WO2002099410A1

WO2002099410A1 - Sensor device and method for indicating oxygen consumption

Info

Publication number: WO2002099410A1
Application number: PCT/US2002/017923
Authority: WO
Inventors: La Shaun Berrien; Gary Binyamin; Hilary S. Lackritz; Petr Vanysek; Mingqi Zhao
Original assignee: Aclara Biosciences, Inc.
Priority date: 2001-06-04
Filing date: 2002-06-04
Publication date: 2002-12-12
Also published as: WO2002099410A9

Abstract

A sensor device for measuring electrochemical, chemical, or biological oxygen demand in a sample is disclosed. The device includes a measurement chamber (14) having at least one chamber dimension of less than 1 millimeter. Also included in the device are working (56) and counter (60) electrodes, and possibly a reference electrode (58) adapted for electrical contact with liquid contained in the chamber. Measurement is accomplished under conditions of electron transfer to or from the components or intermediary to or from the working electrode (56), an electrical circuit connecting this electrode to the counter electrode (60) and possibly the reference electrode (58) and having a power source for applying a voltage potential between the working and counter electrodes, and possibly the working and reference electrodes. A current-responsive means is included in the circuit for measuring the instantaneous or time-averaged electrical current or for integrating the charge flowing through the circuit over a defined period of time, the current or charge being related to the total oxidizable or reducible bio/organic components contained in the sample with the measurement chamber.

Description

Sensor Device and Method for Indicating Oxygen Consumption

This patent application claims priority to U.S. provisional patent application serial number 60/295,908, filed on June 4, 2001 , which is incorporated herein in its entirety by reference.

Field of the Invention

The present invention relates to a method for measuring the electrochemical oxygen demand in a liquid sample, and to devices useful in practicing the method.

Background of the Invention

Oxygen demand is an important parameter for assessing the concentration of organic/biological compounds or components in water resources and in industrial process streams. Because the degradation of organic compounds often requires oxygen, their concentrations can be estimated by the amount of oxygen required to degrade them to a known chemical state. When this oxidation is carried out chemically, the value obtained is called chemical oxygen demand (COD). Biochemical (or biological) oxygen demand (BOD) is its counterpart, cited when the process is carried out using biological entities such as microbes. The conventional methods for COD and BOD determination involve tedious and time-consuming procedures. COD is preferred for estimating organic pollution, but its conventional evaluation methods have several disadvantages such as long analysis time, hazardous reagents that must be disposed of, and high probability of error due to complex procedures dependent upon operator skill. There exists a strong need for oxidizable species determination in the water, food and beverage, paper and pulp, and pharmaceutical industries. It would therefore be highly desirable to provide a device that provides semi-quantitative or quantitative values for oxygen demand that (a) is simple to use, (b) is easy to dispose, (c) is safe and convenient, (d) provides rapid results, (e) provides electrochemical oxygen demand (EOD) values that can be correlated to COD or BOD values, (f) is effective for samples containing between 10 and 15,000 ppm of oxidizable bio/organic material, and (g) is inexpensive.

The present invention is designed to meet these needs. Summary of the invention

The invention includes, in one aspect, a sensor device for measuring electrochemical, chemical, or biological oxygen demand in a sample. The sample may be a liquid. The device includes a measurement chamber having at least one chamber dimension of less than 1 millimeter. Also included in the device are first and second electrodes, designated the working and counter electrodes, and possibly a third electrode, designated the reference electrode, adapted for electrical contact with liquid contained in the chamber. The first working electrode is or contains a catalyst capable of catalyzing the electrochemical change of oxidizable bio/organic components, or a catalyst capable of producing an intermediary species capable of causing chemical change of one or more oxidizable bio/organic components, present in the measurement chamber as part of the analyte sample. Measurement is accomplished under conditions of electron transfer to or from the components or intermediary to or from the working electrode, an electrical circuit connecting this electrode to a the counter and possibly the reference electrode and having a power source for applying a voltage potential between the working and counter electrodes, and possibly a measurement circuit for measuring the potential between the working and reference electrodes. Current flow between working and counter electrodes through the sample solution is sustained by electron donation/acceptance to/from one or more sample components or intermediaries at the counter electrode and by the oxidation/reduction of said one or more bio/organic components at the working electrode. A current-responsive means is included in said circuit for measuring the instantaneous or time-averaged electrical current or for integrating the charge flowing through the circuit over a defined period of time, said current or charge being related to the total oxidizable or reducible bio/organic components contained in the sample within the measurement chamber.

