WO2014118539A1 - Measuring solutions for an electrochemical detection system - Google Patents

Abstract

Provided herein are methods for performing an assay. The assay comprises a first measuring solution comprising a substrate and a first group of cofactors; a second measuring solution comprising the substrate and a second group of cofactors; at least one sensor; and an enzyme that can specifically react with the first and second measuring solutions. The method comprises contacting the at least one sensor with a sample comprising an analyte and at least one standard solution. A first group of the at least one sensor is incubated in the first measuring solution, and a second group of plurality of sensors is incubated in the second measuring solution. From these methods, a calibration curve for each the first and the second measuring solution is obtained. The assay may be an immunoassay, an enzymatic, sandwich, or competitive assay. Components of the assay may be selected based on the assay's sensitivity, measuring range, and differentiation.

Description

MEASURING SOLUTIONS FOR AN ELECTROCHEMICAL DETECTION

SYSTEM

CROSS-REFERENCE

The present disclosure claims priority to U.S. Provisional Application Serial Number 61/758,576 filed January 30, 2013, and entitled "Substrate for an

Electrochemical Detection System," which is incorporated herein by reference in its entirety.

FIELD

The present disclosure generally relates to an electrochemical detection system for conducting electrochemical analysis, and more particularly to an electrochemical detection system that can perform one or more electrochemical analyses using various measuring solutions, or tailoring the properties of measuring solutions to enhance sensitivity and extend the detection range by manipulating the system response to the required detection range concentration of a targeted analyte. This method not only improves the outcome, but makes it possible to perform multiple test formats using sensor arrays by sequentially changing measuring solutions.

BACKGROUND

Electrochemistry is a branch of chemistry that studies chemical reactions occurring in a solution at the interface of an electrode and an electrolyte. The reaction can involve charge transfer between the electrode and the electrolyte. For example, the electrode may comprise a metal or a semiconductor.

Under some circumstances, the chemical reactions discussed above may be driven by applying either an externally derived voltage or a voltage created by the chemical reaction. Under these circumstances, these chemical reactions are known as electrochemical reactions. Moreover, some chemical reactions where electrons are transferred between one or more molecules are known as oxidation / reduction reactions or redox reactions. Generally, electrochemistry relates to situations where oxidation and reduction reactions are separated in space or time and are connected by an external electric circuit that may be used to control or quantify the reaction.

Some electrochemical analyses can be undertaken in a disposable cartridge that may include a reagent for inducing electrochemical reactions controlled, monitored, detected, or quantified by one or more sensors. Some conventional cartridges may be configured to operatively engage a reader device that initiates a protocol, such as via the mechanical actuation of the cartridge. Furthermore, the reader device may receive data signals to produce test results of the reaction occurring within the cartridge.

For example, in the case of some conventional redox reactions for use with a conventional system, such as one employing or not employing a cartridge, one or more portions of the system may be coated with an enzyme, such as peroxidase, or a protein complex comprising an enzyme for use in the reaction. Moreover, in the same system, one or more enzyme substrates may be stored for use in the redox reactions. In particular, in the case of the peroxidase enzymes, hydrogen peroxide or analogues such as hydrogen perborate may be used as a conventional substrate. At least some conventional substrates exhibit a limited capacity for detection. Accordingly, cofactors, such as hydrogen donors (H-donors) for peroxidase reaction, for example o-phenylenediamine, 3,3',5,5'-tetramethylbezidine (TMB), and the like, are employed to make reaction optically or electrochemically detectible. Normally, for peroxidase- based reaction a single cofactor/H-donor is employed for detection, and other compounds may be formulated to obtain the best possible results. Sometimes sufficient results cannot be obtained using the same measuring solution, so some substrate solutions may be formulated with different amounts or ratios of the same cofactor and substrate, such as Fast and Slow TMB, which is particularly challenging for multisensory systems containing several sensors designed for various assays. Moreover, if the multisensory system contains assays of different nature, such as immunoassays and enzymatic assays, the substrate for immunoassays may be an analyte in enzymatic assays.

SUMMARY

The disclosure provides a method for adjusting the sensitivity and range in enzyme-based electrochemical assays, including immunoassays and enzymatic assays. The response of the sensing element may be tailored to particular assays or types or groups of assays by formulating the measuring solution consisting of at least one cofactor or substrate, in particular cofactors such as hydrogen donors for peroxidase-based assays. The disclosure also provides a method for the sequential introduction of measuring solutions into a multisensory system, allowing the sequential collection of results from sensors prepared for various assays of different nature, such as immunoassays and enzymatic assays. At the measuring step, the same substrate may be combined into one multisensory system, such as a fluidic cell.

