Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/27—Association of two or more measuring systems or cells, each measuring a different parameter, where the measurement results may be either used independently, the systems or cells being physically associated, or combined to produce a value for a further parameter
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
  • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
  • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
  • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
  • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
  • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
  • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

Definitions

• the invention relates to a method for producing a device for detecting an analyte, and the device as such and their use.
• a solution containing one or more of the substances to be measured brought by means of a reference electrode to a defined potential.
• a further electrode is added at which the detection can take place. If this electrode is at a potential which is suitable for oxidizing or reducing the analytes, a reaction takes place at the electrode.
• the analytes are oxidised or reduced at the electrode surface and thus generate a current flow that can be measured at the electrode. This current flow is proportional to the Arv number of converted molecules and allows accurate conclusions about the concentration of the molecules in the sample.
• glucose oxidase test which is used for clinical blood sugar determination.
• glucose is catalysed in a bioreactor by means of the enzyme glucose-oxidase to gluconolacetone and hydrogen peroxide.
• the hydrogen peroxide concentration can be measured electrochemically. Since this concentration is proportional to that of glucose, the proportion of glucose can be accurately determined.
• the method is successfully used in numerous tests, it still has some methodological disadvantages that preclude its use in broader fields of application.
• the electrode current and thus the sensitivity of the sensor are always limited by the mass transport of the analyte to the electrode.
• molecules of the analyte that have already reacted at the electrode surface are diffusively replaced with native molecules of the sample.
• the sensors can only be miniaturized to a certain degree. The smaller the electrode surface, the fewer molecules can react to it. Therefore, this method can only be limited in Lab-on-a Chip applications are used.
• the packing density of these sensors is limited to one chip, since each sensor must be contacted by its own interconnect.
• the titanium and chromium layers mentioned serve to adhere the electrode to the substrate or to the passivation. After removal of the sacrificial layer, the design necessary for the redox cycling is achieved in an avidity from two superimposed electrodes.
• the electrodes are each provided with conductor tracks and contact surfaces and aligned parallel to each other. Up to 29 cavities are arranged in such a way in a biochip and switched against the reference electrode.
• the method for detecting provides for the analyte to be brought to the bottom and the top electrode via a microfluidic access from PDMS and to be detected by the voltage change after the voltage has been applied to the test electrode. This method can be used to fabricate a sensor array with multiple sensors.
• the sensor system produced in this way is limited in relation to the maximum achievable spatial resolution.
• a spatially high-resolution electrochemical detection of analytes is not possible because a large number of measuring devices are needed.
• each sensor consisting of the two electrodes must be read out with a separate measuring device. This increases the costs with a high number of pixels and makes the construction of a measuring apparatus considerably more difficult.
• serial data acquisition on the other hand, fewer measuring devices are required, but each sensor nevertheless has to be connected separately with a suitable switch, so that a complex read-out apparatus is likewise required.
• the method for producing a device for detecting an analyte has the following steps. a) On a insulating substrate, a first conductor with Elektt odenfunktion is arranged.
• the conductor track is correspondingly arranged in a line on the substrate.
• the trace advantageously consists of a material such as gold, platinum and the like.
• a plurality of first conductor tracks can be arranged simultaneously.
• the first interconnect and possibly below and above arranged adhesion layers for attaching the first interconnect to the substrate and to the passivation consist of materials which are not removed during the removal of the later-arranged sacrificial layer.
• the first conductive trace is made of a non-etchable material as compared to the sacrificial layer.
• a first thin passivation layer is arranged on the conductor track, that is, the conductor track is passivated.
• the conductor track has in effect no more freely accessible surface but is completely covered by the passivation.
• the substrate is also passivated next to it.
• Passivation is advantageously carried out when using the sensor no vertical or horizontal charge transport.
• an encapsulation of the conductor path is effected by the passivation. Since the conductor track is made of conductive material and forms a sensor together with the electrode to be deposited at this location, this step ensures that in the case of number of formed sensors on the substrate, the individual sensors along a conductor track and to adjacent tracks are not in contact with each other. Since a large number of sensors is formed on each conductor track, it is advantageously effected that the sensors or cavities formed thereon also have no contact with one another along a single conductor track.
• the first passivation is particularly advantageously made of a material that is not removed during the removal of the sacrificial layer arranged thereafter.
• the passivation consists of a non-etchable material compared to the sacrificial layer.
