JP2011226897A - Trace substance detector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a trace substance detector that can improve detection accuracy in detecting a trace substance, such as DNA, by measuring electric current.SOLUTION: A trace substance detector comprises: a measurement chamber 41 that comprises a pair of rooms 45, 47 divided by an ion exchange membrane 43 or an ion permeable membrane, and that houses a sample and buffer fluid; and a pair of electrodes 51, 53 placed in the rooms of the measurement chamber. Electric current generated between the electrodes is measured while voltage is actively controlled to be "0" V or approximately "0" V. While electric current is continuously measured to obtain a current value curve, a time derivative is calculated. Then it is determined whether the calculated time derivative value exceeds a predetermined threshold. When the value exceeds the threshold, it is determined that a reaction is started or that a substance to be detected is in a sample.

Description

  The present invention relates to a trace substance detection device for detecting trace substances such as DNA (deoxyribonucleic acid), and in particular, is configured to detect the presence and amount of trace substances by measuring a current value. In this example, the electric charge generated is detected while the electrode potential is set to “0” V or substantially “0” V, and the time derivative value of the current curve measured continuously is used. In addition, the present invention relates to a device devised so that the presence or concentration and the concentration of a target substance to be detected in a specimen can be detected, thereby improving the detection accuracy.

For example, when detecting a trace substance such as DNA, it is common to detect by a voltage measurement.
Moreover, there exist patent document 1, patent document 2, patent document 3, patent document 4, patent document 5, patent document 6, etc. as what is detected by the measurement of an electric current instead of a voltage measurement.
In the case of this type of current measurement, a reference current value is set and stored in advance, and the reference current value is compared with the measured current value obtained by measuring the current to determine whether or not the threshold has been exceeded. The presence or absence of the target substance to be detected in the sample is determined, and the concentration of the target substance to be detected in the specimen is calculated based on the peak of the measured current value.

JP 2008-2333050 A JP 2007-108160 A JP 2006-3222 A JP 2005-69836 A JP 2008-134255 A JP 2006-275788 A

The conventional configuration has the following problems. Usually, the measurement current value obtained by measurement includes noise. This type of noise is caused by contamination of the electrodes installed in the measurement chamber, impurities other than the detection target contained in the sample, a difference in concentration between the two buffer solutions, and the like. . Since this noise is superimposed on the detection current body, the presence / absence of the detection target substance in the sample is determined based on the threshold value, and the concentration of the detection target substance in the sample is calculated based on the peak value. There was a problem that an error occurred.
In particular, in the initial stage of detection, an offset DC current that is substantially steady or slowly decaying may be measured for the reasons described above, and thus it is impossible to specify the absolute “0” point position. As a result, there is a problem that high-precision detection is impaired.

  The present invention has been made based on such points, and an object of the present invention is to provide a trace substance detection device capable of improving the detection accuracy when detecting trace substances such as DNA by current measurement. There is.

