MXPA00005876A - Sample detection to initiate timing of an electrochemical assay - Google Patents

Abstract

An electrochemical assay includes a method for determining with great accuracy the time at which an applied sample bridges a gap between the electrodes of an electrochemical cell. The method involves applying a constant small current across the gap, while monitoring the potential difference between the electrodes. The time at which the sample bridges the gap is marked by a sharp drop in the potential. A constant voltage is applied after the sample is detected, and the current and/or charge through the sample is monitored over a period of time. From the measured current or charge, the analyte concentration of interest can be calculated.

Description

DETECTION OF SAMPLE TO START THE TIME CONTROL OF AN ELECTROCHEMICAL ANALYSIS BACKGROUND OF THE INVENTION 1. - FIELD OF THE INVENTION The invention relates to an electrochemical device for measuring the concentration of an analyte in a biological fluid; more in particular, to a mechanism to determine the moment in which the fluid provides an electrical connection between the working and reference electrodes of the device. 2. - DESCRIPTION OF THE RELATED TECHNIQUE A variety of medical diagnostic procedures involve analysis in biological fluids, such as blood, urine, or saliva, to determine an analyte concentration in the fluid. Among the analytes of greatest interest is glucose, and strips with reagents in dry phase that incorporate enzyme-based compositions are widely used in clinical laboratories, doctors' offices, hospitals and homes to analyze samples of biological fluids for glucose concentration. . In fact, reagent strips have become the everyday need for many of the estimated 16 million diabetics in the United States. Since diabetes can cause dangerous abnormalities in blood chemistry, it can contribute to vision loss, kidney failure and other serious medical consequences. To minimize the risk of these consequences, most diabetics must analyze themselves periodically, and then adjust, as a consequence, the concentration of their glucose; for example, by controlling diet and / or insulin injections. Some patients should test their blood glucose concentration as often as four or more times a day. It is especially important for diabetics that they must control their diet in order to regulate sugar intake and / or administer insulin injections, and that they should be guided in this regard by frequent blood glucose concentration tests, which have a fast, low cost and accurate system to determine glucose. One type of system for measuring glucose works electrochemically, detecting the oxidation of blood glucose in a strip of dry reagent. The reagent generally includes an enzyme, such as glucose oxidase or glucose dehydrogenase, and a reduction-oxidation mediator (redox), such as a ferrocene or ferricyanide. This type of measurement system is described in US Patent 4,224,125, issued September 23, 1980 to Nakamura and co-inventors; in U.S. Patent 4,545,382, issued October 8, 1985 to Higgins and co-inventors, and in U.S. Patent 5,266,179, issued November 30, 1993, to Nankai and co-inventors, all incorporated herein by this reference. The electrochemical glucose meters can be characterized as columbymmetric, amperometric or potentiometric, depending on whether the system involves the measurement of the load, the current or the potential, respectively, when determining the concentration of glucose. In each case it is important to define the point in time when the blood sample makes contact with the reagent, since an electrical signal must be applied to the strip in the later controlled time period with precision. Nankai and co-inventors, in US Pat. No. 5,266,179, issued November 30, 1993, describe an electrochemical system for measuring blood glucose, in which the time of application of the sample is defined as the moment when there is a fall of resistance between a pair of electrodes to which a constant voltage is applied. White and co-inventors, in U.S. Patent 5,366,609, issued November 22, 1994, discloses the same principle of monitoring the resistance drop between the electrodes to determine when the blood was applied to a strip of dry reagent for glucose. . In both patents a constant voltage is applied between the working and reference electrodes, to follow the resistance changes that are the result of the introduction of a blood sample, to a strip of dry reagent.

