WO2011022173A1

WO2011022173A1 - Bead based electrochemical biosensor

Info

Publication number: WO2011022173A1
Application number: PCT/US2010/043352
Authority: WO
Inventors: Changchun Liu; Haim H. Bau
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 2009-08-21
Filing date: 2010-07-27
Publication date: 2011-02-24
Anticipated expiration: 2012-02-21

Abstract

Provided are bead-based electrochemical biosensor devices suitable for multiplexed, parallel detection of multiple analytes and methods for fabricating and using such devices.

Description

BEAD BASED ELECTROCHEMICAL BIOSENSOR

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/235,891, filed August 21, 2009, which is incorporated by reference in its entirety.

GOVERNMENT SUPPORT

[0002] The research carried out in this application was supported, in part, by a grant from the National Institute of Health (NIH/NIDCR) through grant UO IDEO 17855, and by a grant from the National Science Foundation through grant NSEC DMR-0425780. Pursuant to 35 U.S. C. § 202, the government has certain rights in any patent issuing from this application.

TECHNICAL FIELD

[0003] The disclosed invention relates to the field of electrochemical sensors. The present invention also relates to the field of microscale devices.

BACKGROUND

[0004] Various scientific and patent publications are referred to herein. Each is

incorporated by reference in its entirety.

[0005] Biosensors are used to detect a wide range of analytes in, among other places, the health care industry, food industry, environmental monitoring, and drug development. Many of the detection modalities require the immobilization of the target analytes to a solid substrate. However, challenges still exist in immobilizing various biorecognition molecules, such as enzymes, oligonucleotides, and proteins, to the biosensors' surfaces at high density while retaining bioactivity.

[0006] Micropipette electrodes are often used for electrochemical measurements. The micropipette electrodes are typically fabricated by sealing a platinum wire or a carbon fiber in a pulled glass capillary with epoxy glue. In this method, the glass/fiber interface contains epoxy, which may adversely affect the characteristics of the electrodes. Often, the epoxy provides a leaky seal, which results in high noise, low sensitivity, and short life of the electrodes. Moreover, the epoxy may contaminate the solution, in particular when working with organic solvents. SUMMARY OF THE INVENTION

[0007] This invention describes simple, robust, single bead-based electrochemical biosensors and methods for producing them.

[0008] The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.

[0009] In one aspect, the present invention provides electrochemical sensor devices, comprising at least one electrode capable of being electrically addressed, penetrating a bead comprising one or more catalytic mediators capable of inducing an amperometric response with an analyte. Additional embodiments provide that the electrodes are hermetically sealed with heat- fusion sealed glass, such as not to require any epoxy or glue, and that the beads are highly functionalized. In certain embodiments, the catalytic mediators comprise at least one enzyme complex attached to the bead by immobilized ligands.

[0010] In another aspect, the present invention provides analytical systems and equipment that incorporates these devices.

[0011] Further provided are methods of forming these sensors, comprising providing an electrode and a bead comprising one or more catalytic mediators capable of inducing an

amperometric response with an analyte, wherein the electrode is capable of penetrating the bead and penetrating the bead with the electrode. Further embodiments provide for sharpening of the electrode prior to penetration, and heat fusion of an insulative sleeve over the electrode.

[0012] Still other embodiments of the invention include methods of analyzing a solution containing an analyte with a sensor device comprising at least one electrically-addressable electrode penetrating a bead comprising one or more catalytic mediators capable of inducing an amperometric response with an analyte when submerged in a solution containing the analyte

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments of the invention; however, the invention is not limited to the specific methods, compositions, and devices disclosed. In addition, the drawings are not necessarily drawn to scale. In the drawings:

[0014] FIGURE 1. The fabrication process for a micropipette electrode: (A) The glass capillary micropipette is pulled by a Sutter laser-based P-2000 puller to form a fine tip. (B) The tip of the glass capillary micropipette is trimmed to obtain the opening size of about 30 μm diameter; (C) The etched bare, 25 μm diameter platinum wire is inserted into the glass capillary micropipette with 70 μm length of platinum wire protruding out of the glass. (D) The glass tip of the micropipette is flame-fused to seal the platinum wire inside and provide an insulation layer.

[0015] FIGURE 2. A photograph of the agarose bead-based micropipette biosensor shown next to a US dime. The left inset is an enlargement of the probe's tip, showing the dry, functionalized agarose bead mounted on the platinum electrode. The right inset shows a fluorescent image of the hydrated biotin agarose bead labeled with streptavidin-Alexa Fluor 488 and suspended in PBS solution.

[0016] FIGURE 3. A schematic depiction of the process for mounting an agarose bead on the sharpened micropipette electrode: (A) One drop of agarose bead suspension is placed next to the edge of a glass slide superposed on a second glass slide. (B) The suspension is drawn to the wedge formed by the two glass slides by capillary action, and the agarose beads self-align along the edge of the top glass slide. The micropipette electrode, controlled with a precision XYZ micromanipulator, spears one of the agarose beads while the glass slide provides reactive force; (C) After the mounting of the agarose bead, the micropipette electrode is withdrawn.

[0017] FIGURE 4. Cyclic voltammograms of the micropipette biosensor in PBS (pH 7.4) containing 1.0 mM hydroquinone. Scan rate is 100 mV/s.

[0018] FIGURE 5. A schematic depiction of an agarose bead-based electrochemical assay. (A) A biotin agarose bead-based micropipette (Bio-BMP) biosensor for H₂O₂ sensing. (B) Streptavidin agarose bead based micropipette (SA-BMP) biosensor for DNA sensing. [0019] FIGURE 6. The steady-state current detected with the Bio-BMP sensor as a function of H₂O₂ concentration. The measurements were carried out in a 3.0 mM hydroquinone PBS solution at a fixed voltage of -0.30 V (vs. Ag/AgCl).

