NL2013393B1

NL2013393B1 - Integrated (bio) detection with micro fluidic micro needles for analytical application.

Info

Publication number: NL2013393B1
Application number: NL2013393A
Authority: NL
Inventors: Rassaei Liza
Original assignee: Univ Delft Tech
Priority date: 2014-08-30
Filing date: 2014-08-30
Publication date: 2016-06-21
Also published as: NL2013393A; WO2016032335A1

Abstract

The invention provides a sensor device comprising a substrate, especially a needle with a needle tip, wherein the substrate, especially the needle, even more especially the needle tip, comprises a sensor unit, wherein the sensor unit includes a stacked layer structure including an electrode layer, wherein the stacked layer structure further includes a nanogap dividing the electrode layer in a first electrode and a second electrode with the nanogap in between, wherein the nanogap has a width (w) selected from the range of 10-500 nm, such as from the range of 20-200 nm, and wherein in a specific embodiment the needle further comprises a micro fluidic channel structure with an orifice, wherein the orifice is especially arranged at the needle tip, for delivery or extraction of a fluid.

Description

P100173NL00

Integrated (bio) detection with micro fluidic micro needles for analytical application

FIELD OF THE INVENTION

The invention relates to a sensor device, especially comprising a (micro) needle. The invention further relates to a measurement apparatus comprising such sensor device. The invention also provides the use of electrochemical redox cycling within a sensor unit of such sensor device. Yet, the invention also relates to a method for producing such sensor unit, especially such sensor device. The invention also relates to a design of such sensor unit and sensor device.

BACKGROUND OF THE INVENTION

The use of nano sensors is known in the art. US8585973, for instance, describes a method for the manufacture of a nano-sensor array. A base having a sensing region is provided along with a plurality of nano-sensors. Each of the plurality of nanosensors is formed by forming a first nanoneedle along a surface of the base, forming a dielectric on the first nanoneedle, and forming a second nanoneedle on the dielectric layer. The first nanoneedle of each sensor has a first end adjacent to the sensing region of the base. The second nanoneedle is separated from the first nanoneedle by the dielectric and has a first end adjacent the first end of the first nanoneedle. The base is provided with a fluidic channel. The plurality of nano-sensors and the fluidic channel are configured and arranged with the first ends proximate the fluidic channel to facilitate sensing of targeted matter in the fluidic channel.

SUMMARY OF THE INVENTION A problem with state of the art methods for measuring e.g. neurotransmitters is that such measurements often include an offline measurement, meaning the sample is taken from the subject and analyzed using normal lab equipment, or using micro needles which have an electrode at the end. The electrode, however, may have to be modified using different complicated strategies to be sensitive to analytes of interest in presence of undesired interferences, such as ascorbic acid. Further, the sensitivity is not very high. For environmental samples, although few handheld devices are available, most of the analysis are carried out in the lab and require big sample volumes and expensive equipment.

Hence, it is an aspect of the invention to provide an alternative analysis method and/or analysis device (herein also indicated as “measurement apparatus”) (and attributes that can be used therefore, such as the herein described sensor device or sensor unit), which preferably further at least partly obviate one or more of above-described drawbacks, and which may especially be used for small volumes with high sensitivity and/or selectivity.

In a first aspect, the invention provides a sensor device (“device”) comprising a substrate, especially a needle (even more especially a micro needle) with a needle tip, wherein the substrate, especially the needle, even more especially the needle tip, comprises a sensor unit, wherein the sensor unit includes a stacked layer structure including an electrode layer, wherein the stacked layer structure further includes a nanogap dividing the electrode layer in a first electrode and a second electrode with the nanogap in between, wherein the nanogap has a width (w) selected from the range of 10-500 nm, such as from the range of 20-200 nm, and wherein in a specific embodiment the needle further comprises a micro fluidic channel structure with an orifice, wherein the orifice is especially arranged at the needle tip, for delivery or extraction of a fluid.

As the nanogap especially divides the electrode layer, more especially substantially the entire stacked layer structure, a horizontal nanogap electrode is provided. In contrast to vertical nanogap based sensors, horizontal based nanogap sensors are much easier to make, and can be applied on many more substrates than the vertial nanogap sensors. Also there is more freedom in choosing materials for the respective layers of the stack. Hence, especially the sensor unit comprises a horizontal nanogap (or “horizontal nanogap electrode”). The first electrode and second electrode are thus essentially identical in layer sequence and layer heights as they originate from the same (undivided) stack. The electrode layer is not necessarily divided in two halves (by the nanogap), though in general the cross-section (parallel to the layers) will be substantially equal. The introduction of the nanogap provides two electrodes that have no direct physical contact, and there is (thus) no electrical contact between the two electrodes (unless an external circuit is generated, see below). Optionally, more than two electrodes may be provided.

Further, the present sensor unit may be very sensitive (see also below), more sensitive than prior art sensors. Especially, the sensor unit may make use of electrochemical redox cycling within the nanogap between the first electrode and the second electrode of the sensor unit, espeically for analysis of a first fluid. The term “first fluid” is herein used to indicate the fluid that can be analyzed by the sensor unit; for instance, this can be fluid that is extracted by a micro fluidic channel structure (which may be comprised by the device, see below).