The method adds a tool or dimension to assay development other techniques, helping to reduce the incubation times and to achieve a wider dynamic range, better sensitivity, or differentiation within required range. Although peroxidase is used in description and examples, the method is not limited to peroxidase and maybe applied to other enzymes, substrates or cofactors.

The potentiometric determination of the reaction may be achieved using an enzymatic label in sandwich or competitive immunoassays. In this format, a generated potentiometric response is directly (sandwich) or inversely (competitive) proportional to the amount of enzyme-label attached to the surface of the sensing element as a result of immune-reaction. The measuring solution is a substrate solution comprising a substrate or a cofactor formulated to achieve a response in the measuring range. Each cofactor, substrate, and enzyme contributes to the change of potential of the sensor during an enzymatic reaction.

The method includes choosing the cofactor or a combination of cofactors for the potentiometric response in a particular assay. The method includes one, two, or more constituents with different electrochemical properties at various ratios in the measuring solution. For example, component A enhances lower range of the calibration curve in sandwich assay, while component B increases an overall dynamic range. Formulations with solely or mostly component A would enhance the sensitivity in sandwich assays for a lower measuring range, while the use of solely or mostly component B prevalent would enhance sensitivity in competitive assays where the signal is reversely proportional to concentration of the enzymatic label on the surface. A particular combination of A and B would enhance the response for the range in particular assays. More than two components may be used together. Two solutions with the different properties may also be introduced consecutively, which would allow, for example, improved performance in a cartridge containing at least one sensor prepared for sandwich, competitive, and enzymatic assays within one fluidic cell. In enzymatic assays one or more enzymes may be attached/immobilized on the surface of the sensing element by various means, such as biotin-streptavidin interaction, covalent-binding, passive adsorption, and the like. In this format, a generated potentiometric response is proportional to amount of substrate, which can be either an analyte itself or a product of reaction between the analyte and other reagents in reaction mixture. While such an enzymatic response can be measured on its own, the response can be enhanced and the measuring range can be tailored by components, which undergo electrochemical changes as a result of enzymatic reaction. For example, in case of peroxidase attached to the surface, the enzymatic cascade is constructed to achieve production of peroxide, which reacts with peroxidase and H-donors undergoing electrochemical changes. Such electrochemical change results in enhanced potentiometric response of the sensing element. The magnitude of that response can be tailored to the assay by introducing a specific H- donor at a specific concentration, or by the combination of two or more H-donors at specific ratio.

This method for conducting such assays is unlike other systems employing enzymatic cascade reactions in a bulk solution and measuring the result of the change in the bulk solution or integrating the enzyme or other reaction constituents within the electrode, which uses specific modifications of the sensing element for each single enzymatic assay. In immobilizing the enzyme onto the surface of the sensing element, the reaction occurs on the surface. Immobilizing the enzyme rather than integrating it or other reagents within the sensor allows the properties of the sensing electrode to be left unchanged, and does not use specific preparation or modification of the electrode itself. The close proximity of the enzyme to the sensing element significantly improves results.

Moreover, in some embodiments, the enzyme may be at least one of a peroxidase (e.g., horseradish peroxidase), an oxidase, a laccase, or a catalase. In one aspect, solutions A and/or B may include one or more hydrogen donors, such as, but not limited to, ferulic acid, a ferulic acid derivative, 2-naphthol, ^-iodophenol, p- coumaric acid, a fluorescent dye, a dye, or other chemical, which electrochemical state changes significantly as a result of the enzymatic reaction. In addition, the same concept may be similarly applied to other, conventionally used reduction/oxidation reaction reagents, such as electron donors, generally used as single reagent, rather than in combination with other similar reagents.

An electrochemical detection system may include a platform for performing an assay protocol. The platform includes at least one sensor that is at least partially disposed to fluids and the platform also includes at least one fluid source. In one aspect, at least some sensors may be coated in a receptor material, such as antibodies, streptavidin, and the like. Each sensor may be prepared to target a different analyte. In particular, a first fluid source may contain an analyte (e.g., blood or a blood component). A second fluid source may contain a washing fluid or a reagent. Next, a third fluid source may contain an enzyme solution that includes an enzyme-labeled constituent or an enzyme, such as a peroxidase, an oxidase, a laccase, or a catalase. In some embodiments, the enzyme may or may not be biotinylated. In addition, a fourth fluid may contain a measuring Solution A. The Solution A may include at least one relative enzymatic substrate or enzyme cofactor selected based on measurement sensitivity and measurement range of the assay protocol. In one aspect, in peroxidase-based assays the at least one cofactor, also known as hydrogen donors, may include at least two of ferulic acid, a ferulic acid derivative, 2-naphthol, p- iodophenol, ^-coumaric acid, a fluorescent dye, and a dye.