In some embodiments or applications, chemical adjustments to the sample constitution, for example the ionic strength or pH level of the sample solution, may be necessary to provide rapid, accurate, and/or reliable measurement results. This can be achieved by external chemical treatment of the sample prior to its introduction into the measurement chamber. In a preferred embodiment, any necessary adjustment of sample ionic strength and/or pH is made by including dried chemical reagents in a channel or prechamber that is part of the integrated device and through which the sample must pass before reaching the measurement chamber. Said chemical reagents may be immobilized or contained directly in or on the surfaces of the channel or prechamber, or they may be immobilized on the surfaces of a support matrix material located within said chamber or channel. This support matrix may have a relatively high surface-to-volume ratio in order to provide rapid mixing and dissolution of said chemical reagents. Said support matrix may serve the additional function of the filtration of large particulate material from the liquid sample prior to its entrance into the measurement chamber. A separate particulate filter can also be located prior to the chemical reagent support material in some embodiments.

The device preferably has at least one chamber dimension between 50 and 500 micrometers. The sensor device may also include a cover that encloses the reaction chamber. An opening or a connecting input microchannel for introducing the liquid sample into the reaction chamber and a vent or connecting output microchannel can be present; the sample is moved into the reaction chamber following any chemical pretreatment and prior to analysis.

In a preferred embodiment of this device, the methods and components above yield the ability to generate quantitative measurement results rapidly for electrochemical oxygen demand, chemical oxygen demand, and/or biological/biochemical oxygen demand. The overall device operation and measurement time can total less than thirty minutes, or in other embodiments be as short as five to fifteen minutes. In one embodiment, results described below indicate that after 0.5 seconds, the measurement result from analyte oxidation is similar to results obtained at significantly longer times, but allowing the oxidation to continue improves the signal-to-noise ratio and hence the measurement reliablity.

In a preferred embodiment of this device, the methods and components above yield the ability to generate quantitative results for electrochemical oxygen demand, chemical oxygen demand, and/or biological/biochemical oxygen demand using a disposable, single-use platform.

In a preferred embodiment of this device, the methods and components above yield the ability to generate quantitative results for electrochemical oxygen demand, chemical oxygen demand, and/or biological/biochemical oxygen demand over the entire measurement range.

These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings. Brief Description of the Drawings

Figure 1 is a sectional view of a microfluidics device constructed in accordance with one embodiment of the present invention;

Figures 2a and 2b show two parts of a microfluidics device constructed in accordance with one embodiment of the present invention;

Figure 3 is an exploded view of a microfluidics device constructed in accordance with one embodiment of the present invention; Figure 4 shows the linear curve of concentration versus measured COD value, collected on a commercial system;

Figure 5 shows a linear concentration versus peak current value for an EOD measurement according to one embodiment of the invention;

Figure 6 shows the measured COD/EOD correlation for a commercial chemical COD versus an EOD according to one embodiment of the invention;

Figure 7 shows EOD measured values and a similar sample measured on a commercial system;

Figure 8 shows the charge versus concentration curves at various short times up to 0.5 seconds up to lOOOppm sample; Figure 9 shows a comparison of short time periods to the established five-minute test;

Figures 10 and 11 show cyclic voltammograms illustrating the decrease in concentration following galvanostatic oxidation;

Figure 12 shows the electrochemical titration point according to one embodiment of the invention.

Detailed Description of the Invention

I. Electrochemical Sensor Device

The invention includes, in one aspect, a device for measuring oxygen demand in a liquid sample. Fig. 1 is a sectional view of a device 12 constructed according to a preferred embodiment of the invention. In this embodiment, the device includes a reaction chamber 14 and a sample preparation chamber 16 in contact with two printed circuit board substrates 20 and 21. Circuit board substrates 20 and 21 are held at a fixed distance apart by a pressure sensitive adhesive spacer 28 and 29. In one embodiment, reaction chamber 14 and sample preparation chamber 16 each have a volume from about 1 μl to about 60 μl. Preferably, reaction chamber 14 and sample preparation chamber 16 each have a volume of about 5-10 μl. First and second electrodes (not shown) are printed onto circuit board substrates 20 and 21 and adapted for electrical contact with the liquid sample contained in the reaction chamber. A substrate 24, as illustrated in Fig. 1 , may be provided to house the components of the device 12. A via 26 that is adapted for air/fluid communication with reaction chamber 14 may be formed in substrate 24 and printed circuit board 20, thus providing means for venting the reaction and sample preparation chambers. In one embodiment, via 26 has a volume of about 0.8 μl. A second via 28 that is adapted for fluid communication with sample preparation chamber 16 may be formed in substrate 24 and printed circuit board 20, thus providing an inlet for the liquid sample to reach the sample preparation chamber. Device 12 may also include a circuit connecting the two electrodes. The circuit may include a power source 30 for applying a potential across the two electrodes; current flow in the sample can be sustained by electron donation or acceptance to one or more sample components at the second electrode and by the oxidation or reduction of one or more organic components at the first electrode. A current response element 32 may be included in the device for measuring the current flow over a given time or the total charge during a collection period in theelectrochemical cell, related to the total oxidizable or reducible components contained in the sample. The circuit may be fully integrated with the device and housed within substrate 24. Assembly of device 12 is accomplished by stacking the printed circuit board substrates with pressure sensitive adhesives 28 and 29 to form a sandwich-like structure. Alignment of the layers can be achieved using an alignment mark on the substrates to mark the positions of the pressure sensitive adhesives.