[0020] FIGURE 7: Current response curves of the micropipette biosensor for H₂O₂ in PBS using three different RedOx mediators: potassium ferrocyanide (-0.1 V vs. Ag/AgCl ), KI(-0.4 V vs. Ag/ AgCl), and hydroquinone (-0.3 V vs. Ag/AgCl ).

[0021] FIGURE 8. Plot of amperometric response of the micropipette biosensor vs. concentration of hydroquinone in PBS (pH 7.4) containing 1.5 mM H₂O₂ at -0.30 V vs. Ag/AgCl

[0022] FIGURE 9. Dependence of steady-state current on applied potential. The micropipette biosensor is submerged in pH 7.4 PBS containing 3.0 mM hydroquinone andl.5 mM H₂O₂.

[0023] FIGURE 10. Amplification curves (relative fluorescent units) of real-time PCR for genomic B. Cereus DNA template as functions of the number of PCR cycles. Curves 1, 2, 3, 4, 5, and 6 correspond, respectively, to template masses of 0 (negative control), 0.001, 0.01, 0.1, 1, and 10 ng template.

[0024] FIGURE 11. An agarose gel (2.0%) electrophoresis image of PCR products amplified from real time PCR. The various lanes are cross referenced with (A) above. M is the DNA Marker VIII ladder.

[0025] FIGURE 12. The current detected with the SA-BMP biosensor as a function of the mass of B. Cereus genomic DNA template. The error bars correspond to the scatter of the data obtained in three experiments. The electrochemical detection was carried out in 1.5 mM H₂O₂ and 3.0 mM hydroquinone in PBS solution at a fixed voltage of -0.30 V (vs. Ag/ AgCl counter electrode).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0026] The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. Also, as used in the specification including the appended claims, the singular forms "a," "an," and "the" include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term "plurality", as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0027] It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further, reference to values stated in ranges include each and every value

within that range.

[0028] As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

Terms

[0029] "Adjacent" refers to two or more entities, such as objects, surfaces, or any combination thereof, that reside near, next to, or are in contact with each other and are physically, electrically, or any combination thereof, and may be affected by their proximity to each other. "Directly adjacent" means two or more objects that are in contact with one another.

[0030] "Amperometric" or "amperometry" refers to a chemical analysis by techniques which involve measuring electric currents. This includes the determination of any analyte concentration by measurement of the current generated in a suitable chemical reaction [0031] "Catalytic mediator" refers to one or more species capable of catalyzing a chemical or biological reaction. Enzymes or metallic catalysts are exemplars of catalytic mediators. These are typically attached to the beads by immobilized ligands.

[0032] "Electrical connection" or "electrical communication" refers to two or more entities, such as objects, surfaces, or any combination thereof, wherein a change in the electrical characteristics of one or more of the entities is capable of affecting or of being detected by one or more of the other entities.

[0033] "Electrode" means an object or site capable of passing an electrical current or a magnetic field. The term can also mean an object or site capable of projecting an electrical potential or current or a magnetic field.

[0034] In one aspect, the present invention provides a sensor device comprising at least one electrode capable of being individually electrically addressed, penetrating a bead comprising one or more catalytic mediators capable of inducing an amperometric response with an analyte, when in contact with the analyte. In particular, the amperometric response is communicated to the electrode, so as to be measured.

[0035] In some embodiments, the electrode materials comprises a conducting material, in others a semiconducting material, and in still others, a combination of conducting and semiconducting materials.

[0036] In some embodiments, the electrode is a solid core electrode. In other

embodiments, the electrode can be hollow or comprise a liquid. Additionally, the electrode can comprise a single material, or multiple solid materials. When multiple materials are used, each different material may be arranged spatially so as to form concentric cylindrical layers or shells, or eccentric layers or shells, optionally interspaced by intermediary insulative dielectrics.

[0037] The conductive materials may comprise such materials as carbon, gold, silver, platinum, palladium, aluminum, copper, nickel, chromium, indium, tin, or any combination thereof. Preferred embodiments comprise carbon, gold, silver, platinum, or palladium, and most preferably platinum. One skilled in the art would appreciate the various permutations available with this array of materials. [0038] This invention further teaches that the shaft of the electrode has a diameter in the range of from about 1 nm to about 100 microns, and that this dimension needs to be consistent with the dimensions of the beads into which it is to be penetrated. Still other embodiments for this diameter dimension are those in the range of from about 50 nm to about 1000 microns, in the range of from about 1 micron to about 100 microns, in the range of from about 10 microns to about 200 microns, in the range of from about 10 microns to about 100 microns, and in the range of from about 10 microns to about 50 microns.

[0039] Generally speaking, once the electrode is inserted so as to penetrate the bead, the resulting compression forces provides sufficient strength to the connection so as not to require further adhesive or locking forces. However, one skilled in the art will also recognize that the use of adhesives or other locking mechanisms may be used to provide additional structural integrity. As but one example, for sufficiently fine tips on the electrode, relative to the bead, it may be useful to bend, or provide a hook or barb to the end of the electrode, so as to provide such a locking mechanism, and this concept is embodied in this invention.

[0040] Another characteristic of the electrode, at least as it relates to the bead, is that it is sufficiently rigid, especially at the thicknesses contemplated, that it is capable of actually penetrating the bead. This can be accomplished either by selecting sufficiently stiff or rigid electrode materials, softening the bead material, for example by hydrating, or a combination of the two techniques.

[0041] In some embodiments, the electrode and the bead are in electrical connection with one another. It is contemplated that the electrode is capable of detecting a signal related to the binding of one or more functionalities of the particle to one or more analytes to which the particle is contacted.