In one aspect, the platform may include a fifth fluid source, for example a Solution B including at least one cofactor or substrate. In some aspects, the cofactor or the substrate in the Solution B may be different than the at least one cofactor or substrate in the Solution A, or they may be the same or substantially similar to at least some of components in Solution A, but present in different ratios. Moreover, Solution A may be a sample containing a target analyte, and the analyte may be a substrate or a cofactor for one the enzymes used in the system. Solution B may contain a substrate or a cofactor to another target analyte measured in the subsequent step in the same system by replacing Solution A.

Most substances used in examples, in particular enzyme cofactors, do not produce color change, which is not required for an electrochemical system. Some are used in enzyme-based applications. Any substance used for altering the

potentiometric response using the method described herein is part of this disclosure. Additional objectives, advantages and novel features will be set forth in the description which follows or will become apparent to those skilled in the art upon examination of the drawings and detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is simplified block diagram illustrating the different components of the electrochemical detection system.

FIG. 2 is a simplified illustration showing the cartridge and reader

arrangement.

FIG. 3 is a graph showing calibration curves from experiments using a measuring solution for a wide range of applications (Solution A) and a measuring solution developed for sandwich-type immunoassays with a high sensitivity (Solution B). Both measuring solutions contain multiple hydrogen donors.

FIG. 4 is a linear graph of the data of FIG. 3, displaying a lower portion of the calibration curve with improved sensitivity seen in the conditions. The assay includes Solution B.

FIG. 5 is a graph showing calibration curves from experiments employing the competitive assay using measuring solutions with different cofactors for peroxidase (hydrogen donors), most of which contain multiple hydrogen donors.

FIG. 6 is a graph showing calibration curves from experiments using different cofactors (hydrogen donors) in an enzymatic assay.

FIG.7 is a graph showing two calibration curves for two different assays ran concurrently— immunoassay and enzymatic assay— obtained from multisensory system contained within a single fluidic cell in less than 10 min.

Corresponding reference characters indicate corresponding elements among the view of the drawings. The headings used in the figures should not be interpreted to limit the scope of the claims.

DETAILED DESCRIPTION

Referring to the drawings, an embodiment of the electrochemical detection system is illustrated and generally indicated as 10 in FIG. 1. The electrochemical detection system 10 provides a means for conducting at least one assay protocol on a multiplexed sensor format when operatively engaged to a reader 14. Moreover, in some embodiments, the electrochemical detection system 10 may be configured and arranged so that the at least one assay protocol has a dynamic measurement range and sensitivity. In some embodiments, at least one hydrogen donor may be selected for use during the performance of the assay protocols to provide a range and sensitivity. In another embodiment, the at least one hydrogen donor may be selected for use with certain enzymes and target molecules, as described in greater detail below.

Some embodiments of the electrochemical detection system 10 may operate in a generally similar manner all embodiments of a flow channel and fluidics control component, such as disposable cartridges, but with different physical configurations.

In yet other embodiments, one or more sensors may be used in conjunction with other equipment. For example, the steps may occur in a reaction vessel, such as a microtiter plate, a cell culture vessel, a test tube, or other laboratory equipment, for example a flask, beaker, or the like. The sensors may be contacted with the test solution automatically or manually. Moreover, the test solution may also be added to the sensors. Regardless, the sensors may be employed with any equipment and experimental plan to meet the needs of the user.

In some embodiments, some portions of the electrochemical detection system 10 can be prepared before use to detect electrochemical reactions. For example, one or more of the reagents used by the electrochemical detection system 10 may provide reduced incubation times and achieve wider dynamic range, increased sensitivity, or increased differentiation. Accordingly, some embodiments provide a method for optimizing one or more assay protocols through selecting one or more reagents that have been previously demonstrated to afford a user increased range, sensitivity, or differentiation for a particular assay protocol.

In some embodiments, the sensitivity and range of one or more of the electrochemical assays that can be employed with the electrochemical detection system 10 may be adjusted or optimized by tailoring the response of the sensors. In one aspect, the response of the sensors, such as the potential measured by the sensors, may be tailored for certain assays through carefully selecting the measuring solution (i.e., substrate) used in the final steps of the assay. For example, at least one hydrogen donor or cofactos may be selected for a particular measuring solution for use with a certain enzyme, such as a peroxidase, a laccase, an oxidase, a catalase, a urease, a kinase, a dehydrogenase, and a deiminase in an assay protocol. Suitable peroxidases include, but are not limited to, horseradish peroxidase (HRP), deiodinase, such as iodothyronine diodinase and iodotyrosine deiodinase; eosinophil peroxidase, glutathione peroxidase, such as GPX 1, GPX 2, GPX 3, GPX 4, GPX 5, GPX 6, GPX 7, and GPX 8; haloperoxidase, myeloperoxidate (MPO), hemoprotein, peroxiredoxin, thyroid peroxidase, vanadium bromoperoxidase, and lactoperoxidase. Suitable oxidases include, but are not limited to, laccase, glucose oxidase, monoamine oxidate (MAO), cyctochrome P450 oxidase, NADPH oxidase, xanthine oxidase, L- gulonolactone oxidase, and lysyl oxidase. Suitable kinases (also referred to as phosphotransferases) include, but are not limited to, OH acceptor kinases, such as hexokinase, glucokinase, fructokinase, hepatic fructokinase, galactokinase, phosphofructokinase 1, phosphofructokinase liver-type (PFKL), phosphofructokinase muscle-type (PFKM), phosphofructokinase platelet-type (PFKP),