In a broader aspect, the device can be used for measuring sample BOD, COD, pH, nitrates, ammonia, phosphate, heavy metals, pesticides, and bacteria.

A. Material Substrates In the preferred embodiment, printed circuit boards 20 and 21 are used as the substrate for connections between the electrodes and the electronics subsystem. Traces consisting of 4 μm of Ni followed by 0.1 μm of Au are plated on the circuit boards according to the designated positions of the electrodes. Fluid input/output ports can be drilled through one substrate, such that the inlet port position allows for fluid contact with the electrode on the opposing surface and the outlet port position allows for ventilation of the chambers. The geometry of the drilled port may be compatible with a syringe for use in sample introduction. A recess can be machined in both halves of the boards to accommodate a 3V coin batter for power supply of all electronics. The connections between the electrodes and the electronics and all other inter-component connection may be formed via conductive traces on the circuit boards using standard industry techniques.

Assembly of the circuit boards follows standard industry techniques that include the use of solder paste to attach IC components to the circuit board with pre-plated traces. The boards may then be processed in a reflow IR oven, cleaned and tested.

See, for example, U.S. Patent No. 5,200,051 , which is incorporated herein by reference in its entirety.

In a separate embodiment, the device can be produced from plastic, ceramic

(including amorphous silicon), composites of plastic or plastic and ceramic, and other engineering materials.

B. Reaction and Sample Preparation Chambers

With reference to Figure 1, the preferred embodiment has a reaction chamber 14 and sample preparation chamber 16 that may be separated from each other by a constriction 34 that acts as a stop junction. Constriction 34 may include an opening 36 that allows the fluid sample in sample preparation chamber 16 to flow into reaction chamber 14 when a desired volume is reached. Likewise, opening 36 allows fluid communication between the reaction chamber 14 and sample preparation chamber 16.

Alternatively, the device may not include a constriction; thus, the reaction and sample preparation chambers function as one chamber.

The volume of the electrochemical cell and the exposed electrode area may be important for reproducibility. In the preferred embodiment, both of these parameters are defined by the thickness and the shape of the spacer, which is also used as an active bond between the two parts of the card, holding the opposing electrodes together. The spacer may be 0.1 mm to 0.7 mm thick. In a preferred embodiment, the spacer is 0.5 mm thick. The spacer may have a consistent thickness. The pressure used for sealing may be well controlled. Higher pressure may render the spacer thinner. Also, higher pressure may compress and expand the spacer, which upon releasing the pressure will relax and rebound. In one embodiment, the device includes a stop alignment feature. The stop alignment feature prevents lateral expansion and contraction, described below, which can compromise the function of the device. For example, following retraction, some of the adhesive of the spacer may stay behind on the electrode surfaces, thus rendering them smaller in surface area. In addition, in some spacer designs that have two compartments separated by a narrow channel, the channel may close upon applied pressure and upon its release it may never fully open.

In the preferred embodiment, a pressure sensitive adhesive is used as a spacer between the two circuit board substrates and as a means to form the volume of the reaction chamber. The desired area of the reaction chamber may be punched into a layer of the pressure sensitive adhesive via mechanical die, and the resulting mask may be used over the electrodes to form the reaction chamber. The pressure sensitive adhesive may be compatible with adhesion directly to the electrode surfaces.

In another embodiment, the reaction chamber and sample preparation chamber with connecting channel are formed, via machining, embossing, cold forming or other method common in manufacturing technology, into the device substrate material to a specified depth for control of chamber volumes. A hot melt adhesive or other material known to those skilled in the art may be used to bond the formed substrate to another substrate used to seal the device. Techniques such as thermal lamination, ultrasonic welding and other methods generally practiced in the art may be used to bond the device substrates.

In yet another embodiment, a lateral flow filter of optimal geometry and construction can be used to define the volume of the reaction cell. The filter can be sandwiched between the device material substrates and hold sample in contact with electrodes, thereby forming an electrochemical cell. The filter can also serve as the sample preparation mechanism by pre-loading with reagents, microorganisms or enzymes.

