Device including biosensor and holder

Abstract

A device utilizing biosensors to enable rapid electrochemical sensing of one or more analytes in a container. The device comprises a holder which incorporates at least one reference electrode and at least one sensing electrode. The sensing electrode comprising an electrically conductive substrate which is coated in a first layer of a suitable electron acceptor and subsequently with a second layer incorporating a biorecognition molecule adsorbed or within a suitable electropolymer matrix or carrier.

Claims

1. A device for a container or support, where the container or support is capable of or adapted for receiving a biological sample for use in rapid electrochemical sensing of one or more analytes using biosensors, wherein the device forms at least part of a closure for the container or support and the device comprises a holder for holding at least one sensing electrode and at least one reference electrode, the or each sensing electrode and the or each reference electrode being held and protruding from a sample receiving surface of the holder, and where the or each sensing electrode comprises an electrically conductive substrate comprising platinum, platinum alloy, gold, gold alloy, or carbon, with a first layer on the substrate comprising an electron acceptor, and a second layer comprising a biorecognition molecule adsorbed or within an electropolymer matrix or carrier.

2. The device of claim 1 wherein the device forms at least part of a cap for a container having at least a partial vacuum.

3. The device of claim 1 wherein the holder comprises 2, 3, 4, 5, or 6 sensing electrodes.

4. The device of claim 1 wherein the electron acceptor is Ruthenium Purple.

5. The device of claim 1 wherein the electrically conductive substrate comprises gold of at least 18 carat purity.

6. The device of claim 1 wherein the electrically conductive substrate comprises a platinum alloy with a 90:10 weight/weight ratio of platinum to iridium.

7. The device of claim 1 wherein the electropolymer matrix comprises a sol-gel.

8. The device of claim 7 wherein the sol-gel comprises a mercaptan containing silane, a bifunctional silane, or a combination thereof.

9. The device of claim 1 wherein the or each sensing electrode includes an intermediate layer between the electron acceptor and the electropolymer matrix or carrier, where the intermediate layer comprises a polyaniline or a derivative thereof comprising one or more non-polar substituents.

10. The device of claim 1 wherein the analytes are biomarkers selected from the group consisting of hypoxanthine, adenosine, ATP, inosine, acetylcholine, choline, glucose, glutamate, lactate, and D-serine, and combinations thereof.

11. The device of claim 1 wherein the biorecognition molecule is an antibody or enzyme.

12. The device of claim 1 wherein the analytes are capable of providing indicators of biochemical abnormalities for use in diagnostic or monitoring purposes or indicators of physiological condition in healthy organisms.

13. The device of claim 1 wherein the holder comprises more than one type of sensing electrode for the analysis of more than one analyte.

14. The device of claim 1, where the device optionally forms at least part of a cap for a container having at least a partial vacuum, or where the device or the cap is optionally included in a kit for the detection of one or more analytes; the kit comprising the device or the cap, and a set of instructions for the detection of the one or more analytes, and where the device, the cap, or the kit is adapted for single or disposable use.

15. A cap for a container having at least a partial vacuum comprising the device of claim 1.

16. A kit for the detection of one or more analytes, the kit comprising the device of claim 1, where the device optionally forms at least part of a cap for a container having at least a partial vacuum; and a set of instructions for detecting the one or more analytes.

17. The kit of claim 16 wherein the one or more analytes are capable of being used for fetal monitoring, stroke monitoring, or schizophrenia diagnosis.

18. A method comprising using the device of claim 1 for detecting one or more analytes, where the device optionally forms at least part of a cap for a container having at least a partial vacuum, or where the device or the cap is optionally included in a kit for the detection of one or more analytes; the kit comprising the device or the cap, and a set of instructions for the detection of the one or more analytes.

19. The method of claim 18 wherein at least one analyte has a half-life in the biological sample of less than 10 min.

20. A process for preparing the device of claim 1, the process comprising: i) providing a substrate comprising an electrically conductive substrate surface within a holder; ii) depositing a first layer comprising a suitable electron acceptor; and iii) depositing a second layer comprising an electropolymer matrix with one or more biorecognition molecules absorbed on or included within said second layer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Shows a schematic cross-sectional representation of a device for a container.

(2) FIG. 2: Shows a schematic cross-sectional representation of a device connected to a measurement apparatus for the detection of an analyte.

(3) FIG. 3: Shows a picture of a polyurethane moulded cap.