In order to achieve the above object, a trace substance detection device according to claim 1 of the present invention includes a measurement chamber that includes a pair of chambers partitioned via an ion exchange membrane or an ion permeable membrane and that stores a specimen and a buffer solution, and the above measurement. In a trace substance detection apparatus comprising a pair of electrodes installed in a pair of chambers, a voltage generated between the pair of electrodes is actively controlled to “0” V or substantially “0” V. The generated current is measured, the current is continuously measured to obtain a current value curve, and its time derivative is calculated, and it is determined whether or not the calculated time derivative exceeds a predetermined threshold value by a predetermined amount. When it is determined that the target is exceeded, the reaction is started or it is determined that there is an object to be detected in the sample.
Further, the trace substance detection apparatus according to claim 2 is the trace substance detection apparatus according to claim 1, wherein the time differential is measured for a time exceeding a predetermined amount and whether the time exceeds another threshold. If it is determined whether or not it has been exceeded, it is determined that there is an object to be detected in the reaction start or in the sample.
According to a third aspect of the present invention, there is provided the trace substance detection apparatus according to the first or second aspect, wherein the concentration of the target substance to be detected in the sample is calculated based on the magnitude of the time differential amount. It is characterized by that.
A trace substance detection device according to claim 4 is the trace substance detection device according to any one of claims 1 to 3, wherein the current amount is observed when the time differential value falls below the threshold value. Only when the amount does not fall below a certain value, it is determined that there is a substance to be detected in the sample.
Further, the trace substance detection device according to claim 5 is the trace substance detection device according to any one of claims 1 to 3, wherein the current amount is observed when the time differential value falls below the threshold value. Only when the amount does not fall below a certain value for a set time, it is determined that the target substance to be detected is present in the sample.
Further, the trace substance detection device according to claim 6 determines that the reaction has ended when the time differential value falls below a certain threshold value in the trace substance detection device according to any one of claims 1 to 5. It is characterized by being.
Further, the trace substance detection device according to claim 7 is the trace substance detection device according to any one of claims 1 to 5, wherein it is determined that the reaction has ended when the current value falls below a certain threshold value. It is characterized by being.
The trace substance detection apparatus according to claim 8 is the trace substance detection apparatus according to claim 6, wherein the reaction is terminated when the time differential value is below a certain threshold value and exceeds a set value. It is characterized in that it is determined that it has been performed.
The trace substance detection device according to claim 9 is the trace substance detection device according to claim 7, wherein the reaction is terminated when the current value is below a certain threshold value and exceeds a certain set value. It is what distinguishes.
A trace substance detection device according to claim 10 is the trace substance detection device according to any one of claims 6 to 9, wherein the sum and integral of the current values flowing from the reaction start time to the reaction end time are as follows. The sum is calculated, and the concentration of the target substance to be detected in the sample is calculated based on these values.
A trace substance detection apparatus according to claim 11 is the trace substance detection apparatus according to any one of claims 1 to 10, wherein a timer is operated from the time when the specimen is introduced into the measurement chamber, and the timer is activated. In other words, the presence / absence of the detection target substance in the sample and the detection of the concentration are performed in association with the detection time.