[0042] In yet another embodiment, the shaft of the electrode is coated at least in part, with an insulator, generally (but not restricted to) providing a hermetic seal between the electrode and the insulator. One purpose of this seal is to protect the shaft of the electrode (and connections thereto) from the analyte solution. While this insulative coating may be organic or inorganic, for example, organic polymers, it is preferred that this insulative layer, and any optional material forming the seal between the insulative material and the electrode be comprised of a material or materials which do not leach into the analyte solution. In one embodiment, this insulative material is glass, and its seal with the electrode is provided inserting the electrode through a glass sleeve, and by heat fusing the glass sleeve around the electrode so as to make the desired seal. In some embodiments, the shaft also contains a stopper to control the exposed length of the electrodes.

[0043] Other embodiments of this invention describe the bead as having a cross-sectional dimension in the range of from about 10 nm to about 1000 microns. Other embodiments provide that this cross-sectional dimension be in the range of from about 10 nm to about 500 microns, in the range of from about 1 micron to about 500 microns, in the range of from about 10 microns to about 500 microns, in the range of from about 20 microns to about 200 microns, in the range of from about 10 microns to about 100 microns or in the range of from about 20 microns to about 50 microns. The optimal particle size for a given application will depend on the needs of the user and will be known to those of ordinary skill in the art. Also, as described herein, beads used in chromatography applications are suitable for this instant invention, and sizes associated with these chromatography applications are therefore also suitable here. Again, as described above for the electrode, one skilled in the art will appreciate that this dimension needs to be consistent with the diameter of the penetrating electrode.

[0044] Generally, the bead comprises a porous material, so as to improve the concentration of functional groups to be attached, but the invention is not so constrained. Non-porous beads, suitably functionalized also work in this invention. Suitable matrices of these beads may include inorganic matrices, organic matrices, or magnetic or paramagnetic materials. Suitable inorganic matrices comprise silica or alumina, including silica or alumina hydrogels. Suitable organic matrices generally include organic polymers. Examples of such organic polymer materials include those comprising an acrylic acid derivative, a cellulose, a dextran, a polystyrene, a polyacrylamide, a polystyrene, or agarose, or mixtures or combinations thereof. Agarose bead is especially attractive when used in this invention because of its excellent biocompatibility, nontoxicity, resistance to nonspecific protein binding, and low cost. Beaded agarose bearing functional groups such as amine (- NH₂), carboxylic acid (-COOH), aldehyde (-CHO), thiol (-SH), and hydroxyl (-OH) are the support of choice for many ligands of interest. Furthermore, agarose beads functionalized, among other things, with streptavidin, biotin, and protein A are available from many vendors.

[0045] It should be appreciated that beads used for chromatography are useful as beads in the capacity of this invention. Moreover, the technology for the functionalization of

chromatography beads is highly developed, and microbeads with high density, highly active surface coverage are readily available from various vendors. Hence, in many cases, the use of commercially available beads are attractive for use in this invention.

[0046] In other embodiments of this invention, the bead is functionalized to immobilize or render non-leachable the catalytic mediators. Such functionalization can be accomplished using pendant nitrogen, oxygen, or sulfur containing groups. Non-limiting examples of pendant groups include amine, carboxylic acid, aldehyde, thiol, and hydroxyl functional groups, as well as metal ligands or chelating ligands. These pendants may be attached directly to the bead composition, or linked through oligomeric or polymeric linkages. The nature of these linkage may be organic or biopolymers, for example, peptides, oligonucleotides, or proteins.

[0047] Functionalized particles are available from, for example, Bangs Laboratories, Inc. (www.bangslabs.com, Fisher, Indiana, USA) or Bio-Rad Laboratories, Rockville, Centre, N.Y. Suitable particles may be purchased commercially and modified by the user or, alternatively, synthesized by the user. Methods of selecting, synthesizing, and modifying particles will be known to those of ordinary skill in the art.

[0048] Particle functionalities can be selected based on the analyte or analytes the device will be used to detect, and can be selected based on being complementary to the analytes. Particles can also be chosen based on size, shape, color, the presence of one or more fluorescent dyes of the particle, electrical resistance, or electrochemical properties. As discussed in further detail elsewhere herein, particle functionalities may be used to bind to target analytes and may also be used to identify a particle or its spatial location.

[0049] This invention also teaches that the catalytic mediator is a catalytic redox mediator, which provides for the catalytic oxidation or reduction of the analyte. Such mediators, again including enzyme complexes, may comprise at least one of an antigen, antibody, monomer, oligonucleotide, nucleic acid, ligand, enzyme, enzyme substrate, a metal, or a paramagnetic material. Exemplary examples of such mediators include streptavidins, a synaptobrevins, biotins (e.g., biotin, desthiobiotin, or iminobiotin), or proteins (e.g., Protein A).

[0050] In other embodiments, the materials of construction are biocompatible.

[0051] As will be appreciated, the invention also describes a more complicated piece of analytical equipment or device comprising at least one such sensor. When two or more sensors are used, so as to comprise an array of these devices, this invention teaches that each of the sensor devices may comprise beads of differing compositions. That is, in a device comprising two or more functionalized electrodes, this invention teaches that at least two beads may be functionalized with different catalytic mediators. Similarly, each functionalized electrode may be connected to individual electrical connections so as to allow individual interrogation.

[0052] A plurality of individually addressable and spatially distributed capture electrodes can comprise a substrate, wherein the substrate comprises one or more electrically conductive and individually electrically addressable sites acting as electrodes. Multiple electrode-particle assemblies can be used as multiplexed detector devices. Such multiplexed devices are suitable for simultaneously detecting multiple analytes, where the analytes are complementary to the

functionalities present on the particles.

