JP2016186422A - Temperature calibration method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a temperature calibration method of a minute flow channel device capable of correcting a calibration obtained by one calibration work to a proper calibration value.SOLUTION: The temperature calibration method includes the steps of: after feeding at least one type of reagent which emits a signal at a known temperature to a minute flow channel, calculating a relationship between time and temperature based on the signal of the reagent; estimating temperature of at least a neighboring flow channel at a point when the reagent signal in the minute flow channel as a target of the calibration is obtained; calculating a fluctuation portion in the resistance values from a temperature difference between the minute flow channel as the target and a fluctuation portion; correcting the calculated resistance value of the fluctuation portion with respect to the resistance value of the target minute flow channel; and calibrating the relationship between the temperature in the flow channel of the target minute flow channel and a resistance value of a resistance body by using resistance value after correction.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a temperature calibration method for a chip incorporating a plurality of heaters.

  In recent years, research and development on a technology called a micro total analysis system (μ-TAS) that incorporates all elements necessary for chemical and biochemical analysis on a single chip has been active. The chip is composed of a micro flow path, a temperature control mechanism, a concentration adjustment mechanism, a liquid feeding mechanism, a reaction detection mechanism, and the like, and is generally called a microfluidic device.

  Among them, a DNA analysis device for examining genetic information such as a single nucleotide polymorphism (SNP) of the human genome has attracted attention, and research is being actively conducted.

  There are two steps for analyzing DNA. (1) a step of amplifying DNA and (2) a step of determining DNA.

  In the step of amplifying DNA of (1), a PCR (Polymerase Chain Reaction) method is generally used. This is a technique for amplifying DNA by mixing a primer complementary to a part of DNA to be amplified and an enzyme and applying a thermal cycle. This process requires high-speed temperature control that is accurate and shortens the reaction time. Therefore, a chip in which a heater is formed in the vicinity of the microchannel is preferable.

  FIG. 10 to FIG. 12 show diagrams of configuration examples for explaining a chip incorporating a heater close to the micro flow path. FIG. 10 shows an overall view of the chip, in which 101 is a micro flow path, 102 is a heater, 103 is an electrode pad, and 104 is a lead connecting the electrode pad 103 and the heater 102.

  FIG. 11 is a diagram for explaining the relationship between the heater 102 and the microchannel 101 in the A-A ′ cross section in FIG. 10. Here, there are a total of eight micro flow paths 101 in FIG. 10, but in the following, two channels are shown as representatives because problems due to the influence between adjacent micro flow paths will be described. The microchannel 101 and the heater 102 are arranged at a constant pitch. Next, in FIG. 12, only one channel is extracted and shown as a top view for explaining the actual driving method. The heater 102 is connected to the electrode pad 103 by a lead 104. A micro flow channel 101 is disposed on the heater 102, and an observation region 105 is set in a region located substantially at the center of the heater 102. The observation area 105 is conceptual, and a structure for discriminating the observation area 105 is not provided in the flow path itself. The observation area 105 is an area for observing a signal from the microchannel 101, such as fluorescence intensity, using an observation system (not shown), and it is necessary to accurately grasp and control the temperature of this area. Generally, a metal is used as the heater 102, and the resistance value increases as the temperature increases due to energization. That is, the temperature of the heater 102 itself can be monitored by measuring the change in the resistance value of the heater 102 itself. Electrodes 106 for voltage drop measurement are arranged at both ends of the heater 102, and the resistance value of the heater 102 can be stably and accurately measured by a four-terminal measurement method for monitoring the potential applied to the heater 102. Since the insulating layer separating the microchannel 101 and the heater 102 is as thin as 1 to 2 μm, it is considered that the temperature of the microchannel 101 follows the temperature of the heater 102 well. By measuring the resistance value of the heater 102, the heater 102 has not only a heating function but also a temperature measuring function. Hereinafter, the temperature in the observation region 105 is indicated when it is expressed as the temperature of the microchannel without particular notice.

  As a method for calibrating the temperature in the microchannel, there is a method disclosed in the example of Patent Document 1.