To obtain accurate results, the sample detection procedure should not alter the concentration of the analyte, and several techniques have been described to minimize disturbance of the analyte. Quade and co-inventors, in the German patent application (DDR) 148,387, filed on December 28, 1979, describe an electrochemical measurement using a novel electronic circuit, which allows rapid switching between the potentiostatic (applied constant voltage) and the galvanostatic modes (constant current applied), while also allowing a reduction in the number of electronic components. It is a goal of the circuit to minimize the disturbance of the sample before starting a measurement. Bartels and co-inventors, in the German patent application (DDR9 208,230, filed on November 24, 1981), describes an electrochemical measurement that also attempts to minimize sample disturbance.The measurement device includes a circuit using a diode for To minimize the flow of current before starting the measurement, without using an additional amperometric control circuit, in addition, the circuit commutes to the potentiometric mode in a precise and fast way, Littlejohn and co-inventors, in the US patent 4,940,945, issued on 10 July 1990, describes a portable device that can measure the pH of a blood sample.The apparatus detects the presence of a sample in a cell, injecting a constant current between a filling electrode, outside the sample chamber, and one of two electrodes inside the chamber, when the impedance decreases by at least two orders of magnitude, the meter recognizes that provided enough sample and emits a sound. The filling electrode of the circuit including the two electrodes is then segregated inside the sample cell, and the measurement is performed potentiometrically.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for measuring the concentration of analyte in a sample of a biological fluid that is applied to an electrochemical diagnostic strip, of the type including juxtaposed working and reference electrodes. The method comprises: (a) applying a predetermined constant current source between the working and reference electrodes; (b) monitor a potential difference across the electrodes; (c) apply the sample to the strip; (d) determining a sample detection time, noting when the potential difference falls below a predetermined threshold voltage; (e) applying a predetermined constant voltage to the sample; (f) measuring an electrical response at a predetermined time after applying the constant voltage; and (g) calculate the analyte concentration using the measured electrical response. A meter for measuring the concentration of analyte in a sample of a biological fluid that has been applied to a diagnostic strip comprises, in electrical communication: (a) means for applying a predetermined current between the working and reference electrodes; (b) means for monitoring a potential difference across the electrodes; (c) means for determining when the potential difference falls below a predetermined threshold voltage to indicate detection of the sample; (d) means responsive to the detection of the sample, to apply a predetermined constant voltage to the sample; (e) means for measuring a resultant electrical response; and (f) means for calculating the analyte concentration using the measured electrical response. The present invention provides a method and apparatus for electrochemically measuring the concentration of analyte, which includes: defining with great precision the moment in which a sample that is applied to the reaction zone of an electrochemical diagnostic strip, establishes a bridge in the separation between the electrodes. The determination of the moment of application of the sample (more precisely: the time of detection of the sample; these terms are used interchangeably), it allows for greater accuracy and precision of the analysis carried out on the sample. An advantage of the method of the present invention for determining the moment of application of the sample, is that applying a small, constant current to detect the sample, minimizes the disturbance of the sample, compared to the methods of the art. previous, that they applied a constant voltage. Using this procedure, applying a sample causes a current that exceeds a defined threshold to start time control. Since the sampling rate is limited, the current will typically be substantial before the sensor recognizes that the threshold has been exceeded. When a large current is observed, a correspondingly large disturbance is observed in the mediator. This could lead to an inaccurate measurement, especially at low analyte concentrations. The method of the prior art, of applying a constant potential to detect the application of the sample, has another disadvantage, in that the initial current generally decreases as the analyte concentration decreases. Thus, it is more difficult to determine an initial sample detection time for samples with low analyte content. Following the same reasoning, if it is set too low under the current threshold, it can be falsely triggered by noise. To further complicate this, the presence of a high concentration of red blood cells also decreases the initial current.

The concentrations of analyte and red blood cells do not affect the method of the present invention. Similarly, noise is also not a significant problem, since the detection trip is a large change in the signal voltage.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of applied current and voltage measured against time, illustrating the sample detection process of the present invention. Figure 2 is a graph of applied voltage and resultant current response, versus time, for a method of analysis of the present invention. Figure 3 is a plot of applied voltage and current response versus time, for an alternative method of analysis of the present invention. Figure 4 is a load versus time graph for another alternative analysis method of the present invention. Figure 5 illustrates an electrochemical device suitable for use in the method of analysis of the present invention. Figure 6 is a diagram of a circuit suitable for use in the present invention.

