KR20100006410A - Bio-sensor having sensing electrode and measuring method using the same - Google Patents

Abstract

The present invention relates to a biosensor in which a sensing electrode is formed and a method for analyzing a sample using the sensing electrode. The sensing electrode is formed on a lower insulating substrate to extend a reference electrode from the working electrode to the sample passage region, and through the sample inlet. The measuring device can detect the exact filling time of the injected sample, and in the end, the biosensor and the analysis method of the sample can be obtained more precisely by performing sample analysis using the starting point of the actual filling of the sample. It is about.

Description

Bio-sensor having sensing electrode and measuring method using the same

The present invention relates to a biosensor and a method for analyzing a sample using the same, and more particularly, to form a sensing electrode extending from the working electrode to the sample passage region to the rear of the working electrode on the lower insulating substrate, and using the sample inlet using the same. The measuring device can detect the exact filling time of the injected sample, and in the end, the biosensor and the analysis method of the sample can be obtained more precisely by performing sample analysis using the starting point of the actual filling of the sample. It is about.

A biosensor is a system that converts information into a useful signal that can be recognized, such as color, fluorescence, and electrical signals, by using biological elements or mimicking biological elements when obtaining information from a measurement object.

In particular, biosensors using biological enzymes have excellent sensitivity and specificity of reaction, and thus they are expected to be applied in a wide range of fields, such as the medical / medical field, process measurement in the bio industry, environmental measurement, and stability evaluation of chemicals.

Investigating chemical components in vivo is very important medically, and biomedical sensors are widely used to analyze biological samples such as blood in the medical diagnostic field.

Among them, enzyme assay biosensors using specific reactions of enzymes and substrates or enzymes and inhibitors are most widely used in hospital and clinical chemistry analysis because they are easy to apply and use, have excellent measurement sensitivity, and have fast results. .

Enzyme assays for examining chemical components in vivo can be classified into colorimetric methods for observing light transmittance before and after enzymatic reaction by spectroscopic methods and electrode methods for measuring electrochemical signals.

Among them, the color development method requires longer measurement time and requires a large amount of samples, and it is difficult to analyze important biomaterials due to measurement errors due to turbidity of biological samples.

Therefore, in recent years, an electrode system composed of a plurality of electrodes is formed on a plastic film (insulating substrate) by etching, screen printing, sputtering, etc. Electrode method for quantitatively measuring substances has been applied to many biosensors using enzymes.

In the case of using the electrode method, a separate measuring device for reading information about the biomaterial from the biosensor is required, and the measuring device includes a socket electrically connected to the thin film electrode of the biosensor. When the thin film electrode of the biosensor is inserted into the measuring device through the insertion hole, the thin film electrode is connected to the terminal formed in the socket of the measuring device. Then, the measuring device receives information about the biomaterial to be analyzed while the measuring device is turned on. do.

One of the widely applied biosensors of the electrode method is a blood glucose meter, and anyone can easily collect their own blood and measure the amount of glucose (blood sugar) in the blood.

In insulin-dependent patients who need to measure blood glucose in real time two or three times a day, the fingertip is usually pierced with a needle-shaped lancet, and the blood glucose meter is used to collect the sample (blood) and the reactants in the biosensor. The blood glucose value is measured from the electrical signal generated by the electrochemical reaction.

In other words, the enzyme reaction layer composed of hydrophilic polymer, oxidoreductase, and electron acceptor is fixed on the electrode system of the biosensor, and the user injects a sample (blood) containing a substrate (glucose) through the sample inlet of the biosensor. By contacting the enzyme reaction layer, the enzyme reaction layer dissolves it, the substrate of the sample and the enzyme (Enzyme) is reacted, and the substrate is oxidized and the electron acceptor is reduced. The oxidation current obtained by electrochemically oxidizing the reduced electron acceptor can be measured through a measuring instrument to determine the concentration of the substrate contained in the sample.

1 and 2 are basic configuration diagrams of an electrochemical biosensor, and a configuration of a conventional biosensor will be described with reference thereto. 1 is an exploded perspective view, Figure 2 is an assembled perspective view.

As shown in the drawing, the biosensor 10 is formed by stacking a long working electrode 12 and a reference electrode 13 on an upper surface (which is an inner surface) of the lower insulating substrate 11. In addition, the analysis reagent, that is, the reaction enzyme layer 14 is fixed to cross the working electrode 12 and the reference electrode 13. The electrodes 12 and 13 may be formed by a thin film forming method such as etching, screen printing, sputtering, or the like.

In addition, the spacers 15 and 16 are stacked on the lower insulating substrate 11 on which the electrodes 12 and 13 are formed so that a sample (a sample such as blood) can be appropriately administered to the entire reaction enzyme layer 14. The upper insulating substrate 17 is stacked on the upper side of the upper and lower insulating substrates 15 and 16, and the upper and lower insulating substrates 11 and 17 spaced apart from each other by the spacers 15 and 16 have a capillary structure above the enzyme reaction layer 14. A phosphor sample passage 18 is formed.