As described above, the trace substance detection device according to claim 1 of the present invention includes a measurement chamber that includes a pair of chambers partitioned via an ion exchange membrane or an ion permeable membrane and that stores a specimen and a buffer solution, and the measurement chamber. In a trace substance detection device comprising a pair of electrodes installed in a pair of chambers, a voltage generated between the pair of electrodes is generated while being actively controlled to “0” V or substantially “0” V To measure the current, continuously measure the current to obtain a current value curve and calculate its time derivative, determine whether or not the calculated time derivative exceeds a predetermined threshold, When it is determined that it has exceeded, it has been configured to determine that there is an object to be detected in the reaction start or in the sample, so it was determined simply by comparing the measured current with the reference current as in the past Compared to the case The accuracy of discrimination and detection can be improved.
Further, the trace substance detection apparatus according to claim 2 is the trace substance detection apparatus according to claim 1, wherein the time differential is measured for a time exceeding a predetermined amount and whether the time exceeds another threshold. If it is determined whether or not it is exceeded, it is determined that there is a detection target in the reaction start or in the sample, so that the accuracy of determination and detection can be further improved.
According to a third aspect of the present invention, there is provided the trace substance detection apparatus according to the first or second aspect, wherein the concentration of the target substance to be detected in the sample is calculated based on the magnitude of the time differential amount. Thus, the concentration of the detection target substance in the sample can be detected with high accuracy.
A trace substance detection device according to claim 4 is the trace substance detection device according to any one of claims 1 to 3, wherein the current amount is observed when the time differential value falls below the threshold value. If it is determined that there is a substance to be detected in the sample only when the amount does not fall below a certain value, the accuracy of determination and detection can be further increased.
Further, the trace substance detection device according to claim 5 is the trace substance detection device according to any one of claims 1 to 3, wherein the current amount is observed when the time differential value falls below the threshold value. Since it is configured to determine that the target substance to be detected is present in the sample only when the amount does not fall below a certain value within a set time, the accuracy of determination and detection can be further increased.
Further, the trace substance detection device according to claim 6 determines that the reaction has ended when the time differential value falls below a certain threshold value in the trace substance detection device according to any one of claims 1 to 5. Since it is configured, the end of reaction can be detected with high accuracy.
A trace substance detection device according to claim 7 is configured to determine that the reaction has ended when the current value falls below a certain threshold value in the trace substance detection device according to any one of claims 1 to 5. Therefore, the end of the reaction can be detected with higher accuracy.
The trace substance detection apparatus according to claim 8 is the trace substance detection apparatus according to claim 6, wherein the reaction is terminated when the time differential value is below a certain threshold value and exceeds a set value. Therefore, the detection accuracy of the end of the reaction can be improved.
The trace substance detection device according to claim 9 is the trace substance detection device according to claim 7, wherein the reaction is terminated when the current value is below a certain threshold value and exceeds a certain set value. Therefore, the detection accuracy of the end of the reaction can be improved.
A trace substance detection device according to claim 10 is the trace substance detection device according to any one of claims 6 to 9, wherein the sum and integral of the current values flowing from the reaction start time to the reaction end time are as follows. -Since the sum is calculated and the concentration of the detection target substance in the sample is calculated based on those values, the concentration of the detection target substance in the sample can be calculated with high accuracy. .
A trace substance detection apparatus according to claim 11 is the trace substance detection apparatus according to any one of claims 1 to 10, wherein a timer is operated from the time when the specimen is introduced into the measurement chamber, and the timer is activated. Since the determination of the presence or absence of the detection target substance in the sample and the detection of the concentration are performed in association with the detection time according to the above, a series of determination and detection can be performed with high accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the 1st Embodiment of this invention, and is a systematic diagram which shows the structure of the whole trace substance detection apparatus. It is a figure which shows the 1st Embodiment of this invention, and is a circuit diagram which shows the structure of the measurement circuit in the control apparatus of a trace amount substance detection apparatus. It is a figure which shows the 1st Embodiment of this invention, and is a circuit diagram which shows the structure of the current amplification part of the measurement circuit shown in FIG. 2 in detail. It is a figure which shows the 1st Embodiment of this invention, and is a circuit diagram which shows the structure of the differentiating circuit of the measuring circuit shown in FIG. 2 in detail. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the 1st Embodiment of this invention, and is a figure which shows typically the DNA of the state by which the magnetic bead was couple | bonded with one end and the oxidoreductase was couple | bonded with the other end. It is a figure which shows the 1st Embodiment of this invention and is a schematic diagram for demonstrating reaction in a measurement chamber. It is a figure which shows the 1st Embodiment of this invention and is a figure which shows the current waveform actually measured. It is a figure which shows the 1st Embodiment of this invention, and is a figure which shows the waveform which reduced the noise from the actually measured current waveform with a low-pass filter. It is a figure which shows the 1st Embodiment of this invention, and is the figure obtained by performing differentiation on the waveform diagram shown in FIG. It is a figure which shows the 2nd Embodiment of this invention, and is a characteristic view which shows an electric current apparatus and a difference, respectively. It is a figure which shows the 2nd Embodiment of this invention, and is a characteristic view which shows the actual difference value. It is a figure which shows the 2nd Embodiment of this invention, and is a characteristic view which shows a noise reduction difference value.

Hereinafter, a first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a system diagram showing a configuration of a trace substance detection apparatus according to this embodiment. First, there is a tube 1. A sample injection unit 3 is provided at one end side (left end in FIG. 1) of the tube 1, and a sample injector 5 is installed in the sample injection unit 3. The sample 6 is injected by the sample injector 5. This specimen 6 contains a substance to be detected. In addition, a buffer solution injection unit 7 is installed in the tube 1 near the sample injection unit 3, and the buffer solution injection unit 7 is filled with a buffer solution 9. The buffer solution injection part 7 is connected to the tube 1 via a branch tube 11, and a first pushing valve 13 is attached to the branch tube 11.

  The buffer solution 9 is, for example, a liquid obtained by adding an electron mediator such as dichloroindophenol to a mixture of an aqueous solution of potassium dihydrogen phosphate and an aqueous solution of dipotassium hydrogen phosphate. The electron mediator is for mediating the movement of electrons between the oxidoreductase and the electrode.

