WO2001033206A1 - Microscopic combination amperometric and potentiometric sensor - Google Patents

Microscopic combination amperometric and potentiometric sensor Download PDF

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Publication number
WO2001033206A1
WO2001033206A1 PCT/US2000/030265 US0030265W WO0133206A1 WO 2001033206 A1 WO2001033206 A1 WO 2001033206A1 US 0030265 W US0030265 W US 0030265W WO 0133206 A1 WO0133206 A1 WO 0133206A1
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sensor
electrode
device according
amperometric
electrodes
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PCT/US2000/030265
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French (fr)
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Hal C. Cantor
Timothy D. Strong
Richard B. Brown
Robert W. Hower
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Advanced Sensor Technologies, Inc.
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327Biochemical electrodes electrical and mechanical details of in vitro measurements
    • G01N27/3275Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry

Abstract

According to the present invention, there is provided an electronic device for monitoring active biological operations including a support substrate, at least one amperometric sensor, and at least one potentiometric sensor.

Description

MICROSCOPIC COMBINATION AMPEROMETRIC

AND POTENTIOMETRIC SENSOR

BACKGROUND OF THE INVENTION

1 FIELD OF THE INVENTION

The present invention relates to an amperometπc and potentiometπc sensor array More specifically, the present invention relates to a microscopic amperometπc and potentiometπc sensor array 2 DESCRIPTION OF RELATED ART

As array systems grow relatively large, the efficient operation of the system becomes more critical Efficient interfacing of an array based system with electrical connections off-chip raise pin or contact limitation issues Further, constraints regarding effective chip or array size present issues relative to the selection and size of components for inclusion on the chip or substrate Often times, various selections must be made to provide an effective optimization of advantages in the overall design

One proposed solution for the control of an array of electrodes utilizing less than one individual dedicated connection per electrode or test site is

provided in Kovacs U S patent application Ser No 08/677,305, entitled "Multiplexed Active Biological Array", filed July 9, 1996 The array is formed of

a plurality of electrode sites A typical electrode site includes an electrode, a driving element coupled to the electrode for applying an electrical stimulus to the electrode, and a local memory coupled to the driving element for receiving and storing a signal indicative of a magnitude of the electrical stimulus to be applied to the electrode Multiple embodiments are disclosed for selectively

coupling a signal value through coaction of a row line and a column line for storage in the local memory In this way, the values at the various electrodes in the array may differ from one another

In Fiaccabπno, G C , et al , "Array of Individual Addressable Microelectrodes", Sensors and Actuators B, 18-19, (1994) 675-677, an array of n2 electrodes are connected to 2n pins, plus 2 additional pins for signal output and bulk bias The row and column signals drive series connected transistors to provide a single value to a working electrode This system does not enable the switching of two or more electrodes simultaneously at different potentials

In Kakerow, R et al , "A Monolithic Sensor Array of Individually Addressable Microelectrodes", Sensors and Actuators A, 43 (1994) 296-301 , a monolithic single chip sensor array for measuring chemical and biochemical parameters is described A 20*20 array of individually accessible sensor cells is provided The sensor cells are serially addressed by the sensor control unit One horizontal and one vertical shift register control selection of the sensor cells Only one sensor cell is selected at a time As a result, multiple sites may not be activated simultaneously

Yet another concern is the ability to test an electronic device prior to

application of a conductive solution on the device As devices or chips become more complicated, the possibility of a manufacturing or process defect generally increases While visual inspection of the device may be performed, further electrical and chemical testing may ensure an operational device is provided to the end user

As is apparent from the preceding discussion, numerous attempts have been made to provide effective techniques to monitor multi-step, multiple molecular, analytical, and biological reactions However, for the reasons stated above, these techniques are "piece-meal", limited and have not effectively optimized solutions In many situations it is necessary to monitor both potentiometπcally and amperometπcally over an area to monitor electrochemical signals and concentration gradients However, these various approaches are not easily combined to form a system which can carry out a complete cellular metabolic analysis and/or DNA diagnostic assay Despite the long-recognized need for such a system, no satisfactory solution has been proposed previously

It would therefore be useful to develop an interdigitated amperometπc and potentiometπc sensor array

SUMMARY OF THE INVENTION

According to the present invention, there is provided an electronic device for monitoring active biological operations including a support substrate, at least one amperometπc sensor, and at least one potentiometπc sensor

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings

wherein:

Figures 1 A and B is a schematic diagram of a sensor array with rectangular electrode geometry, Figure 1A is a three electrode conformation where the counter electrode serves the dual function as potentiometric electrode and Figure 1 B is a four electrode conformation;

Figure 2 is a cross section image of the working and reference electrodes; Figure 3 is a legend showing distinctions between platinum, silver and the contact;

Figure 4 is a schematic showing a 2μm contact size;

Figure 5 is a schematic showing a 4μm contact size;

Figure 6 is a schematic showing an 8μm contact size; Figure 7 is a schematic showing a 32μm contact size;

Figure 8 is a schematic showing a 100μm contact size;

Figure 9 is a picture showing an Xμm contact size;

Figure 10 is a block diagram for the present invention;

Figures 11 is a photomicrograph of an Xμm electrode size; Figure 12 is a photomicrograph of a 32μm electrode;

Figures 13 is a photomicrograph of a 100μm electrode which is in complete scale with the above figures;

Figure 14 is a schematic showing the amperometric circuitry of the present invention,

Figures 15 A-C are schematics of the ultra-low noise potentiometπc

electrode instrumental amplifier block,

Figure 16 is a schematic of the ultra-low noise amperometric current-to-

voltage conversion block,

Figure 17 is a schematic of an amperometric sensor function generator

block,

Figure 18 is a schematic of an amperometric controller functional block,

Figure 19 is an image of the carrier printed circuit board and the sensor controlling/monitoring circuitry constructed on bread boards,

Figure 20 is an amperometric dose response curve for dopamine which oxidizes at approximately 300mV v Ag/AgCI reference, 650mV signal remains unaltered,

Figure 21 is a cyclic voltammogram of dopamine in conditioned culture medium,

Figure 22 is superimposed traces of oxidatively derived current versus time transduced by all 16 amperometric sensors in the array simultaneously,

Figure 23 is an image of the six foot Baker EdgeGARD horizontal laminar flow hood encased with stainless steel screen and plastic sheeting to provide a Faraday cage free of electronic noise with constant temperature environment,

Figure 24 is a photomicrograph of nearly confluent hNT cells cultured on a 4 μm electrode size sensor array, and