[0053] As will be apparent to those of ordinary skill in the art, there are innumerable permutations of functionalities that may be used on beads. By careful design, the user may assemble a broad library of particles bearing identifying functionalities, thus enabling the assembly of sensors that may be customized to detect any number of targets or analytes. It is envisioned that the plurality of electrodes is suitable for use as a multiplexed device capable of simultaneously detecting multiple analytes, wherein the analytes are complementary to the sensing functionalities present on particles in known spatial locations.

[0054] This invention also includes embodiments wherein these sensor devices are attached to and in electrical communication or connection with electrical devices. Such devices may comprise a meter capable of detecting electrical signals, a current source, a sensor, a controller, a computer, or any combination thereof. As such, the invention also describes larger analytical systems comprising at least one of these sensor devices. Therefore, also provided are devices made according to the method, wherein the devices are used as probes, sensors, detectors, or any combination thereof. Such devices can be electronic, optoelectronic, or electromechanical devices, or any combination thereof.

[0055] In suitable embodiments, at least one electrode is capable of applying a current, an electric potential, a magnetic field, or any combination thereof. In some configurations, at least one electrode is capable of applying an alternating current, a direct current, a variable current, or any combination thereof. The electrode detection cell may be a one, two or three electrode system. When a two or three electrode system, the counter electrode may be concentric or eccentric with the working electrode, separated from the working electrode with at least one insulating layer.

[0056] This invention also teaches methods for forming or making these sensor devices. One such method includes providing an electrode and a bead comprising one or more catalytic mediators capable of inducing an amperometric response with an analyte, wherein the electrode is capable of penetrating the bead, and penetrating the bead with the electrode.

[0057] The assembly of the beads the electrodes have been discussed above, but another way for facilitating this penetration is to sharpen the electrode at the end intended to penetrate the bead. This can be done physically, for example through abrasion or chemical techniques. One preferred embodiment described herein is to hone this point electrochemically. Additionally, beads may be hydrated or otherwise softened to facilitate the penetration of the electrode.

[0058] Also, as described above, where the relative dimensions of the bead and the electrode permit, the penetrating tip of the electrode may be further shaped by bending, hooking, or barbing the end of the electrode intended to penetrate the bead.

[0059] An additional embodiment comprises attaching an insulative layer over at least part of the shaft of the electrode. Again, this insulative material may be organic or inorganic. A preferred embodiment is to use glass, and to insert the electrode through a preformed glass sleeve, and fuse the glass to the electrode using heat.

[0060] This invention also teaches a method of analyzing a solution containing an analyte with a sensor device comprising at least one electrically-addressable electrode penetrating a bead comprising one or more catalytic mediators capable of inducing an amperometric response with an analyte when the sensor device is submerged in the solution containing the analyte. The system which allows such methods of analysis is also within the purview of this invention.

[0061] The methods of the invention are particularly described in the specific examples that follow. The examples set forth herein are illustrative only, and are not intended to limit the scope of the invention. Those skilled in the art will appreciate that modifications to these examples can be performed without detracting from the spirit of the invention. EXAMPLES

Example I: Chemical Sensor

Reagents and materials

[0062] Borosilicate glass pipe (OD=I. Omm, ID=O.75, Length=3 inches) was purchased from World Precision Instruments, Inc. (Sarasota, FL, USA). ImmunoPure^® Immobilized D-Biotin agarose beads and high capacity streptavidin agarose beads were obtained from Pierce

Biotechnology (Rockford, IL, USA). Prior to the beads' use, the beads' suspension was vortexed for 1-2 minutes to separate aggregates into individual beads. Bare platinum wire (25 μm in diameter) was obtained from A-M Systems Inc. (Everett, WA, USA). Hydrogen peroxide (30% w/v), potassium ferrocyanide, potassium iodide (KI), hydroquinone, sodium nitrite, and phosphate buffer solution (PBS, pH 7.4) were purchased from Sigma (St. Louis, MO, USA). Streptavidin-horseradish peroxidase conjugate and streptavidin-Alexa Fluor 488 were purchased from Invitrogen (Carlsbad, CA, USA). Anti-dig-HRP conjugate was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). All reagents were of analytical grade and used without further purification. An Ag/ AgCl reference electrode (Orion, USA) was used throughout the experiments.

Electrochemical etching of the platinum wire

[0063] The bead-based micropipette biosensor's fabrication required a platinum electrode with a sharp tip to allow the mounting of the agarose bead. The platinum electrode tip's diameter reduction was achieved with electrochemical etching. The shape of the tip depended on the etching time and the applied potential difference between the working platinum and the counter electrode. When the voltage exceeded 5.0 V, the etching process was too fast and the resulting tip exhibited an irregular shape. When the voltage ranged from l.OV to 1.5 V, a smooth, sharp tip was obtained. When the voltage was less than 0.5 V, the etching process was exceedingly slow. The selected etching voltage of 1.3 V provided a reasonable compromise between etching time and tip

smoothness. FIGURE 1C shows a typical platinum electrode with a sharpened tip.

[0064] Prior to etching, the bare platinum wire was rinsed with ethanol and water. Then, the platinum wire's tip was gently inserted into the saturated NaNO₂ etching solution with a micromanipulator. An AC voltage of 1.3V, which provided a reasonable compromise between etching time and tip smoothness, was applied between the platinum wire and a platinum foil counter electrode submerged in the solution for 15 minutes. Bubbles formed at the Pt tip/solution interface during the electrochemical etching. After 15 minutes of etching, the applied voltage was removed, and the etched platinum wire was dipped for 2 s in a 3:1 HCl / HNO3 solution to clean the surface. Then the wire was thoroughly rinsed with DI water.

