resolution. Other methods known in the art to separate the different sensors may also be used within the scope ofthe present invention. Figure 6 shows one possible way of the the working principle of a sensor of the present invention. Probes (10) may be immobilised in the insulating areas ofthe channels according to probe immobilization methods known in the art as such epoxy linkage, carbodiimide, reductive amination, cyanogen bromide, succinimide. carbodiimidazole. tresyl and tosyl chloride, divinyl chloride, maleimide, hydrazide, iso(thio)cynates and more preferred silanization with amino silanes, epoxysilanes, thiocyanato silanes, isocyanato silanes, succinic anhydride silanes, sulfhydryl silanes, caprolactam silanes and so on. However, in other measurement configurations and/or set- ups, the probes can be selectively immobilised: -only on metallic surfaces, for example by adsorption of sulfur containing moieties at a Au layer, - or all over the active test site area: on the insulating and the conductive layers, for example by a sequential immobilisation process or by plasma polymerisation of an organic layer exhibiting reactive groups, hke amino, sulfhydril, aldehydes, carboxyl, hydroxyl and so on. The probes in the context ofthe present invention may be, by way of example and not by way of limitation, enzymes (with affinity for specific substrates), oligonucleotides (with affinity for specific DNA and RNA fragments), antibodies (with affinity for specific antigens), antigens (with affinity for specific antibodies), or any other component of a analyte/coanalyte complex. By applying an electrical signal, i.e. voltage or current, on the bonding pad (3) and bonding pad (2) an electric field arises, resulting in electric field lines (11). If the analyte to be detected (12) is in a sample solution it will bound to the specific probes (Figure 6b), resulting in a change in the electric field (11) in contrast with the situation depicted in Figure (6a). This change can be quantitated by measuring the impedance at the proper frequency and/or dc bias. By preference this electrical measurement is an impedance analysis, which can devolve in a measurement of resistance, capacitance, dielectric loss and/or reactance over a frequency range, including or not dc bias, or a combination of these techniques. Due to the sub-micron dimensions of the channels and due to the shape of the electrodes ( emerging from the deposition of metal under an angle), the electric fields (11) strongly penetrate in the region with the immobilised probes (11). An even stronger confinement ofthe electrical fields in the region of interest would be achieved in case when a second insulating layer is put on top ofthe (4) and (5) planes. In this way the electrical field lines probe more the interior ofthe channels where the bound analyte occupies most ofthe space. When fabricating a sensor according to the present invention with the smaller dimensions (sub 100 nm) larger molecules that have to be detected might not enter the channels anymore due to steiic hindrance. Figure 7 shows schematically how this problem can be overcome. A sensor is to be fabricated that can be spht in two. The left side ( 15) ofthe sensor consists of an anay of sensors with the interdigitated electrode stmcture, fabricated in the same way as discussed above. The right hand side ( 16) has tlie min-or image ofthe sensor array and is covered with immobilized probes ( 10). The sample solution comprising the molecular stmctures and elements that are to be detected is put (incubated) on top ofthe right hand side ( 16). Certain molecules ( 12) will bind to the probes (10). After this recognition process, the sensor is closed by folding (Fig. 7b). The apphcation of mechanical force brings the probes ( 10) and molecules (12) close enough to the interdigitated electrode stmcture, so that eventually a difference in impedance ofthe incubated versus a not incubated structure can be measured. The present invention describes sensors which are suitable for real time measurements, i.e. the binding process during different incubation steps, with or without different condition changes, like for instance temperature. The present invention also allows very flexible measurement set-ups such as analyte immobilisation on the surface ofthe sensing device and recognition of certain probes apphed in the solution phase. The described technological process and the high sensor miniaturisation also allow the constmction of microdiagnostic devices. Sensor anays comprising different probes can be fabricated in the way just described, to result in microdiagnostic devices. Said integrated microdiagnostic devices are capable of simultaneous detection of a multitude of parameters, i.e. multiparameter testing. This is of particular importance for limited sample situations like in the case of neo-natals blood samples, for reliable diagnosis requirements hke in the case of transplantation immnology, autoimune diseases or blood-infections and ultimately for screening assays. Such juxtaposed microdiagnostic anays have the additional advantage that they can process parallely and simultaneously several different samples.