In this case, the working electrode 12 and the reference electrode 13 are insulated by the spacers 15 and 16, and the sample passages formed by the upper and lower insulating substrates 11 and 17 by the spacers 15 and 16. The inlet of (18) becomes the sample inlet 18a into which the sample is injected. In addition, the ends of the working electrode 12 and the reference electrode 13 are exposed at the connecting end of the biosensor so that when the biosensor 10 is inserted into a measuring instrument (not shown), it can be connected to a terminal formed in the socket of the measuring instrument. .

As described above, the electrical signal generated by the reaction between the component (analyte, for example, blood glucose) and the reactive enzyme layer 14 in the sample while the working electrode 12 and the reference electrode 13 are electrically connected to the measuring device. Will be detected. At this time, the redox reaction of the components in the sample and the reactive enzyme layer 14 takes place at the working electrode 12, the reaction result of which generates a predetermined current by the electrochemical mechanism at the working electrode 12, this current When is applied to the meter, the meter reads the current applied from the biosensor 10 and quantitatively analyzes the corresponding components in the sample and displays the result on the display.

Hereinafter, the operation and measuring method of the measuring instrument when the biosensor is inserted will be described in more detail.

FIG. 3 illustrates a biosensor and a measuring device. The biosensor 10 is a schematic view of only the lower insulating substrate 11 and the electrodes 12 and 13 in a plan view, and the internal structure of the measuring device 20 is illustrated. Shown in the block diagram.

4 is a waveform diagram showing the waveform of the operating voltage and the current flowing through the working electrode, (a) shows the operating voltage applied by the measuring device 20 to the working electrode 12 of the biosensor 10, (b) shows the current flowing through the working electrode 12 as the sample is introduced.

In the measuring device 20, the operation / control unit 21 causes the operation voltage generation unit 22 to apply a predetermined operating voltage to the working electrode of the biosensor 10, and the current / voltage conversion unit 23 operates. The current input via the electrode 12 is converted into a voltage, and then input to the A / D converter 24. The A / D converter 24 converts the voltage into a digital signal, thereby calculating and controlling the controller 21. Enter

At this time, the operation / control unit 21 reads the value of the current flowing through the working electrode 12 from the digital signal input from the A / D conversion unit 24 and measures the concentration of the analyte (blood glucose, etc.). The control unit 21 stores data on the relationship between the concentration of the analyte and the current.

The operation and measuring method of the measuring device will be described in more detail. First, when the biosensor 10 is inserted into the measuring device 20, an operating voltage is applied to the working electrode 12 of the biosensor. At this time, the operation / control unit 21 recognizes the insertion of the biosensor 10 and causes the operating voltage generator 22 to transmit a predetermined operating voltage (eg, 300 mV) to the working electrode 12 of the biosensor 10. To be authorized.

Subsequently, when a sample such as blood is injected into the reaction enzyme layer through the sample inlet of the biosensor 10, an analyte in the sample reacts with the reaction enzyme layer to generate a charge, and a voltage applied to the working electrode 12. This charge causes current to form.

As the reaction between the analyte and the reaction enzyme layer proceeds, the current is increased as shown in FIG. 4 (b). When the current is increased to a predetermined size, the operation / control unit 21 is operated. Causes 22 to apply no voltage to the working electrode 12 (releasing the operating voltage).

Thereafter, there is a predetermined delay time, and since the operating voltage becomes 0 V, charges generated by the reaction between the analyte and the reactive enzyme layer do not flow through the working electrode but are collected around the working electrode.

As such, the current value decreases from the time when the application of the operating voltage is released, and electric charges are generated by the reaction until the application of the operating voltage is resumed, but the current does not flow.

After a predetermined delay time has elapsed while the operating voltage is released, the calculation / control unit 21 of the measuring device 20 causes the operating voltage generator 22 to reset the predetermined operating voltage (300 mV) to the biosensor 10. To the working electrode 12.

At this time, the charge gathered around the working electrode flows through the working electrode at one time while the operating voltage is applied again, so that the peak current Ip3 appears at the time when the operating voltage is applied as shown in FIG.

Then, the current value gradually decreases, and the calculation / control unit 21 calculates the concentration of the analyte by reading a value im of the current flowing through the working electrode 12 at a predetermined time t4 and then displays the result. (25).

In FIG. 4, t0 represents the time when the biosensor is inserted, t1 represents the time when a sample such as blood is injected through the sample inlet, t2 represents the time when the measured current reaches the ith current value, and t3 represents a predetermined delay. It represents the time when the operating voltage is applied again after time. T4 also represents the time to read the current value (im) to determine the concentration of the analyte.

That is, when the biosensor 10 is inserted into the measuring device 20 at the time t0, the operation / control unit 21 recognizes this and applies an operating voltage (eg, 300 mV) to the working electrode 12. When the sample is injected at t1 time, the current value gradually increases, and then at t2 time when the current value rises to ith, the operation / control unit 21 releases the voltage.

Thereafter, the operation / control unit 21 applies a working voltage to the working electrode 12 again at a time t3 with a predetermined delay time, and a peak current Ip3 appears by applying the working voltage again at t3 time, and then the current value gradually increases. Descend.

The concentration of the analyte is then calculated by reading the current value (im) at time t4 and comparing it with the stored data.