A conventional heater temperature calibration method disclosed in Patent Document 1 will be described with reference to FIGS. 15 and 16. As a material used for the heater, platinum is often used because of its good linearity of resistance value change with respect to temperature change. Since each heater actually manufactured has a manufacturing error, in order to accurately obtain the flow path temperature from the measured heater resistance value, a TCR (temperature resistance coefficient: temperature change coefficient) estimated from the design value of the heater 102 is used. If the ratio of resistance change) is used, an error occurs, and calibration work is necessary. An artificially synthesized DNA reagent is often used for the calibration work. That is, a fluorescent reagent that can emit fluorescence only when it is incorporated into DNA in a double helix is commercially available. A reagent in which this is mixed with DNA is used. As the temperature rises, the DNA double helix is gradually broken and separated into double strands. The fluorescence intensity decreases as the double helix dissociates, and is completely quenched when separated into double strands. The temperature at which this fluorescence disappears depends on the binding strength of DNA, and artificial DNA is synthesized so that it is quenched at a specific known temperature. FIG. 15 shows that the microchannel 101 is mixed and filled with two types of artificial DNA reagents synthesized so that fluorescence is quenched at two different temperatures (T1, T2), and the voltage applied to the heater 102 is gradually increased. The result of measuring the change in resistance value and recording the change in fluorescence intensity (vertical axis) while increasing the temperature (horizontal axis) of the microchannel 101 is shown. After this calibration work, the temperature coefficient obtained by the calibration work is used. As can be seen from FIG. 15, the fluorescence intensity itself has a gently downward-sloping curve because the luminous efficiency decreases as the temperature increases. Here, there is a site where the fluorescence intensity changes sharply at known dissociation temperatures T1 and T2 of the artificial DNA. FIG. 16 shows the signal obtained by differentiating the signal obtained in FIG. 15 once with respect to the temperature and inverting the sign. Sharp signals are obtained at known dissociation temperatures T1 and T2. Using the corresponding resistance values R1 and R2 at the time when the signals of T1 and T2 are obtained, a coefficient of a linear expression indicating the relationship between the resistance value and the microchannel temperature is obtained. That is, after obtaining the slope of the linear equation from the temperature difference between T1 and T2 and the resistance value difference between R1 and R2, the intercept can be obtained using one of the values. That is, T = K1 × R + K2 between the temperature T [° C.] and the resistance value R [Ω].
K1 = (T2-T1) / (R2-R1)
K2 = (R2 / T1-R1 / T2) / (R2-R1)
Is established.

JP 2013-51969 A

  However, the above-described conventional calibration method has problems specific to microfluidic devices. That is, the micro flow path of a certain channel is not only heated only by the supply of heat from the heater immediately below, but also affected by the inflow of heat from the heaters of adjacent channels arranged in close proximity. More specifically, there is a manufacturing variation in the resistance value of the heater and the TCR before calibration, that is, the coefficients K1 and K2 may vary from channel to channel. Since these values are not exactly known before calibration, the heater temperature during calibration cannot be accurately grasped, and the temperature of each channel cannot be controlled at exactly the same temperature. That is, the relationship between the resistance value and the fluorescence intensity change in FIG. 15 is obtained in a state where the heater temperatures of adjacent channels are not uniform. Here, for example, when there is a variation in the amount of heat generated due to a variation in the heater resistance values of the nth and (n + 1) th channels, the temperature distribution in the heater is different depending on the temperature difference with the heater of the adjacent channel. 17A, 17B, and 17C are diagrams schematically showing the difference in heat distribution when the temperatures of adjacent channels are different. The solid line □ indicates the n-th heater of interest, and the dotted line □ indicates the (n−1) -th and (n + 1) -th heater surfaces adjacent to each other, and the distribution of the amount of heat due to the heat generation and propagation of each heater Is indicated by gray scale contour lines. (A) when three channels are driven at the same temperature, (b) when (n-1) th and (n + 1) th channels are driven at a lower temperature than the nth channel, (C) shows a case where the (n−1) th and (n + 1) th channels are driven at a higher temperature than the nth channel. The temperature distribution in the heater is determined by the amount of heat from the adjacent heater in addition to the amount of heat generated by itself, that is, as can be seen from FIGS. 17 (a), (b), and (c), n The temperature distribution in the lateral direction of the second heater and the temperature distribution in the vicinity of the end in the heater longitudinal direction change. In this configuration, the temperature in the flow path is observed by the change in resistance value due to the temperature change of the heater. However, if the temperature distribution in the heater changes, the resistance value distribution in the heater changes accordingly. This also indicates that the temperature in the observation region is not uniquely determined by the temperature distribution. Therefore, if the relationship between the temperature and resistance value (calibration value) is obtained with the conventional calibration technique and the heater is driven based on the calibration value, the temperature difference between adjacent channels may be different from the temperature difference during calibration work. An error was left in the relationship between the heater resistance value and the flow path temperature. On the other hand, it is possible to obtain the correct calibration value by repeating the calibration work using the acquired calibration value and repeating until the fluctuation of the calibration value falls within the regulation value. There was a problem that it took a long time. Accordingly, an object of the present invention is to correct a calibration value obtained by one calibration operation to an appropriate calibration value.