In the invention, amongst others use is made of electrochemical redox cycling. This approach, as amongst others described by Lemay et al. (Anal. Bioanal. Chem. 394: 447-456, incorporated herein by reference), relies on two electrodes to reduce and oxidize redox-active molecules repeatedly and reversibly. Each analyte molecule may thus contribute several thousand electrons to the faradic current, amplifying the detected signal. Electrochemical redox cycling is naturally suited for use in nanogap configuration since the degree of amplification increases by decreasing the separation between the two electrodes. Hence, especially the nanogap width is in the range of 20-200 nm. In such nanogap configuration each target molecule may travel between electrodes repeatedly, reversibly and changes its charge state with each electrode interaction. In conventional voltammetry techniques, a potential (most commonly a DC potential that varies linearly in time) is applied to a single working electrode to oxidize or reduce species present nearby in solution, and the resulting current from the redox process is measured as a function of potential. The information about the chemical identity of the analyte molecule (position of the features on the potential axis) as well as the concentration (magnitude of the current at a certain potential) can be obtained from the resulting voltammogram. Electrochemical redox cycling requires at least two working electrodes (the herein indicated first electrode and second electrode). The electrodes are biased at sufficiently cathodic and anodic potentials relative to the formal potential of the given reaction versus a reference electrode. The product of the reduction at one electrode can diffuse to the other electrode where it can undergo oxidation and vice versa. For instance, the sensor unit can be used, for analysis of the presence of a predetermined (bio)molecule in said first fluid. For instance, the (bio)molecule may comprise a neurotransmitter, such as a neurotransmitter selected from the groups of: amino acids, such as one or more of glutamate, aspartate, D-serine, γ-aminobutyric acid (GABA), glycine; amines, such as one or more of dopamine (DA), norepinephrine (noradrenaline; NE, NA), epinephrine (adrenaline), histamine, serotonin (SE, 5-HT); trace amines: such as one or more of phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, Ν,Ν-Dimethyltryptamine; peptides, such as one or more of somatostatin, substance P, cocaine and amphetamine regulated transcript, opioid peptides; and others, such as one or more of acetylcholine (ACh), adenosine, anandamide, nitric oxide, etc. The fluid may include more than one (bio)molecule of interest, which can be detected by the sensor device or especially measurement apparatus.

As indicated above, the sensor unit can be very sensitive. Even samples of less than 10 μΐ may be anaylised. Especially, the sensor unit can be used, for analysis of the presence of a predetermined (bio)molecule in said first fluid, wherein the fluid may even have a volume of 1 nanoliter or less, especially less than 0.1 nanoliter.

With the sensor device (comprising the sensor unit), especially when including a micro fluidic channel structure, the sensor device may also be used for the analysis of the first fluid, especailly on the presence of a (bio)molecule, wherein the analysis is performed as function of the delivery via the micro fluidic channel structure of a second fluid. Hence, the influence of e.g. a drug in brain fluid (first fluid), especially cerebrospinal fluid (CSF), may e.g. be evaluated as function of introduction of a drug in a second fluid. Instead of or in a addition to a drug, also another (bio) molecule may be introduced with a second liquid, and the response may be sensed (from the first fluid) with the sensor unit.

The use of the sensor device may (in an embodiment) be (used) for in vivo sensing or (in another embodiment) be (used) for ex vivo sensing. The use may also include sensing in an apparatus, like a chormatographic apparatus, like an LC (such as LC, HPLC, such as LC-MS, or HPLC-MS), in an electrophoresis device, etc.. The sensor device may also be used for low volume samples and for chemical forensic applications, etc.

The term “sensor unit” may in embodiments also refer to a plurality of (identical or different) sensor units. For instance, the sensor device may include a plurality of sensor units, like two or more different sensor units at a needle tip or other substrate. The term “sensor device” may in embodiments also refer to a plurality of (identical or different) sensor devices. For instance, the measurment apparatus may include a plurality of sensor devices, like two or more different sensor devices which may optionally be used to measure (in) the same first fluid.

Hence, the invention also provides a method integrating a measurement of analytes and an extraction or injection of a fluid, the method comprising (i) providing the sensor device as described herein, (ii) connecting an electronic measuring device to the sensor unit, (iii) providing a fluid transport through the micro fluidic channel structure, and (iv) measuring an analyte. The term “measuring” and similar terms herein may include a qualitative measurement and optionally also a quantiative measurement. Of course, the outcome of a measurement or analysis may also be that the analyte is below the detection limit, and may thus be considered not to be avialable in the (first) fluid. Such method may e.g. be used for online analysis of the effect of local drug delivery (e.g. via delivery with a second fluid) in the brain, the method comprises injecting a selected amount of a selected drug locally in a brain or in brain tissue and measuring e.g. the concentration of neurotransmitters.

The term “fluid” in “first fluid” or “second fluid” especially refers to a liquid, such as a body liquid like blood or brain liquid. However, the fluid may also include (waste) water, or a fluid (carrier) from a chromatograph, etc. For instance, the sensor device may also be used for measurement of an environmental pollutant, such as in water, or for food control.