phosphofructokinase 2, riboflavin kinase, shikimate kinase, thymidine kinase, ADP- thymidine kinase, NAD+ kinase, glycerol kinase, pantothenate kinase, mevalonate kinase, pyruvate kinase, deoxycytidine kinase, fructose-6-phosphate 1- phosphotransferase (PFP), diacylglycerol kinase, phosphoinositide 3 kinase (Class I PI 3, abd Class II PI 3), sphingosine kinase, and glucose- 1,6-bisphosphate synthase; COOH acceptor kinases, such as phosphoglycerate aspartate kinase; N acceptor kinases such as creatine; P0₄ acceptor kinases, such as phosphomevalonate kinase, adenylate kinase, nucleoside-diphosphate kinase, uridylate kinase, guanylate kinase, and thiamine-diphosphate kinase; and diphosphotransferases (P₂O₇), such as ribose- phosphate diphosphokinase and thiamine diphosphokinase. Suitable dehydrogenases include, but are not limited to, aldehyde dehydrogenase acetaldehyde dehydrogenase, alcohol dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, pyruvate dehydrogenase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3 -phosphate dehydrogenase, sorbitol dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, ad malate dehydrogenase. Suitable deiminases include, but are not limited to, arginine deiminase.

Some exemplary hydrogen donors include, but are not limited to, ferulic acid, a ferulic acid derivative, 2-naphthol, p-iodophenol, p-coumaric acid, phenolics, and a dye. Suitable dyes including, but are not limited to, Alexa Fluor 488, fluorescein isothiocyanate (FITC), JAM™, JOE™, TET™, Alexa Fluor 532, HEX™, TAMRA™, Cy3, Cy3.5, ROX™, Alexa Fluor 594, Texas Red^®, Alexa Fluor 633, Cy5, Alexa Fluor 647, Cy5.5; an Atto dye, such as Atto 488, Atto 520, Atto 532, Atto Rho6G, Atto 550, Atto 565, Atto 590, Atto 594, Atto Rhol 1, Atto 630, Atto Rhol4, Atto 647, Atto 647N, Atto 655, Atto 680, and Atto 670; a Ponceau dye, such as Ponceau 2R, Ponceau 4R, Ponceau 6R, Ponceau S, and Ponceau SX; and the like. In exemplary embodiments, the dye may be Atto 590, Atto 655, or Ponceau S. The hydrogen donors may be selected based on the results of testing which hydrogen donors more greatly impact the enzymatic reaction. In some aspects, different ratios of hydrogen donors may be selected for use in an optimized assay protocol.

For example, the potentiometric determination of a reaction of an enzyme may be achieved using an enzymatic label in either a sandwich or a competitive immunoassay. In this format, a generated potentiometric response may be directly (sandwich) or inversely (competitive) proportional to the amount of enzyme label attached to the surface of the sensor, such as from being bound by one or more antibodies. The measuring solution or substrate may be selected from the larger group of test solutions to achieve a response in a measuring range by enhancing the measuring sensitivity for very low concentrations of target molecule or by targeting better discrimination between high concentrations of enzymatic label on the sensor. In some embodiments, achieving the desired response during the assay may be achieved through the use of two or more hydrogen donors with different

electrochemical properties at certain ratios in the measuring solution.