The delay time, that is, the time from t2 to t3, is generally referred to as an incubation time. The charge gathered around the working electrode 12 during the incubation time flows at the same time as the operation voltage is applied at the time t3, thereby peaking. The current Ip3 is shown.

While it was described that the operating voltage is not applied during the incubation time in the measurement process as described above, a predetermined low voltage (eg, 74 mV) is applied during the incubation time as disclosed in Patent Publication No. 2005-96490 (2005.10.6). You may.

In this case, the waveform diagram of the operating voltage and the waveform diagram showing the current flowing in the working electrode are shown in FIG. 5, and (a) indicates that the measuring device 20 is applied to the working electrode 12 of the biosensor 10. The operating voltage is shown, and (b) shows the current flowing through the working electrode 12 as the sample is introduced.

Incubation time may be avoided. Measurement results can be obtained by analyzing a current signal in which the current value reaches a peak and then gradually decreases as the reaction current increases after the sample is introduced.

On the other hand, ith can be said to be a current value which is set in advance by assuming that the current value of the sample is sufficiently filled in the sample passage (sample filled state). The voltage is released at t2 when this current value is reached, and this t2 time is a start point for obtaining a measurement result.

That is, the operation / control unit 21 obtains a result by reading the current value im at a time t4 time after the incubation time elapses from this time as t2 time of detecting the ith current as a start time. The detected t2 time must be correctly recognized by the operation / control unit 21.

The start point when the sample is sufficiently filled in the sample passage is because accurate measurement results can be obtained, and the arithmetic / control unit 21 assumes that ith current value is reached when the sample is filled in the sample passage by a sufficient amount. When the ith current value is reached, it is recognized as the start time and the measurement result is obtained.

The sample injected into the sample inlet reaches the first electrode, the working electrode 12, and the second electrode, the reference electrode 13, within a short time in the sample passage on the reaction enzyme layer by capillary phenomenon. When the first electrode is reached, it must be sufficiently filled in the sample passage so that a normal reaction occurs and accurate measurement is possible.

However, the conventional method of recognizing a start time by sensing a specific current value has the following problems.

Since there may be a difference in the time when the biosensor reaches the ith current value, the time when the ith current value is reached is not a precise time point when the actual sample reaches the second electrode (reference electrode) and is sufficiently filled.

That is, even if ith current value is reached, the actual sample may not have reached the second electrode sufficiently, and if the measurement is performed in the state that the sample is not sufficiently filled, an error may occur in the result.

Therefore, it is necessary for the operation / control unit 21 to accurately detect whether the actual sample reaches the second electrode and is sufficiently filled, and it is necessary to recognize the exact time point at which the sample is sufficiently filled as the start time.

In the case of using a conventional biosensor, it is simply recognized as a start time when the ith current value is reached, and it is not accurate whether the time is actually a time when the sample is sufficiently filled in the sample passage.

After the actual sample is injected into the sample inlet and starts to reach the working electrode, the reaction current appears. After that, the reaction current rises and the ith current value can be reached even if the sample touches only a part of the reference electrode through the working electrode.

When the ith current value is reached, it is possible to know whether the sample has passed the working electrode to reach the reference electrode and is sufficiently filled, or stopped as soon as it touches the reference electrode, or is partially filled, or stopped partially in the middle of the reference electrode. none.

In addition, the flow rate of the sample into the sample passage may vary according to the flow characteristic of the sample, and the flow characteristic of the sample may affect the measurement result value. That is, measurement errors may occur depending on the flow characteristics of the sample.

Therefore, the measurement error correction according to the flow characteristics of the sample at the time of analysis can be corrected by the inflow velocity difference. It is also possible to infer the nature of the sample from the difference in inflow rate.

However, in the conventional biosensor, it is impossible to correct the measurement error according to the flow characteristics because the rate at which the sample is introduced into the sample passage is not known.

Therefore, the present invention was invented to solve the above problems, by forming a sensing electrode on the lower insulating substrate extending the reference electrode from the working electrode to the sample passage region to the rear, the sample injected through the sample inlet It provides a biosensor and an analytical method for analyzing a sample that can accurately detect the point of fullness of the measuring device, and in the end, perform a sample analysis by using the point of time when the sample is actually filled. There is a purpose.

In addition, the present invention is able to detect the initial time the sample is injected and the time when the sample is completely filled by the added electrode of the biosensor, it is possible to estimate the flow characteristics and the inflow rate of the sample, correction of the measurement results The purpose of the present invention is to provide a biosensor and sample analysis method that can be used to obtain more accurate results.

In order to achieve the above object, the present invention, the working electrode and the reference electrode formed on the lower insulating substrate so as to pass through the sample passage and configured to fix the reaction enzyme layer across the working electrode and the reference electrode along the sample passage In the biosensor,

A sensing electrode extending from the working electrode to a sample passage region downstream of the reference electrode from the working electrode with respect to the sample flow direction, so that a sample flowing along the sample passage flows through the working electrode and the reference electrode; It provides a biosensor formed with a sensing electrode, characterized in that configured to reach through the sensing electrode region in turn.