DETAILED DESCRIPTION OF THE INVENTION This invention relates to an electrochemical method for measuring an analyte concentration in a biological fluid. In the interests of brevity, the following description emphasizes the measurement of glucose concentration in whole blood samples; however, people who are ordinary experts in the medical diagnostic technique will recognize how the description can be adapted to perceive other analytes (such as cholesterol, ketone bodies, alcohol, etc.) in other fluids (such as saliva, urine, fluid). interstitial, etc.). The electrochemical (amperometric) method for measuring an analyte concentration in an aqueous sample involves placing the sample in a reaction zone in an electrochemical cell having two electrodes having an impedance that is suitable for the amperometric measurement. The analyte is allowed to react directly with an electrode or redox reagent to form an oxidizable (or reducible) substance in an amount corresponding to the analyte concentration. The amount of oxidizable (or reducible) substance is then determined electrochemically. This type of analysis must precisely define the point in time at which the sample is detected in the reaction zone. This allows an electrochemical waveform (ie, a voltage) to be immediately applied after the sample has been applied, and accurately defines an incubation period or reaction time. In turn, this improves the accuracy and precision of the analysis, as described below. The present invention provides an improved method and apparatus for determining the detection time of the sample. The method involves applying a small constant current source through the electrode of an electrochemical diagnostic strip and monitoring the potential difference between the electrodes. Since there is a dry space between the electrodes, a negligible current initially flows. When the sample is applied to the strip and it fills the gap, the measured voltage decreases rapidly, which causes the test time to start. By thus recognizing that the sample has been applied, the apparatus switches from a constant current mode to a constant voltage mode. In constant voltage mode, current or load is measured as a function of time to allow the concentration to be calculated. This technique minimizes the error introduced in the signal response, by means of the time control initiation circuit and, thus, allows low detection limits. The electronic components are simple and inexpensive. Figure 1 is a graph of applied current and measured voltage, illustrating the sample detection process of the present invention. Before the zero moment (that is, before introducing the sample), a constant current (here, for example, 1 μA) is applied between the electrodes, but a negligible current flows. A smaller current reduces the disturbance and is preferred, particularly for a small concentration of analyte. The voltage measured by the circuit supply voltage is determined; in this case, 5 volts. When the sample is introduced into the cell (at time zero), the applied current between the electrodes can flow and the measured voltage drops rapidly. When the voltage drops before a threshold voltage, the device switches from the applied constant current to the applied constant voltage. Figure 2 is a graph illustrating the applied potential and the current measured as a function of time after the detection of the sample. The sample is detected at time t = 0, and a voltage is applied between the working and opposite electrodes immediately afterwards. As a result, current flows between the electrodes. The current after a predetermined time is a measure of the analyte concentration, once the system has been calibrated, using samples having known concentrations of analyte. The predetermined time duration is not critical. In general it is at least about three seconds when the fluid is blood and the analyte is glucose. That duration generally provides enough time to dissolve reagents and reduce an amount of mediator that is easily measurable. With all the same things, at high content of hematocrit, longer times are necessary. In practical terms, generally a user is interested in having a reading as quickly as possible. Ten seconds is a typically satisfactory time, with no motivation to wait longer. Of course, once a predetermined time is set, precise and accurate results require that the same time is used each time. In any case, the precision of the current determination depends on the precision of the determination of t = 0. Figure 3 illustrates a graph of the measured current and the applied voltage versus time, in an alternative method. In this method a second voltage pulse is applied through the electrodes after the predetermined time. In general, the second pulse is applied after the predetermined time (to minimize the total measurement time), but a delay is permissible. Again, reproducible results require reproducible procedures; thus, in this method also, it is important to determine precisely the point t = 0. The second pulse causes a positive peak in the current through the electrodes, followed by a decaying current. The analyte concentration can be determined, after the system has been calibrated, from the decay rate, either alone or in combination with the current measurement illustrated in Figure 2. In general, the current decays exponentially over a period of time. which starts approximately one second after applying the second pulse, and which continues for at least several seconds afterwards. Figure 4 illustrates the method of Figure 3, in which the load was measured, instead of the current. As with the graph of Figure 3, the analyte concentration can be determined from the total charge at a fixed time and / or from the decay rate, after the second voltage is applied.