  The invention according to the present application corrects the resistance value of calibration data acquired in one calibration operation by estimating the temperature difference between adjacent channels at the time of calibration signal acquisition, and uses the corrected data for temperature control during actual operation. It is characterized by using.

  According to the invention of the present application, even in a state before calibration, calibration with suppressed errors can be performed by aligning the influence of adjacent channels with the state after calibration.

When the temperature of the nth channel is controlled to 50 ° C., 70 ° C. or 90 ° C., the temperature difference between the (n + 1) th channel adjacent to the nth channel and the nth heater is supplied. The simulation results on the relationship between the energy changes are shown. The simulation result regarding the influence which the temperature deviation of an adjacent flow path gives is shown. The simulation result which shows that the magnitude | size of the misunderstanding of both ends of a heater always has a fixed relationship according to the temperature deviation with the (n + 1) th channel is shown. It is a figure which shows the signal of the artificial DNA used in the calibration work of a present Example. It is a signal obtained by differentiating the change of the fluorescence signal intensity once with respect to time. It is the figure which expanded a part of FIG. It is a schematic diagram for description. Data based on actual measurements are shown. It is a graph which shows the temperature error at the time of the peak of the fluorescence signal in each case of the presence or absence of the correction | amendment of this invention. It is a figure for demonstrating the chip | tip which incorporated the heater adjacent to the microchannel. FIG. 11 is a diagram for explaining a relationship between a heater 102 and a micro flow channel 101 in the A-A ′ cross section in FIG. 10. It is the figure which extracted one channel explaining the chip | tip which incorporated the heater adjacent to the microchannel. It is a graph which shows the relationship between the temperature of the artificial DNA for calibration, and fluorescence intensity. 14 is a graph obtained by differentiating the graph of FIG. 13 and inverting the sign. It is a figure which shows the relationship between resistance value and a fluorescence intensity change. It is a figure which shows the relationship between a resistance value and the differential value of a fluorescence intensity change. It is a schematic diagram for demonstrating the difference in heat quantity distribution.

  The present invention provides a temperature calibration method for a microchannel device. The microchannel device of the present invention has a plurality of microchannels. In the temperature calibration method of the present invention, the resistor is provided by providing the plurality of microchannels and a resistor adjacent to each microchannel, and passing a current through the resistor to raise the temperature of the microchannel. Calculate the temperature from the resistance value.

  In the temperature calibration method of the present invention, at least one kind of reagent that emits a signal at a known temperature is introduced into the microchannel, the temperature is increased at a known rate, and the resistance value from which the signal is emitted from the reagent is determined as the temperature. Is considered. The temperature calibration method of the present invention includes a step of obtaining a relationship between time and temperature based on a reagent signal for each microchannel, and at least a point in time when a reagent signal of the microchannel to be calibrated is obtained. A step of estimating the temperature of the adjacent flow channel, a step of obtaining a resistance value variation from a temperature difference between the target micro flow channel and the adjacent micro flow channel, and a micro flow channel targeted for the obtained resistance value variation And a step of calibrating the relationship between the temperature in the channel of the target microchannel and the resistance value of the resistor using the corrected resistance value.

  In the temperature calibration method of the present invention, it is particularly preferable that the resistor is longer than the length of the channel in the region in the region where the reagent signal in the microchannel is acquired.

  Hereinafter, the present invention will be described in detail.

  The following examples illustrate the present invention more specifically.

  As a result of diligent investigation on the thermal behavior of a microdevice having a heater close to the microchannel, the inventor has come to understand that there is a useful relationship between the temperature difference between adjacent channels and the heater resistance value. .