In another aspect, the present invention is provided with a sensing electrode extending to insert at least a portion of the lower insulating substrate in the sample passage region downstream of the reference electrode relative to the sample flow direction from the working electrode, the sample flowing along the sample passage is Using a biosensor configured to reach the sensing electrode region in turn through a working electrode and a reference electrode,

Detecting the peak current occurring when the sample reaches the sensing electrode region in the measuring device into which the biosensor is inserted, and then detecting the peak current as a time when the sample is filled in the biosensor Provided is a method for analyzing a sample using a biosensor having electrodes.

Here, the time at which the peak current is sensed is used as a start time, and then the current value at a specific time is read, and the analytes in the sample are analyzed using the same.

In another aspect, the present invention, in the biosensor configured to form a long working electrode and the reference electrode in the lower insulating substrate to pass through the sample passage and to fix the reaction enzyme layer across the working electrode and the reference electrode along the sample passage,

A reference electrode is formed on the lower insulating substrate by branching to a first reference electrode portion and a second reference electrode portion passing through a sample passage region, and a sample passage region between the first reference electrode portion and the second reference electrode portion from the working electrode. And sensing electrodes each extending to insert at least a portion of the sample passage region downstream of the second reference electrode portion with respect to the sample flow direction, so that the sample flowing along the sample passage passes the working electrode and the first reference electrode. The present invention provides a biosensor having a sensing electrode, wherein the sensing electrode is configured to pass through the first sensing electrode and the second reference electrode in order to reach the second sensing electrode region.

In addition, the present invention is formed by branching the first reference electrode portion and the second reference electrode portion through which the reference electrode passes through the sample passage region on the lower insulating substrate, and between the first reference electrode portion and the second reference electrode portion from the working electrode. And a sensing electrode extending to insert at least a portion of the sample passage region and the sample passage region downstream of the second reference electrode portion with respect to the sample flow direction, so that the sample flowing along the sample passage passes through the working electrode and the sample. Using a biosensor configured to reach the second sensing electrode region in order through the first reference electrode portion, the first sensing electrode, and the second reference electrode portion;

After detecting the peak current appearing when the sample reaches the second sensing electrode region in the measuring device in which the biosensor is inserted, the time at which the peak current is detected is used as the time when the sample is filled in the biosensor. It provides a method for analyzing a sample using a biosensor formed with a sensing electrode.

Here, the time at which the peak current is sensed is used as a start time, and then the current value at a specific time is read, and the analytes in the sample are analyzed using the same.

In addition, the sensor detects a peak current occurring when the sample reaches the first reference electrode region after passing through the working electrode, and then the time at which the peak current is detected is the initial time when the sample is injected into the biosensor. It is characterized by using.

In addition, the inflow rate of the sample is estimated by using the measuring time of the peak current when the sample reaches the first reference electrode region and the peak current when the sample reaches the second sensing electrode region. do.

Accordingly, in the present invention, by forming a sensing electrode extending from the working electrode to the sample passage region to the rear of the working electrode on the lower insulating substrate, it is possible to detect the exact filling time of the sample injected through the sample inlet by using the measuring device As a result, a more accurate measurement result can be obtained by performing sample analysis by using the time point at which the sample is actually filled as a start time point.

In addition, the present invention is able to detect the initial time the sample is injected and the time when the sample is completely filled by the added electrode of the biosensor, it is possible to estimate the flow characteristics and the inflow rate of the sample, correction of the measurement results It can be used to obtain more accurate results. In particular, the flow characteristics and inflow rate of the sample obtained by using the sensing electrode of the present invention has the possibility of being utilized in various ways during the analysis of the sample.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

When the working electrode, which is the first electrode, is extended to the sample passage region behind the reference electrode, which is the second electrode (downstream in the sample flow direction), the sample sufficiently passes the working electrode and the reference electrode in the sample passage. By experimentally confirming that the peak current appears while touching the extension region (sensing electrode region) of the working electrode in a filled state, it is possible to obtain a more accurate measurement result if the point of time when the peak current appears as a start point. The invention has been completed.

When the peak current appears, the sample is injected into the sample passage and then passes through the working electrode and the reference electrode in order to reach the extension region of the working electrode, so that if the sample reaches the extension region of the working electrode It may be considered that a sufficient amount of sample is filled in the passage.

6 and 7 are perspective views illustrating a first embodiment of a biosensor in which a sample filling detection electrode is mounted according to the present invention. FIG. 6 is an exploded perspective view and FIG. 7 is an assembled perspective view.

As shown, the biosensor 10 of the present invention is characterized in that the sample filling detection electrode 12a formed by extending the working electrode 12 has a main feature. The lower insulating substrate 11 and the spacer ( 15, 16, the upper insulating substrate 17, and the reaction enzyme layer 14 is the same configuration as in the prior art and will not be described.

Basically, the working electrode 12 and the reference electrode 13 are formed to be stacked on the upper surface of the lower insulating substrate 11, based on the flow direction of the sample injected from the sample inlet 18a into the sample passage 18. The first electrode (upstream electrode) becomes the working electrode 12, and the second electrode (downstream electrode) becomes the reference electrode 13.

At this time, the end of the working electrode 12 and the reference electrode 13 is connected to the end of the biosensor so that the biosensor 10 can be connected to a terminal formed in the socket of the meter when the biosensor 10 is inserted into the meter (not shown). Are exposed from.