Moreover, in some embodiments, at least some components of the

electrochemical detection system 10 may include paired bioreceptors, such as chemical or biological compounds, materials, or other structures configured to bind together when adjacent. In some embodiments, at least some sensors may be at least partially coated in streptavidin and at least a portion of the enzyme may be either biotinylated or conjugated to a molecule that is biotinylated. As a result, when a solution that includes the biotinylated enzyme is added to the streptavidin-coated sensors, the biotin-streptavidin interaction affixes the enzyme to the sensors. In other embodiments, the sensors may be at least partially coated in an antibody that binds to an epitope present within a sample solution. For example, a free thyroxin antibody bound to the sensor can bind free thyroxin within a sample. In yet other

embodiments, the bioreceptors may be any other bioreceptor, for example one or more aptamers, desired by the user or manufacturer. In some embodiments, the method for optimizing the assay protocols may begin by assembling one or more solutions of possible hydrogen donors. These donos are the test solutions from which the final measuring solution or substrate is selected. In other words, the test solutions of hydrogen donors may be evaluated to determine which solution provides the user with most desirable signal profile of potential measurements resulting from the assay. In some embodiments, these solutions of likely hydrogen donors may include one or more of the hydrogen donors mentioned above. These solutions of hydrogen donors may each include one or more hydrogen donors. Moreover, these test solutions may include other substances such as enzymatic substrate, such as hydrogen peroxide for a system using peroxidase.

Moreover, the constituent hydrogen donors of these test solutions may be selected for their known impact on the reaction rate of an enzyme. Hydrogen donors of unknown affinity for any given enzymatic reaction may also be included. For example, a hydrogen donor known to have a desirable impact on the reaction rate of an enzyme may be selected for at least one test solution. In addition, some test solutions may include the same or similar constituent hydrogen donors present in the test hydrogen donor solutions at different ratios.

After preparing the test hydrogen donor solutions, one or more assay protocols may be performed according to some previously mentioned embodiments. In particular, the different test hydrogen donor solutions may be used as the measuring solution or substrate during the final steps of the protocol. Specifically, the sensors may be immersed within the measuring solution and potential values recorded by a reader (e.g., the reader 14) for comparison. Once the recorded potential values are available, the measurement range and measurement sensitivity or differentiation associated with each test solutions of hydrogen donors may be compared to select the best solution for that assay. For example, potential measurements taken from sensors immersed in the "best" test solution exhibits a greater measuring range and improved measuring sensitivity, relative to some or all other conditions.

The following paragraph is an example of a potentially beneficial combination of hydrogen donors in a measuring solution and how this combination may impact an assay. Hydrogen donor A may improve detection at a lower range of a calibration curve of the assay and hydrogen donor B may increase the overall dynamic range. Formulations with only hydrogen donor A or formulations where hydrogen donor A is the predominant component of the measuring solution would have enhanced sensitivity at the lower end of the measuring range. The use of only hydrogen donor B or formulations where hydrogen donor B is the predominant component of the measuring solution would exhibit enhanced sensitivity in competitive assays, where the signal is reversely proportional to the concentration of the enzymatic label on the sensor. A measuring solution that includes a particular combination of hydrogen donors A and B could enhance the response (i.e., the potential detected by the sensors) in the range for any particular assays. In addition, more than two hydrogen donors may be used for further enhancement. Moreover, two measuring solutions with different properties (e.g., different hydrogen donors or hydrogen donors in different ratios) may also be introduced consecutively, which may also improve the performance of both sandwich and competitive assays.

As a result of the selecting one or more hydrogen donors in known ratios for the measuring solution or substrate, some embodiments of the electrochemical detection system offer improvements relative to some convention systems. For example, because of the improved sensitivity and wide dynamic range afforded by the optimized measuring solution, robust data may be generated with the sensors in a relatively short time, for example about 10 minutes or less, such as less than about 5 minutes, or less than about 1 minute. This time represents a significant improvement over some conventional system, which may take between about 1 hour and about 3 hours to complete.

In some embodiments, the method may be conducted in a single chamber. In other embodiments, the assay may further comprise a detection platform. The detection platform may be selected from the group consisting of automated robotic system, integrated cartridge-based system, and manual microtitre plate system.

The assay may be performed on an aqueous sample. Suitable examples of aqueous samples include, but are not limited to, blood, serum, plasma, urine, tears, central nervous system fluid, interstitial fluid, and milk. The sample may be extracted from a solid before analysis. Example of solids include, but are not limited to tissue (such as skin, liver, kidney, heart, lung, or brain tissue), grain (such as wheat, rye, barley, and rice), stools (fecal matter), meat (such as the flesh of pigs, cows, poultry, deer, and the like), food (such as grains, meat, fish, fruits, vegetables, and foodstuffs made from these materials), and feed (such as feed for dogs, cats, livestock, and the like).

When introducing elements of the present disclosure or the embodiments(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. The terms comprising plurality do not exclude the method where there is only "one of.

EXAMPLES

The following examples detail some manners in which one skilled in the art may employ some embodiments of the electrochemical detection system 10. The following examples are not intended to be limiting of the disclosure and the claims, but rather an illustrative discussion regarding some uses of the system.