DETAILED DESCRIPTION OF THE INVENTION

(4) Referring to FIG. 1, the device encompasses at least one reference electrode (1) and at least one sensing electrode (2). The device may comprise 2, 3, 4, 5 or 6 sensing electrodes. In one embodiment of the invention the device comprises four sensing electrodes. In a further embodiment the device comprises four sensing electrodes and two reference electrodes.

(5) The electrodes are incorporated into the holder (3) and protrude into the interior of the container (4). It will be appreciated by the skilled reader that the protrusion of the electrodes into the container is sufficient so that they are in contact with any biological sample within the container. In one embodiment the protrusion of the electrodes is in the range 10 to 150 μm. In a further embodiment the protrusion is around 100 μm. It will also be appreciated that the container could be inverted or tilted to enable this contact to be made, and therefore that the device may seal the container to enable inversion with retention of the contents. In one embodiment of the invention the device forms part of a cap for a container. In a further embodiment, the device forms part of a cap for a vacutube or vacutainer. In another embodiment, the device is a cap for a vacutube.

(6) Each sensing electrode (2) comprises an electrically conductive substrate, which may be made from platinum, platinum alloy, gold, gold alloy or carbon. In one embodiment the substrate comprises gold wire. In another embodiment of the invention this substrate comprises gold of at least 18 carat purity. In a further embodiment the substrate comprises a platinum alloy with a 90:10 ratio of platinum:iridium (weight:weight).

(7) This substrate is coated in a first layer (5) which comprises an electron acceptor and a second layer (7) which comprises a biorecognition molecule adsorbed or within a suitable electropolymer matrix or carrier. In one embodiment of the invention the electron acceptor for the first layer is ruthenium purple, KFeRu(CN).sub.6 or Fe.sub.4[Ru(CN).sub.6].sub.3. In a further embodiment, the electron acceptor is ruthenium purple and the electropolymer matrix or carrier comprises a sol-gel. In a further embodiment the sol-gel comprises one or more silicon based compounds. In another embodiment the sol-gel comprises a mercaptan containing silane and/or a bi-functional silane.

(8) The biorecognition molecule may be, for example, an antibody or an enzyme. Suitable antibodies or enzymes include those which recognise biomarkers, for example hypoxanthine, adenosine, ATP, inosine, acetylcholine, choline, glucose, glutamate, lactate and D-serine. Purines such as adenosine, inosine and hypoxanthine are suitable biomarkers for the indication of a stroke or Ischaemia. Different purines plus glutamate and/or D-serine are combinations that may be used to try to discriminate different sources of ischaemia.

(9) It will be appreciated by the skilled person that the device is suitable for the electrochemical sensing of analytes with a range of half-life values, including those with a short half-life in the biological sample, for example ATP, adenosine, inosine or hypoxanthine. Analytes with a short half-life in the biological sample include those with a half-life of less than 5 minutes, for example less than 2 minutes, less than 1 minute, less than 30 seconds and less than 15 seconds.

(10) An additional layer (6) may be used between the first layer comprising an electron acceptor (5) and the second layer comprising a biorecognition molecule adsorbed or within a suitable electropolymer matrix or carrier (7). The additional layer (6) may function to protect and preserve the electron acceptor. In one embodiment the additional layer comprises a polyaniline. In a further embodiment the additional layer is a derivative of a polyaniline comprising one or more non-polar substituents.

(11) It should be noted that the relative dimensions of the electrodes and the layers coating the electrodes are as shown in FIGS. 1 and 2 for ease of illustration only.

(12) More than one sensing electrode may be used to allow measurement of more than one analyte simultaneously, and to incorporate control sensors that lack the enzymes to detect specific analytes thus increasing the quality of the data. It will be appreciated that, in order to enable the simultaneous detection of more than one analyte, the sensing electrodes may comprise different bio-recognition molecules.

(13) The at least one reference electrode (1) is made from an electrically conductive substrate. The electrically conductive substrate may be coated to form a layer (8). In one embodiment the reference electrode comprises a silver wire. In a further embodiment the reference electrode is silver wire coated with AgCl. The at least one reference electrode (1) and at least one sensing electrode (2) may comprise different electrically conductive substrates. In one embodiment of the invention the at least one reference electrode comprises silver wire and the at least one sensing electrode comprises gold wire. In one embodiment of the invention the diameter of the wire is in the range 150-250 μm. In a further embodiment the diameter of the wire is around 200 μm. The electrodes are attached to supporting legs (9) which are embedded within the holder (3). It will be understood by the skilled person that the supporting legs are made from an electrically conductive substrate. In one embodiment of the invention the supporting legs are made from a nickel alloy.