  FIG. 1 shows the temperature difference between the (n + 1) th channel and the nth channel and the nth heater when the temperature of the nth channel is controlled to 50 ° C., 70 ° C. or 90 ° C. This is a graph of the relationship between changes in the energy input to. The horizontal axis indicates the temperature deviation based on the temperature of the nth flow path of the (n + 1) th flow path, and the vertical axis indicates the energy input to the corresponding nth heater. Symbols indicated by triangles are results at 50 ° C. control, symbols indicated by squares are results at 70 ° C. control, and symbols indicated by diamonds are results at 90 ° C. control. The vertical axis indicates the value normalized with the maximum power in the examined range. As can be seen from the graph, when the temperature of the (n + 1) th channel is higher than that of the nth channel when the nth channel is controlled at a constant temperature, the energy input to the nth heater is suppressed. It has been. Conversely, when the temperature of the (n + 1) th channel is lower than that of the nth channel, a large amount of energy is input to the nth heater. This indicates that the temperature of the nth channel is not determined only by the temperature of the latest heater but is also influenced by the heat generation amount of the adjacent (n + 1) th heater.

  Furthermore, as a result of examining the influence of the temperature deviation of the adjacent channel by simulation, the following was found. FIG. 2 shows the result of examining the distribution in the longitudinal direction of the heater for the temperature at the center in the width direction of the nth heater. The abscissa indicates the position in the heater longitudinal direction, and the ordinate indicates the same profile for the observation regions 105 of the nth and (n + 1) th flow paths at the same 90 ° C. and the longitudinal temperature distribution. With reference to the temperature distribution of the nth heater at the time, the broken line is the temperature difference from the nth heater when the (n + 1) th flow path is 5 ° C higher, and the solid line is 5 ° C for the (n + 1) th flow path The distribution of the temperature difference with the n-th heater when the temperature is low is shown. Although the observation region 105 has the same temperature, a difference appears in the temperature distribution near the end of the heater.

  The resistance value distribution of the heater also changes according to this temperature distribution. That is, when the overall resistance value of the heater is measured and the temperature is monitored, the observation region shows a constant temperature, but the actual temperature is in the flow path according to the temperature of the (n + 1) th flow path. Indicates that a distribution occurs.

  In this way, when a value is observed as a constant resistance value, but there is actually a difference in the temperature distribution in the flow path, the value indicating this variation as a resistance value is referred to as a “mistake”. And

  As a result of investigations, it was found that the magnitude of the misunderstanding at both ends of the heater always has a constant relationship according to the temperature deviation from the (n + 1) th channel, regardless of the temperature target value to be controlled. FIG. 3 is a graph showing the relationship. The horizontal axis shows the temperature deviation of the (n + 1) th flow path, and the vertical axis shows the error value of the resistance value at the nth heater.

  In the description so far, the relationship with the adjacent channel on one side has always been discussed. Since the interaction occurs between adjacent channels on both sides, correction must be made on the channels on both sides. Further, although the influence of adjacent channels exceeding one channel is weaker than that of the most adjacent channel, it can be considered that accuracy can be improved if correction can be made according to the temperature difference.

  Next, the temperature calibration method of the present invention will be described with reference to actually observed numbers. 4 and 5 are diagrams relating to the artificial DNA signal used in the calibration work of this embodiment. FIGS. 4 and 5 and FIGS. 13 and 14 are based on the same experiment but differ in that in FIGS. 13 and 14, the horizontal axis is indicated by temperature, while FIGS. 4 and 5 are indicated by time.

  FIG. 4 is a graph showing the fluorescence intensity. The artificial DNA used in this figure was designed and manufactured to melt at 70 ° C and 90 ° C. The horizontal axis in FIG. 4 indicates the time when heating is performed at a constant temperature gradient, and the vertical axis indicates a value normalized with respect to the luminance of the fluorescence.

  FIG. 5 shows a signal obtained by differentiating the change in the fluorescence signal intensity once with respect to time. FIG. 6 is an enlarged view of a part of FIG. Comparing the peaks of the differential curves, the peak of the (n + 1) th channel is shifted slightly later than the nth channel.