In an alternate embodiment, the device has a multi-chamber design. Two or more chambers may be advantageous for on-card addition of reagents to the sample, wherein a plurality of reagents for catalysis, conditioning, sensitizing, stability, etc. could be placed in compartments away from the electrodes. Thus, after mixing with the sample, it could be introduced into the reaction chamber. In addition, via 26 or via 28, or both, can contain immobilized microorganisms or enzymes that can degrade the organic analytes. Thereafter, the products of the predigestion can be introduced into said chambers.

C. Electrodes

In the preferred embodiment, the electrodes are screen printed onto the printed circuit boards over plated conductive traces described previously. The electrodes can detect organic compounds in the liquid sample. The electrodes may be flexible and modified to have the desired composition, resistance, and geometry, which is standard in the art. One or more integrated electrocatalysts may be provided on the electrode surface to catalyze the oxidation or reduction of organic compounds in the solution. In one embodiment the catalyst is a metal oxide. The metal oxide may be selected from the group consisting of Cu₂O, CuO, PbO₂, RuO₂, SnO₂, PbO₂, NiO, and TiO₂.

In another embodiment, the catalyst is a metal or metal oxide alloy. In yet another embodiment, the catalyst is a metal complex. The catalyst can be added to the working electrode by conductive ink in a controlled manner known to those of skill in the art. The reference electrode or counter electrode or both can be Ag/AgCI, Pt, Au, or other material known to those skilled in the art. The electrode layout can be designed to maximize the consumption or reaction time of organic compounds in the solution. The electrode layout can also be designed to yield the desired signal level.

In other embodiments, alternate techniques for depositing the electrodes, such as vapor deposition, photolithography, spin coating chemical formation and related techniques standard in the art may also be used.

Two or three electrodes may be used in this device, where the third electrode is used to minimize potential drift and solution resistance caused by excessive current through the reference electrode. As shown in Figure 3, the device in the preferred embodiment consists of a three electrode sandwich-structure device 50. Two substrates 52 and 54 sandwich the device 50. The electrodes may use conductive ink or conductive ink composites as the working electrode 56, reference electrode 58, and/or counter electrode 60. In one embodiment, the electrodes have conductive traces 64 and 65 in contact with the electrodes. Alternatively, the electrodes do not have conductive traces underneath. Figures 2a and 2b show two parts 70 and 72 of the device 50 following assembly.

The electrodes can be manually or automatically printed onto a plastic substrate for catalyst screening. Insulated wires can then be attached to the electrodes and cured. Holes can then be drilled through the counter electrode. D. Electrode Coating

In the preferred embodiment, the electrodes do not have a coating, as described in Example 2. Alternatively, the electrodes are provided with an electrode coating. The electrode coating is a permeation layer that provides spacing between the electrode surface and the liquid sample and allows solvent molecules, small counter-ions, and electrolysis reaction gases to freely pass to and from the electrode surface. It is possible to include within the permeation layer substances which can reduce the adverse physical and chemical effects of electrolysis reactions, including, but not limited to, iron complexes for O₂ and peroxides. The thickness of the electrode coating can range from approximately 1 nm to 100 μm, with 2 nm to 10 μm being the most preferred. Electrode coating permeation layers are discussed in U.S. Patent No. 6,238,624, which is incorporated herein by reference in its entirety.

In one embodiment, the purpose of the said coating layer can be to prevent or minimize electrode fouling.

The sentence above indicates the presence an antifouling layer which may or may not be overcoated on electrodes in all cases. Said layer does not significantly alter the heterogeneous electrochemistry or any associated chemistries and transport, but does prevent degradation in signal due to the inactivation of electrode surface area due to species in solution, products of the reaction, or other species formed by their combination.

E. Electronic Circuit Design

In the preferred embodiment, the electronic subsystem may consist of a potentiostat, an integration circuit, a microprocessor, a thermistor and a output display element. The function of the potentiostat is to maintain a constant potential across the electrochemical cell. The integration circuit builds up charge from current flow through the electrochemical cell onto a capacitor, which is then discharged and reset by the microprocessor after a specified voltage across the capacitor is reached. The microprocessor also counts the number of discharge/reset cycles and sums them to determine the total charge.