  A first reagent injection part 15 and a second reagent injection part 17 are connected to the tube 1 via branch tubes 19 and 21, respectively. The first reagent injection part 15 is filled with a first reagent 23. The second reagent injection part 17 is filled with the second reagent 25. The first reagent 23 is provided with a part that specifically binds to the substance to be detected in the specimen 6 and has magnetic particles (magnetic beads). The second reagent 25 is provided with a portion that specifically binds to the substance to be detected in the specimen 6 and has an oxidative or reductase enzyme. A second pushing valve 27 is attached to the branch tube 19 and a third pushing valve 29 is also attached to the branch tube 21.

  The tube 1 is provided with a reaction unit 31, and a temperature control heater 33 is installed in the reaction unit 31. The reaction unit 31 is provided with an ultrasonic vibration device 35. The temperature control heater 33 realizes a temperature environment suitable for the reaction. Further, the stirring effect is enhanced by the ultrasonic vibration device 35. The reaction unit 31 is provided with a first magnetic force control unit 37. The first magnetic force control unit 37 adsorbs and holds the substance to be detected (DNA) in the specimen 6 to which magnetic beads are attached. Further, a fourth pushing valve 39 is attached to the tube 1 on the left side of the temperature control heater 33 in FIG.

A measurement chamber 41 is connected to the other end of the tube 1. An ion exchange membrane 43 is installed in the measurement chamber 41, and a pair of chambers 45 and 47 are defined and formed by the ion exchange membrane 43. Electrodes 51 and 53 are installed in these chambers 45 and 47. The measurement chamber 41 is configured to be vibrated by an ultrasonic vibration device that is the same as or different from the ultrasonic vibration device 35 provided in the reaction unit 31.
In this embodiment, the ion exchange membrane 43 is used, but an ion permeable membrane may be used.

  Branch tubes 53 and 55 are connected so as to sandwich the measurement chamber 41. These branch tubes 53 and 55 are assembled, and a pump 57 is connected thereto. Further, a fifth pushing valve 59, a sixth pushing valve 61, and a seventh pushing valve 63 are attached to the branch tube 53, the tube 1, and the branch tube 55. A second magnetic force control unit 65 is installed in the measurement chamber 41.

  In addition, a control device 71 is installed, and a measurement circuit 73 is provided in the control device 71. Various devices already described, the first pushing valve 13, the second pushing valve 27, the third pushing valve 29, the fourth pushing valve 39, the fifth pushing valve 59, the sixth pushing valve 61, the seventh The pressing valve 63, the temperature control heater 33, the ultrasonic vibration device 35, the first magnetic force control unit 37, the pump 57, and the second magnetic force control unit 65 are all controlled by the control device 71. Further, current measurement and the like via the electrodes 51 and 53 are controlled by the measurement circuit 73 of the control device 71.

According to the above configuration, first, the first pushing valve 13, the fourth pushing valve 39, and the fifth pushing valve 59 are opened, and the pump 57 is driven. As a result, a negative pressure is generated in the tube 1, and the buffer liquid 9 in the buffer liquid injection unit 7 is sucked and filled in the tube 1. In this state, the sample 6 is injected from the sample injector 5 through the sample injection unit 3.
Next, the second pushing valve 27, the third pushing valve 29, the fourth pushing valve 39, and the fifth pushing valve 59 are opened, and the first reagent injection unit 15 A small amount of the first reagent 23 and the second reagent 25 are drawn from the two-reagent injection unit 17. At the same time, the first pushing valve 13 is opened and the buffer solution 9 is drawn out from the buffer solution injection part 7. As a result, the specimen 6, the buffer solution 9, the first reagent 15, and the second reagent 25 are introduced into the reaction unit 31 and mixed.

  Next, the fourth pushing valve 39 and the fifth pushing valve 59 are closed to cause the sample 6 to react with the first reagent 15 and the second reagent 25. At that time, temperature adjustment is performed by the temperature control heater 33 and ultrasonic vibration is applied by the ultrasonic vibration device 35. As a result, a temperature environment suitable for the reaction is provided, and a stirring / mixing action works. By the reaction of the specimen 6 with the first reagent 15 and the second reagent 25, the magnetic beads are bound to one end of the DNA as the substance to be detected and the oxidoreductase is bound to the other end. This is shown in FIG. FIG. 4 is a diagram showing a state in which a magnetic bead 83 is bound to one end of DNA 81 and an oxidoreductase 85 is bound to the other end.