The sensor device as described herein especially comprises the sensor unit. This sensor unit may in a specific embodiment be integrated with a needle. For instance, the sensor unit may be attached to the needle. The needle is a specific example of a substrate. As indicated above, the sensor device may optionally comprise a plurality of sensor units. The sensor unit may also be applied on other substrates and may also be used in other applications than herein described especially in relation to a needle, even more especially described herein in relation to a needle with a micro fluidic channel structure.

The needle especially comprises a micro needle, i.e. having a tip that can be used to penetrate the skin, or even bone or scalp. Especillay, the micro needle has (a part having) a diameter of less than 200 pm over a length of at least 300 pm. Especially, the micro needle includes a tip having these dimensions. The tip may be used to penetrate a human or animal body, such as skin, skull, bone, etc..

In a specific embodiment, the sensor unit comprises a base layer, optionally an adhesion layer, an electrode layer, and optionally a (first) isolation layer, which are especially arranged as stack (on the base layer). Note however that optionally one or more layers may be arranged to the base layer, opposite to the adhesion layer, optionally one or more layers may be arranged between any set of two layers (selected from the base layer, the adhesion layer, the electrode layer, and the isolation layer), and optionally one or more layers may be arranged to the isolation layer, opposite of the electrode layer. Hence, the set or stack of the adhesion layer, the electrode layer, and the isolation layer indicate in essence the necessary layer, but one or more further layers may be available. In general, the adhesion layer may be available to allow adhesion of the electrode layer on the base layer (or support). Further, in general also the isolation layer will be avialable. With suitable substrates, an (additional) base layer may not necessary and the stack may be provided on the substrate (whereby the substrate can be considered a type of base layer).

The base layer (and/or substrate) may especially comprise one or more materials selected from the group consisting of silicon, silicon oxide, mica, quartz, glass, and a polymer. Hence, a substrate of such material may be used. When e.g. a needle is used as substrate, such needle may comprise e.g. steel. In such instance, on the substrate the base layer may be provided. The base layer especially comprises an insulating material. Hence, the base layer can also be used as (second) isolation latyer The polymer as base layer may especially be polyimide (PI). Note that when a base layer is provided on a substrate, this base layer will (thus) in general comprise an electrically insulating material. Alternatively or additionally, on the base layer a (second) insulating layer may be provided. Hence, the substrate may be any material and the base layer, when applied, is especially selected from the herein indicated materials. Instead of the term “insulating layer”, and similar terms, also the terms “insulation layer” or “insulating layer”, or similar terms, may be used.

On the base layer, a stack may be provided of the adhesion layer, the electrode layer, and the isolation layer, though optionally the stack may include more layers (see above). Hence, the stack layer structure comprises the in an embodiment the adhesion layer, the electrode layer, and the isolation layer. In yet another embodiment, the stack layer structure comprises the electrode and the isolation layer.

In an embodiment, the adhesion layer may especially comprise one or more materials selected from the group consisting of titanium, chromium and tantalum. Also combinations, such as alloys, may be used. Alternatively or additionally, the adhesion layer may comprise a polymer. When a polymer is used as adhesion layer, this polymer layer will in general comprise an insulating material.

The electrode layer, which may especially be arranged on the adhesion layer, may especially comprise one or more materials selected from the group gold, carbon, graphene, graphite, platinum, ruthenium, iridium, silver, rhodium, palladium, indium tin oxide, boron doped diamond, copper, zinc, and an electrically conductive polymer. Also combinations, such as alloys, may be used. Would an electrically conductive polymer be used as electrode layer on a polymer adhesion layer (or directly on a polymer base layer), then these polymers are of course different, as they have different functionalities.

The insulating layer (or isolation layer), which may especially be arranged on the electrode layer, comprises an electric isolating material, especially one or more materials selected from the group of silica, silicon nitride and a polymer, especially PMMA. The insulating layer may especially be applied to allow the electrode surfaces facing each other be accesible to the fluid and the other part of the electrodes not being accesible to the fluid. In this way, redox cycling and electrochemical reaction may only takes place in the nanogap. Herein, the terms “isolation layer” (or “insulating layer”) or “first isolation layer” (or “first insulating layer”) refer to the isolation layer on the electrode layer (or in fact on the first and the second electrode), i.e. an insulating layer further away from the substrate or base layer. The optional second isolation layer may be arranged anywhere between the electrode layer and the substate, in order to provide electrical isolation of the electrode layer (or in fact on the first and the second electrode) from the substrate, which may optionally electrically be conductive (like a steel micro needle).

Optionally, one or more layers between the base layer and the stacked layer structure may be available, such as an insulating layer. Hence, one or more layers may be arranged between the base layer and the stacked layer structure. Further, optionally one or more layers between the electrode layer and the adhesive layer may be avialable (when the adhesive layer is available. Hence, one or more layers may be arranged between the electrode layer and the adhesive layer. Further, optionally (i) one or more layers between the insulating layer and the electrode layer, and/or (ii) one or more layers on the insulating layer may be available (i.e. opposite of the electrode layer). Hence, (i) one or more layers may be arranged between the insulating layer and the electrode layer, and/or (ii) one or more layers may be arranged on the insulating layer.