• the passivation layer is then locally limited, z. B. punctiform by a suitably designed mask z. B. opened by etching, so that the conductor is limited locally, z. B. is exposed selectively. Lithographic methods can be used for this purpose.
• a sacrificial layer is arranged on the conductor track.
• the sacrificial layer is necessary for the formation of the later nanocavity between the electrodes.
• the sacrificial layer is preferably etchable and consists z. B. chromium or other etchable material.
• an electrode for. B. also arranged from gold.
• the task of the electrode is to form a sensor together with the section of the first conductor track opposite the sacrificial layer. Since the first printed conductor always has an electrode function due to the choice of the material of gold, platinum and so on, it is ensured that a sensor can be formed between the electrode and the first printed conductor.
• steps c) to e) are carried out particularly advantageously in a single lithography step, so that a perfect alignment of the sacrificial layer and the electrode on the first conductor track takes place at the location of the sensor.
• the steps of methods e) and f) may be performed in a single lithographic step using the same mask.
• a second interconnect is then arranged orthogonally to the first interconnect on the electrode, wherein the second interconnect preferably the electrode only at de contacted the edge.
• the trace is preferably lithographically deposited so as to have an opening at the location of the electrode. With the separation of steps e) and f), the electrode and the conductor are separated from each other. This advantageously has the effect that the electrode and the sacrificial layer can be deposited with the same mask in a single lithography step.
• the second interconnect and possibly below and above arranged adhesion layers for attaching the second interconnect to the first and then arranged second passivation consist in an advantageous embodiment of the invention of materials that are not removed in the removal of the sacrificial layer.
• the second trace is made of a non-etchable material as compared to the sacrificial layer.
• a second passivation layer is then arranged on the electrode as well as on the second conductor track and preferably also on the first passivation layer.
• the purpose and purpose of the second passivation are identical to that of the first passivation.
• the passivation takes place in particular over the entire surface of the substrate and the entire layer structure.
• the second passivation layer and the electrode are opened at least at one point, so that the sacrificial layer arranged below the electrode is exposed. As a result, at least one aperture for the sensor is provided by which the analyte can reach the sensor electrodes.
• the sacrificial layer is then removed. This provides the nanocavity.
• the steps a) - i) can be performed several times in succession or simultaneously. It can, for. B. a plurality of first conductor tracks are arranged in parallel to each other simultaneously on the substrate and a plurality of electrodes are arranged simultaneously. The same applies to the remaining process steps, such. B. the arrangement of the second interconnects.
• a checkerboard-like sensor field is formed, in which a sensor with two electrodes for forming a nanocavity is formed at each crossing point of a first and a second conductor track.
• the first passivation layer on the first conductor track is not removed during the removal of the sacrificial layer. As a result, a cavity is formed exclusively in the region of the point of intersection between a first printed conductor and a second printed conductor.
• Fabrikaüonsrea For the implementation of the sensor according to the invention, a novel Fabrikaüonslui is used.
• Wolfrum et al. and Kätelhön et al. describe in their publications fabrication processes in which the interconnects are attached by means of adhesive layers of chromium to the adjoining layers. This is necessary in order to remove the adhesion layers together with a chromium sacrificial layer and thus to obtain electrodes of the desired material that are not covered with an adhesion layer.
• step b) has been introduced in claim 1 in the present invention. This passivation allows the separation of the nanocavities from each other.
• Adhesion layers can be attached to the substrate and to the passivation. These remain untouched during the subsequent removal of the sacrificial layer as well as the passivation.
• an electrode pair which is used for redox reactions on the analyte, is formed between the (top) electrode deposited at the point of intersection and the section of the first printed conductor at this point. Since the nanocavity with gap S is accessible from the outside only via the aperture, an analyte can penetrate from above (see figure) and be successively reduced and oxidized by diffusion to the electrodes, depending on which of the two electrodes a positive or negative Voltage is applied.
• the method is advantageously characterized by the choice of a sacrificial layer which is etchable. Etching can be carried out in a wet-chemical or dry-chemical way.
• steps c) to e) are carried out in a single lithographic step using only one mask. This guarantees an exact alignment of the electrode on the sacrificial layer above the first conductor track. This ensures that the electrode and the opposite section of the first conductor track are exactly aligned with each other and thus form a sensor for detecting the analyte.
• steps e) and f) may be performed in a single lithographic step using the same mask.