  Next, the first magnetic force control unit 37 is turned on to open the first pushing valve 13, the fourth pushing valve 39, and the fifth pushing valve 59, and the temperature is adjusted by the temperature control heater 33. In addition, while the ultrasonic vibration is applied by the ultrasonic vibration device 35, the buffer liquid 9 continues to flow. As a result, the DNA 81 as the substance to be detected in the specimen 6 is adsorbed and held by the first magnetic force control unit 37, and the others are washed away.

  Next, the first pushing valve 13, the sixth pushing valve 61, and the seventh pushing valve 63 are opened and suctioned by the negative pressure of the pump 57, thereby being attracted and held by the first magnetic force control unit 37. The DNA 81 as the detection target substance after the reaction is introduced into the measurement chamber 41. Then, the sixth pushing valve 61 and the seventh pushing valve 63 are closed to perform measurement. After the measurement, the first pressing valve 13 and the seventh pressing valve 63 are opened, and the measured sample 6 and the like are aspirated and washed away by the negative pressure by the pump 57. As a result, the pump 57 itself is also cleaned.

Here, the reaction in the measurement chamber 41 will be described with reference to FIG. A chemical reaction in the measurement chamber 41 when an oxidase (for example, glucose oxidase) is used as the oxidoreductase 85 will be specifically described. In the chamber 45 above the measurement chamber 41 (upper side in FIG. 5), an oxidase 96 bound to the DNA 81 is present. In the measurement chamber 41, an oxidizable substance 98 and an electron mediator 100 added to the buffer solution are also present. The oxidizable substance 98 is oxidized by the oxidase 96 and releases electrons 102 and cations 104. The electrons 102 are captured by the electron mediator 100, and as a result, the electron mediator 100 is charged “negatively”. The electron mediator 100 charged “negatively” moves to the electrode 53 side having a relatively “positive” potential. On the other hand, the cation 104 passes through the ion exchange membrane 47, is installed on the chamber 47 side, moves to the electrode 53 side having a relatively “negative” potential, and receives the electrons 102. As a result, a potential difference is generated between the chamber 45 and the chamber 47 across the ion exchange membrane, and a current flows between the electrode 51 and the electrode 53. By measuring this current, the DNA 81 is detected and quantified.

Further, the present invention is not limited to the above example, and it is conceivable to use a reductase instead of the oxidase 95 as the oxidoreductase 85. In this case, a substance to be reduced is used instead of the substance to be oxidized 98, and electrons are transferred from the electrode 51 to the reductase through the electron mediator to cause a reduction reaction. Therefore, the electrode 51 that receives electrons from the outside is a “positive” electrode, and the electrode 53 that emits electrons to the outside is a “negative” electrode.
The measurement of the substance to be detected can also be performed by measuring the voltage between the electrodes 51 and 53.

  Next, the configuration of the measurement circuit 73 provided in the control device 71 will be described with reference to FIG. First, there is a current amplification unit 91, and a differentiation circuit 92 and an A / D converter 93 are connected to the current amplification unit 91 via a switch 90. When the switch 90 is switched to the differentiation circuit 92 side (in this embodiment, the switch 90 is switched to the differentiation circuit 92 side), the signal amplified by the current amplifier 91 is differentiated from the differentiation circuit 92. And then analog / digital converted by the A / D converter 93 and input to the microcomputer 95. The microcomputer 95 is connected with an I / O 97 and a memory 99. The I / O 97 is connected with an input means 101 and a closing sensor 103.

The configuration of the current amplifier 91 will be described in more detail with reference to FIG. The current amplifier 91 includes an operational amplifier 105 and a resistor 107. The resistor 107 functions to convert the generated current into a voltage in cooperation with the operational amplifier 105. The + side terminal Sp is connected to one electrode 51 side in the measurement chamber 41, and the − side terminal Sn is connected to the other electrode 53 side in the measurement chamber 41. The + side terminal Fp and the − side terminal Fn are terminals for current flow.
Incidentally, reference numeral 109 in the figure is a display unit incorporated in the control device 71 shown in FIG. 1, and is for displaying the measurement result.