Figures 25 A and B are a photomicrographs wherein Figure 25A is a light field and Figure 25B is a dark field illumination of hNT cells on day 4 of

incubation

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention provides an electronic device 10 for monitoring active biological operations and/or analyte chemical reactions having a support substrate 12, at least one amperometric sensor 14, and at least one potentiometπc sensor 16 The sensors of the present invention can detect neuronal action potentials and the resulting release of neurotransmitting and/or hormones The sensors can also detect the diffusion, dispersion, degradation, and re-uptake of neurotransmitters, hormones and/or other cellular metabolites

By "support substrate", it is meant a substrate on which a sensor array is placed or integrated This can be made of, but is not limited to, ceramic, glass and silicon "Coulometry" is the determination of charge passed or projected to pass during complete or nearly complete electrolysis of an analyte, either directly on the electrode or through one or more electron transfer agents The current, and therefore analyte concentration is determined by measurement of charge passed during partial or nearly complete electrolysis of the analyte or, more often, by multiple measurements during the electrolysis of a decaying current and elapsed time Once the hydration shell has been established around the electrode, the decaying current results from the decline in the local

concentration of the electrolyzed species caused by the electrolysis A "counter electrode" refers to an electrode paired with the working electrode, through which passes an electrochemical current equal in magnitude

and opposite in sign to the current passed through the working electrode In the context of the invention, the term "counter electrode" is meant to include counter electrodes which can have the dual function as a potentiometric reference electrode (i e a counter/potentiometπc electrode)

An "amperometric electrochemical sensor" is a device configured to detect the presence and/or measure the concentration of an analyte via electrochemical oxidation and reduction reactions on the sensor These reactions are transduced to an electrical signal that can be correlated to an amount or concentration of analyte

"Electrolysis" is the electrooxidation or electroreduction of a compound either directly at an electrode or via one or more electron transfer agents An example of this includes, but is not limited to, using Glucose Oxidase to catalyze Glucose oxidation creating oxidized Glucose and Peroxide, where the Peroxide is being measured

The term "facing electrodes" refers to a configuration of the working and counter electrodes in which the working surface of the working electrode is disposed in approximate apposition to a surface of the counter electrode A compound is "immobilized" on a surface when it is physically entrapped on or chemically bound to the surface

The "measurement zone" is defined herein as a region of the sample chamber sized to contain only that portion of the sample that is to be interrogated during the analyte assay

A "non-leachable" or "non-releasable" compound is a compound which does not substantially diffuse away from the working surface of the working and/or counter electrodes for the duration of the analyte assay

A "redox mediator" is an electron transfer agent for carrying electrons between the analyte and the working electrode, either directly, or via a second electron transfer agent

A "reference electrode" is an electrode used to monitor and account for voltage drop due to medium resistance in amperometric sensors, and supplies a reference potential for comparison in potentiometric electrodes

A "second electron transfer agent" is a molecule which carries electrons between the redox mediator and the analyte (See example above)

"Sorbent material" is material which wicks, retains, or is wetted by a fluid sample in its void volume and which does not substantially prevent diffusion of the analyte to the electrode

A "counter electrode" is an electrode at which analyte is electrooxidized or electroreduced with or without the agency of a redox mediator

The "working electrode" supplies the potential source to affect oxidation/reduction A "working surface" is that portion of the working electrode which is coated with redox mediator and configured for exposure to sample

Electrochemical detection, specifically amperometry, has been used in

the past in relatively unsophisticated applications, for example detecting and quantifying eluted molecules at the end of chromatographic columns (Kissinger et al, 1984) The mam limitations of amperometry are its low specificity and sensitivity The present invention takes advantage of this technique's speed

and overcomes its limited specificity and sensitivity First, to enable the amperometric sensors to detect multiple neurotransmitters independently, the proposed system employs two particular forms of amperometry, cyclic and constant voltage voltammetry Second, utilizing a micro-screen printing device, such as a New Long LS-15TV, several different selectivity membranes can be applied over the individual sensors to eliminate background measurement of unwanted compounds (such as ascorbic acid) and impart specificity onto the microscopic electrodes comprising the sensor (Goldberg et al, 1994) Finally, by encapsulating the multi-site sensor array leads with silicon nitride, a substrate upon which neurons can be made to readily attach, the sensor array is in very close apposition to the secreting neurons allowing measurement of the relatively high neurotransmitter concentrations in the immediate vicinity of the axon, prior to degradation, dilution, dispersion, and re-uptake

An amperometric process, cyclic voltammetry, is a technique whereby a cyclically repeated triangular waveform of potential is applied between the working and counter electrodes Individual analytes, such as neurotransmitters, have characteristic oxidation and reduction potentials based on their chemical moieties (Adams, 1969, Dryhurst et al, 1982) When the voltage between the

electrodes reaches the oxidation potential of a particular neurotransmitter, that molecule oxidizes Oxidation is a process whereby an electron is stripped from the molecule The counter electrode, absorbs the oxidatively produced

electrons, effectively transducing chemistry into electricity The flow of electrons

per unit of time is current, which is proportional to the number of molecules being oxidized The voltage at which this oxidatively produced current is

obtained provides information useful for identifying the analyte such as

neurotransmitter, hormone or cellular metabolite being measured (Dryhurst et al, 1982, Baizer et al, 1973)

At solid stationary microelectrodes operating under conditions of cyclic voltammetry the peak current in microamperes, ip, is given for a reversible

electrode reaction by the Randles-Sevcik equation (Rand'-^s et al, 1948)

= 2.687 x l05 n3 2AD1/2Cv1 2

where = the number of electrons transferred

A = the electrode area in cm

D the diffusion coefficient of the electroactive species in cm per

second

C the bulk concentration of the electroactive species in mil moles

per liter v = the scan rate of the applied cyclic voltage sweep in volts per second

Cyclic voltammetry has several advantages over other amperometric

techniques such as constant voltage voltammetry During each cycle, the

potential on the working electrode reverses and electrically cleans the electrode

of molecules adsorbed during the previous cycle The technique is quantitative for both oxidation and reduction (i e the measurement of biogenic amines or oxygen respectively) (Bard et al , 1980) Cyclic voltammetry is capable of providing further confirmation of the identity of an analyte by measuring its reduction potential as well as its oxidation potential (Oldham et al , 1989, Heineman et al , 1989) As the electrode potential is scanned toward a negative potential, a cathodic peak is obtained due to reduction of the analyte, Ox, to form a reduced metabolite, Red, according to the following equation