Particularly, in the biosensor of the present invention, the working electrode 12 extends in the longitudinal direction of the substrate 11 and then extends in the transverse direction and again toward the opposite side in the longitudinal direction to pass through the sample passage region. .

As such, the working electrode 12 extends around the end of the reference electrode 13 and passes through the sample passage region downstream of the reference electrode based on the sample flow direction, wherein the extended electrode 12a passes through the sample passage region. ) Serves as a sensing electrode that senses that the sample is sufficiently filled in the sample passage.

That is, the extended electrode (hereinafter referred to as a sensing electrode) detects that the sample is sufficiently filled in the sample passage after passing through the working electrode 12 and the reference electrode 13 after the sample is injected through the sample inlet 18a. As electrodes, they are formed with the same width or smaller width than the existing working electrodes 12 on the upstream side of the reference electrode 13. As will be described later, the measuring device detects the full state of the sample from the signal appearing when the sample touches the sensing electrode 12a region, and recognizes this time as a start time.

The sensing electrode 12a extends from the working electrode 12 and is at least partially inserted into the sample passage region. The sensing electrode 12a may extend to completely pass through the sample passage region, or may not extend through the sample passage region. The end of the sensing electrode 12a may extend in the passage area.

In addition, the sensing electrode 12a may be formed in the form of a strip or a thin line having a reduced width compared to the existing working electrode 12 upstream of the reference electrode 13, and the working electrode 12 and the reference electrode 13 ), It may be formed by a thin film forming method such as etching, screen printing, sputtering, or the like.

In the case of using the biosensor of the present invention, the peak current appears when the sample reaches the sensing electrode 12a region while the sample is sufficiently filled in the sample passage 18 through the working electrode 12 and the reference electrode 13. This will be described below with reference to FIGS. 8 and 9.

8 and 9 are waveform diagrams showing the waveform of the operating voltage when the biosensor of the present invention is used, and the current flowing through the working electrode. The operating voltage and current conditions appearing after insertion of the biosensor and application of the operating voltage and sample introduction are shown.

In each drawing, (a) is a waveform diagram of an operating voltage, in which a solid line does not apply an incubation time, and a dotted line shows a state in which a voltage is released at a start time when an incubation time is applied, and 'I' In the state where the voltage is released at the start time (actual sample filling time) t2 'when the biosensor of the present invention is applied,' II 'is a start time point (ith current value reaching time) (t2) when the conventional biosensor is applied. ) Shows the state where the voltage is released.

(b) is a waveform diagram showing the current flowing to the working electrode when the incubation time is not applied, that is, when the voltage is not released, and (c) shows a current state rapidly falling due to the voltage release when the incubation time is applied. The waveform diagram shown. Here, 'I' represents the application of the biosensor of the present invention, and 'II' represents the application of the conventional biosensor.

8 illustrates an example in which the incubation time proceeds in the 0V state by completely releasing the operating voltage, and FIG. 9 illustrates an example in which the incubation time proceeds by lowering the operating voltage to a predetermined voltage.

First, when the biosensor of the present invention is applied, a sample is injected through the sample inlet 18a and then passes through the working electrode 12 and the reference electrode 13 in the sample passage 18 to the sensing electrode 12a. At the point of contact, that is, at the time t2 ', as shown in FIGS. 8 and 9 (b) and (c), a current peak state (indicated by a circle' B ') that rapidly increases and then falls down is shown. This is a phenomenon only appearing in the biosensor of the present invention, in particular, a current peak phenomenon exhibited by the sensing electrode 12a extending from the working electrode 12. That is, when the sample passes the working electrode 12 past the reference electrode 13 and reaches the area of the sensing electrode 12a downstream thereof, the peak current Ip2 'is instantaneously displayed, and then the normal reaction state and The current value is displayed.

As such, the t2 'time at which the peak current Ip2' appears is the point at which the sample passes through the working electrode and the reference electrode to reach the sensing electrode region, so that a sufficient amount of the sample can cause a normal reaction with the working electrode of the sample passage. At this point, the reference electrode is filled.

Therefore, the operation / control unit of the measuring device may detect the peak current Ip2 'appearing at the time t2' and output a start signal, and a typical measurement process may be performed using the time t2 'as the start time.

For example, the operation / control unit has an incubation time by completely releasing the operating voltage to 0 V or lowering it to a predetermined voltage at the time t2 ', which is the point at which the sample is actually filled (see' I '), and then applying the operating voltage again (Fig. 4). The concentration of the analyte can be measured by reading the current value (im in FIG. 4) at a specific point in time (t4 time in FIG. 4).

In the biosensor of the present invention, the peak current (Ip2 ') takes a form in which the response current rises to the normal state after a momentary rise as the sample touches the sensing electrode region. More specifically, first, a momentary change occurs in the area where the sample and the working electrode meet at the moment when the sample touches the sensing electrode 12a extended from the working electrode 12. Since the sensing electrode 12a is an extended electrode connected from the working electrode 12, the sample is actually a part of the working electrode disposed downstream of the reference electrode 13, so that the sample is located in the sensing electrode 12a region. A momentary increase in the area of the working electrode area in contact with the sample occurs when the sensing electrode is part of the working electrode. Therefore, the peak current Ip2 'is displayed by adding the increase in the reaction current which is indicated by the instantaneous change of the area of the working electrode region to which the sample touches, and the increase in noise that occurs when the sample touches the working electrode region.