In one embodiment, a the selected microprocessor enables programming with multiple conversion algorithms and variable reaction time settings. The circuit can also contain an RS232 connection for download or upload of data to the microprocessor. The electronic subsystem of the device can include a thermistor that measures the temperature of the electrochemical cell during the reaction and calibration tables and correlation algorithms are used to process data and provide the appropriate output to the display driver for the desired output display. In a different embodiment, the reaction cell is maintained at a given temperature above room temperature using an on-chip resistive heater. This alleviates the need for calibration of the measured value for differences in temperature. This device may or may not have a differential amplifier contained in the circuit for amplifying the current difference produced in the two or more reaction chambers in proportion to the difference in total oxidizable or reducible components contained in the two or more reaction chambers.

F. Output Display Element

In the embodiment shown in Figure 1 , the electronic circuit is integrated with the printed circuit board to form a handheld device that can indicate the content of the organic compounds or dissolved oxygen concentration in the sample. Thus, the device consists of a complete functional unit with no secondary hardware. The indicator can be based on the concept of measuring current or integrating current over time, i.e., measurement of charge. In one embodiment, the indicator is an output display element. This output display element can be in the form of a light emitting diode (LED display), a liquid crystal display (LCD display) or a range selection meter.

In another embodiment, the indicator can be based on colorimetric methods. In one embodiment, the electrochemically generated current, after amplification in a current amplifier, can be routed through a wedge-shaped resistive foil heater. As the resistance of the heater varies with distance, the most resistive tip of the wedge will heat to higher temperature than the wider end. A heat sensitive dye strip may be placed on top of the heater. Upon temperature increase to a certain value the color of the dye changes; the higher the current, the farther on the wedge heater the temperature will reach the dye color trigger point. Thus, current in the system will be proportional to the length of the discolored dye strip.

In yet another embodiment, the indicator can be based on electrochemistry. In one embodiment, the electrochemically generated current, after amplification, is routed through a separate charge measuring electrochemical reaction cell. One electrode, the anode, may be a film of a metal, which can be easily electrochemically dissolved, layered on a transparent window. The metal can be, for example, copper or silver. The second electrode may be near the window, but placed so that the window is not obstructed. The cell is filled with a solution, either prefilled, or derived from the aqueous test sample. The metal film consists of steps of increasing thickness. As current passes through the thin electrode, its surface is gradually removed, eventually removing the thinnest step first, entirely. Further current flow will remove the next steps of the deposited metal. The window denudation will result in appearance change from that of a shiny mirror surface to a dull and dark color of the material inside the electrochemical charge measuring electrochemical reaction cell. Thus, the indicator connected with the amplifier directly gives out the level of the COD or BOD in the solution. The final data may be given in numbers or as levels, such as "very good", "moderately clean," "polluted," or "very polluted."

G. Fluid Delivery Method In one embodiment, fluid delivery to the reaction and sample preparation chambers can be achieved via mechanical actuation. In the preferred embodiment, actuation can occur via injection syringe.

In other embodiments, fluid delivery to the reaction and sample preparation chambers can be achieved via more novel methods using wicking and capillary action, diaphragm mechanism or other positive displacement pumping mechanism.

H. Device Enclosure or Package

The device may have an enclosure or package. This enclosure can be made from any of the class of polymer or plastic materials that are economically molded or formed. The enclosure will be formed such that the electrochemical sensor device can be placed inside for the protection of the electronic components from the environment. The enclosure will have inlet and outlet vias that correspond and accurately align with those on the device. Silicone gasket material can surround these vias, so as to create a fluid tight seal at the inlet and outlet ports. The enclosure will have a backing that can be attached by standard fastening techniques. The enclosure may also have a transparent window aligned over any output display element, such that the display can be visualized through the enclosure.

In a separate embodiment, the electrochemical cell and the electronics subsystems and output display elements are separate entities. In this embodiment, the material substrates, reaction and sample preparation chambers and electrodes with conductive lead make up a single disposable device. A semi-permanent handheld device houses the electronics and output display elements. These devices can be integrated into a single sensor device for the measurement of EOD, COD or BOD. Each disposable electrochemical cell would perform a single or single set of measurements and be discarded while the handheld electronic device would be used for analysis of numerous cells.

II. Oxygen Demand Measurement Method In one aspect, the invention includes a method for measuring the electrochemical, chemical, or biological oxygen demand in a liquid sample. The first step of the method involves adding the sample to the reaction chamber to fill the reaction chamber in the device with the sample. In one embodiment, the sample is injected, via syringe, into the reaction chamber through the inlet port and air is expelled through the outlet. A syringe may be used to introduce the liquid sample into the sample preparation chamber or reaction chamber via channel 26 or channel 28, and air is thus expelled out through channel 26 or channel 28. In one embodiment the syringe incorporates a means for pH adjustment and filtration of the sample. Alternatively, means for sample pH adjustment and filtration are incorporated into device 12. In an alternative embodiment sample is introduced into the cell and possibly mixed with previously deposited reagents using a lateral flow membrane and wicking through the matrix. These filters can be made out of a variety of materials which are stable in the presence of the needed reaction reagents and known to people skilled in the art. In another embodiment, the cell is designed such that liquid is introduced into the cell via capillary action without the aid of a filter, having cell dimensions, channel dimensions, and possibly surface treatments known to those skilled in the art.