Ox + ne → Red

where ne is the number of electrons transferred in the reaction (Hush et al, 1971 ) The voltage sweep then reverses direction and scans towards a positive potential If the scan rate is sufficiently rapid, some of the Red produced by the

cathodic sweep can still be in the vicinity of the electrodes and are reoxidized to Ox, producing the anodic peak (Adams, 1969) For completely reversible reactions, the anodic and cathodic peak potentials are separated by the

potential increment

Eanodlc -Ecathodlc = 0.059 / ne Volts

where ne is the number of electrons involved in the oxidation and reduction (Oldham et al , 1989) If the electrode reaction is not completely reversible, i e stable intermediate reaction products are produced, then the peak potentials are separated by a characteristic, but more than expected value (Oldham et al, 1989, Hush et al, 1971 ) This is the case for ascorbate the oxidation of ascorbate produces dehydroascorbate, a fairly stable product (Adams, 1969, Oldham et al, 1989, Rose, 1989) For totally irreversible reactions, one of the peaks disappears completely Independent of the extent of reversibility, the anodic/cathodic peak voltage difference is constant for any particular voltage scan rate, and in addition to oxidation voltage, it can be used to determine analyte identity (Rose et al, 1989, Wang et al, 1997)

Amperometric techniques are very sensitive to the voltage scan rate When slow scan rates (< 200 mV/sec) are used, all of the analyte in the immediate vicinity of the electrodes is oxidized Sensor measurements are then limited by the diffusion of unoxidized analyte to the electrode surface This

leads to diffusion limiting current, and results in defined oxidation peaks

centered around the oxidation potential of the analyte High scan rates (> 500 mV/sec) do not permit enough time for diffusion to occur When medium to high scan rates are used, the oxidized molecule remains in the vicinity of the electrodes and can be reduced back to its native form This aids in identification of the analyte as discussed previously

Cyclic voltammetry provides the ability to measure the concentrations of several molecules sequentially in a single scan, as long as their oxidation potentials differ For example, the concentrations of dopamine, norepinephπne, serotonin, and ascorbate can all be monitored sequentially from a mixture of these compounds, the value of oxidatively-deπved current flow is captured at the characteristic potentials for each analyte Once the identity of the analyte is confirmed using cyclic voltammetry, high speed measurements (> 20 kHz) can be achieved by utilizing constant voltage voltammetry

Constant voltage voltammetry, as the name implies, employs a single operating potential to effect oxidation This technique is used most commonly by investigators since it is the simplest to implement, both in terms of the controlling circuitry, and especially in the data acquisition phase One major advantage to constant voltage voltammetry is that the sensor can be sampled at a very high rate allowing elucidation of the dynamics of neurotransmitter secretion, degradation, and re-uptake Unfortunately, the drawback to this technique is that it lacks specificity since all molecules within the vicinity of the electrodes whose oxidation potential is less than that applied to the electrodes oxidize and contribute to the value of the measurement

To overcome this limitation, selective molecular access to the electrodes

can be provided by depositing membranes on them Several different classes of membranes are available for use Several ion exchange materials, such as Nafion, poly(vιnylpyπdιne), and poly(ester sulfonic acid) act as ion exclusion membranes (Wang et al , 1997, Su et al , 1990, Runnels et al , 1999, Brazell et

al , 1987, Wiedeman et al, 1990, Wang et al , 1990), allowing only uncharged molecules to gain access to the electrodes Nafion is commonly used to greatly reduce the background signal generated by ascorbate ion, which is ubiquitously present in neural tissue Additionally, various mixtures of cellulose acetate were prepared which act as size exclusion membranes, allowing only specific molecular weight species to gain access to the electrodes This is critical when monitoring dopaminergic neurons Dopamine is quickly degraded to dopac, homovanillic acid, and other break down products (Cahill et al, 1996) While only dopamine is active as a neurotransmitter, all of the break down products oxidize at potentials very close to the parent molecule Using a variety of decreasing size exclusion membranes on the sensors in the array, one can determine the concentration of the parent molecule (i e dopamine), as well as each of its break down products, uniquely

While many electroactive molecules are found in biological systems, very few oxidize at low potentials, i e less than 900 mV vs an Ag/AgCI reference electrode (Dryhurst et al, 1982) Fortunately, most neuronal and endocrine products are among the very few low-voltage oxidizing molecules and can therefore be measured without interference using this technique (Adams et al,

1982, Dryhurst et al, 1982, Dryhurst et al, 1982) Neurotransmitters produced by certain neurons are not electroactive, however, they are stored in vesicles and packaged with granms and chromogranins, protective anti-oxidant molecules that sacπficially prevent neurotransmitter degradation (Winkler et al , 1992, Bassetti et al , 1990, Huttner et al , 1991 , Konecki et al , 1987) If the neuronal product is not oxidizable (such as magnocellular neurons in the hypothalamus) the co-secreted molecules can be measured Fortunately, these

protective agents are secreted with the neurotransmitters concomitantly and stoichiometπcally (Scammell et al, 1993, Hinkle et al, 1992)

In one embodiment, the small volume of the in vitro analyte sensors of the present invention are designed to measure the concentration of an analyte in a portion of a sample having a volume less than about 1 μL, preferably less than about 0 1 μL, more preferably less than about 0 01 μL, and most preferably less than about 0 001 μL The analyte of interest is typically provided in a solution or biological fluid, such as blood or serum

A small volume, in vitro electrochemical sensor 20 of the invention generally includes a working electrode 22, a counter electrode 25, a reference electrode, and a sample chamber 26 The sample chamber 26 is configured so that when a sample is provided in the chamber the sample is in electrolytic contact with both the working electrode 22, the counter electrode 25, and the reference electrode 24 This allows electrical current to flow between the electrodes to effect the electrolysis (electrooxidation or electroreduction) of the

analyte

The counter and/or working electrode can be formed from a molded

carbon fiber composite or can consist of an inert non-conducting base material, such as polyester, upon which a suitable conducting layer is deposited The conducting layer should have relatively low electrical resistance and should be

electrochemically inert over the potential range of the sensor during operation Suitable conductors include gold, carbon, platinum, irπdium, and palladium, as well as other non-corroding materials known to those skilled in the art The electrode and/or conducting layers are deposited on the surface of the inert material by methods such as vapor deposition or printing

A tab 22 is provided on the end of each electrode 23 for easy connection of the electrode to external electronics such as a voltage source or current measuring equipment Other known methods or structures may be used to connect the electrodes 22 to the external electronics