According to the above configuration, a DC voltage of approximately 10 to 30 mV is generated when the external impedance is sufficiently high due to a current component including noise generated in the measurement chamber 41. In the case of the present embodiment, first, this voltage is detected via the + side terminal Sp and the − side terminal Sn. Next, a current is passed from the operational amplifier 105 through the resistor 107 in the direction from the positive terminal Fp to the negative terminal Fn so as to cancel the generated voltage. In this way, the generated voltage is offset to prevent a decrease in measurement accuracy.
Incidentally, only a very small amount (several picoamperes) of current flows between the + side terminal Sp and the − side terminal Sn, so that a so-called “drop voltage” does not occur.

  Next, determination of presence / absence of a detection target and detection of concentration in the present embodiment will be described. In the case of the present embodiment, first, a current is continuously measured to obtain a current value curve. Further, a time differential value is obtained by time differentiation of the current value curve. In order to obtain this time differential amount, the differentiating circuit 92 already described is provided. The differentiating circuit 92 is configured as shown in FIG. That is, the differentiating circuit 92 includes a resistor 121, a capacitor 123, an operational amplifier 125, a capacitor 127, and a resistor 129. The differential current is obtained by differentiating the measured current waveform by the differential circuit 92 having such a configuration.

  Hereinafter, a specific description will be given. First, a measurement current waveform as shown in FIG. 7 is assumed. FIG. 7 is a graph showing time variation of the current value with the horizontal axis representing time and the vertical axis representing current value. By applying a low-pass filter to the measured current waveform shown in FIG. 7 to reduce noise, a diagram as shown in FIG. 8 can be obtained. FIG. 8 is also a graph showing time variation of the current value, with time on the horizontal axis and current value on the vertical axis. Next, the differential waveform as shown in FIG. 9 can be obtained by differentiating the diagram shown in FIG. FIG. 9 is a diagram showing time variation of the differential value with time on the horizontal axis and differential value on the vertical axis. As can be seen from FIG. 9, the differential value is significantly “positive” from around time 30 to 85. This indicates that a reaction is occurring.

In the case of the present embodiment, it is determined whether or not the time differential value exceeds a predetermined threshold value by a predetermined amount. When it is determined that the time differential value has exceeded, the reaction starts or the target object in the sample 6 is detected. It is determined that DNA 81 is present.

Further, the concentration of DNA 81 as the detection target substance in the specimen 6 is calculated based on the magnitude of the time differential amount. Further, it is determined that the reaction has ended when the time differential value falls below a certain threshold value. Also, a timer is activated from the time when the sample 6 is put into the measurement chamber 41, and the presence / absence of the detection target substance in the sample is detected and the concentration is detected in association with the detection time by the timer.
The introduction of the sample 6 into the measurement chamber 41 is detected by the introduction sensor 103 described above.

As described above, according to the present embodiment, the following effects can be obtained.
First, since active control is performed so as to cancel the DC voltage generated in the measurement chamber 41, the current can be detected with high accuracy, and the detection accuracy of the trace substance can be increased.
In addition, chemical noise with relatively weak power is attenuated immediately by the above active control. However, when DNA 81 as a target substance to be detected exists in the specimen 6, a clear current peak can be seen. Highly accurate detection is possible.
Further, since the redox reaction proceeds rapidly, the time required for detection is shortened.
In the case of the present embodiment, since the voltage is actively measured to measure the current, so-called “drop voltage” does not occur, and higher detection accuracy can be realized. it can.
In the case of this embodiment, as in the prior art, simply by comparing the reference current value with the measurement current, it is possible to determine the presence or absence of DNA 81 as the detection target substance in the sample 6 and calculate the concentration. The determination of the presence / absence of DNA 81 as the detection target substance in the sample 6 and the calculation of the concentration are performed using the time differential value of the current value curve measured continuously. Therefore, the accuracy of discrimination and detection can be improved.