Sealing of the micropipette electrode

[0065] Flame-fused sealing offered a highly efficient method for sealing micropipette electrodes, averted problems associated with the commonly used epoxy and glue-based sealing, and provided a hermetic seal. Borosilicate glass was chosen as the pipette material because of its relatively low melting temperature. The flame produced by a common cigarette lighter provided a sufficiently high temperature to fuse the pulled borosilicate glass capillary around the platinum wire. It is important to complete the fusion process within a short period of time (~ls). Prolonged exposure of the glass tip to the flame (>ls) may deform the glass tip and may cause glass beading. FIGURE ID, shows an image of the micropipette electrode after fusion.

[0066] The end of a borosilicate glass capillary (OD=I.0mm, ID=O.75mm,

Length=76mm) was pulled with a micropipette puller (Sutter Instrument, Novato, CA, USA) into a fine, blunt taper. To conveniently insert the etched bare platinum wire (25 μm in diameter) into the glass capillary micropipette, the tip of the pulled glass capillary was cut with a glass pipette cutter (Sutter Instrument, Novato, CA, USA) to obtain a tip diameter of 30μm. The etched platinum wire was cleaned by sonication for 5 min, rinsed with acetone, alcohol, and distilled water, and dried by exposure to room air. Then, the platinum wire was inserted into the glass capillary through the pulled, cut end until 70μm of platinum was left exposed outside the glass. Finally, the tip of the glass capillary was inserted into the flame of a cigarette lighter for one second. The glass softened and fused around the platinum wire to form a tight, hermetic seal. The sealing process was reliable and reproducible. To provide a good electrical contact for electrochemical detection, aluminum foil was wrapped around the distal end of the glass capillary with the end of the platinum wires inserted between the layers of aluminum foil (FIGURE 1).

[0067] FIGURE 2 shows a photograph of an agarose bead-based micropipette biosensor. The left inset shows the mounted, speared agarose bead when the bead is dehydrated. The right inset shows a fluorescent image of a mounted, hydrated biotin agarose bead after incubation with streptavidin- Alexa Fluor 488 for 10 minutes and suspension in PBS solution. The functionalized agarose beads could be hydrated and dehydrated with little or no loss of activity.

Fixing single agarose bead to the tip of a micropipette electrode

[0068] Since the agarose bead is soft and porous, it is relatively easy to puncture it with the sharp platinum tip of the micropipette electrode. Hydrated agarose beads were speared since they are considerably larger than the dehydrated ones. It is necessary, however, to immobilize the beads, to facilitate spearing. To this end, it is helpful to advantage of capillary forces to align the agarose beads along the wedge formed by two glass slides (FIGURE 3). A water drop, laden with beads, was placed next to the top glass plate. The suspension flowed into the wedge between the two plates by capillary action, and the agarose beads aligned along the top glass plate's edge. Then, a glass capillary micropipette electrode with a sharp platinum tip was navigated with a precision XYZ micromanipulator (Eppendorf TransferMan NK 2, Hamburg, Germany) to puncture one of the agarose beads. Once speared, the agarose bead was withdrawn, and the bead-based biosensor was ready for use

Sensor Characterization

[0069] The electrochemical measurements were carried out with a HEKA EPC-10 patch clamp amplifier (HEKA Electronic Lambrecht, Germany) coupled to a desktop computer for data acquisition. Since the electric current associated with the electrochemical experiments is small, a two electrode arrangement was adopted and an Ag/ AgCl electrode was used as the reference/counter electrode (Cui et al, 2007; Schwarz et al, 2001). The micropipette biosensor was evaluated by cyclic voltammetry using 1.0 mM hydroquinone in PBS solution as the electroactive agent (FIGURE 4).

Electrochemical detection of hydrogen peroxide

[0070] The detection of hydrogen peroxide (H₂O₂) is important in clinical diagnostics, the chemical and pharmaceutical industry, and environmental control. Amperometric biosensors based on horseradish peroxidase (HRP) have emerged as the most convenient tools for H₂O₂ detection due to their simplicity, high sensitivity, and selectivity. To improve the performance and long-term stability of the enzyme electrode, effective immobilization of HRP to the transducer surface is of great importance.

[0071] A Bio-BMP biosensor was designed, fabricated, and tested for hydrogen peroxide detection. Commercial biotin agarose beads, which have high binding affinity to streptavidin-HRP were used as the enzyme immobilization substrate for hydrogen peroxide biosensing. FIGURE 5A depicts the operating principle of the Bio-BMP biosensor for H₂O₂ detection. To immobilize the horseradish peroxidase (HRP) enzyme to the biotin agarose bead's surface, the tip of the Bio-BMP biosensor was incubated in a solution, consisting of 1.5 μg/mL streptavidin-HRP suspended in PBS, for 30 minutes. Subsequently, the tip of the Bio-BMP biosensor was washed with PBS solution for 10 minutes. Then, the Bio-BMP biosensor was dipped in the reaction solution, which contained 3.0 mM hydroquinone in PBS solution and to which 1% H2O2 portions were added. The H₂O₂ portions were prepared from a 30 % stock H₂O₂ solution.

[0072] The immobilized HRP enzyme catalyzed the reaction of hydroquinone with H₂O₂. The optimization of the experimental parameters is described below. The amperometric response of the electrode as a function OfH₂O₂ concentration was determined in the presence of 3.0 mM hydroquinone in PBS and a reduction potential of -0.3 V vs. the Ag/ AgCl reference electrode. Over 95% of the steady-state current was achieved within 5s, which may be ascribed to the proximity of the Pt electrode to the RedOx mediator. FIGURE 6 depicts the steady state current as a function of the peroxide concentration. The symbols and solid line correspond, respectively, to experimental data and a best fit line. The Bio-BMP biosensor exhibits a linear response dI/dC~0.4 (nA/DM), where / is the steady state current and C is the concentration OfH₂O₂, in the range from 1.0 x 10 ⁶ to 1.2 x 10 ⁴M H₂O₂ with a correlation coefficient of 0.998 (n=3). A detection limit of 5.3 XlO ⁷ M is estimated at the signal-to-noise ratio of ~3.