In FIG. 8 and FIG. 9B, the portion indicated by the circle 'A' shows a current state in which noise reflected when the sample meets the reference electrode 13 is reflected.

Referring to (c), a clear time interval exists between the conventional start time t2 and the start time t2 'of the present invention. Conventionally, the time t2 at which the reaction current value reaches the predetermined current value ith is a start time. However, in the present invention, a sufficient amount of the sample is filled through the reference electrode 13 to reach the sensing electrode 12a. One point (a point of time at which the peak current is detected) t2 'is taken as a start point, so that a more accurate point at which the actual sample is sufficiently filled can be a start point, and thus a more accurate measurement result can be obtained. The time from t2 to the ith value until t2 'when the peak current is detected is the time to fill up until the sample is full.

In the biosensor 10 of the present invention as described above, the sensing electrode 12a extending from the working electrode 12 and positioned downstream of the reference electrode 13 may completely pass through the sample passage region as shown in the illustrated example. However, as briefly mentioned above, the sensing electrode 12a is an electrode into which at least a portion or more of the sensing electrode 12a is inserted, and the sensing electrode 12a is present in the sample passage region instead of passing through the sample passage region. It can also be implemented in the form.

In addition, in the biosensor 10 of the present invention, the sensing electrode 12a may be formed to have the same width or a smaller width than the width of the working electrode 12. When the width is small, the sensing electrode 12a looks like a real visual line. It may be in the form of an electrode, it may be formed in a width of approximately 100 ~ 200 ㎛. Here, it is not preferable because the peak current is too weak or may give an error to the measurement result when it is out of the range of 100 to 200 μm.

On the other hand, Figure 10 is a perspective view showing the electrode structure in the second embodiment of the biosensor according to the present invention, Figure 11 and Figure 12 is a waveform of the operating voltage when using the biosensor of the second embodiment the current flowing through the working electrode This is a waveform diagram showing the operating voltage and current waveform diagram at the beginning of the measurement. The operating voltage and current conditions appearing after insertion of the biosensor and application of the operating voltage and sample introduction are shown.

First, referring to the electrode structure, the reference electrode 13 is formed on the upper surface of the lower insulating substrate 11 by branching into the first reference electrode part 13a and the second reference electrode part 13b, and the first reference electrode part. 13a and the second reference electrode portion 13b are formed to pass through the sample passage region side by side at a predetermined interval. In the drawing, the first reference electrode part 13a is disposed upstream with respect to the sample flow direction, and the second reference electrode part 13b is disposed downstream.

In addition, the sensing electrodes 12a and 12b extending from the working electrode 12 in the same direction as the first embodiment are formed, and the sensing electrodes also branch into two electrodes and extend into the sample passage region.

Both sensing electrodes 12a and 12b are formed to extend from the working electrode 12. Hereinafter, for the sake of clarity, the upstream sensing electrode 12a as the first sensing electrode and the downstream sensing electrode ( 12b) will be referred to as a second sensing electrode.

As illustrated, the first reference electrode portion 13a is positioned downstream of the working electrode 12, and the first sensing electrode 12a is disposed between the first reference electrode portion 13a and the second reference electrode portion 13b. The second sensing electrode 12b is positioned downstream of the second reference electrode part 13b.

Accordingly, the working electrode 12, the first reference electrode part 13a, the first sensing electrode 12a, the second reference electrode part 13b, and the second sensing electrode 12b based on the sample flow direction in the sample passage region. It is arranged in the order of, and when the sample is introduced to pass through these electrode areas in order.

In the biosensor 10 of the present invention as described above, the first and second sensing electrodes 12a and 12b extending from the working electrode 12 and located downstream of the reference electrode 13 are sample passages as shown. Although it is possible to completely pass through the region, as in the first embodiment, at least a part or more of the electrode is inserted into the sample passage region, and the end of each sensing electrode 12a, 12b does not exit through the sample passage region and passes through the sample passage. It is also possible to implement in the form existing in the area.

At this time, in the case of using the biosensor of the second embodiment, looking at the current waveform diagram, as shown in FIGS. 11 and 12 (b) and (c), a total of three peak currents Ip1, Ip1 ', and Ip2 after sample introduction. ') Appears.

First, t0 denotes the time point at which the biosensor is inserted (the voltage application start time point), t1 denotes the time point at which the sample is first injected (time point where the working electrode 12 meets the region), and t2 denotes the conventional start time point (conventional voltage). Release or step down time), t2 'represents a start time of the present invention. Here, t2 'is a time point at which the third peak current Ip2' appears, which is a time point at which the sample touches the second sensing electrode 12b area. In the present invention, this time is sensed as a time point when the sample is sufficiently filled. Will be used.

Among the three peak currents Ip1, Ip1 ', and Ip2', the first peak current Ip1 appears when the sample reaches the region of the first reference electrode portion 13a past the working electrode 12, and the second peak current. (Ip1 ') appears when the sample touches the area of the first sensing electrode 12a past the first reference electrode portion 13a, and the third peak current Ip2' is the first sensing electrode 12a and the second. Appears at the point of contacting the second sensing electrode 12b area past the reference electrode portion 13b.