Figure 5 illustrates a "thin layer" device 10 that is suitable for use in the methods described above. The substrate 12 is a base 14 of polyester, on which a coating 16 of Pd, which forms the working electrode, has been deposited, typically by cathodic deposition. A dry reagent, consisting of regulator, mediator and enzyme, is deposited near one end 18 of the electrode. The separating layer 20 has adhesive on two sides, which has a cutout 22 defining the electrochemical cell. Typically the separator has a thickness of less than about 200 μm. The upper layer 24 is the polyester layer 26, on which an Au coating 28, which forms the reference electrode, has been deposited, also typically by means of cathodic deposition. A device of the type described above can use a glucose oxidase (GOD) / ferricyanide system to determine glucose concentrations by means of the following reactions, in which GOD * is the reduced enzyme.

Reaction 1: glucose + GOD - gluconic acid + GOD * Reaction 2: GOD * + 2ferricyanide - GOD + 2ferrocyanide Ferricyanide. { [Fe (CN) 6] 3".} Is the mediator, which returns the GOD * to its catalytic state.GOD, an enzyme catalyst, will continue to oxidize the glucose as long as an excess of mediator is present.The ferrocyanide. { . [Faith (CN) 6] 4"} it is the product of the total reaction. Ideally there is no ferrocyanide initially, although in practice there is often a small amount. After the reaction is complete, the concentration of ferrocyanide (measured electrochemically) indicates the initial concentration of glucose. The total reaction is the sum of reactions 1 and 2: Reaction 3: GOD glucose + 2-ferricyanide - gluconic acid + 2-ferrocyanide "Glucose" refers specifically to β-D-glucose. Details of this system are described in the application of TCP No. WO 97/18465, incorporated herein by reference. Figure 6 illustrates a modality of circuits suitable for practicing this invention. Initially a constant current source is applied to the strips with the switch 105 in position 1. The current source consists of the operational amplifier 104, the voltage reference 102 and the resistors 101 and 103. The current is determined by the ratio of reference voltage 102 to resistor 103. Resistor 101 is used to generate the required excitation. Operational amplifier 110 and resistor 109 are used as a current-to-voltage converter. Initially, without sample in the strip, the resistance between points 107 and 108 is very large, and the current passing through the strip is negligible. The output voltage of the operational amplifier 104 (V1) is high in this condition. When a sample is applied to the strip its resistance drops significantly and, since a constant current flows through the strip, V1 falls. V1 is fed to the microprocessor 112, through the converter 111 from analog to digital. The microprocessor 112, which recognizes this reduced voltage as sample detection, switches at 105 to position 2, to disconnect the strip from the current source and connect it to the voltage source 106. In this condition a chronoamperometric measurement can be achieved. measuring the output voltage of the operational amplifier 110 (V2). This voltage is proportional to the current that passes through the strip. The following example demonstrates the present invention, but is not intended in any way to be limiting.