Comparison of various electrochemical mediators

[0073] Three different RedOx mediators were tested: potassium ferrocyanide, KI, and hydroquinone. In the presence of hydrogen peroxide, the HRP enzyme that is immobilized to the agarose bead catalyzes the oxidation of the RedOx mediator. The potential of the biosensor's working electrode was fixed at -0.1 V, -0.4 V, and -0.3 V against the Ag/ AgCl counter/reference electrode to monitor the reduction of the enzymatically generated ferricyanide, iodine, and quinone species, respectively. The responses times of the biosensors for all the RedOx mediators were remarkably fast, ranging from 4 to 15 s. However, hydroquinone showed the best analytical performances, namely the lowest detection limit and the highest sensitivity (FIGURE 7). Thus, hydroquinone was selected as the RedOx mediator in all the experiments reported below.

Concentration of the RedOx Mediator

[0074] The effect of the hydroquinone concentration on the agarose bead-based micropipette biosensor's response was studied in the presence of 1.5 mM H₂O₂ (PBS, -0.3 V vs. Ag/ AgCl) by varying the concentration of the hydroquinone from 0 to 5 mM. When the

concentration of the hydroquinone increased, the current increased and reached a saturation value at 3.5 mM hydroquinone concentration (FIGURE 8). Further increases in the hydroquinone concentration did not alter the current significantly. The pattern described above is typical of a mediator-based sensor. When the mediator concentration is low, the enzyme electrode's response is limited by the enzyme-mediator's kinetics. At high mediator concentrations, the response is limited by the enzyme-substrate's kinetics. In light of this problem, the concentration of hydroquinone was fixed at 3.0 mM in all subsequent experiments.

Optimization of the Reduction Potential

[0075] The magnitude of the applied potential strongly affects the amperometric response of the electrochemical sensors. The impact of the applied potential on the amperometric response of the Bio-BMP biosensor to H₂O₂ were systematically studied The experiments were carried out in the presence of 3.0 mM hydroquinone and 1.5 mM H₂O₂ in PBS solution. The steady-state reduction current increased gradually as the applied electrode potential decreased from -0.10 to -0.40 V

(FIGURE 9). At more negative potentials, there may have been interfering reactions from other electroactive species in the solution. Therefore, the potential of -0.30 V was selected as the appropriate working potential for the biosensor.

Stability and reproducibility of single agarose bead-based micropipette biosensor

[0076] The reproducibility of the current response of a particular Bio-BMP biosensor was examined at a H₂O₂ concentration of 1.0 mM to obtain a relative standard deviation (RSD) of 2.1% (n=6). To ascertain the variability in the current response from biosensor to biosensor, the performance of six similarly fabricated biosensors was compared. The standard deviation of the measurements carried out with the six sensors was 14%. This relatively large scatter among similar probes is attributed primarily to variations in the size of the agarose beads that were supplied by the manufacturer. The beads' uniformity can be improved by sieving the beads. Non-uniformities in the manufacturing process may have also resulted in variations in the exposed area of the Pt electrodes, which may have contributed to variations in the probes' performance. These variations can be greatly reduced with an appropriate mounting jig. Biosensors stored in a refrigerator at 4°C for two weeks retained about 95% of their initial current response.

Example II: Biosensor

Genomic DNA isolation and real time PCR amplification

[0077] A 0.2 mL B. cereus bacteria solution (1 x 10⁹ cells/mL, a gift from Dr. D.

Malamud, New York University), a spore-forming Gram-positive soil bacterium associated with food-borne diseases, was centrifuged at 14,000^χg for 2 min. The centrifuged pellet was then washed twice with ImL of PBS and resuspended in 0.2 mL of PBS for DNA extraction. Genomic DNA was extracted with QIAGEN DNeasy™ Tissue kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocol and was stored at -20 ⁰C until use. The DNA template purity and amount were determined with a NanoDrop ND- 1000 spectrophotometer (NanoDrop Technologies,

Wilmington, DE). To prepare the PCR mixture, Illustra PuRe Taq Ready-To-Go PCR beads (GE Healthcare, Piscataway, NJ) containing 2.5 units of DNA polymerase, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 200 μM dNTPs, carbohydrate stabilizers, and BSA were used. A series of 10 fold dilutions of whole B. cereus genomic DNA (5,224,283 bp), ranging from O.OOlng to 10ng, were used as PCR templates. The negative control contained the same mixture as in the above, but without the DNA template. For real time PCR reaction, SYTO Green 9 fluorescence dye

(Invitrogen, Carlsbad, CA, USA) was added into the PCR mix as a fluorescence indicator. The forward and reverse primers, at 0.3M concentration, were, respectively, 5'Biotin-TCT CGC TTC ACT ATT CCC AAG T-3' and 5'Digoxigenin-AAG GTT CAA AAG ATG GTA TTC AGG-3'. The primers target a 305 bp specific gene fragment. The real time PCR was carried out in a Peltier Thermal Cycler PTC-200 (Bio-Rad DNA Engine, Hercules, CA, USA). The DNA amplification was initiated with a denaturation step at 95⁰C for 120sec, followed by 35 amplification cycles (95⁰C, 20sec; 55⁰C, 30sec; 72⁰C, 23sec), and terminated with an extension step at 72⁰C for another 120sec. [0078] After real time PCR, a portion of each PCR-amplified sample was loaded into a lane of a 2.0% agarose gel. Electrophoresis of the amplified DNA fragments was carried out in TE (Tris-EDTA) buffer at a constant voltage of 140 V for 30 minutes. Afterward, the gel was stained with ethidium bromide and was visualized by UV illumination by standard techniques. DNA marker VIII (Roche Diagnostic, Indianapolis, Indiana, USA) was used to determine the sizes of the DNA products. All DNA manipulation procedures were carried out with standard molecular biology protocols.