Example 1— The Impact of Changing the Hydrogen Donors in the Measuring Solution

The following experiment was performed to initially determine how changing the constituents of the measuring solution could impact the potentials measured during the assay. Biotinylated horseradish peroxidase (HRP) was prepared in a dilution series in a 20% StabilZyme™ solution (provided by Surmodics, Eden Prairie, MN) designed for use with HRP. Specifically, 0, 0.001, 0.01, 0.1, 1, 10, and 10 ng/mL solutions of HRP were prepared. In addition, two measuring solutions were prepared: Solution A comprising 0.5 mg/mL ferulic acid and 0.4 mg/mL p- iodophenol in a 1 :0.8 ratio, and Solution B comprising 2 mg/mL ferulic acid. The cofactors were H-donors for the enzyme peroxidase.

After preparing the dilutions and two measuring solutions, two groups of sensors were incubated with respective HRP-biotin dilutions for 15 minutes. During this incubation period, the biotin of the HRP bound to at least some surfaces of the streptavidin-coated sensors. The sensors were washed in a trough to remove excess HRP. Finally, each sensor was incubated in one of the two measuring solutions, and the potential was measured and recorded. Referring to Table 1 and FIGS. 3 and 4, the data reveal that each solution differently affected the potential detected during the reaction. In particular, Solution A exhibited a greater measuring range over the different concentrations of HRP (Figure 3), while Solution B offered greater sensitivity (FIGS. 3 and 4); that is, a greater ability to distinguish between the different lower concentrations of HRP. Accordingly, either solution could be used in the assay protocol, depending on whether a greater range or a higher sensitivity was desired. For example, if the assay was a sandwich- style assay with a greater measuring range, Solution A that afforded the greater measuring range may be more desirable. If the assay was a sandwich- style assay with a greater sensitivity, Solution B that afforded the greatest sensitivity may be more desirable.

Table 1

Example 2— The Impact of Different Hydrogen Donors in Competitive Assays The following experiment was conducted using a deoxynivalenol (DON, vomitoxin) assay. The sensors used were first coated with anti-DON antibodies and stabilized. Also, a dilution series of DON was prepared at 0, 3.3, 33, 333, and 3333 ng/mL. The conjugate was diluted 1 : 1000 in a StabilZyme™ HRP solution and the test hydrogen donor solutions were prepared. Specifically, the first test hydrogen donor solution was 0.1 mg/mL ort/zo-phenylenediamine ("Sol opd" in Table 2 and FIG. 5). The second test hydrogen donor solution (labeled "Sol A" in Table 2 and FIG. 5) comprised 0.5 mg/mL ferulic acid and 0.4 mg/mL /?-iodophenol (as in Example 1). The third test hydrogen donor solution (labeled "Sol C" in Table 2 and FIG. 5) comprised 0.1 mg/mL 2-naphthol and 0.1 mg/mL ferulic acid. Finally, the fourth test hydrogen donor solution (labeled "Sol D" in Table 2 and FIG. 5) comprised 0.1 mg/mL 2-naphthol. After preparing the reagents, 30 μΙ_, conjugate dilution was mixed with 300 μΙ_, of the respective DON dilution. Each resulting mixture was incubated in the presence of a respective antibody-coated sensor for 10 minutes at room temperature.

Thereafter, the sensors were washed and incubated in one of the four test hydrogen donor solutions and the individual potentials were measured and recorded.

Referring to Table 2 and FIG. 5, each of the four test hydrogen donor solutions was shown to create a different signal profile. In particular, use of the first test hydrogen donor solution, ort/zo-phenylenediamine, led to a potential profile with only a limited measurement range. On the other hand, the third and fourth test hydrogen donor solutions exhibited a much greater measurement range and sensitivity, relative to the first and second test hydrogen donor solutions. Finally, the second test hydrogen donor solution, which is known to be useful for a wide range of assays, exhibited a medial range of potentials. Sol A is the same solution as Solution A used in the first example.

Table 2

Example 3— The Impact of Different Hydrogen Donors on Enzymatic Assay

Unlike the above examples, some assay protocols used an enzymatic cascade. These cascades may involve multiple reactions that culminate a potential detected by the sensor. For example, in the case of an enzymatic cascade that uses a peroxidase as the final reaction, the enzymatic cascade is constructed to produce peroxide. The concentration of peroxide is proportional to the analyte and, therefore, is a target for measurement. The peroxidase then reacts with the peroxide and any hydrogen donors (cofactors) within the local environment. The resulting electrochemical changes associated with the reaction lead to an enhanced potentiometric response detected by the sensors. For example, creatinine concentration may be assessed using a sensor system that involves an enzymatic cascade system. In particular, this system uses a peroxidase as the final reporting enzyme in the cascade. This enzymatic cascade system for creatinine was used to assess the impact of different hydrogen donor solutions on the potential detected by the sensors in this system. In this case, the two test hydrogen donor solutions used ort 20-phenylenediamine (labeled "OPD" in FIG. 6) and a solution of ^-coumaric acid. As illustrated by the data in FIG. 6, different hydrogen donors (coafctors) resulted in a different signal profile for the potentials measured by the sensors. In particular, /?-coumaric acid exhibited a greater dynamic measuring range than did ort 20-phenylenediamine.