EXAMPLE The circuit of Figure 6 was established, wherein strip S is a thin layer electrochemical glucose strip, of the type shown in Figure 5, having electrodes Pd and Au. The Pd electrode was coated with a regulator layer, glucose dehydrogenase (PQQ) and ferricyanide. A small, non-interfering, constant current (around 1 μA) was applied between the working electrode and the counter / reference electrode, from the dried glucose strip. Because the strip was dry, the resistance between the working electrode and the counter / reference electrode was essentially infinite. After a whole blood sample was applied through the cell, a drop in voltage was observed. A threshold of approximately 50 to 500 mV initiated the starting time (a threshold of approximately 300 mV is preferred). After the sample was detected, the instrument switched from applying a constant current to applying a constant voltage. The measurement of the current through the sample is a function of time, allowed to calculate the glucose concentration. Those of skill in the art will understand that the foregoing description and example are illustrative of the practice of the present invention.; but in no way restrictive. Variations of the detail presented here can be made, without departing from the scope and spirit of the present invention.

Claims (15)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for measuring the concentration of an analyte in a sample of biological fluid, which is applied to an electrochemical diagnostic strip, of the type including juxtaposed work and reference electrodes, characterized in that it comprises: (a) applying a source of constant current, predetermined, between the working and reference electrodes; (b) monitoring a power difference through the electrodes; (c) apply the sample to the strip; (d) determining a sample detection time, noting when the potential difference falls below a predetermined threshold voltage; (e) applying a predetermined constant voltage to the sample; (f) measuring an electrical response at a predetermined time after applying the constant voltage; and (g) calculate the analyte concentration, using the measured electrical response.

2. The method according to claim 1, further characterized in that the measured electrical response is the current through the sample at the predetermined time.

3. The method according to claim 1, further characterized in that the measured electrical response is the load that passes through the sample from the sample detection time, up to the predetermined time.

4. - The method according to claim 1, further characterized by additionally comprising applying a second predetermined voltage, after the predetermined time, and measuring a second electrical response, after applying the second predetermined voltage.

5. The method according to claim 4, further characterized in that the second electrical response is the speed with which the current decays through the sample.

6. The method according to claim A, / -. and, /) further characterized in that the second electrical response is the load Y passes through the sample during a predetermined time interval, after the second voltage is applied.

7. A meter for measuring an analyte concentration in a sample of a biological fluid that has been applied between a working and reference electrode of a diagnostic strip, characterized in that it comprises, in electrical communication: (a) means to apply a predetermined current between the working and reference electrodes; (b) means for monitoring a potential difference across the electrodes; (c) means for determining when the potential difference falls below a predetermined threshold voltage to indicate sample detection; 8d) means responsive to sample detection to apply a predetermined constant voltage to the sample; (e) means for measuring a resultant electrical response; and (f) means for calculating the analyte concentration, using the measured electrical response.

8. The meter according to claim 7, further characterized by the means for measuring an electrical response resulting in an ammeter.

9. The meter according to claim 7, further characterized in that the means for measuring a resulting electrical response is a coulometer.

10. The meter according to claim 7, further characterized in that it comprises means for applying a second predetermined voltage to the sample; and a means for measuring a second electrical response.

11. The meter according to claim 10, further characterized in that the means for measuring a second electrical response resulting is an ammeter.

12. The meter according to claim 10, further characterized in that the means for measuring a second electrical response is a coulometer.

13. A method for measuring an analyte concentration in a sample of a biological fluid that is applied to an electrochemical diagnostic strip, of the type that includes juxtaposed working and reference electrodes; further characterized by comprising: (a) applying a predetermined constant current source between the working and reference electrodes; (b) monitor a potential difference across the electrodes; (c) apply the sample to the strip; (d) determining a sample detection time, noting when the potential difference falls below a predetermined threshold voltage; (e) applying a predetermined constant voltage to the sample; (f) applying a second predetermined voltage to the sample after a predetermined first time; (g) measuring an electrical response at a predetermined time, after the first predetermined time; and (h) calculate the concentration of the analyte using the measured electrical response.

14. The method according to claim 13, further characterized in that the measured electrical response is the rate of decay of the current through the sample.

15. The method according to claim 13, further characterized in that the measured electrical response is the load that passes through the sample during a predetermined time interval, after the second predetermined voltage is applied.