Electrochemical detection of PCR products of B. cereus bacteria

[0079] The doubled-labeled amplicons of B. cereus genomic DNA templates were detected with the SA-BMP biosensor. The method of labeling the amplicons is described below. PCR amplicons of a 10 fold dilution series of B. cereus genomic DNA templates (ranging from 0.00 Ing to 10 ng), were used as the analytes in the SA-BMP biosensor experiments. The assay is depicted schematically in FIGURE 5B. The immobilization of the double labeled amplicons was achieved by submerging the tip of the SA-BMP biosensor in a PCR tube containing amplicons suspended in PBS for 10 minutes at room temperature. The biotin end of the ds-DNA amplicon bonded to the surface of the SA-BMP biosensor's streptavidin bead. Subsequent to two washing steps with PBS solution (10 minutes each) to remove nonspecifically bound DNA, the tip of the SA-BMP biosensor was submerged in blocking PBS solution (2% BSA and 0.1% Tween 20) for 10 minutes and then in anti- dig-HRP solution (1 : 5,000 dilution). The anti-dig-HRP bonded with the 3' dig end of the ds-DNA amplicon. Two washing steps with PBS buffer solution, lasting 10 minutes each, were then performed to remove unbound anti-Dig-HRP. The SA-BMP biosensor was then transferred into an electrochemical reaction cell containing 1.5 mM H₂O₂ and 3.0 mM hydroquinone for amperometric measurements. To determine the amplicon concentration, the current was measured at the fixed voltage of -0.30 V (vs. the Ag/AgCl reference electrode). The duration and number of the washing steps and reaction times were not optimized.

Detection of PCR amplicons of B. Cereus genomic DNA

[0080] To evaluate the reliability and applicability of the agarose bead-based micropipette biosensor for detecting biomolecules, a sequence of experiments were carried out in which the amplification products of PCR were detected and compared the results of the electrochemical detection with standard agarose gel electrophoresis imaging. DNA diagnosis has enormous applications in various fields including molecular biology, clinical diagnostics, agriculture, forensic science, and pathogen detection. Electrochemical biosensors for DNA detection have received considerable attention because electrochemical transducers offer a simple, sensitive, and inexpensive platform to detect nucleic acids binding. Here, instead of DNA hybridization, the amplicons were functionalized with dig and biotin. Thus, this method of DNA detection is similar to protein detection.

[0081] In these samples, the concentration of the B. cereus genomic DNA template ranged from lpg to IOng. The templates were amplified in a PCR machine. Additional details are provided below. FIGURE 10 depicts the amplification curves of real-time PCR for a 10 fold dilution series of B. cereus DNA template. The Figure shows the relative fluorescence intensity as a function of the number of cycles. The curves labeled with 1, 2, 3, 4, 5, and 6 correspond, respectively, to template concentrations of 0 (negative control), 0.001, 0.01, 0.1, 1, and 10 ng of genomic DNA. The real time PCR detected amplicons down to 0.0 Ing of B. cereus DNA template after 36 cycles. FIGURE 11 provides images of gel electrophoresis of the various PCR amplicons. The various lanes in

FIGURE 11 correspond to the template concentrations of FIGURE 10. Lane M is the DNA ladder that provides a marker for the DNA size and confirms the presence of the expected 305 bp amplicons.

[0082] FIGURE 5B depicts the operating principles of the streptavindin agarose bead-based micropipette (SA-BMP) biosensor used to detect PCR-amplified B. Cereus DNA sequences of 305- bp length. The biotin primer and dig primer used in the PCR amplification produced biotin and dig labeled B. Cereus DNA amplicons, which directly bonded to the streptavidin agarose bead of the SA-BMP biosensor through their biotin functionalization, avoiding the need for an intermediary biotin capture probe. Subsequently, the anti-dig HRP complex bonded to the dig end of the DNA amplicon. The sensor's current depended on the concentration of the bound HRP. Hydroquinone was used as the RedOx mediator (3.0 mM in PBS) and 1.5 mM H₂O₂ as the substrate for the HRP enzyme.

[0083] FIGURE 12 depicts the steady-state current detected with the SA-BMP biosensor as a function of the B. Cereus DNA template concentration (prior to amplification). At low DNA concentrations, the current increased monotonically as the concentration increased. The signal, however, saturated at higher DNA concentrations (template mass of about IOng). The behavior depicted in FIGURE 12 is consistent with first order kinetics which predicts the number of bound complexes to be proportional to R is the number of binding sites (likely

reduced due to steric hindrance), K_A is the affinity constant, and C is the analyte concentration. As shown in FIGURE 12, the SA-BMP biosensor can detect the amplicons of 0.00 Ing (1 pg) DNA template of B. Cereus, which exceeds the detection ability of the conventional gel electrophoresis by a factor of -10.

Claims

What is Claimed:

1. A sensor device comprising at least one electrode capable of being electrically addressed, penetrating a bead comprising one or more catalytic mediators capable of inducing an amperometric response with an analyte in the presence of the catalytic mediator and analyte.

2. The sensor device of claim 1 wherein the catalyzed mediators comprise at least one enzyme complex attached by immobilized ligands.

3. The sensor device of claim 2 wherein the ligands are proteins and/or oligonucleotides.

4. The device of claim 1 wherein the amperometric response is communicated to the electrode.

5. The device of claim 1 wherein the electrode comprises a conducting material, a

semiconducting material, or any combination thereof.