In the preferred embodiment, five geometric variations of microscopic amperometric and potentiometric sensors were designed and constructed in a 4 x 4 array on silicon utilizing CMOS technology The amperometric and potentiometric sensors are general purpose devices which can be modified to detect and quantify a wide range of analytes (cellular products) depending upon the electronic method of operation at the amperometric sensors and upon the selection of membrane lonophores, enzymes, antibodies and for the potentiometric sensors As mentioned above, membranes can also be utilized on the amperometric sensors to confer added specificity

Five conformations of sensor arrays, each incorporating 49 electrodes,

were constructed on silicon using CMOS processing with electrode sizes of 2, 4, 8, 32, and 100 μm respectively Three electrodes compose each amperometric sensor (reference, working, and counter) A diagram of a sensor array is presented in Figure 1 Table 1 contains a process for forming the sensor array

The resulting sixteen sensor sites are arranged into a 4 x 4 array The surface of the counter and working electrodes are platinum to provide a

polaπzable contact to solution The platinum layer forming these electrodes also forms the interconnecting leads for each electrode The reference electrode included in each sensor site and the global reference electrode are coated with silver (Ag) and electrolytically chloπdized to provide reversible Ag/AgCI electrodes Alternatively, a fabrication process was also used to chloπdize the Ag reference electrodes in a batch-wise manner This fabrication process entails using a reactive ion etch (RIE) plasma as a chloride source This technique allows wafer-level chloπdation of all reference electrodes within each sensor array at once, prior to separating the silicon wafer into individual chips This methodology eliminates the necessity to provide electrolysis current during chloπdation and improves the accuracy and precision of the silver chloride fabrication process

In this implementation, electrode orientation was altered from site to site This provides the capability to combine electrodes from adjacent sites to act as a single larger electrode providing more flexibility for studying the effect of electrode size on the electrochemical response curves

A single platinum electrode can be used as a working electrode for the

entire array, replacing the working electrode present in each amperometric sensor In this manner, each of the former working electrodes is available to function as a potentiometric electrode This provides the capability to monitor 16 amperometric sensors (both with chrono-amperometry and cyclic voltammetry) and 16 potentiometric sensors simultaneously The only limitation this produces is that each amperometric sensor has to cycle in concert for the cyclic voltammograms This is not an issue since concerted cycling is generally employed Further, the physical distance between the amperometric and potentiometric electrodes is very small (4-20 μm depending upon the electrode size) providing for example the ability to monitor neuronal action potentials and neurotransmitter release from a single cell Alternate configurations include producing arrays of four electrode units, three of which are used for amperometry while the fourth is used for potentiometric action potential determinations

The sensor arrays are fabricated in a three-mask process with two (and/or three or four - see below) metal electrode layers, silver (Ag) and platinum (Pt) Since Ag and Pt are difficult to etch using wet chemistry, a resist lift-off process is used to pattern them, however wet chemistry can be employed to etch the Pt, Ag, or any other metals used The lift-off process provides an additional advantage in allowing the use of layered materials in the metal structure to modify electrode properties and still allows for patterning to occur in one step (see cross section presented in Figure 2)

Previous experimentation has shown a tendency for the silver to delaminate from underlying metal layers, and silicon nitride To prevent this, a thin layer of titanium (Ti) is deposited in order to enhance adhesion Chromium (Cr) has also been employed as a adhesion layer, however it is not as adhesive as Titanium and requires special care during the lift-off process The advantage of Cr is that it provides less reactivity to solution than is observed when the Ti is exposed to solution, i e in failure mode

The metal lift-off step has been improved by utilizing a short undercut etch to promote easier release of excess material After depositing the lift-off photoresist and patterning it, a short etch is performed on the underlying dielectric This etch is a wet HF acid etch of the silicon dioxide in the case of the platinum metallization and a reactive ion etch (RIE) of the silicon nitride for the silver (Ag) sites This etch performs two functions First, since the resist tends to be undercut in a wet etch, a small gap is formed under the resist edge After metal deposition, the gap forms a natural breakline during lift-off that prevents metal, coating the sidewalls of the resist, from remaining behind on the device forming vertical spikes or walls These spikes are very difficult to cover completely in later dielectric depositions and can form short circuits to solution Second, if the etch is timed properly, it also planaπzes the sensor topography by submerging the metal layer into the dielectric 38

Next, a 650 nm thick layer of silver is applied with a 20 nm layer of titanium as an adhesion promoter The final lift-off is performed in acetone Early work using Microposit 1112A photoresist remover (Shipley, Marlboro, MA,

U.S A ) caused discoloration of the silver reference electrodes Acetone is less reactive to the silver, however, it lacks surfactants to prevent re-deposition Some care is required to insure that all of the waste metallization is completely removed

Standard silicon methods for encapsulating the Ag layer have been found

to tarnish the silver Because of this, the Ag layer is deposited on top of a PECVD silicon nitπde layer Since wet etches for silicon nitride are difficult to control and react with metallization used in circuit processes, an RIE machine is used for the etching of this dielectric Due to the vertical directional nature of an RIE, it does not provide the undercut and gap described above in a wet etch However, the steep sidewalls created in the dielectric enhance the lift-off effect and with the addition of the recessed metal layer the result is that the surface is more planer than if no etch is performed If CMOS circuitry is not intended to be present on the chip, an undercut etch can be performed after the RIE which aids in the liftoff process

Other materials can be utilized to encapsulate the Pt conducting lines Some of the materials that can be used include low temperature oxide (LTO), nitride, Parahne, spin-on-glass, Polyimide, and TEFLON™ to name a few

Evaporators presently used to deposit platinum for these devices have a maximum allowable thickness of 1000A Combined with the fine line-width for metallization on the smaller devices, this factor causes a large resistance in series with the electrodes To reduce this resistance an alternate metallization technology was devised After depositing the titanium adhesion layer, gold is evaporated onto the surface A second adhesion layer of titanium is applied

and followed by a platinum layer to provide the polaπzable interface to solution Patterning is unchanged as the lift-off procedure can be used to pattern all layers at once The gold provides low resistance and is compatible with

platinum The resistance in these devices is an order of magnitude lower than devices not incorporating gold, depending on the thickness of the gold layer

applied

There are several other metahzation schemes that can be employed to reduce the resistance of the interconnect lines Some of these include Copper (Cu), Tungsten (W), and Aluminum (Al) as the underlying layer rather than Au Cu is becoming widely used in the integrated circuit (IC) fabrication processing industry to reduce the resistance of the interconnect lines W has a very high melting point and is very hard marking it physically more compatible with the Pt layer, though it possesses a higher resistance than the Au Al has a lower resistance than Pt and is the standard metal used in the IC industry Because either Ti or Ti/W will act as a metal diffusion barrier, many different metals can be used to reduce the resistance of the lines Care must be taken to choose a metal that will have a low resistance and not create a potential dependant resistance or create a thermocouple However, if circuitry is employed on the sensor chips, the Pt interconnect lines will be very short, and this problem becomes irrelevant The complete step-by-step process of sensor manufacture is presented in Table 1