As indicated above, the stacked layer structure is divided into two parts with a nanogap interposed between the two parts, with one part comprising said first electrode and with the other part comprising said second electrode, wherein the surfaces of the two electrodes facing each other are mutually of equal size, wherein the width of the nanogap is selected from the range of 10-500 nm, especially 15-300 nm, such as 20-200 nm. With such nanogaps, effective electrochemical redox cycling may be obtained. The nanogap may be substantially have rectangular cross-sections though the edges may be (slightly) slanted (due to the carving out of the nanogap with e g. e-beam lithography. The length of the nano gap may in embodiments be at least 200 nm, such as at least 500 nm, like especially in the order of 1 pm or more, like at least 10 pm, such as in the range of 0.5-100 pm. The height of the nanogap may in embodiments be in the order of 50 nm - 5 pm, such as 50-1000 nm (i.e. 50 nm - 1 pm). Especially, the length of the nanogap is larger, like at least 5 times larger than the width of the nanogap. Optionally, the nanogap is a through hole (i.e. even through the substrate/base layer).

The sensor unit may be arranged adjacent to the micro fluidic channel structure. As the nanogap is very narrow, the nanogap may be a side channel of a larger micro fluidic channel structure. The term “micro fluidic channel structure” may refer to a structure comprising at least one micro fluidic channel. However, the structure may include more than one channel. For instance, the channel structure may include a channel for delivering a fluid and a channel for extracting a fluid. Especially, the channel structure includes an orifice at a needle tip, when a needle is applied. The term “orifice” may also refer to a plurality of orifices, e.g. an orifice for extraction of a fluid and an orifice for introduction of a fluid. In such embodiments, more than one orifice may be arranged at the needle tip.

In an embodiment, the needle comprises a hollow needle comprising a micro fluidic channel, or especially the micro fluidic channel structure. However, the micro fluidic channel structure may also be arranged at the surface of the needle. The sensor unit may be arranged anywhere at a substrate, such as a needle. When the sensor unit is used to measure in the first fluid, then especially the sensor unit may be arranged at the tip. However, when the sensor device is configured to extract a fluid and then measure, the sensor unit may be configured anywhere downstream from an orifice of the channel structure. Optionally, a single channel may (consecutively) be used for extraction and delivery.

The invention provides the sensor unit per se; the invention also provides a sensor unit comprised by a needle, which is an embodiment of the sensor device.

The sensor device will in general also include a reference electrode. The reference electrode will especially be configured to be at a relative short distance, such as e.g. at a distance smaller than 5 mm, like smaller than 2 mm, such as smaller than 0.1 mm, from the sensor unit. However, for for instance non-needle applications larger distances may also be possible. Hence, especially the reference electrode is configured to allow the reference electrode and sensor unit be in contact with the same fluid. Optionally, the reference electrode may extend into the nanogap. The reference electrode is not in direct physical contact with first electrode and second electrode. The reference electrode may e.g. be selected from the group consisting of an Ag/AgCl electrode, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper (II) sulfate electrode (CSE), a silver chloride electrode, a pH-electrode, a palladium-hydrogen electrode, a dynamic hydrogen electrode (DHE), and an iridium oxide reference electrode, etc.

Hence, the invention provides an integrated (bio) detection with micro fluidic micro needles for analytical application. The invention further provides a new design of such sensor unit and sensor device.

In a further aspect, the invention also provides a measurment apparatus comprising the sensor unit. In a specific embodiment, the invention provides a measurement apparatus comprising (i) an electrical power source, (ii) an analysis unit, and the sensor unit as defined herein, especially the sensor device as defined herein, wherein the electrical power source is configured to apply a potential difference between the first electrode and the second electrode (versus the reference electrode), and wherein the analysis unit is functionally coupled to the first electrode and the second electrode for measuring an electrical parameter. The electrical parameter may be selected from the group consisting of a voltage change, a current, a current change, a resistance, a resistance change, etc. Further, optionally the potential difference may be modulated with one or more frequencies. Further, optionally the potential difference may be varied in time. Hence, the electrical parameter may also be measured as function of time and/or modulation(s). The electrical power source and analysis unit may be a single device having the above indicated functionalities. The electrical power source and analysis device are - during use of the measurment apparatus - functionally coupled to the first electrode and the second electrode, and also to the reference electrode. When the measurment apparatus comprises a plurality of sensor devices or sensor units, the electrical power source and analysis device are are - during use of the measurment apparatus - functionally coupled to the first electrodes and the second electrodes, and also to the reference electrode(s).

Further, as indicated above such sensor device comprised by such measurement apparatus may (thus) also include the micro fluidic channel structure, etc. Hence, in a specific embodiment the measurment apparatus may further comprising a micro fluidic unit for controlling - during use of the measurement apparatus - delivery or extraction of said fluid. The phrase “for controlling delivery or extraction of said fluid” may also refer to controlling delivery and (controlling) extraction of said fluid.