Next, a second embodiment of the present invention will be described with reference to FIGS. In the case of the first child embodiment, the current is continuously measured to obtain a current value curve, and the current value curve is time-differentiated to obtain a time differential value. In the case of the second embodiment, “difference” is calculated and discriminated.
In the case of the first embodiment, the switch 90 is switched to the differentiation circuit 92 side in the circuit diagram of FIG. 3, but in the case of the second embodiment, A / D conversion is performed. It is switched so that a signal is directly input to the device 93 side.
The “difference” is as follows.
That is, when there is a certain discrete value x (i), the difference value d (i) is defined by the following equation (I).
d (i) = x (i + 1) -x (i) --- (I)

In the following, a specific description will be given. First, a measurement current waveform and its difference value data as shown in FIG. 10 are assumed. FIG. 10 is a graph showing the time change of the current value with time on the horizontal axis and time with the vertical axis, and simultaneously showing the change in difference value with the number of samples on the horizontal axis and the difference value on the vertical axis. is there. FIG. 11 shows only the difference value data extracted. FIG. 11 is a diagram showing changes in the difference value with the number of samples on the horizontal axis and the difference value on the vertical axis. Furthermore, noise reduction processing is performed on the difference value data shown in FIG. 11 to remove noise, and a diagram as shown in FIG. 12 is obtained. FIG. 12 is a diagram showing the change of the difference value with the number of samples on the horizontal axis and the difference value on the vertical axis. It can be seen from FIG. 12 that the reaction occurs around 25 samples.

In the case of the present embodiment, it is determined whether or not the difference value exceeds a predetermined threshold value by a predetermined amount. When it is determined that the difference value has exceeded, the reaction starts or the DNA 81 as the detection target in the sample 6 is detected. It is determined that there is.

Further, the concentration of DNA 81 as the detection target substance in the specimen 6 is calculated based on the magnitude of the difference value. Further, when the difference value falls below a certain threshold value, it is determined that the reaction has ended. Also, a timer is activated from the time when the sample 6 is put into the measurement chamber 41, and the presence / absence of the detection target substance in the sample is detected and the concentration is detected in association with the detection time by the timer.
The introduction of the sample 6 into the measurement chamber 41 is detected by the introduction sensor 103 described above.

Therefore, the same effect as in the case of the first embodiment can be obtained.

The present invention is not limited to the first and second embodiments.
For example, in the case of the above-described embodiment, the explanation has been given by taking the example of active control to be “0” V. However, the present invention is not limited to this, and control to be substantially “0” V. For example, control that is within ± 1 mV is also conceivable.
It is also conceivable to apply a reverse potential.
The circuit configuration is not limited to that shown in FIGS. 2, 3, and 4, and various examples are conceivable.
In the case of the first embodiment, the presence / absence of the detection target substance in the sample is determined based on whether or not the time differential value exceeds a predetermined amount. However, the present invention is not limited to this. It is not something. For example, it measures the time when the time differential exceeds a threshold by a predetermined amount, determines whether or not the time exceeds another threshold, and when it is determined that the time exceeds, the reaction starts or the object to be detected in the sample It may be possible to determine that there is.
Further, it is conceivable to observe the amount of current when the time differential value is below the threshold value, and to determine that there is a substance to be detected in the sample only when the amount of current does not fall below a certain value.
Also, the current amount is observed when the time differential value falls below the threshold value, and it is determined that there is a target substance to be detected in the sample only when the current amount does not fall below a certain value within a set time. Can be considered.
Further, it may be determined that the reaction has ended when the current value falls below a certain threshold.
Further, it may be determined that the reaction has ended when the length of time that the time differential value is below a certain threshold exceeds a certain set value.
Further, it may be determined that the reaction has ended when the length of time during which the current value is below a certain threshold exceeds a certain set value.
It is also conceivable to calculate the sum, integral, and sum of the current values that flow from the reaction start time to the reaction end time, and calculate the concentration of the detection target substance in the sample based on these values. .
In the case of the first and second embodiments, the measurement chamber has been described as an example of a type having both a chamber in which a reaction is performed and a chamber in which a reaction is not performed. It is not limited to that. For example, a chamber in which a reaction is performed and an electrode is provided, and a chamber having the characteristics of an ion exchange membrane or an ion permeable membrane that is directly or indirectly covered with a resin or a membrane and that has an electrode. It may be a measurement chamber having an arrangement.
The direct means that the resin of the ion exchange membrane or the ion permeable membrane is directly applied to the electrode, and the indirect means that a water-containing resin or the like is provided between the electrode and the resin of the ion exchange membrane or the ion permeable membrane. This means a configuration that intervenes.
In the first and second embodiments, the case where the electron mediator is placed in the buffer liquid has been described as an example. For example, the third reagent is placed beside the second reagent injection portion. An injection unit may be provided, and an electronic mediator may be placed therein, and the injection valve may be opened to inject.