  FIG. 7 is a schematic diagram for explanation. A point surrounded by a solid circle on the left side indicates that the signal peak of the artificial DNA in the observation region 105 of the flow path is T1, and at this time, the time is t1. A point surrounded by a solid circle on the right side indicates that a signal indicating that the temperature has reached T1 is observed at time t2 from the (n + 1) th flow path. At time t2, the nth flow path is already at a temperature higher than T1. In other words, when the nth channel is at temperature T1, the (n + 1) th channel is at a temperature lower than temperature T1 (dotted circle). In such a state, the resistance value includes an error.

  Therefore, in the present invention, the correction operation describes the temperature of the flow path as a function of time, and corrects the data of the nth flow path to correct the (n + 1) th and (n-1) th flow adjacent to each other. Estimate the temperature of the road and correct the reading error of the resistance value obtained from the estimated temperature deviation.

  FIG. 8 shows actually observed data. In this data, artificial DNA uses a reagent that gives a signal at 70 ° C. and 90 ° C. “Time when peak was observed” indicates the time when the fluorescence peak of the artificial DNA was observed in each channel. T1 is 70 ° C. and T2 is 90 ° C.

  From the peak time obtained for each flow path, an expression of a linear function with respect to the time of the flow path temperature is obtained, and the time when the n-th channel shows a peak signal (27.23597 seconds at 70 ° C., 47. The flow path temperature at 38929 seconds) was obtained by fitting it to its linear function. As a result, for example, when the n-th flow path is 70 ° C., the (n−1) -th flow path is 70.06654 ° C. and the (n + 1) -th flow path is 70.23280 ° C. at the same time. . From this, when the n-th flow path is 70 ° C., the (n−1) -th channel has a temperature deviation of 0.06654 ° C. and the (n + 1) -th channel has a temperature deviation of 0.23280 ° C. I understand.

In the flow channel dimension in this example, the amount of error in the resistance value corresponding to the temperature difference between the adjacent flow channels is
dR = −0.0015 dT + 0.0006
Since the temperature deviation is known, the temperature deviation is converted into an error in the resistance value.

  The correction amount adds up the effects of both sides and corrects the resistance value when the peak of the fluorescence signal is observed. From the corrected resistance value and the known channel temperature indicated by the artificial DNA, a coefficient of a relational expression between the channel temperature and the heater resistance value is obtained.

  The calibration work was compared with and without correction for a chip with 8 channels. A calibration work for confirmation was performed by obtaining a relational expression between the flow path temperature and the heater resistance value without correction. The peak of the fluorescent signal after calibration should be observed at a known temperature, but has some error. In parallel, the same data was corrected using the correction method of the present invention, and the relationship between the channel temperature and the heater resistance value was determined. In FIG. 9, the horizontal axis represents the temperature error at the peak of the fluorescence signal observed in the second calibration operation when calibrated without correction, and the horizontal axis represents the fluorescence signal when calibrated by performing the correction of the present invention. The temperature error at the peak is plotted on the vertical axis. It can be seen that the error is reduced by the correction method of the present invention.

Claims (2)

A temperature calibration method for a micro-channel device having a plurality of micro-channels, wherein a plurality of micro-channels and a resistor adjacent to each micro-channel are provided, and a current is passed through the resistor to control the temperature of the micro-channel. A temperature calibration method for a microchannel device, characterized in that the temperature is calculated from the resistance value of the resistor by raising the temperature,
At least one kind of reagent that emits a signal at a known temperature is introduced into the microchannel, the temperature is increased at a known rate, and the resistance value at which a signal is emitted from the reagent is regarded as the temperature,
Obtaining a relationship between time and temperature based on a reagent signal for each microchannel;
Estimating the temperature of at least the adjacent channel when the reagent signal of the microchannel to be calibrated is obtained;
Obtaining a resistance value variation from a temperature difference between a target microchannel and an adjacent microchannel;
Correcting the obtained resistance value variation for the resistance value of the micro-channel,
A method for calibrating a temperature of a micro-channel device, comprising a step of calibrating a relationship between a temperature in a micro-channel of a target micro-channel and a resistance value of resistance using a corrected resistance value.   The temperature calibration method according to claim 1, wherein the resistor is longer than the length of the channel in the region in the region for acquiring the reagent signal in the microchannel in the microchannel device.

Images