In a preferred embodiment, the sample resides in the cell/chamber or cells/chambers previously defined, containing electrodes on a substrate joined with an adhesive layer. In another embodiment a single catalyst or plurality of catalysts may be embodied in each and/or all chambers to allow for maximum oxidation/reduction efficiency leading to enhanced device performance. In yet another embodiment the catalyst(s) may be side-by-side, in different or the same region of the device, or employed as interdigitated electrodes. Multiple catalysts may be used in a single array unit. In a preferred embodiment the measurement occurs over the specified time period and output is obtained. In one embodiment, the sample volume is between 1 and 10 μl. In a preferred embodiment, the sample volume is 5 μl. In one embodiment, the sample is diluted in the device. On-board sample dilution allows the concentration range to be expanded. In another embodiment, the surfaces exposed to the sample are treated with a surface treatment.

In an embodiment, amperometry is used as described in the embodiment below. Additionally, potentiometry and/or impedence measurements (which may or may not require electron transfer) may be used with equal benefit and result. In a preferred embodiment potential can be applied across the electrodes, wherein current flow in the sample is sustained by electron donation to one or more sample components at the second electrode and by the oxidation of said one or more organic components at the first electrode. The total current flow in the circuit is then measured and integrated over time to yield charge obtained from oxidation of sample in 5 minutes, which is related to the total oxidizable components contained in the sample. An algorithm, as described in Example 2C may be used to correlate the electrochemical signal to the COD or BOD values measured using commercial instruments. In one embodiment the measured electrochemical signal is current. In another embodiment, the electrochemical signal measured is charge. In one preferred embodiment, measurement results can be obtained between 5 and

30 minutes following sample injection. In another preferred embodiment, sample results may be obtained between 10 and 20 minutes. In yet another preferred embodiment, sample results may be obtained in less than 15 minutes. The range of the detection can be from 10 to 15,000 ppm. In one embodiment, the range of detection is from 10 to 15,000 ppm. In another embodiment, the range of detection is between 10 and 3,000 ppm.

In one preferred embodiment, the electrode-containing cell may be disposed of, with the residing sample, when the measurements are complete while retaining the electronics component "handle". In a separate embodiment, the electrode-containing cell is incorporated into a system containing integrated electrodes, all of which, including the residing sample, would be disposed of when the measurements are complete. In one embodiment, an enzyme, or mixture of enzymes may be used, either in free solution or immobilized in channels (via beads, patches, sol-gels, hydrogels, etc known to those skilled in the art). The enzyme system would be capable of converting (through a variety of well-known reactions including, e.g. oxidation, reduction, hydrolysis, etc.) some or all analytes in solution from non-electroactive or analytes in solution with little electroactivity into electroactive species making the device more sensitive and capable of indicating more analytes.

In another embodiment, a microorganism, or mixture of microorganisms, in a variety of states, e.g. active, inactive, killed, lyophilized, dormant, or the like, either in free solution or immobilized in channels, e.g. via beads, patches, sol-gels, hydrogels, etc. as is known to those skilled in the art. The microorganism system would be capable of converting completely or partially, e.g. through digestion, some or all analytes in solution from non-electroactive analytes or analytes with little electroactivity into electroactive species making the device more sensitive and capable of detecting more analytes.

An additional embodiment allows for a partitioning of an analyte from an aqueous phase to an organic liquid or organic solid phase containing electrolyte and previously described electrodes. The aprotic solvent allows for an increased potential window during electrochemical measurements, alleviating possible interference of water oxidation in aqueous solvent and the ability to oxidize a broader list of analytes.

Yet another embodiment allows for the generation in situ of oxidizing reagents for the chemical oxidation of analytes in solution. The appropriate reactants would be in solution and species such as, but not limited to, hypochlorous acid or ozone can be generated electrochemically and then go on to react with organic analytes in solution.

From the foregoing, it can be seen how various objects and features of the invention are met.

III. Examples The following examples further illustrate the invention described herein and are in no way intended to limit the scope of the invention.