  The present invention relates to a trace substance detection apparatus for detecting trace substances, and in particular, in a device configured to detect the presence or amount of trace substances by measuring a current value, the potential of an electrode is set to “0”. The target substance to be detected in the sample is configured so as to detect the amount of generated charges while V or approximately “0” V, and using the time differential value of the current curve measured continuously. It is suitable for the detection of DNA (deoxyribonucleic acid), for example, which is designed to detect the presence or absence and concentration thereof, thereby improving the detection accuracy.

5 Sample 6 Target substance 41 Measurement chamber 43 Ion exchange membrane 45 Chamber 47 Chamber 51 Electrode 53 Electrode 71 Controller 73 Control circuit 91 Current amplifier 105 Operational amplifier 107 Resistance

Claims (11)

A measurement chamber having a pair of chambers partitioned via an ion exchange membrane or an ion permeable membrane and containing a specimen and a buffer solution, and a pair of electrodes installed in the pair of chambers of the measurement chamber In trace substance detection equipment,
Measure the current generated while actively controlling the voltage generated between the pair of electrodes to "0" V or substantially "0" V,
When the current is continuously measured to obtain a current value curve and its time derivative is calculated, it is determined whether or not the calculated time derivative exceeds a predetermined threshold value, and if it is determined that it has exceeded A trace substance detection apparatus characterized in that it is determined that there is an object to be detected in a reaction start or in a sample. The trace substance detection device according to claim 1,
Measure the time when the time differential exceeds the threshold by a predetermined amount, determine whether the time exceeds another threshold, and if it is determined that the time has exceeded, the reaction start or the target object to be detected in the sample A trace substance detection device characterized in that it is determined to be present. In the trace substance detection apparatus according to claim 1 or 2,
A trace substance detection apparatus characterized in that the concentration of a detection target substance in a specimen is calculated based on the magnitude of the time differential amount. In the trace amount substance detection device according to any one of claims 1 to 3,
A trace substance detection apparatus characterized by observing an amount of current when the time differential value falls below the threshold and determining that there is a substance to be detected in the specimen only when the amount of current does not fall below a certain value. In the trace amount substance detection device according to any one of claims 1 to 3,
A current amount is observed when the time differential value falls below the threshold value, and it is determined that there is a substance to be detected in the sample only if the current amount does not fall below a certain value within a certain set time. Trace substance detection device. In the trace substance detection apparatus according to any one of claims 1 to 5,
A trace substance detection device characterized by determining that the reaction has ended when the time differential value falls below a certain threshold value. In the trace substance detection apparatus according to any one of claims 1 to 5,
A trace substance detection device characterized by determining that the reaction has ended when the current value falls below a certain threshold value. The trace substance detection device according to claim 6,
A trace substance detection device characterized in that when the length of time for which the time differential value is below a certain threshold value exceeds a certain set value, the reaction is terminated. The trace substance detection device according to claim 7,
A trace substance detection device characterized in that when the length of time during which the current value is below a certain threshold value exceeds a certain set value, the reaction is terminated. The trace substance detection device according to any one of claims 6 to 9,
The total, integral, and sum of current values that flow from the reaction start time to the reaction end time are calculated, and based on these values, the concentration of the target substance to be detected is calculated. Trace substance detection device. The trace substance detection device according to any one of claims 1 to 10,
A minute amount characterized in that a timer is started from the time when the sample is put into the measurement chamber, and the presence or absence of a detection target substance in the sample is detected or the concentration is detected in association with the detection time by the timer. Substance detection device.

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