Claims: CLAIMS 1 Sensoi comprising - an insulating layei with a plurality of interspaced channels therein having essentially the same direction, said channels having a bottom and at least two opposite sidewalls along said d ection , - a metal coating being applied on at least part of one of said two opposite side¬ walls of essentially each channel and on at least part ofthe top ofthe insulating layei in between said channels thereby foπning part of an impedimetnc device compnsing 2 electrodes 2 Sensoi accoiding to claim 1 furthei composing hills, with said hills - bemg located at the end(s) ofthe planes between said channels. - having a heigth which is laigei than the width ofthe channels, and, - ovei lapping the plane between said channels and part of two adjacent channels 3 A sensoi according to claims 1 oi 2 furthei comprising probes foi binding to moleculai stmctures present in a sample to be tested, said probes being appUed to either the insulating part ofthe channels and/oi to the surface ofthe electrodes 4 A sensoi accoiding to any of claims 1 to 3 wheiein said insulatmg layei is applied on a base lavei substrate 5 A sensoi according to claim 4 wheiein said substiate is a silicon wafei 6. A sensor according to any of claims 1 or 5 wheiein the dimensions of said channels are within a range of 10 nm to 500 nm. 7. A sensor according to claim 6 wherein said channels are 100 nm deep and 100 nm wide, said channels being interspaced by 100 nm. 8. A sensor accoiding to any of claims 1 to 7 wherein said insulating layer is a thermally grown SiO, . 9. A sensor according to any of claims 1 to 7 wherein said insulating layer is a polymer. 10. A method of producing a sensor, said method comprising the steps of : - forming a plurality of interspaced channels in a insulating layer, said channels having essentially the same dhection, said channels having a bottom and at least two opposite side- alls along said direction and optionally hills as defined in claim 2; - depositing a metal layer on said insulating layer while aligning said insulating layer with respect to the metal deposition source such that the bottom of said channels and the sidewalls of said channels along the deposition direction are shadowed and not covered by metal to thereby form an impedance; and, - optionally applying probes for binding to molecular stmctures present in a sample to be tested, said probes being applied to either the insulating part ofthe channels and/or to the surface ofthe electrodes. 1 1. A method according to claim 10 wherein the angle of metal deposition with respect to said insulating layer is smaller than 90°. 12 A method accordmg to claims 10 oi 11 furthei comprising the step of applying said insulating layer on a base layei substrate 13 A method according to any of claims 10 to 12 wherein said substrate is a sihcon wafer and said insulating layei is thermally grown Si0 2 14 A method accoiding to any of claims 10 to 13 wheiein the step of formmg said channels is executed using micioelectronics patterning techniques 15 A method according to claim 14 wheiein the step of forming said channels is executed by a photolithographic pi ocess 16 method according to any of claims 10 to 15 wheiein said insulating layei is a polymei. 17 A method according to claim 16 wherein said polymer is stmctuied by miciostmctuie moulding 18 A sensoi obtainable by a method according to any of claims 10 to 17 19 A sensoi appaiatus foi identifying moleculai stmctuies within a sample solution, comprising - a plurality of sensors accoiding to any of claims 1 to 9 oi 18; - optionally an additional insulating layei applied above said sensoi in oidei to confine the electrical field in said channel between the two sepaiated parts of said metal layei , - means foi applying a voltage on the metal coatings, and - means foi measuring the electrical pioperties 01 the impedance in between the electiodes of the sensois to determine which piobes have bonded to (an) associated taiget molecule(s) 20 A sensoi appaiatus according to claim 19 furthei comprising connection means to said metal coatmgs, said connection means hording the active area of said sensoi and being oriented essentially peipendiculai with respect to said direction and said voltage being appUed on said connection means 21 An aoay of sensois, said aoay being a geometric configuration ofthe sensois accoiding to any of claims 1 to 9 or 18, the different sensois ofthe aoay being essentially paiallel one to anothei along said duection 22 Method foi identifying molecular structures with in a sample solution comprising the steps of - applying said sample solution to a plurality of sensois, according to any of claims 1 to 9 or 18 oi 21, each sensor having one or more probes appUed therein to bond with an associated target moleculai stmcture, - applying an electionic signal to the sensor; and - measuring the electical properties ofthe sensoi to deteπnine which probes have bonded to (an) associated target moleculai stmctuιe(s) such that a pluiality of different taigets can be detected 23 A method according to claim 22 wherein said sensoi have one oi moie types of oligonucleotide piobes applied therein 24 A method according to claim 22 wheiein said sensoi have one oi moie types of antibody probes applied therein 25 A method accoiding to claim 22 wherein said sensor have one oi more types of antigen piobes applied theiein 26 A method according to claim 22 wheiiein said sensoi one or moie types of peptide piobes apphed theiein 27 A method according to any of claims 22 to 26 wherem said probes is (are) covalently oi non-covalently attached to said sensoi 28 A sensoi according to any of claims 1 to 9 oi 18 comprising one or moie probe(s) apphed to eithei the insulating part ofthe channels and/oi to the surface ofthe electiodes, with said piobe being as defined in any of claims 22 to 26 29 A sensoi appaiatus according to any of claims 1 oi 20 also comprising one oi more types of piobes applied to said sensois as defined in claim 28 30 A sensor which is spht in two parts and comprising - a first part comprising an aoay of sensois accoiding to claim 21, - a second part comprising an aoay of containers to which probes may be attached, and, - with said first and said second parts being bi ought mto contact in such a wa that said aoay of containeis coπesponds to said aπay of sensois

Inventors: BAERT KRIS, ROSSAU RUDI, VAN GERWEN PETER, BAERT, KRIS, ROSSAU, RUDI, VAN GERWEN, PETER
Applicants: BAERT KRIS, GERWEN PETER VAN, IMEC INTER UNI MICRO ELECTR, INNOGENETICS NV, ROSSAU RUDI, BAERT, KRIS, INNOGENETICS N.V., INTERUNIVERSITAIR MICRO-ELECTRONICA CENTRUM (IMEC), ROSSAU, RUDI, VAN GERWEN, PETER
Application year: 1996
Main classification: G01N27
IPC codes: C12Q1, G01N27, G01N33