6. The device of claim 1 wherein the electrode is a solid core electrode.

7. The device of claim 1 wherein the electrode comprises carbon, gold, silver, platinum,

palladium, aluminum, copper, nickel, chromium, indium, tin, or any combination thereof.

8. The device of claim 1 wherein the shaft of the electrode has a diameter in the range of from about 10 nm to about 200 microns.

9. The device of claim 1 wherein the shaft of the electrode has a diameter in the range of from about 10 nm to about 100 microns.

10. The device of claim 1 where the tip of the electrode penetrating the bead is hooked or barbed

11. The device of claim 1 wherein the shaft of the electrode is coated at least in part, with an insulator.

12. The device of claim 11 wherein the coating provides a hermetic seal between the electrode and the insulator.

13. The device of claim 1 wherein the shaft contains a stopper to control the exposed length of the electrode.

14. The device of claim 12 wherein the coating providing the hermetic seal is inorganic.

15. The device of claim 11 wherein the insulator is glass.

16. The device of claim 1 wherein the bead has a characteristic cross-sectional dimension in the range of from about 20 nm to about 500 microns.

17. The device of claim 1 wherein the bead has a characteristic cross-sectional dimension in the range of from about 20 nm to about 200 microns.

18. The device of claim 1 wherein the bead comprises a porous material.

19. The device of claim 1 wherein the bead comprises an inorganic matrix, a magnetic material, or an organic polymer.

20. The device of claim 19 wherein the inorganic matrix comprises silica or alumina.

21. The device of claim 19 wherein the organic polymer comprises an acrylic acid derivative, a cellulose, a dextran, a polystyrene, a polyacrylamide, a polystyrene, or agarose.

22. The device of claim 1 wherein the bead is functionalized to immobilize or render non- leachable the catalytic mediators.

23. The device of claim 1 wherein the bead is functionalized to immobilize or render non- leachable the at least one enzyme complex.

24. The device of claim 1 wherein the bead is functionalized by pendant nitrogen, oxygen, or sulfur containing groups.

25. The device of claim 1 wherein the bead is functionalized by amine, carboxylic acid,

aldehyde, thiol, hydroxyl, metal ligands or chelating ligands.

26. The device of claim 1 wherein the functionalizing linkages are oligomeric or polymeric.

27. The device of claim 1 wherein the functionalizing linkages are proteins.

28. The device of claim 1 wherein the catalytic mediator is a catalytic redox mediator.

29. The device of claim 1 wherein the enzyme complex can directly catalyze the electrochemical oxidation or reduction of the analyte.

30. The device of claim 1 wherein the catalytic mediator comprises at least one of an antigen, antibody, monomer, oligonucleotide, nucleic acid, ligand, enzyme, enzyme substrate, a metal, or a paramagnetic material.

31. The device of claim 2 wherein the enzyme complex comprises at least one of an antigen, antibody, monomer, oligonucleotide, nucleic acid, ligand, enzyme, enzyme substrate, a metal, or a paramagnetic material.

32. The device of claim 1 wherein the catalytic mediator comprises a streptavidin, a

synaptobrevin, a biotin, or protein A.

33. The device of claim 32 wherein the biotin is biotin, desthiobiotin, or iminobiotin.

34. The device of claim 1 wherein the electrochemical detection cell is a two electrode system or a three electrode system.

35. The device of claim 34 wherein the counter electrode is concentric or eccentric with the working electrode and separated from the working electrode with an insulating layer.

36. The device of claim 1, further comprising an electrical device.

37. The device of claim 36, wherein the electrode is in electrical connection with the electrical device.

38. The device of claim 36, wherein the electrical device comprises a meter capable of detecting electrical signals, a current source, a sensor, a controller, a computer, or any combination thereof.

39. The device of claim 1 wherein the device comprises an array of functionalized electrodes.

40. The device of claim 1 comprising two or more functionalized electrodes wherein at least two beads are functionalized with different catalytic mediators.

41. The device of claim 1 comprising two or more functionalized electrodes wherein at least two beads are functionalized with at least two different enzymes.

42. An analytical system comprising a device of any one of claims 1-41.

43. A method of forming the sensor device of any one of claims 1-41 comprising:

providing an electrode and a bead comprising at least one catalytic mediator capable of inducing an amperometric response with an analyte in the presence of the catalytic mediator, wherein the electrode is capable of penetrating the bead; and

penetrating the bead with the electrode.

44. A method of forming a sensor device comprising

penetrating the bead with the electrode.

45. The method of claim 43 or 44 wherein the at least one catalytic mediator is an enzyme

complex attached to the bead by an immobilizing ligand.

46. The method of 43 or 44 wherein the electrode is sharpened at the end intended to penetrate the bead.

47. The method of 46 wherein the electrode is sharpened electrochemically

48. The method of claim 43 or 44 further comprising bending, hooking, or barbing the end of the electrode intended to penetrate the bead.

49. The method of claim 43 or 44 further comprising fusing an insulative layer over at least part of the shaft of the electrode.

50. The method of claim 49 wherein the insulative layer is glass and the fusing is induced by heat.

51. A method of analyzing an solution containing an analyte comprising

providing a sensor device comprising at least one electrode capable of being electrically addressed, penetrating a bead comprising at least one catalytic mediator capable of inducing an amperometric response with an analyte; and

contacting the sensor device with the solution containing the analyte.

52. The method of claim 51 wherein the at least one catalytic mediator is an enzyme complex attached to the bead by an immobilizing ligand and the amperometric response is induced in the presence of the catalytic mediator.

53. A method of analyzing an solution containing an analyte comprising

providing a sensor device of any one of claims 1-41; and

contacting the sensor device with the solution containing the analyte.