Initially, five geometric variations of microscopic amperometric and potentiometric sensors were designed and constructed in a 4 x 4 array on

silicon utilizing CMOS technology The amperometric and potentiometric sensors arrays were constructed and tested in vitro During the in vitro experimentation, it was demonstrated that the sensor arrays were capable of monitoring the metabolism of single mammalian neurons, both amperometπcally and potentiometπcally in concert, for periods up to 75 days without any

performance degradation

EXAMPLES General Methods:

Two printed circuit boards (PCB) (Figure 19) 30 were designed and constructed to provide a simple and easy to use interface between the sensor bearing ceramic carrier and the potentiometric and amperometric circuitry The PCBs contain a zero insertion force (ZIF) 32 socket that allows rapid swapping of the ceramic carriers, and a series of connector rails 34 that provide access to each individual electrode within the sensor array The PCB 30 is a three-layer board with the inner layer constituting a ground plane to help reduce environmental noise

In an effort to increase the amount of useful data that could be acquired from the sensors, it was decided that a single, external, platinum electrode would replace the working electrode present in each amperometric sensor The only limitation this produced was that each amperometric sensor would have to cycle in concert for the cyclic voltammograms This was not an issue since concerted cycling was intended The main advantage provided by this alteration was the availability of each of the former working electrodes to act as

potentiometric electrodes In this manner, it was possible to monitor 16 amperometric sensors (both with constant voltage and cyclic voltammetry) and 16 potentiometric sensors simultaneously Further, the physical distance between the amperometric and potentiometric electrodes was very small (4 to

20μm depending upon the electrode size) providing the ability to monitor cellular action potentials and neurotransmitter release in extremely close proximity to each other Alternatively, the amperometric sensors can be cycled independently, in which case the proximity of the electrodes must be carefully monitored to prevent interference between the electrodes

The amperometric and potentiometric circuitry was constructed on breadboards allowing manipulation of the components responsible for gam, filtering, etc (Figure 19) Using this technique, a variety of circuitry parameters were easily altered and optimized to provide relatively noise free data from the individual electrodes within each of the sensor arrays

The 16 channels of output from the potentiometric sensor circuitry were connected to 16 channels of input on a high speed, 16-bιt, National Instruments a/d board (PCI-MIO16XH) housed in a PowerPC Macintosh computer Similarly, the 16 channels of output from the amperometric sensor circuitry were connected to 16 channels of input on another high speed, 16-bιt, National Instruments a/d board (NB-MIO16XH) housed in a separate Macintosh computer A NB-MIO16XH board, housed in a second computer, was utilized to achieve the goal of monitoring from 32 total sensors, 16 amperometric and 16

potentiometric, simultaneously Data synchronization was assured by sharing a single clocking signal between both National Instruments boards A digital trigger was used to initiate acquisition in the two a/d converters within 100 nanoseconds of each other

The maximum sampling rate of the NB was less than that of the PCI

board Therefore, the PCI board was used to monitor the potentiometnc sensors that transduced the neural action potentials (the highest speed component) while the NB board was used to monitor the amperometric sensors that transduced the secreted neurotransmitters (the slower speed component) The sample rates were maintained at the maximum that each board would allow while maintaining whole number multiples of each other, simplifying direct comparison of data from each computer system When monitoring each of the 32 sensors, a/d sampling rates were 6kHz for the potentiometnc sensors and 2kHz for the amperometric sensors When cellular activity was detected on a potentiometπc/amperometπc set of sensors, that subset of sensors was monitored at higher sampling rates For example, 16 sensor (8 potentiometnc and 8 amperometric) were monitored at 12kHz for the potentiometnc and 4kHz for the amperometric When a single potentiometric and amperometric pair were monitored, the potentiometnc rate was 60kHz and the amperometric rate was 20kHz A second PCI-MIO16XH board can be placed in a single computer to provide a 1 1 ratio of sampling rate between the potentiometric and amperometric sensors

The high speed, 16-bιt, d/a converter in the PCI-MIO16XH board was

utilized as the voltage source for the amperometric controller circuitry Software was written to provide a variety of user-selectable schemes for cyclic voltammetry (selectable parameters included control of voltage range and cycle frequency) and for constant voltage voltammetry (selectable parameters included control of initial and final voltage composing the voltage step function

as well as delay before onset and duration of the step function) The biological testing of the sensors was accomplished using hNT

neuronal cells Once the sensor array is secured within the ceramic carrier, a layer of inert Dow Corning Si cone RTV Sealant 732 (World Precision Instruments, Inc Sarasota, FL) or any similar inert sihcone or other sealant is applied leaving a 2 mm x 2 mm window over the actual sensor array hNT neuronal cells require a basement membrane matrix for cell attachment to the sensor surface Poly-D-lysine and Matπgel matrix (Becton Dickinson Labware, Bedford, MA) were used to provide a basement membrane for the cells

The sensor array was first coated with poly-D-lysme to promote bonding of the Matπgel matrix Sterile poly-D-lysine, at a concentration of 10μg/ml in distilled water, was applied to each of the 2mm x 2mm exposed sensor arrays and allowed to incubate at room temperature for 2 hours The poly-D-lysine solution was then aspirated with a sterile pipette The sensor arrays were placed at an incline with lids off in a sterile laminar flow hood and allowed to dry

for 1 5 hours Matπgel matrix was thawed overnight in a refrigerator, and then diluted to

1 40 in cold DMEM/ F12 (Life Technologies, Rockville, MD) 20μl of the

Matπgel matrix was applied to each sensor array and spread evenly using a Pasture pipette The solution was allowed to completely dry at room temperature in a sterile laminar flow hood The Matπgel application was then

repeated

The sensor arrays could be stored for at least 2 months with one coat of poly-D-lysine and one coat of Matπgel matrix The final coat of Matπgel matrix

must be applied on the day of use Under the microscope, a dry coated sensor appeared to have a fine, frost-like mesh Care was taken to avoid opaque clots indicating the Matπgel matrix concentration was too high

The sensor arrays were tested for their ability to monitor neurotransmitters simultaneously and independently These measurements were conducted using hNT conditioned DMEM/F12 culture medium (Stratagene,

La Jolla, CA), the same medium used throughout the biological experimentation

In all cases, the medium was equilibrated for temperature and CO2 in a mammalian cell incubator for 30 minutes prior to testing