(14) The holder (3) comprises an inert and insulating material. In one embodiment of the invention the cap comprises a polyurethane resin.

(15) Referring to FIG. 2, the at least one reference electrode (1) and the at least one sensing electrode (2) are positioned by the holder (3) to protrude into the interior of the container (4) and to make contact with a biological sample (11). The holder additionally enables electrical connection of the electrodes (1, 2), via the supporting legs (9) to a docking station (12) which in turn connects the electrodes via wires (13) to a measuring device (14).

(16) Each sensing electrode is connected to one channel on the docking station. A further channel is required for the reference electrodes. Each channel is connected to a measuring device (14). In one embodiment of the invention the measuring device is a potentiostat able to measure currents in the pA to nA range. One skilled in the art would understand that said potentiostat should be capable of at least two-electrode operation.

EXAMPLES

(17) Formation of a Cap with Integrated Sensing and Reference Electrodes

(18) (A) Moulding of a Polyurethane Cap

(19) An example of a polyurethane cap is shown in FIG. 3. This picture shows gold wire tipped legs (21), silver wire tipped legs (22), a polyurethane cap (23) and a supporting collar (24).

(20) Gold wire is attached to four nickel alloy legs (length 2.5 cm) and silver wire is attached to two nickel alloy legs (length 2.5 cm). The six legs are placed in a supporting collar (24) which aligns the legs with holes in the bottom of a mould. The mould defines the shape of the cap and has three holes on each side to correspond to the desired position of the electrodes. The legs are positioned so that there is one silver wire tipped leg (22) and two gold wire tipped legs on each side (21), with the silver wire tipped electrodes in the central position.

(21) Fast casting polyurethane resin (approximately 1.5 mL) is injected into the mould using a syringe. The cap (23) is then removed from the mould after a period of 15 minutes at room temperature and then cured for a period of one day.

(22) (B) Electrodeposition of Silver Chloride onto the Surface of the Silver Wire Electrodes.

(23) The cap is connected to a multi-socket board and then dipped into an electrochemical bath containing AgCl in HCl (1 M). The electrochemical bath is equipped with a Pt foil counter electrode and a Ag/AgCl reference electrode. A constant galvanostatic current of 10 mAcm.sup.−2 is applied to anodically plate the silver wire with AgCl.

(24) (C) Electrodeposition of Ruthenium Purple (RP) on the Surface of the Gold Wire Electrodes.

(25) The cap connected to a multi-socket board is dipped into an electrochemical bath, equipped with a Pt foil counter electrode and a Ag/AgCl reference electrode, and filled with a mixture of FeCl.sub.3 (1 mM) KCl (40 mM, pH 2), K.sub.4Ru(CN).sub.6 (1 mM) and KCl (40 mM, pH 2). The channels connected to the gold wires are subjected to scanning cyclic voltammetry from −0.2 to +0.7 V for forty cycles at 50 mV/s. The resulting ruthenium purple modified cap is heated at 80 degrees for 12 hours. The cap is further subjected to scanning cyclic voltammetry (2-4 cycles, −0.2 to +0.7 V at 50 mV/s) in a solution of RuCl.sub.3 (1 mM containing 1 mM KCl, pH2) to further stabilize the ruthenium purple film on the gold wires.

(26) (D) Formation of a Polyaniline Layer.

(27) In examples where a polyaniline layer is used, the ruthenium purple modified gold wires are dipped into an electrochemical bath containing 10 mM aniline plus 0.5 M H.sub.2SO.sub.4 and 0.5 M KCl, followed by electrochemical cycling between −0.2 and +1.3V for 7 cycles at 100 mV/s.

(28) (E) Electrodeposition of a Sol Gel Layer.

(29) The multi-socket board is transferred onto a multiwall plate, which is filled with pre-hydrolysed silane mixture either with or without desired enzymes. The central AgCl coated silver wire on each side of cap is employed as reference and counter electrode for gel formation on ruthenium purple modified gold wires. The sol gel layers are preferably entrapped by electro-deposition at −0.9 to +1.3 V or 6 μA for 10 to 30 seconds.

(30) Formation of a Cap with Different Sensing Electrodes

(31) Formation of a cap with more than one type of biosensor may be carried out by the control of the circuit to each sensing electrode during the electrodeposition of the sol gel layer. The gold wires are connected and controlled with different channels of a multi-socket board. Each channel may be switched on forming a biosensor on the surface of a specific gold wire. Different types of biosensor can therefore be prepared using a series of pre-hydrolysed silane mixtures with different enzymes and applying appropriate potential or current through each circuit in turn.