• the opening of the second passivation layer and the electrode with holes in a hexagonal arrangement is particularly advantageous.
• the person skilled in the art will balance this between preserving the sensor surface on the one hand, because material of the top electrode is removed during the formation of the holes, and an accessibility of the nanocavity for the analyte on the other hand.
• Several small holes with a diameter on the nanometer scale eg up to 250 nm
• ensure that the analyte to be detected can easily diffuse between the electrodes via the holes in the gap S, and that at the same time the very large electrode area (FIG. eg up to 100 ⁇ m diameter), the detection is ensured by successive redox reactions of the analyte.
• Several holes particularly advantageously reduce the response time of the sensor.
• multiple holes also improve the response of the sensor to rapid changes in analyte concentration near a measuring crossover point, such as might be expected in the delivery of neurotransmitters through neurons located thereabove. Since in this case there is only an extremely short exposure of the sensor by the analyte and only the analyte molecules that are actually located within the nanocavity between the electrodes can be detected, the size of the sensor response scales above all with the gap length of the opening to the nanocavity , That is, a strongly localized extension of the gap opening through many small openings is basically advantageous for the detection of short, positive concentration pulses.
• the device for the detection of analytes is characterized in that between at least two orthogonally extending conductor tracks in the crossing point a arranged in self-contained nanocavity for receiving the analyte between the interconnects, wherein above and below a gap S for forming the nanocavity two opposing regions of the first and second interconnects electrodes for a sensor forming the detection of an analyte by sequential oxidation and reduction of the analyte at the electrodes.
• the electrode is made of the same material as the second conductor and is arranged in the same plane as this.
• the nanocavity is self-contained, since apart from the openings formed in step h) it has no further inlets or outlets. In particular, the nanocavities are not in lateral connection with other nanocavities. A connection between the nanocavities can only be made via the sensor inputs (aperture).
• the interconnects are advantageously passivated with the exception of the crossing points.
• a plurality of crossing points of a plurality of orthogonally arranged conductor tracks are arranged in the device.
• a nanocavity is formed between two orthogonal printed conductors.
• There is no connection between adjacent nanocavities in particular no connection via a diffusion of the analyte except via the sensor input (aperture) itself.
• the measurement takes place in each case at the crossing points of the conductor track, wherein the redox cycling effect is used.
• the signals at the individual crossing points can be read out either serially or line by line.
• a plurality of parallel electrodes (A) are respectively set to an oxidizing or reducing potential while an orthogonal electrode (B) is set to a reducing or oxidizing potential. All other electrodes are either not connected or set to the potential at which no redox cycling occurs. Thus, redox cycling occurs simultaneously at all crossing points of (A) and (B). The redox cycling currents can then be measured in parallel on the electrodes (A) for the respective crossing point, whereas the sum of the redox currents of (A) is applied to electrode (B).
• the advantageous use of the device is in the detection of neurotransmitters as analytes.
• the device Since the device is passivated as described, it is also biocompatible. Nerve cells can be cultured by applying proteins to the surface of the device directly on the device. The released neurotransmitters are detected in real time.
• Figure 1 Inventive manufacturing process. Short description: After the deposition of the first gold conductor 2 on the substrate 1, a thin SiO 2 passivation 3 is applied (FIG. 1 a). This is opened at the later intersection of the first 2 and second 6 tracks by reactive ion etching ( Figure lb). In the same structuring step, a chromium sacrificial layer 4 and a thin layer are then introduced into the opening Layer of the electrode material gold 5 deposited. Subsequently, the upper conductor 6 and a further passivation layer 7 is applied. The further, second passivation and the electrode are opened at the points of intersection to the lower interconnects by means of reactive ion etching (FIG.
• FIG. 1 The fabrication process is given by way of example in FIG. 1 to form a single intersection point.
• the top view is on the left, the cross section is shown on the right in the picture.
• the dotted line represents the location of the cut.
• the deposition of the lower conductor 2 (width B: 1 to 100 ⁇ m, in the present case 5 ⁇ m, thickness: 30 nm-1 ⁇ m, in the present case for example 150 ⁇ m) takes place by means of optical lithography and lift-off.
• an adhesion layer of titanium (not shown) having a width of 5 ⁇ m and a thickness of 7 nm is first deposited on the oxidized wafer 1.
• the gold layer 2 is arranged (FIG. 1a).
• FIG. 1b shows the deposition of a thin passivation 3 by means of PE-CVD.