Dose response curves for dopamine (Sigma-Aldπch Co , St Louis, MO) were generated by performing cyclic voltammetry utilizing the microscopic sensor arrays Oxidatively derived currents generated at the unique oxidation potential for dopamine, approximately 500mV versus silver/silver chloride reference, were stored and plotted as a dose response curve in Figure 20

Since the acetylcholme is not oxidized at low voltages, the dose response curves for dopamine were unaffected by the presence of even super- physiological concentrations of acetylcholme Independent of the concentration

of acetylcholme, the value of the oxidatively derived current at 500mV varied in a linear manner with dopamine concentration The peaks in oxidatively derived current can be visually discerned (Figure 21 ) The peak at 500mV corresponds to the oxidation of dopamine The other two peaks at 650mV and 1080mV remained essentially constant in amplitude regardless of neurotransmitter concentrations and are most likely

attributable to components within the DMEM/F12 culture medium When phenol red was not present in culture medium, the peak at 1080mV disappeared The molecular source of the peak at 650mV is currently under investigation Again, the value of the oxidatively derived current at these voltages did not significantly vary in any of the dose response, control, or cellular experiments (Figure 21 ) During the generation of dose response curves, transduction properties of individual sensors within the sensor array were compared to one another to insure uniformity Since the electrode structures are created using electron beam lithography, physical parameters of each electrode, such as size and surface area, are nearly identical The uniform transduction properties afforded by the uniform physical geometries are demonstrated by nearly identical cyclic voltammograms seen at each of the 16 amperometric sensors (Figure 22)

In another embodiment of the present invention, a six-foot EdgeGARD horizontal laminar flow hood (The Baker Company, Sanford, Maine) was modified for use in data acquisition The hood was lined with stainless steel small mesh screen The screening, when grounded, provided a nearly electronic noise-free environment within the hood, essentially mimicking an

expensive Faraday cage The front opening of the laminar flow hood was also covered with the screen material An access door in the form of a retractable flap was constructed for easy access to the experimental setup (Figure 23) This is not required for the sensor array to function properly, but can be used in association with the sensor of the present invention

The laminar flow hood was also modified to provide a heated, constant temperature environment (37C) to maintain normal physiological activity within the cultured cells Towards this goal, the hood was lined with an additional layer of plastic sheeting to make the inner chamber nearly air tight A standard hair dryer, controlled by an inexpensive digital temperature regulator (Fisher Scientific, Chicago, IL), was used as the heat source The hair dryer was located outside of the Faraday cage to minimize electronic noise at the sensor arrays The heated air was transported to the interior through a 4-ιnch diameter metal dryer duct The thermocouple probe for the temperature controller was placed in close proximity to the cells This inexpensive setup provided a constant temperature environment with approximately 0 5C temperature fluctuations

Most important, the experimental setup allowed rapid transfer and electrical connection of the culture chambers with integrated sensor arrays to the electronic circuitry Using this setup, one could easily monitor dozens of culture chambers with integrated sensor arrays daily hNT cells were procured from Stratagene (Catalog #204104, Lot

#0980822) in a highly purified, frozen state The hNT cells were thawed and plated, according to Stratagene recommended procedures, on the sensor arrays at a concentration of approximately 8 x 105 cells/cm2 Several cultures were prepared as previously reported with the exception of plating hNT cells These cultures served as controls

Electrochemical monitoring of the cultured hNT cells began the day following plating At this point, day 1 , the cells had spread out into a monolayer with approximately 70-80% confluence (Figure 24) Note that the cells spread almost exclusively upon the sensor array coated with Matπgel matrix as opposed to the sihcone No measurable activity was observed until day 4

By day 4, the hNT cells resembled primary neuronal cultures morphologically and in density of process outgrowth and, like primary neurons, exhibited elaborate processes that differentiated into axons and dendπtes (Figure 25) On day 4, one culture was observed to spontaneously exhibit action potentials and neurotransmitter release By day 5, one third of the cultures were observed to spontaneously exhibit action potentials and neurotransmitter release Finally, by day 6, twenty-four of the twenty-eight cultures were observed to spontaneously exhibit action potentials and neurotransmitter release Experimentation was continued for a total of 75 days Approximately every 5 days, one culture was taken apart and observed microscopically Of the cultures remaining, all those that were previously active remained active for the entire 75 days Even though the cultures were still active, experimentation had to be terminated at the end of the 75th day due to lack of available funds

Selective molecular access to the electrodes can be provided by

depositing membranes on them Several different classes of membranes are available for use Nafion acts as a cation exchange membrane (Brazell et al , 1987), allowing only uncharged molecules to gam access to the electrodes Additionally, various mixtures of cellulose acetate can be prepared which act as size exclusion membranes, allowing only specific molecular weight species to gam access to the electrodes This is critical when monitoring large bioagent molecules Often, large bioagent molecules degrade into a variety of breakdown products It is possible that only the parent bioagent exerts a biological effect and the break down products do not, however, several of the break down products may oxidize at potentials very close to the parent molecule when monitored amperometπcally Using a variety of decreasing size exclusion membranes on the sensors in the array, the concentration of the parent bioagent molecule can be determined as well as each of its break-down products uniquely

One goal of the present invention is to augment semiconductor sensor technologies with new formulations of membranes containing lonophores, antibodies and enzymes, to enable the array to monitor a wide range of biological analytes, environmental toxins, as well as standard blood chemistries (i e electrolytes, antibodies, steroid and protein hormones, anesthetics, a variety of herbicides, medicinal drugs, drugs of abuse, etc ) Other complex molecules, such as neurotoxins and molecules of biological warfare can be detected by immobilizing antibodies and/or enzymes on the surface of an lon-

selective membrane, via ELISA assays, or through the production of amperometπcally detectable reaction products catalyzed by enzymes causing the formation of electroactive molecules, such as hydrogen peroxide, from the parent molecule

One example of monitoring complex molecules with a membrane is a

catalyzed inhibitory reaction involved in the measurement of the pesticide Paraoxon Because organophosphates inhibit the reaction mechanism causing the breakdown of various chohne compounds, catalyzed by a variety of cholmesterase enzymes, these can be used to monitor the presence of Paraoxon The membranes must adhere well to the silicon surface to prevent detachment of the membrane during sampling, rinsing, and washing of sensors in the sampling chamber When silicon based, optical detectors are to be utilized, slight pealing of the membrane can significantly alter the performance of the device Membranes that adhere strongly to the silicon surface provide the sensors with a long, useful, lifetime