Here, the peak currents Ip1 'and Ip2' appearing at the point of contact between the region of the first sensing electrode 12a and the region of the second sensing electrode 12b are exhibited by the same action as the peak current of the first embodiment.

By arranging a total of five electrodes in the sample passage region as described above, a total of three peak currents Ip1, Ip1 ', and Ip2' appear. In particular, in the case of the last peak current Ip2 ', the sample is the working electrode 12 and the reference electrode. Through (13), a current signal indicating sufficient filling (sample filled state) is obtained. That is, the second sensing electrode 12b is used as an electrode for detecting the fullness of the sample.

As a result, the operation / control unit may detect the peak current Ip2 'represented by the second sensing electrode 12b to recognize that the sample is filled with a sufficient amount of the biosensor, and may output a start signal at this point. .

In addition, in the second embodiment, the calculation / control unit of the measuring device detects the peak current Ip1 at the initial time t2a at which the sample is injected and the peak current Ip2 'at the time t2' at which the sample is filled. In addition, since the time measurement between the two time points t2a and t2 'is possible while the distance between the electrode positions at which each peak current appears is known, it is possible to confirm the flow characteristics and the inflow rate of the sample.

If the time until the sample is completely filled (time between t2a when the peak peak current appears and t2 'when the peak peak current appears) is measured, the measured time is determined by the difference in reactivity (flow characteristics of the sample). It can be used as a means for obtaining a correction coefficient that can correct the resulting error error.

In more detail, the inflow rate of the sample is different according to the flow characteristics of the sample, it is possible to infer the nature of the sample through the difference in inflow rate. In addition, the flow characteristics of the sample may affect the measurement result and cause an error.If the difference of the reaction according to the properties of the sample is quantified in advance through an experiment and stored in the measuring device, the difference of the response is finally corrected by measuring the speed. can do.

For example, when blood is used as a sample, when the red blood cell count in the blood is low, the solubility of the reagent (reactive enzyme layer) is increased and the reactivity is increased, resulting in a high result. Conversely, higher levels of red blood cells in the blood decrease the solubility and lower the reactivity. In addition, there is an inverse relationship between the red blood cell level and the inflow rate of blood. By measuring the inflow rate of blood by a meter, the red blood cell level can be corrected to correct the difference in reactivity.

Accordingly, in the second embodiment of the present invention, the time t2 'filled with the sample can be accurately detected and used as a start time, thereby obtaining accurate measurement results. In addition, since the time from the initial time point t2a when the sample is injected to the time when the sample is completely filled (t2 ') can be known, the inflow rate of the sample can be obtained therefrom to correct the measurement result.

In this way, in the biosensor according to the present invention, a sample injected through a sample inlet using a sensing electrode (connected integrally from the working electrode) extending from the working electrode to the sample passage region from the working electrode to the lower insulating substrate ( Accurate filling time of the sample, such as blood, can be detected by the measuring instrument. As a result, a more accurate result can be obtained by performing a sample analysis using the starting point of the actual filling of the sample.

In addition, by the added electrode of the biosensor, the time point at which the sample is injected and the time point at which the sample is completely filled can be detected by the measuring device, thereby enabling estimation of the flow characteristics and the inflow rate of the sample. It is possible to obtain more accurate results.

In the above, a specific preferred embodiment according to the present invention has been described. However, it is to be understood that the present invention is not limited to the above-described embodiments, and the above-described embodiments merely represent some of various embodiments to which the principles of the present invention are applied. Those skilled in the art to which the present invention pertains may make various changes without departing from the spirit of the technical idea of the present invention described in the claims below.

1 and 2 is a basic configuration of a conventional electrochemical biosensor,

3 is a diagram illustrating a biosensor and a measuring instrument;

4 and 5 is a waveform diagram showing a waveform of the operating voltage and the current flowing in the working electrode when using a conventional biosensor,

6 and 7 are a perspective view showing a first embodiment of a biosensor with a sample filled detection electrode according to the present invention,

8 and 9 are waveform diagrams showing the waveform of the operating voltage and the current flowing in the working electrode when using the biosensor of the first embodiment;

10 is a perspective view showing an electrode structure in a second embodiment of a biosensor according to the present invention;

11 and 12 are waveform diagrams showing the waveform of the operating voltage and the current flowing in the working electrode when using the biosensor of the second embodiment.