The assay protocol included the immobilization of the peroxidase measuring the hydrogen peroxide substrate on the surface of the sensor. The close proximity of the enzyme to the sensing element improved results significantly. In other electrochemical systems, the measured response was normally derived from the bulk solution.

Example 4— Immunoassay and Enzymatic Assay Carried Out Concurrently in the Same Fluidic Cell

In this example, the combination of two different assays performed concurrently in the same fluidic cell is demonstrated. The outcome is illustrated in FIG. 7 as a two-scale graph for two calibration curves. The total time was less than 10 minutes.

The data derived from multiple runs using two working electrodes on the triple-sensor system as in FIG. 2. One sensor was coated with antibodies to HbAlc (diabetes marker) and another sensor was coated with peroxidase. The samples were neat whole blood containing various concentrations of HbAlc and glucose. The conjugate for immunoassay was monoclonal antibody F7C2 conjugated to

horseradish peroxidase using Lightning-Link^® conjugation kit (Innova Biosciences), diluted 1 :250 in 0.02 M 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES) buffer, pH 8 containing 0.1% bovine specific antigen (BSA). The measuring solution for the glucose assay comprised 4 mg/mL /?-coumaric acid and

0.1% BSA in 0.2 M HEPES buffer.

First, 10 μΐ. of the measuring solution for glucose assay was combined with

80 μΐ. whole blood sample and introduced into the multisensory system, similar to the model presented in FIG. 2, comprising a fluidic cell with a volume of about 6 μΐ., sensors prepared for the assays as described above, and a reference electrode. This mixture (25 μΐ.) was aspirated over 30 s, and then kept static while the enzymatic glucose reaction occurred and while the analyte for the immunoassay (HbAcl) was captured by the antibodies on the surface of the second electrode. Glucose in the sample reacted with glucose oxidase producing hydrogen peroxide. Horseradish peroxidase immobilized on the surface reacted with hydrogen peroxide, where its concentration was proportional to the analyte glucose. The change in electrochemical properties of the cofactor for the reaction with /?-coumaric acid contributed to the change of potential of the electrode with immobilized enzyme. The reading for the enzyme-cascade assay was taken from that electrode against the reference electrode at about 3 minutes after introducing the mixture into fluidic cell.

Second, the previous mixture was displaced by pumping 9 μΙ_, air through fluidic cell, followed by 25 μΙ_, conjugate aspirated and incubated over 310 seconds (about 5 minutes).

Third, the conjugate was displaced by pumping 9 μΙ_, air through fluidic cell, followed by aspirating 60 μΙ_, measuring solution containing 0.03% hydrogen peroxide and 1 mg/mL /?-coumaric acid. The results for the immunoassay were taken at 60 seconds.

It should be understood from the foregoing that, while particular embodiments have been illustrated and described, various modifications can be made thereto without departing from the spirit and scope of the disclosure as will be apparent to those skilled in the art. Such changes and modifications are within the scope and teachings of this disclosure as defined in the claims appended hereto.

Claims

CLAIMS:

1. A method for performing an immunoassay protocol,

the immunoassay comprising:

a first measuring solution comprising a substrate and a first group of

cofactors;

a second measuring solution comprising the substrate and a second group of cofactors;

at least one sensor; and

an enzyme that can specifically react with the first and second measuring solutions;

the method comprising:

contacting the at least one sensor with a sample comprising an analyte and at least one standard solution;

incubating a first group of the at least one sensor in the first measuring solution, and incubating a second group of the at least one sensor in the second measuring solution; and

obtaining a calibration curve for each the first and the second measuring solution.

2. The method of claim 1, further comprising selecting the measuring solution based on the immunoassay's sensitivity, measuring range, and differentiation in the at least one standard solution.

The method of claim 1, wherein the first measuring solution and the second measuring solution each comprises at least one different cofactor.

The method of claim 1, wherein the first measuring solution and the second measuring solution each comprises the same group of cofactors in different ratios.

5. The method of claim 1, wherein the cofactors in the first group of cofactors are different from the cofactors in the second group of cofactors.

6. The method of claim 1, wherein at least one cofactor in the first group of cofactors is the same as at least one cofactor in the second group of cofactors.