Some examples of membranes include, but are not limited to, Cellulose Acetate, Poly-Urethane/Poly-Vmyl Chloride, and Si cone Rubber Each of these membrane compositions possess differing properties as related to enzyme and antibody immobilization and adherence to the silicon nitride surface of microscopic solid-state chemical sensors Several methods are available for immobilizing enzymes and antibodies on the surface of the membranes Additional techniques amenable to monitoring organophosphorous containing compounds, including Paraoxon, can also be used Enzymes have been incorporated into a hydrophihc polyurethane membrane and deposited on

top of hydrophobic polyurethane membranes (Cho et al , 1999), promoting adhesion to the sensor surface Additionally, enzymes can be immobilized on the membrane surface that cause local changes in pH in the presence of toxins,

which can be monitored utilizing a potentiometnc electrode (Mulchandani et al ,

1999). The first method to be employed for the detection of organophosphorus compounds such as Paraoxon is to monitor the inhibition of the reaction catalyzed by butyrylchohnesterase, which breaks down butyrylchohne into chohne and butyric acid Paraoxon has been shown to inhibit this reaction linearly in proportion to its concentration (Campanella et al , 1996) Membrane adhesion has typically been a significant problem effecting useful lifetime of solid-state chemical sensors Many of the membranes utilized for traditional chemical sensors do not adhere well to the silicon-nitride surface, reducing the yield and lifetime Membrane adhesion is tested using a Q-Test II adhesion analyzer Membrane adhesion is a critical factor and must be optimized to provide stable electrochemical properties in a flow system The fluid flow system, necessary for sample delivery, calibration, washing, and regeneration of the sensors, tends to cause pealing of the membrane

To improve membrane adhesion, treatments and modifications of the sensor's sihcon-nitπde surface are examined in order to improve membrane/sihcon-nitπde cross-linking These efforts vastly extend the useful lifetime of commercial devices

Membranes can be deposited using a set of micropipettes accurate to

20nL of volume or through the use of commercial systems accurate to 325 pL of volume (Pacuard, CT) Other methods known to those of skill in the art can also be used to deposit the membrane For example, one device, a New Long LS- 15TV screen printing system for patterning membranes and epoxies onto sensor surfaces can print with +/-5 micron alignment and 25 to 50 micron minimum feature size

Due to the multitude of electrical connections (wirebondmg of the sensor chip to the ceramic carrier, electrical connections at the zero insertion force socket, cabling to the potentiometric and amperometric circuitry, breadboard connections of the circuitry, and finally cabling to the analog-to-digital converter) there was a component of external electronic interference present in data transduced by the sensors Through the use of analog bandpass filters and oversamp ng techniques the noise was significantly reduced Additionally, the signals were digitally filtered during data analysis

Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number Full citations for the publications are listed below The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the

nature of words of description rather than of limitation

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Table 1.

Step Name Specification Comments

Starting Mateπal: 4" Si wafers <100> p-type 1 -3 Ω-cm

"RCA Procedure

• 100:1 H20:HF, 30 sec

Pre-Furnace

• 5-1 :5 H20:NH4OH:H202, 75 ° C, 10 mm Clean

• 6.1:1 H20 HCL.H2O2, 75 ° C, 10 mm

• Cascade Rinse until resistance peaks

The lower level oxide layer specifications are

TCA Oxide flexible

Lower • DWDTCA 5/200/5 mm (dry/wet/dry) 1100 ' C, Oxide 10 mm setting time Target. 1 2μm of SiQ2

Wafers exposed using new EV Aligner

• N1A 16 sec, default contact mode, devices under 6 μ not resolved

"Platinum (PT)" Mask • N1 B 16 sec, hard and vacuum contact

• 1827 PR, 4 sec resist, 4k RPM mode, better resolution

• 30 mm softbake, 90 ° C • N1 F 18 sec, default contact mode

Lithography

• 16 sec exposure (see Comments), 1.1 mm Thinner resist is probably necessary to resolve (Platinum) develop smaller features on mask

• hardbake Target. 2 7μm of PR

GaAs Bay Asher

Cleans the areas that will retain metal of any

Descum • 80 W, 250 mT, 1 m resist residue Target -300 nm of PR removed

This recesses the interconnect making it planar It also creates a break in the deposited metal to enhance lift-off The deeper etch for

Buffered HF Etch N1 F is to compensate for the thicker metal

Recess

• N1 A, N1 B 1 2 m etch, 5 mm rinse Dl deposited on it

Etch

• N1 F 5 4 m etch, 5 mm rinse Dl for Platinum Assumed etch rate 1 kA/min

Figure imgf000037_0001

Different metallization was used for the die to try and decrease interconnect resistance

• N1A, N1 B Ti/Pt 222/na A

EnerJet Evaporator Ti/Pt or Ti/Au/Ti/Pt • 1 F Ti/Ag/Ti/Pt 222/4009/206/1007 A

Interconnect

• Base Pressure 2(10)"8 T Deposition Target 0 2/1 kA or 0 2/4/0 2/1 kA

Figure imgf000037_0002

Resolved features are difficult below 8 μm

1112A Lift-off Ti/Pt Resistance measured as -800 Ω between pad

• Heat 10 m and sites

Lift-off • Apply 10 mm

• Ultrasonic 2 mm and repeat until pattern is '' ^Λ defined Table 1 (cont).

of low silane

end. Etch to compenclear con¬

PECVD nitride is very uneven gas flows PECVD

Figure imgf000038_0001
Table 1 (cont).

to leave a nitride around the

size this lift-off

because in that 11 2A visibly cause a DC rather random

Figure imgf000039_0001

REFERENCES

1 Kissinger, P T and Heineman, W R , Laboratory Techniques in

Electroanalytical Chemistry, Marcel Dekker, Inc 1984 2 H D Goldberg, R B Brown, D Liu, M Meyerhoff, "Screen printing a technology for the batch fabrication of integrated chemical-sensor arrays," Sensors and Actuators B, (21 ), 1994, pp 171 -183

3 Adams, R N Electrochemistry at Solid Electrodes, Marcel Dekker, Inc New York, 1969

4 Dryhurst, G , Kadish, K M , and Renneberg, R , Biological Electrochemistry, Academic Press, 1982 5 Baizer, M M , ed, Organic Electrochemistry, An Introduction and