• the thickness may be 50 nm to 2 ⁇ , 400 nm were applied in the present embodiment.
• FIG. 1b also shows the opening of the passivation 3 at a future crossing point.
• the diameter of the opening can be 0.8 to 80 ⁇ ⁇ and carried out via reactive ion etching. In the present case was worked with a diameter of 4 ⁇ .
• the deposition of a chromium sacrificial layer 4 directly into the opening ( Figure lc).
• the thickness can be 10 nm to 1 ⁇ m, in this case 50 nm.
• the layer 4 has the same diameter as the opening and again takes place by means of optical lithography and lift-off.
• a thin top electrode 5 is deposited directly on the sacrificial layer of chromium 4, by means of optical lithography and lift-off.
• the thickness of the electrode 5 is between 10 and 100 nm, in the present case 30 nm. It has the same diameter as the sacrificial layer 4. It makes sense to follow the steps of FIGS. 1b (formation of the opening), 1c (arrangement of the sacrificial layer) and 1d (arrangement the top electrode 5) in a lithography step and only with a lift-off perform. Although this makes the lift-off a little more difficult, but it ensures an absolutely precise alignment of the sacrificial layer 4 and the electrode 5 above the conductor 2 to each other. In the next step, the deposition of the upper conductor 6 takes place with a width of z. B.
• the passivation 7 (FIG. 1f) is brought about by means of PE-CVD.
• the thickness may be between 50 nm and 2 ⁇ m, in the present exemplary embodiment 800 nm Si0 2 were deposited.
• the holes were brought about with electron beam lithography.
• the holes are to be adapted to the size of the sensor 2, 5 in the crossing point.
• the resulting aperture allows the penetration of the analyte to the electrodes of a sensor in this way, but advantageously not laterally via inner channels from sensor to sensor.
• openings 8 are also possible.
• the design of the openings 8 has an influence on the response times and on the efficiency of the sensor. Many densely seeded small ones Holes 8 relative to the upper electrode 5 improve the response time of the sensor by rapid diffusion as opposed to a single small hole 8. Thus, faster measurements are possible. For this, the efficiency of the redox cycling is somewhat reduced because the sensor surface is reduced by the removal of material of the top electrode 5. A large single hole 8 also improves the response time, but reduces the gain of the redox cycling due to the lower effective electrode area 5, 2 at the point of intersection.
• a gap S gives the width of the nanocavity between the open top electrode 5 and the opposite section of the bottom one
• the substrate 1 is a 100 mm Si wafer with a 1 ⁇ thick Si0 2 passivation layer.
• the thickness plays a minor role. It should be chosen so that there is sufficient insulation.
• the printed conductors 2, 6 are applied by means of electron beam evaporation and structured by means of lift-off.
• the following protocol is followed: spin on the LOR3b TM paint at 3000 rpm and cure on a hotplate for 5 min at 180 ° C. Then the nLof 2020 TM paint is spin-coated at 3000 rpm and cured on a hotplate for 90 s at 15 ° C. Exposure takes place through a mask in the Mask Aligner. The development of the coatings is done in MIF326 TM for 45 s. The lift-off of the metal layer takes place in acetone.
• the protocol is performed several times with the following layers deposited.
• the first, lower tracks comprise 150 nm gold on 7 nm titanium as the adhesion layer.
• the upper second traces include 7 nm titanium, 10 nm gold, and again 7 nm titanium.
• Passivations of Si0 2 and / or Si 3 N 4 are deposited by PE-CVD and have thicknesses between 50 and 800 nm. This passivation is then patterned with AZ 5214e TM and reactive ion etching using the following protocol.
• the coating AZ 5214e TM is spin-coated at 4000 rpm and curing on a hotplate for 5 min at 90 ° C. It is exposed.
• the coating is developed in MIF326 TM for 60 s and reactive ion etching at 200 W, 20 ml / s CHF 3 , 20 ml / s CF 4 and 1 ml / s 0 2 .
• this lacquer is used both for the opening of the passivation and for the lift-off of the sacrificial layer 4 and the upper electrode 5 with acetone. These have thicknesses of 50 nm (chromium) and 20 nm (gold), respectively.
• the sacrificial chromium layer 4 is removed wet-chemically with a chrome etch TM solution.
• a chrome etch TM solution For this purpose, the sensor field is covered with the etching solution for about 30 minutes and then rinsed with water.

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