7. The method of claim 1, wherein the cofactors are any substance, which can react with the enzyme and provide a beneficial response in a particular immunoassay.

8. The method of claim 1, wherein the enzyme is selected from the group

consisting of peroxidase, oxidase, laccase, catalase, urease, kinase, dehydrogenase, and deiminase.

9. The method of claim 1, wherein the cofactor in at least one measuring solution comprises one or more selected from the group consisting of ferulic acid, 2- naphthol, ^-iodophenol, ^-phenylphenol, phenylphenol, ^-coumaric acid, a dye, and any derivative thereof.

10. The method of claim 1, wherein the immunoassay is a sandwich, competitive, or sequential immunoassay.

11. The method of claim 1, wherein the first measuring solution is circulated over the first and second groups of sensors followed by the second measuring solution being circulated over the first and second groups of sensors, and the results are collected during each circulation.

12. A method for performing an assay protocol using at least two assays,

the at least two assays comprising:

a first measuring solution comprising a first substrate and a first group of cofactors;

a second measuring solution comprising a second substrate and a second group of cofactors;

a first plurality of sensors prepared for a first assay;

a second plurality of sensors prepared for a second assay; a first enzyme that can specifically react with the first measuring solution; and

a second enzyme that can specifically react with the second measuring solution;

the method comprising:

contacting the first and second pluralities of sensors with a sample

comprising an analyte and at least one standard solution; incubating the first plurality of sensors in the first measuring solution, and the second plurality of sensors in the second measuring solution; and obtaining a calibration curve for each the first and the second measuring solution.

13. The method of claim 12, wherein the first enzyme is the same as the second enzyme.

14. The method of claim 12, wherein the first substrate is the same as the second substrate.

15. The method of claim 12, wherein the first and second assays are sandwich immunoassays.

16. The method of claim 12, wherein at least one of the assays is competitive or sequential immunoassay.

17. A method for performing an enzymatic assay protocol,

the enzymatic assay comprising:

a first group of cofactors;

a second group of cofactors;

a plurality of sensors; and

an enzyme that can react with the first and second groups of cofactors; the method comprising:

contacting a first group of the plurality of sensors with the first group of cofactors; contacting a second group of the plurality of sensors with the second group of cofactors; and

obtaining a calibration curve for each group of the plurality sensors.

The method of claim 17, wherein the enzymatic assay contains more than one enzyme.

The method of claim 17, further comprising selecting the group of cofactors that provides the greater sensitivity in the enzymatic assay.

The method of claim 17, further comprising selecting the group of cofactors that provides the greater measuring range.

The method of claim 17, further comprising selecting the group of cofactors that provides improved sensitivity, measuring range, or differentiation in the measurement.

The method of claim 17, wherein the first group of cofactors and the second group of cofactors each comprise at least one different cofactor.

The method of claim 17, wherein the first group of cofactors and the second group of cofactors each comprise the same group of cofactors at different ratios.

The method of claim 17, wherein at least one cofactor in the first group of cofactors is different from at least one cofactor in second group of cofactors.

The method of claim 17, wherein no cofactor in the first group is the same as in a cofactor in the second group of cofactors.

The method of claim 17, wherein the cofactors are any substance, which can react with the enzyme and provide a beneficial response in a particular enzymatic assay.

The method of claim 17, wherein the enzyme is immobilized on the surface of the sensor before incubation.

The method of claim 27, wherein the enzyme is immobilized by passive adsorption, covalent binding, of biotin-streptavidin interaction.

The method of any of claims 17-28, wherein the substrate is an analyte or the product resulting from an enzymatic conversion of an analyte.

The method of claim 29, wherein the analyte is urea, glucose, or ammonium.

The method of any of claims 17-28, wherein the enzyme is selected from the group consisting of peroxidase, oxidase, laccase, catalase, urease, kinase, dehydrogenase, and deiminase.

The method of any of claims 17-28, wherein the cofactor is at least one selected from the group consisting of ferulic acid, 2-naphthol, ^-iodophenol, ^-phenylphenol, ^-coumaric acid, a dye, and any derivative thereof.

The method of any of claims 1-28, wherein the method is conducted in a single chamber.

The method of any of claims 1-28, wherein the assay further comprises a detection platform.

The method of claim 34, wherein the detection platform is selected from the group consisting of automated robotic system, integrated cartridge-based system, and manual microtitre plate system.

The method of any of claims 1-28, wherein the assay is performed on an aqueous sample selected from the group consisting of blood, serum, plasma, urine, tears, central nervous system fluid, interstitial fluid, and milk.

37. The method of claim 36, wherein the sample was extracted from a solid before analysis.

38. The method of claim 37, wherein the solid is selected from the group

consisting of tissue, grain, stools, meat, food, and feed.