Guide, Dekker, New York, 1973

6 Randies, J E B , Trans Faraday Society, 44 327, 1948 7 Bard, A J , and Faulkner, L R , Electrochemical Methods

Fundamentals and Applications, J Wiley and Sons, Inc , New York, 1980

8 Oldham, K , and Philip, R , Doing Things By Halves Semidifferentation in Voltammetry, current Separations, 9 3-5, 1989

9 Heineman, W R , and Kissinger, P T , Cyclic Voltammetry The Electrochemical Equivalent of Spectroscopy, Current Separations, 9 15-18, 1989 10 Hush, N S , ed , Reactions of Molecules at Electrodes, Wiley

(Interscience), New York, 1971

11 Rose, R C , The Ascorbate Redox Potential of Tissues A Determinant or Indicator of Disease? NIPS, 4 190-195, 1989

12 Wang, J , Pamidi, P V , Cepπa, G Basak, S , Rajeshwar, K , Overoxide poly(pyrrol-co[3-(pyrrol-1 yl)-propanesulfonatel])-coated platinum electrodes for selective detection of catecholamine neurotransmitters, Analyst, 122(9) 981 -4, 1997

13 Su, M T , Dunwiddie, T V , Gerhardt, G A , Combined electrochemical and electrophysiological studies of monoamme overflow in rat hippocampal slices, Brain res, 518(1 -2) 149-58, 1990

14 Runnels, P L , Joseph, J D , Logman, M J , and Wightman, R M ,

Effect of pH and surface functionalities on the cyclic voltammetric responses of carbon-fiber microelectrodes, Anal Chem, 71 (14) 2782-9, 1999 15 Brazell, M P Feng, J , Kasser, R J , Renner, K J , and Adams, R.N , An improved method for Nafion coating carbon fiber electrodes for in vivo electrochemistry, J Neurosci Meth , 22 167-172, 1987

16 Wiedeman, D J , Basse-Tomusk, A , Wilson, R L , Rebec, G b , and Wightman, R M , Interference by DOPAC and ascorbate during attempts to measure drug-induced changes in neostraital dopamine with nafion coated, carbon-fiber electrodes, J Neuroscience Methods, 35(1 ) 9-18, 1990

17 Wang, J , Rayson, G D , LuZ L , Wu, H , Coated amperometric electrode arrays for multicomponent analysis, Anal Chem 62(18) 1924-7, 1990

18 Cahill, P S , Walker, Q D , Fmnegan, J M , Mickelson, G E , Travis, E R , Wightman, R M , Microelectrodes for the measurement of catecholammes in biological systems, Anal Chem, 68(18) 3180-6, 1996

19 Adams, R N , and Marsden, C D , Electrochemical detection methods for monoamme measurements In Vitro and In Vivo In Handbook of Psychopharmacology, Vol 15, pp 1 -74, Eds L I Iversen, S D Iversen and S H

Snyder, Plenum Press New York, 1982

20 Dryhurst, G , Kadish, K M , Scheller, F , and Renneberg, R , Electrochemistry of Ascorbic Acid, In Biological Electrochemistry, Volume 1, pp 256-276, Academic Press, New York, 1982

21 Dryhurst, G Kadish, K M , Scheller, F , and Renneberg, R , Electrochemistry of Catecholammes, In Biological Electrochemistry, Volume 1 , pp 116-177, Academic Press, New York, 1982

22 Wmkler, J , and Fischer-Colbπe, R , The Chromogranins A and B The First 25 Years and Future Perspectives, Neuroscience, 49 497-528, 1992

23 Bassetti, M , Huttner, W B , Zanini, A , and Rosa, P , Co- localization of Sectetogranms/Chromogranms with Thyrotropm and Luteinizing

Hormone in Secretory Granules of Cow Anterior Pituitary, Journal of Histochemistry and Cytochemistry, 38 1353-1363, 1990

24 Huttner, W B , Gerdes, H H , and Rosa, P , The Granin (Chromogranin/Secretogranm) Family, Trends in Biochemical Sciences, 16 27-

30, 1991

25 Konecki, D S , Benedum, U M Gerdes, H H , and Huttner, W B , The Primary Structure of Human Chromogranin A and Pancreastatin, Journal of Biological Chemistry, 262 17, 026-17, 030, 1987 26 Scammell, J G , Granins Markers of the Regulated Secretory Pathway, Trends in Endocrinology and Metabolism, 4(1 ) 14-19, 1993

27 Hmkle, P M , Scammell, J G , and Shanshala E D , Prolactm and secretogranin-ll, a Marker for the Regulated Pathway, are Secreted in Parallel by Pituitary GH4C1 Cells, Endo, 130 3502-11 , 1992

28 Dryhurst, G , et al , 1982 29 Cho YA, Lee HS, Cha GS, Lee YT, "Fabrication of butyrylcho nesterase sensor using polyurethane-based ion-selective membranes," Biosens loelectron 1999 Apr 30, 14(4) 435-8

30 Mulchandani P, Mulchandani A, Kaneva I, Chen W, "Biosensensor for direct determination of organophosphate nerve agents 1

Potentiometnc enzyme electrode," Biosens Bioelectron 1999 Jan 1 ,14(1 ) 77-85

31 Campanella L, Colapicchioni G, Favero G, Sammartino MP, Tomassetti M, "Organophosphorus pesticide (Paraoxon) analysis using solid state sensors," Sensors and Actuators B 33(1996 25-33)

Claims

What is claimed is
1 An electronic device for monitoring analytical reactions and active
biological operations, comprising a support substrate, at least one amperometric sensor attached to said substrate, and at least one potentiometnc sensor located on said substrate with said amperometric sensor
2 The device according to claim 1 , wherein said support substrate being selected from the group consisting essentially of silicon, glass, and ceramic 3 The device according to claim 1 , wherein said amperometric sensor includes at least one reference electrode, at least one working electrode, and at least one counter electrode
4 The device according to claim 3, wherein said amperometric sensor cycles in concert with a cyclic voltammogram 5 The device according to claim 4, wherein said sensors cycle independently
6 The device according to claim 3, wherein said amperometric
sensor operates with a constant voltage
7 The device according to claim 1 , wherein said potentiometric sensor includes at least two reference electrodes.
8. The device according to claim 1 , wherein said amperometric sensor and said potentiometric sensor are in close proximity.
9. The device according to claim 1 , wherein said device is made of material selected from the group consisting of platinum, silver, iridium, and gold.
10. The device according to claim 1 , wherein said device is made using CMOS technology.
11. The device according to claim 1 , wherein said device is etched using a lift-off process.
12. The device according to claim 9, wherein said lift-off process is made by a reactive ion etch.
PCT/US2000/030265 1999-11-02 2000-11-02 Microscopic combination amperometric and potentiometric sensor WO2001033206A1 (en)

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