Example 1. Comparable Performance of the AquaCard Device with Established Industry Methods

Results obtained with the SP2 device described below were compared to COD results obtained with the commercial Orbeco-Hellige 975MP spectrophotometer in combination with the Accu-test EPA approved micro-COD reagent vials, both purchased from Bioscience, Inc. (Bethlehem, PA). The sample consisted of a mixture of glucose, sucrose, glycerol, glycine, and EDA. Figure 4 shows the linear curve of concentration versus measured COD value, collected on the Bioscience system. Briefly, 2.5mL of sample was added to the commercially available reagent tubes and incubated at 150°C for 2 hours. The COD value of the sample was then measured on the appropriately calibrated Orbeco-Hellige reader. Figure 5 shows a linear concentration versus peak current value. The currents reported are the peak currents obtained during a cyclic voltammogram for the sample in a SP2 cell. The peak current is the maximum current obtained during a potential sweep of the sample in contact with said electrodes and is a common electrochemical technique. In Figure 6 the measured COD data is related to the peak current. The graph shows a linear correlation between the measured, EPA- approved COD value and the EOD measurement.

Example 2. Calibration of EOD Data.

A. Preparation of the electrochemical cells

A generation of electrochemical cells (SP2) was prepared. All the cards used were prepared in the same controlled manner to give validity to the further statistical treatment needed to obtain calibration curves. The working electrode was screen printed carbon ink containing an electrooxidation catalyst which is an active catalytic material. Both the counter and reference electrodes were screen printed strips of silver/silver chloride, a material well suited both for making conductive paths and as a steady potential reference electrode. The working electrode and counter/reference pair were printed on different circuit board pieces and assembled facing each other, separated by a defined spacer. This spacer determines the volume of liquid, which is in immediate contact with the electrodes. The shape of the spacer is the "snowman" design.

B. Solution introduction, measurement method and device performance The calibration solution was introduced to the cell through a predrilled hole in the card by a syringe. All external conditions were kept identical for the entire test. The method for obtaining information about a sample is based on electrochemical oxidation of the compounds at the working electrode. The total charge flowing through the system in the course of 5 minutes is recorded and can be correlated to the conventional EPA- approved COD value measured for the sample using the Bioscience, Inc. reagent vials and an Orbeco-Hellige 975MP spectrophotometric reader.

Four compounds (mannitol, glucose, glycerol and glycine) were used in the calibration study, either alone or as a binary mixture. For the single compounds, eight concentrations were measured: 20, 50, 100, 200, 500, 1000 ppm. In addition, a blank, i.e., no analyte (0 ppm) was also measured. The solutions were prepared with the appropriate reagents added prior to introduction into the cell. Each compound and each concentration were measured three times, always on a fresh card, which was not previously exposed to any solution. The approach eliminates any previous electrode contamination, closely reflects the intended use of the AquaCards, and shows card-to- card repeatability. Figure 7 shows the EOD measured values (purple) and the similar sample measured on the Bioscience system (blue). The established correlation curve was derived as described below.

C. Calibration calculation To perform the calculation it was first necessary to convert the concentration of the test compounds into the corresponding theoretical values of COD. The derivation of the calibration algorithm below is based on data obtained from electrochemistry of single compounds.

The purpose of the algorithm is to find an "average" function that converts the measured charge to a value of COD. Because the sensitivities for different compounds are not exactly the same and the experimental data have a degree of scatter, an exact relationship for the conversion is not possible. Instead, a calibration curve is calculated that fulfills the following conditions: (a) it is a monotonic function; and (b) the sum of errors for all points, measured as their distance from the calibration curve, is minimal. Several minimization approaches can be used; however, minimization of weighted sum of squares of the errors was selected for the final calibration. The curve to which the data points were fit was a second order polynomial. The analytical form of the line is:

C_cod = 5.31 x 10⁵ Q² + 1.13 x 10⁴ Q - 14.095 where C_∞d is the calculated concentration in units of ppm COD and Q is the measured charge that has passed through the cell under the standard experimental conditions. The conditions in this test were applied potential 0.5 V Ag/AgCI and duration 300 sec (5 minutes).

The actual calibration curve used for converting measured charge to discrete ppm values for the LED display is shown in Table 1. The conversion was discrete at this phase because the electronic display itself is discrete, showing ranges in logarithmic steps. These values will be entered in the lookup table of the microprocessor.

Table 1.

Example 3: Multiplicity of Methods

A. Chronocoulometric Consumption. Evaluating Measurement Time Analysis of the established five minute charge collection method was performed. Time periods from 0.01 sec to 600sec were graphed (Figs. 8 and 9). Fig. 8 shows the charge v. concentration curves at various short times up to 0.5 seconds up to lOOOppm sample. Fig. 9 shows the comparison of short time periods to the established five- minute test. These results show that after 0.5seconds, the effects of analyte oxidation are similar at all times but the longer the oxidation time the larger the signal-to- background ratio.