In yet a further aspect, the invention also provides a method for producing the sensor device as defined herein, the method comprising: (i) providing a (needle like) substrate, wherein the substrate especially comprises one or more materials selected from the group consisting of silicon, silicon oxide, mica, quartz, glass, and a polymer (such as polyimide); (ii) optionally providing an adhesion layer, especially comprising one or more materials selected from the group consisting of titanium, chromium, tantalum, and a polymer, on at least part of the substrate; (iii) providing an electrode layer, on at least part of the adhesion layer, the electrode layer especially comprising one or more materials selected from the group gold, carbon, graphene, graphite, platinum, ruthenium, iridium, silver, rhodium, palladium, indium tin oxide, boron doped diamond, copper, zinc, and an electrically conductive polymer; (iv) optionally providing an insulating layer on the electrode layer comprising electric isolating material, especially comprising one or more materials selected from the group of silica, silicon nitride and a polymer (especially PMMA); and (v) generating a nanogap in the thus obtained stack (i.e. the layer stack including the electrode layer and especially one or more of (a) the adhesion layer at one side of the electrode layer and (b) the insulating layer at the other side of the electrode layer) to provide two separate electrodes (with a nanogap in between), the gap especially having a width (w) selected from the range of 10-500 nm, wherein in specific embodiments the (needle like) substrate comprises a micro fluidic channel structure or wherein after generation of the thus obtained sensor unit a micro fluidic channel structure is applied to the (needle like) substrate.

The gap may in specific embodiments be provided by one or more of e-beam lithography, focused ion beam lithography and stepper lithography, etc..

The substrate may in an embodiment comprise a base layer. In such embodiment, the base layer especially comprises one or more materials selected from the group consisting of silicon, silicon oxide, mica, quartz, glass, and a polymer. On the base layer or substrate, an adhesive layer may be arranged. Thereon, the electrode layer may be arranged, and thereon the insulating layer. In this way the stack is provided. The generation of the layers may be done with techniques known in the art, like conventional layer depositon techniques. Furhter, lithography may be used. The method may further include arranging a reference electrode on the substrate (optionally on a base layer) and/or on the final insulating layer. Further, the method may include arranging electrical connections, like conductive pads, to the sensor device to allow a functional coupling between the electrodes and an electrical power source and/or an anysis unit. In this way, an electrode system may be created, including the first electrode, the second electrode, a reference electrode and contacts. In comparison to vertical nanogap sensors, the horizontal nanogap sensor production requires less lithographic steps. For instance, the vertical nanogap sensor production requires at least 4 photolithographic steps, such as 4-6 steps, whereas the horizontal nanogap sensor may in embodiments require only 2-3 photolitographic steps.

The term “substantially” herein, such as in “substantially consists”, will be understood by the person skilled in the art. The term “substantially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially may also be removed. Where applicable, the term “substantially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. The term “comprise” includes also embodiments wherein the term “comprises” means “consists of’. The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices herein are amongst others described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation or devices in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention further applies to a device comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterising features described in the description and/or shown in the attached drawings.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Furthermore, some of the features can form the basis for one or more divisional applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

Figs.la-lc schematically depict some embodiments and aspects;

Figs. 2a-2b schematically depict some embodiments and aspects in more detail; and

Fig. 3 shows the SEM picture being a basis for Fig. la.

The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Fig. la schematically depicts an embodiment of a sensor unit 100, wherein the sensor unit 100 includes a stacked layer structure 110 (herein also indicated as stack) including an electrode layer 115. The stacked layer structure 110 further includes a nanogap 116 dividing the electrode layer 115 in a first electrode 115a and a second electrode 115b with the nanogap 116 in between, wherein the nanogap 116 has a width w, such as selected from the range of 10-500 nm. The height of the nanogap 16, indicated with reference h, is indicated above. Reference 111 indicated a base layer, which may be a substrate 1110 or a layer on such substrate 1110. As can be seen from Fig. la, the length of the nanogap 116 is in this schematically depicted embodiment larger than the height (h) or width (w). The stack 110 schematically depicted in Fig. la is also depicted in Fig. lb, with an adhesion layer, an intermediate electrode layer, and the (final) insulating layer.

Reference 1110 thus indicates a substrate, such as a (steel) needle (see also below); reference 113 indicates an insulating layer (herein also indicated as second insulating layer), such as S1O2, etc. This insulating layer 113 may be necessary when the base layer 111 / substrate 1110 are electrically conductive. Reference 112 indicates an adhesion layer, such as Ti. Such layer may be necessary when the electrode layer 115 does not adhere well enough to the base layer 111 or to the insulating layer 113. Reference 115 indicates the electrode layer (see above), which may e.g. comprise gold and/or platinum. The redox cycling between the two electrodes 115a and 115b is schematically indicated with the arrows within the nanogap 116. Reference 117 indicates an isolation layer (herein also indicated as first insulating layer). Electrode contacts are -for the sake of clarity - not depicted. However, this is state of the art technology. The lower part of the Fig. la is based on a photograph (SEM picture), which is also shown in Fig. 3. The insulating layer 117 is used to shield the electrodes from he environment except for the electrode surfaces arranged opposite to each other which (amongst others) define the nanogap 116. Reference 170 indicates the surface of the substrate 1110 or base layer 111. Fig. la is a schematic drawing. In general, the isolation layer 117 will cover the entire electrode layer, and also the edges thereof (not shown in these schematic drawings). In this way, only the electrode surface in the nanogap 116 is available for electrode reactions.