<Explanation of symbols for the main parts of the drawings>

10: biosensor 11: lower insulation board

12: working electrode 12a: sensing electrode (or first sensing electrode)

12b: second sensing electrode 13: reference electrode

13a: first reference electrode portion 13b: second reference electrode portion

14: reactive enzyme layer 15, 16: spacer

17: upper insulating substrate 18: sample passage

18a: sample inlet 20: measuring instrument

21: operation / control unit 22: operating voltage generator

23: current / voltage converter 24: AD converter

25: display

Claims (13)

The working electrode 12 and the reference electrode 13 are formed long on the lower insulating substrate 11 to pass through the sample passage 18, and the working electrode 12 and the reference electrode 13 are formed along the sample passage 18. In the biosensor configured by fixing the reaction enzyme layer 14 across, The lower insulating substrate 11 is provided with a sensing electrode 12a extending to insert at least a portion of the sample passage region downstream from the reference electrode 13 based on the sample flow direction from the working electrode 12. Biosensor with a sensing electrode, characterized in that the sample flowing along the passage (18) is configured to pass through the working electrode (12) and the reference electrode (13) in order to reach the area of the sensing electrode (12a). The method according to claim 1, The sensing electrode (12a) is a biosensor with a sensing electrode, characterized in that extending to pass completely through the sample passage area. The method according to claim 1, The sensing electrode (12a) is a biosensor with a sensing electrode, characterized in that the end is formed extending in the form that does not pass through the sample passage region in the sample passage region. The method according to claim 1, The sensing electrode (12a) is a biosensor with a sensing electrode, characterized in that formed by having the same or smaller width than the width of the working electrode (12). The lower insulating substrate 11 is provided with a sensing electrode 12a extending to insert at least a portion or more into the sample passage region downstream of the reference electrode 13 from the working electrode 12 with respect to the sample flow direction. Using a biosensor 10 configured to cause the sample flowing along the passage 18 to pass through the working electrode 12 and the reference electrode 13 in order to reach the sensing electrode 12a region, After detecting the peak current Ip2 'that appears when the sample reaches the sensing electrode 12a region in the measuring device 20 into which the biosensor 10 is inserted, the peak current Ip2' is detected. Method of analyzing a sample using a biosensor with a sensing electrode, characterized in that the point of time (t2 ') is used as the point of time when the sample is filled in the biosensor (10). The method according to claim 5, A bio-electrode with a sensing electrode, characterized in that the time t2 'at which the peak current Ip2' is detected as a start point is read thereafter and the analyte in the sample is analyzed using the current value at a specific time t4. Sample analysis method using a sensor. The working electrode 12 and the reference electrode 13 are formed long on the lower insulating substrate 11 to pass through the sample passage 18, and the working electrode 12 and the reference electrode 13 are formed along the sample passage 18. In the biosensor configured by fixing the reaction enzyme layer 14 across, A reference electrode 13 is formed on the lower insulating substrate 11 by branching into a first reference electrode portion 13a and a second reference electrode portion 13b passing through a sample passage region, and the first electrode 13 is formed from the working electrode 12. At least a portion of the sample passage region between the first reference electrode portion 13a and the second reference electrode portion 13b and the sample passage region downstream of the second reference electrode portion 13b with respect to the sample flow direction are inserted. Each of the sensing electrodes 12a and 12b is formed to extend, and the sample flowing along the sample passage 18 allows the working electrode 12, the first reference electrode part 13a, the first sensing electrode 12a, and the second to flow through the sample passage 18. The biosensor having a sensing electrode, characterized in that configured to reach the region of the second sensing electrode (12a) through the reference electrode (13b) in turn. The method according to claim 7, The sensing electrode (12a) is a biosensor with a sensing electrode, characterized in that extending to pass completely through the sample passage area. The method according to claim 7, The sensing electrode (12a) is a biosensor with a sensing electrode, characterized in that the end extends in the form that does not pass through the sample passage region exists in the sample passage region. The reference electrode 13 is formed on the lower insulating substrate 11 by branching into the first reference electrode portion 13a and the second reference electrode portion 13b passing through the sample passage region, and the first electrode from the working electrode 12 is first formed. At least a portion of the sample passage region between the reference electrode portion 13a and the second reference electrode portion 13b and the sample passage region downstream of the second reference electrode portion 13b with respect to the sample flow direction are respectively inserted. Elongated sensing electrodes 12a and 12b are provided, and a sample flowing along the sample passage 18 flows through the working electrode 12, the first reference electrode part 13a, the first sensing electrode 12a, and the second reference. By using the biosensor 10 configured to reach the second sensing electrode 12b region in turn through the electrode portion 13b, After detecting the peak current Ip2 'that appears when the sample reaches the region of the second sensing electrode 12b in the measuring device 20 in which the biosensor 10 is inserted, the peak current Ip2' is Method of analyzing a sample using a biosensor with a sensing electrode, characterized in that the detected time (t2 ') is used as a time point when the sample is filled in the biosensor (10). The method according to claim 10, A bio-electrode with a sensing electrode, characterized in that the time t2 'at which the peak current Ip2' is detected as a start point is read thereafter and the analyte in the sample is analyzed using the current value at a specific time t4. Sample analysis method using a sensor. The method according to claim 10, The peak current Ip1 is detected by the measuring device 20 when the sample passes the working electrode 12 and reaches the first reference electrode part 13a. Method of analyzing a sample using a biosensor with a sensing electrode, characterized in that to use the time (t2a) as the initial time when the sample is injected into the biosensor (10). The method according to claim 12, The peak current Ip1 at the time when the sample reaches the region of the first reference electrode 13a and the peak current Ip2 'at the time when the sample reaches the region of the second sensing electrode 12b are detected by the measuring device 20. A method for analyzing a sample using a biosensor with a sensing electrode, characterized in that to estimate the inflow rate of the sample using the time (t2a, t2 ').

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