B. Galvanostatic Measurements The technique of monitoring potential at a constant current as a possible alternative to the current method was investigated. The established sp2 cells described above were used. Similar to the chronoamperometric technique , the galvanostatic method oxidizes glucose in solution (Figs. 10 and 11 ). The cyclic voltammograms (Figs. 10 and 11 ) show the decrease in concentration in the form of a decrease in peak current from the initial filling (red) and the final scan (blue) after galvanostatic oxidation.

There is evidence that galvanostatic oxidation can yield desired dynamic ranges similar to the chronoamperometric methods, forcing a current through the cell while monitoring potential until a titration point is reached. The titration point represents the exhaustion of one analyte, the sample, to an analyte with a higher oxidation potential, in this case water. The point is the time at which the analyte in solution is exhausted and to maintain current values a species with higher potential must be oxidized. It is commonly called the electrochemical titration point (shown in green in Fig. 12 for 10OOppm glucose). Lower concentrations of glucose which had been exhausted earlier in the measurement come to a similar potential value for the desired current flow near the oxidation point of aqueous solutions.

Claims

IT IS CLAIMED:

1. A sensor device for measuring chemical or biological or biochemical oxygen demand in a liquid sample, comprising a measurement chamber having at least one chamber dimension of less than 1 millimeter; working and counter electrodes adapted for electrical contact with liquid contained in the chamber; coating or comprising said working electrode, a catalyst capable of catalyzing the electrochemical change of one or more oxidizable or reducible bio/organic components in the measurement chamber, under conditions of electron flow to the components from the first electrode; a circuit connecting the two electrodes and having a power source for applying a voltage potential across the two electrodes, wherein current flow in the sample is sustained by electron donation to one or more sample components at the second electrode and by the oxidation of said one or more organic components at the first electrode, and a current-response element in said circuit for measuring the total current flow in the circuit, related to the total oxidizable components contained in the sample.

2. The sensor device of claim 1 , wherein said at least one chamber dimension is between 50 and 500 micrometers.

3. The sensor device of claim 1 , further including a cover that encloses the reaction chamber, and provides an opening for introducing the liquid sample into the reaction chamber, which then is carried by capillarity through the reaction chamber.

4. The sensor device of claim 1 , wherein said electrodes are coated with an electrolytic coating which allows ion flow between the electrode and sample fluid, but prevents direct contact of the organic sample components with the electrodes.

5. The sensor device of claim 1 , wherein said current-response element comprises a wedge-shaped resistive foil heater capable of generating heat in proportion to said current; and a heat sensitive dye strip positioned on said heater for displaying a plurality of colors corresponding to the generated heat.

6. The sensor device of claim 1 , wherein said current-response element comprises a charge measuring electrochemical reaction cell having a transparent window; an anode disposed on said window, said anode being composed of a metal film of increasing thickness which is capable of dissolving in proportion to said current; and a cathode positioned in said reaction cell.

7. The sensor device of claim 6, wherein said metal film is selected from the group consisting of copper and silver.

8. The sensor device of claim 1 for use in chemical oxygen demand, wherein the response element is responsive to current from a single sample-containing reaction chamber.

9. The sensor device of claim 1 for use in measuring the biological oxygen demand in a sample, further comprising a second reaction chamber; third and fourth electrodes adapted for electrical contact with the liquid sample contained in the second reaction chamber; and a differential amplifier contained in the circuit for amplifying the current difference produced in the two reaction chambers in proportion to the difference in total oxidizable components contained in the two reaction chambers.

10. The sensor device of claim 9, which further comprises an electrolytic coating on the electrodes which allows ion flow between the electrodes and sample fluid, but prevents direct contact of the organic sample components with the electrodes, and coating said third electrode, an enzyme capable of catalyzing the oxidation of one or more oxidizable organic components in the second reaction chamber, under conditions of electron flow to the components from the third electrode.

11. The sensor device of claim 9, which further comprises an electrolytic coating on the electrodes which allows ion flow between the electrodes and sample fluid, but prevents direct contact of the organic sample components with the electrodes, and coating said third electrode, a microorganism capable of catalyzing the oxidation of one or more oxidizable organic components in the reaction chamber, under conditions of electron flow to the components from the third electrode.

12. A method for measuring chemical or biological oxygen demand in a liquid sample, comprising adding the sample to the reaction chamber of claim 1 , thus to fill the reaction chamber in the device with the sample, applying a voltage potential across the two electrodes, wherein current flow in the sample is sustained by electron donation to one or more sample components at the second electrode and by the oxidation of said one or more organic components at the first electrode, and measuring the total current flow in the circuit, related to the total oxidizable components contained in the sample.