Fig. lb schematically depicts a specific application, wherein a light transmissive substrate 1110, such as glass or MICA is applied. Hence, here the base layer 111 may be the substrate 1110. Or the substrate 1110 is considered to provide the base layer 111. Reference 50 indicates a fluorescence microscope, which is in this picture schematically indicated to measure in fluorescence mode. However, transmissive and reflective modes may also be applied. Reference 120 indicates a reference electrode out of plane for clarity. Reference 130c indicates an insulated electrical wire in electrical contact with the reference electrode 120.

Each layer may independent from the other layers include multi-layers. Further, for instance, on top of the (first) insulating layer 117, more layers may be available. Hence, one or more layers may be arranged to the isolation layer, opposite of the electrode layer. Alternatively or additionally, also further layers may be available between this isolation layer 117 and the electrode layer 115, etc.

As can be seen in Figs, la and lb, the layers at both side of the nanogap are substantially identical in order and layer heights. This is a specific aspect of the horizontal nanogap sensor (or horizontal nanogap electrode). The stacks 110 in Figs la and lb are by way of example substantially identical. In the embodiment of Fig. la, an insulating layer 113 is arranged between the substrate 1110 and the stack, whereas I nthe embodiment of Fig. lb this layer is absent, e.g. because the substrate 1110 may be an isolator.

Figs, la/lb and the other drawings depict the nanogap as gap with a botthe, being e.g. the substrate 1110 or baselayer 110. However, optionally the nanogap is a through hole (i.e. even through the substrate/base layer).

Fig. lc schematically depicts an embodiment of the measurement apparatus 2. Reference 30 indicates an electrical power source and reference 35 indicates an analysis unit. Note that these may also be included in a single apparatus. References 140 indicate contact pads, for functional connection with the electrodes 115a and 115b and the reference electrode 120. There may be insulated electrical connections 130 with the respective electrodes, with reference 130a indicating a first insulated electrical connection or wire with the first electrode 115a, with reference 130b indicating a second insulated electrical connection or wire with the second electrode 115b, and with reference 130c indicating a third insulated electrical connection or wire with the reference electrode 120. Reference 200 refers to a micro fluidic structure, and reference 201 to a channel comprised by such structure. Reference 202 indicates an orifice. The channel structure may e.g. be used to introduce a fluid. Reference 40 indicates a micro fluidic unit, configured to control one or more of introduction and or extraction of a fluid (second fluid and first fluid, respectively). Also the micro fluid unit 40 may optionally be integrated in the analysis unit 35. In figure lc, by way of example a needle 10 as substrate 1110 is depicted. The needle includes a tip 11. By way of example the sensor unit 100 is shown in an enlargement, in a cross-sectional view thereof. Further, by way of example only the first electrode 115a and second electrode 115b are indicated at the tip 11.

Figs. 2a and 2b schematically depict embodiments of the (micro) needle 10 with needle tip 11. The channel(s) 201 may be enclosed by the needle or may be provided to the needle surface, as is the case in fig. 2b. Reference 202 indicates an orifice for fluid extraction or introduction.

Hence, in summary, miniaturized analytical devices are required for on-field environmental monitoring or clinical point-of-care. The current on-chip electro analytical systems rely on microelectrodes. Although their small sizes facilitate their incorporation in analytical systems and miniaturization, the small size results in difficulty to measure low analyte concentrations due to the low signal to noise level. A way to circumvent such problems is to employ electrochemical redox cycling in nano confinement space. A resultant high sensitivity is due to the repetitive oxidation and reduction of a redox molecule achieved by closely spacing two independently biased electrodes (one biased at oxidation and the other at reduction potentials). Due to the Brownian motion, one molecule shuttles between the electrodes and contributes to the current several thousand times before leaving the nanogap. As the current is proportional to the number of molecules (concentration) contributing to the signal, it can be, therefore, used for quantitative detection of molecules. The potential benefits of paired microelectrodes in gap devices include higher sensitivity and selectivity. The feedback current is free of capacitive background current. This method is the base for, e.g., the electrochemical scanning electron microscope, and recently vertical nanogap sensors. The vertical nanogap devices need sophisticated fabrication process, the electrode materials are limited to mostly platinum and gold widely available in the cleanrooms, introducing surface corrosion and low stability under harsh anodic and cathodic conditions. Next to that, their vertical structures results in the buckling of the top electrode and their structure and complicated fabrication hinders their integration with other techniques such as optical spectroscopy.

The invention provides a horizontal electrochemical nanogap device that can be integrated on different platforms for a variety of applications. Because of the fabrication method, these new devices allow the use of other electrode materials such as carbon or graphene in addition to gold and platinum. Horizontal electrodes do not have the problem of buckling of one electrode. Integrating the horizontal nanogap sensor on a micro needle, enables for instance real-time electrochemical monitoring of neurotransmitters metabolism in the brain, the measurement of analytes in cells or organelles, or the measurement of different biomolecules in different body fluids. Integration in other platforms may be used for applications such as environmental monitoring, food safety control, etc.

Neurotransmitters are the primary chemical messengers secreted from neurons that relay, amplify, and modulate signals to target cells, and accordingly regulate brain function. A number of psychiatric and neurodegenerative diseases and several types of drug and alcohol abuse are directly connected to abnormalities in neurotransmitters metabolism. The action of neurotransmitters depends on their overall levels as well as their short burst of activity. Therefore, the real time monitoring of neurotransmitters in extracellular space of the brain and in vivo monitoring of their concentration dynamics within neural tissue are crucial especially in various neurological disorders, such as Parkinson's disease, Alzheimer's disease, depression, addiction and chronic pain.

One of the advantages of the nanogap electrochemical redox cycling is the measurement of chemically reversible redox molecules in presence of chemically irreversible interferences. Thus, clinically important neurotransmitters such as dopamine, serotonin, and glutathione can be detected in presence of ascorbic acid - being a major interference in monitoring neurotransmitters - despite the fact that the concentration of ascorbic acid is much higher.

The electrode layer may comprise metals like gold, platinum, etc., see also above. It also may comprise carbon and graphene. Carbon materials are more inert compared to platinum and gold and thus provide wider potential window and can be used for a wider range of applications. Moreover, carbon electrodes can be easily modified with a wide variety of molecules, e.g., diazonium molecules, mainly due to strong carbon bonds. The surface modification can be used to tune the selectivity for the redox molecules with similar oxidation or reduction potential.

In a specific embodiment a membrane can be added to the entrance of the nanogap device. Although the small gap size already behaves as a nano filter, in order to avoid possible blocking of the electrode during implanting the device in brain tissues, a membrane such as cellulose acetate or other membranes is added to the entrance of the nanogap devices. Preferably the charge of the membrane can be manipulated, to selectively control the molecules entering the nanogap device.

The structure of the proposed micro needles sensors allows further integration of a microfluidic channel. The microfluidic channel allows controlled injection of, e.g., interested drug dosage. This way neurotransmitter’s fluctuation upon controlled injection can be studied. One such example is in in situ monitoring of drugs such as clozapine as antipsychotic drug for treatment of schizophrenia. The integrated micro needle/nanogaps with a microfluidic channel enables real-time monitoring of clozapine at the point of care so that its dosage may be regulated according to the test results. Therefore, in yet another embodiment the nanogap-integrated micro needle sensor, further comprises a microfluidic channel.

In yet another embodiment the nanogaps senor system is integrated with other analytical tools, such as optical tools, preferably a fluorescent microscope. Using this kind of embodiments enables the profiling of the chemical reactions, e.g., pH change for protonated redox active molecules such as catechol/quinone, indigo carmine, and hydroquinone/benzo-quinone during redox cycling using pH sensitive fluorescent dyes.

The invention further applies to a method to fabricate one or more of the devices described in the description.

For fabricating a sensor comprising electrodes of carbon material, the carbon electrodes are fabricated of a photoresist followed by vacuum carbonization. The carbonization temperature and pressure are optimized to obtain high quality conductive carbon electrodes (glassy carbon electrodes). The nanogap in the graphene material is also provided with different methods such as electro burning (creating a gap by removing atom by atom carbon), e-beam, focused ion beam lithography or stepper lithography.

The new devices can be characterized by electrochemical methods using redox model molecules such as Ru(NH3)62+/3+ and Fe(CN)63’/4’ to evaluate the nanogap performance and its robustness. Experiments in the lab have confirmed the robustness and high signal amplification similar to those nanogap sensors in a nanofluidic channel.

If desired, the pH in the gap can also be modulated for few milliseconds using pulse potentials by solvent electrolysis.

The small gap between the two electrodes allows studying chemically highly irreversible redox processes with short lived reaction intermediates. For example nitric oxide can be oxidized to [nitric oxide]+ with a nanosecond life time. However, in presence of phosphate buffer pH 7, it can produce nitrosonium phosphate intermediate with a life time of 1 ms that can go under redox cycling. As nitric oxide is a product of decomposition of S-nitroso-L-Gluthathione, this can be potentially used for the analysis of S-nitroso-L-Gluthathione in blood.

These new types of electrochemical nanogap sensors are particularly important for applications in multi analytes detection in complex media such as blood analysis, food control, environmental or clinical monitoring. The proposed sensor is attractive as it results in a rapid on-site or point of care measurement without the need for pretreatment and offline laboratory equipment.

References 1 sensor device 2 measurement apparatus 10 needle 11 needle tip 30 electrical power source 35 analysis unit 40 micro fluidic unit 50 fluorescent microscope 100 sensor unit 110 stacked layer structure 111 a base layer 112 adhesion layer 113 insulating layer (second insulating layer) 115 electrode layer 115a first electrode 115b second electrode 116 nanogap 117 insulating layer (first insulating layer) 120 reference electrode 130 insulated leading wire 130a insulated leading wire first electrode 130b insulated leading wire second electrode 130c insulated leading wire reference electrode 140 contact pad 170 surface of the substrate/base layer 200 micro fluidic channel structure 201 micro fluidic channel 202 orifice 1110 substrate

Claims

A sensor device (1) comprising a needle (10) with a needle point (11), the needle point (11) comprising a sensor unit (100), the sensor unit (100) comprising a stacked layer structure (110) which comprises an electrode layer ( 115), the stacked layer structure (110) further comprising a nanogap (116) which divides the electrode layer (115) into a first electrode (115a) and a second electrode (115b) with the nanogap (116) in between, the nanogap (116) has a width (w) selected from the range of 10-500 nm, and wherein the needle (10) further comprises a micro-flow channel structure (200) with an opening (202) at the needle tip (11) for administration or extraction of a fluid.

The sensor device (1) according to claim 1, wherein the sensor unit (100) comprises: i) a base layer (111) which is one or more of the materials selected from the group consisting of silicon, silicon oxide, mica, quartz, glass, and a polymer; ii) a stacked layer structure (110) on the base layer (111), with optionally one or more layers between the base layer (111) and the stacked layer structure (110), the stacked layer structure (110) comprising: (1) an adhesive layer (112) ) comprising one or more materials selected from the group consisting of titanium, chromium, tantalum, and a polymer; (2) an electrode layer (115) on the adhesive layer (112) comprising one or more materials selected from the group consisting of gold, carbon, graphene, graphite, platinum, ruthenium, iridium, silver, rhodium, palladium, indium tin oxide, boron doped diamond, copper, zinc, and an electrically conductive polymer, with optionally one or more layers between the electrode layer (115) and the adhesive layer (112); (3) an insulating layer (117) on the electrode layer (115) comprising electrically insulating material, preferably one or more materials selected from the group of silica, silicon nitride, and a polymer, with optionally (i) one or more layers between the insulating layer (117) and the electrode layer (115), and / or (ii) one or more layers on the insulation layer (117); wherein the stacked layer structure (110) is divided into two parts with a nanogap (116) arranged between the two parts, one part comprising the first electrode (115a) and the other part comprising said second electrode (115b), the mutually opposite adjacent surfaces of the two electrodes (115a, 115b) are of equal size, the width (w) of the nanogap (116) being selected from the range of 10-500 nm.

The sensor device (1) according to any of the preceding claims, wherein the needle (10) comprises a hollow needle (10), which comprises the micro-flow channel structure (200).

A measuring device (2) comprising (i) an electrical power supply (30), (ii) an analysis unit (35) and the sensor device (1) according to any one of the preceding claims, wherein the electrical power supply (30) is configured for applying a potential difference between the first electrode (115a) and the second electrode (115b), and wherein the analysis unit (35) is operatively coupled to the first electrode (115a) and the second electrode (115b) for measuring an electrical parameter.

The measuring device (2) according to claim 4, further comprising a micro flow unit (40) for controlling administration or extraction of said fluid.

Use of redox cycling in the nanogap (116) between the first electrode (115a) and the second electrode (115b) of the sensor unit (100) according to any of claims 1-5 for analysis of a first fluid.

The use according to claim 6 for analyzing the presence of a predetermined (bio) molecule in said first fluid.

The use of claim 7, wherein the bio (molecule) comprises a neurotransmitter or drug.

The use according to any of claims 6-8, wherein the volume of the fluid is 1 microliter or less, in particular less than 1 nanoliter.

The use of any of claims 6-9, wherein the sensor unit (100) comprises a horizontal nanogap (116).

The use according to any of claims 6-10, wherein the analysis is performed as a function of the administration of a second fluid via the micro-flow channel structure (200).

A method for manufacturing the sensor device (1) according to any of claims 1-3, wherein the method comprises: i) providing a needle-like substrate (1110), wherein the substrate (1110) one or more materials selected from the group consisting of silicon, silicon oxide, mica, quartz, glass, and a polymer; ii) providing an adhesive layer (112) comprising one or more materials selected from the group consisting of titanium, chromium, tantalum, and a polymer on at least a portion of the substrate (1110); iii) providing an electrode layer (115) on at least a portion of the adhesive layer (112), the electrode layer (115) comprising one or more materials selected from the group consisting of gold, carbon, graphene, graphite, platinum, ruthenium, iridium, silver , rhodium, palladium, indium tin oxide, boron doped diamond, copper, zinc, and an electrically conductive polymer; iv) providing an insulating layer (117) on the electrode layer (115) comprising an electrically insulating material, preferably one or more materials selected from the group of silica, silicon nitride, and a polymer; v) and generating a nanogap in the stack thus obtained to provide two separate electrodes, wherein a width (w) of the nanogap (116) is selected from the range of 10-500 nm, wherein the needle-like substrate (1110) ) comprises a micro-flow channel structure (200) or wherein the micro-flow channel structure (200) is applied to the needle-like substrate (1110) after generating the sensor unit (100) thus obtained.

The method of claim 12, wherein the nanogap (116) is provided by one or more of electron beam lithography, focused ion beam lithography, and stepper lithography.