CN1858201A - Controling device and method for polymerase chain reaction chip array - Google Patents

Abstract

The invention discloses a control device and a control method of a polymerase chain reaction chip array. There are no heaters and sensors inside the PCR chip, and the chip is placed on the upper surface of the heat exchanger in the form of an array. The four liquid storage tanks respectively store the liquid of corresponding temperature, and the circulating pump and eight solenoid valves make the liquid of corresponding temperature circulate in the heat exchanger, so that the temperature of each chip is close to the temperature required for the reaction. A flexible printed circuit board is arranged above the chip array, and heaters and sensors corresponding to each chip are fabricated on the board to complete dynamic fine-tuning of the temperature of each chip. Divide the upper surface of the heat exchanger into several temperature zones, select a chip in each temperature zone as a sample chip, build a temperature sensor and fill it with liquid, use its measured value as a feedback signal to simultaneously control all chips in the temperature zone, and the computer through the interface The circuit implements feedforward-cascade control and statistical process.

Description

A control device and control method for a polymerase chain reaction chip array

technical field

The present invention relates to a control device and a control method of a biochip, in particular to a control device and a control method of a polymerase chain reaction chip (polymerase chain reaction chips, PCR chip) array.

Background technique

Polymerase chain reaction is an enzymatic method for rapidly amplifying specific DNA fragments in vitro, and is widely used in various fields of molecular biology. Its basic principle is to use DNA polymerase to rely on the characteristics of DNA templates to induce polymerization in vitro with a pair of primers complementary to the sequences on both sides of the DNA fragment to be amplified. °C), then anneal to the primer at low temperature (typical temperature 55 °C), and then extend at medium temperature (typical temperature 72 °C). This process is repeated in cycles, usually 20-40 cycles, and the amount of specific DNA is multiplied exponentially, which is convenient for further analysis and detection of DNA molecules. The current general-purpose equipment is a desktop PCR amplification instrument, which can simultaneously amplify dozens of samples, and it takes several hours to perform an amplification test.

With the rise of MEMS technology, PCR chips based on semiconductor and microfabrication technology have become a research hotspot. Due to the small volume, extremely small mass and large specific surface area of the PCR chip, it can essentially make up for the shortcomings of the desktop PCR amplification instrument, achieving high heating and cooling rates (up to more than ten degrees per second), short cycle times, and high temperature control accuracy. , The purpose of good dynamic repeatability. Generally, a PCR chip can be used to complete an amplification test within 30 minutes. Another outstanding advantage of the PCR chip is that it can save precious samples, and the required samples are only a few microliters to tens of microliters. "Microsystem Design" ((US) Senturia (Senturia, S.D.); Translated by Liu Zewen, Electronic Industry Press, 2004) describes two major categories of PCR chips, one is continuous flow PCR chips ( or airspace-type PCR chip), and the other type is micro-reaction tank PCR chip (or time-domain PCR chip, batch-type PCR chip). The two types of PCR chips each have their own advantages. The dominant position in the market is currently unknown. The invention is aimed at micro-reaction tank PCR chip.

"Microsystem Design" also describes a temperature control method for micro-reaction tank PCR chips, which integrates heaters and sensors inside the chip, which essentially solves the problem of large inertia and large hysteresis of macroscopic temperature parameters. However, the complex structure of the chip, many production processes, high production cost, and low yield rate lead to high chip prices, and generally only one or a few samples can be amplified at a time. At this stage, it is difficult to replace conventional PCR amplification instruments on a large scale; "Sensors and Actuators B, the ninth issue of 2000, "A rapidmicro-polymerase chain reaction system for hepatitis C virus amplification", discloses another temperature control method and control device for PCR chips in micro-reaction tanks. The chip itself does not contain heaters, Instead, the PCR temperature cycle of a chip is completed by an external temperature control device. The heating and cooling devices used are Peltier devices, and the temperature control strategy is proportional-integral-derivative control, that is, PID control. The PCR chip has a simple structure, simplifies the production process, reduces the production cost, and improves the yield and reliability of the chip. However, due to only relying on the external temperature control device in the macroscopic sense, although the heat capacity of the chip itself is very low and the specific surface area is high, due to the large inertia and large hysteresis of the external temperature control device, the speed and accuracy of temperature control are still low. Limited, the heating and cooling rates are 4°C/s and 2.2°C/s respectively, and only one chip can be cycled each time. Compared with conventional PCR amplification instruments, it still lacks obvious advantages. The existence of the above problems is One of the important reasons why micro-reaction tank PCR chips have not been widely promoted.

Contents of the invention

The purpose of the present invention is to solve the problems existing in the temperature control of the existing micro-reaction tank PCR chip. The invention provides a new control method and forms a control device according to the method, so that multiple low-cost micro-reaction tank PCR chips which do not contain heaters and sensors are subjected to rapid and accurate temperature control at the same time.

In order to achieve the above object, the present invention adopts the following technical scheme: a control device for a polymerase chain reaction chip array, which includes a heat exchanger, and a polymerase chain reaction chip with at least one microcavity is placed on the upper surface of the heat exchanger. The reaction chip, namely the PCR chip, is characterized in that: there are multiple PCR chips, and they form a PCR chip array on the upper surface of the heat exchanger; a flexible printed circuit board is arranged above the PCR chip array, and a flexible printed circuit board is made on the flexible printed circuit board. A heater and sensor array corresponding to the chip array; at the same time, the upper surface of the heat exchanger is divided into several temperature zones, each temperature zone corresponds to a group of PCR chips in the PCR chip array, and a PCR chip is selected in each temperature zone As a sample chip for measurement and control, place a miniature temperature sensor in the microcavity of the sample chip and fill it with a liquid that has similar thermal properties to the PCR reaction solution; electrical components such as temperature sensors in each sample chip, heaters on flexible printed circuit boards, and sensor arrays They are respectively connected with the computer control system through the interface circuit.

The flexible printed circuit board is formed with a plurality of units composed of heaters and sensors, each unit corresponds to a PCR chip, and these units form a heater and sensor array corresponding to the PCR chip array; the flexible printed circuit board is arranged Above the PCR chip array and in contact with each PCR chip.

The inside of the heat exchanger is a flowing heat exchange medium, the heat exchanger is a combination of heat conduction material and heat insulation material, the bottom and the inlet and outlet of the heat exchange medium are heat insulation materials, and the rest are heat conduction materials, and the heat conduction material forms several grooves In contact with the heat exchange medium; the upper surface of the heat exchanger is divided into several temperature zones to ensure that the temperature uniformity of the upper surface of each temperature zone is within a certain allowable range; at least one temperature sensor is set at the fluid inlet of each temperature zone The upper surface of the heat exchanger in each temperature zone corresponds to a number of PCR chips, one of which is a sample chip for measurement and control and the rest are chips for actually performing PCR reactions; the heat exchanger is connected to four liquid storage tanks and circulation pumps through pipelines, There are eight solenoid valves on the pipeline, and the four liquid storage tanks are respectively equipped with temperature sensors, heating elements or refrigeration elements. These electrical elements are connected to the computer control system through the interface circuit, and each solenoid valve is turned on and off under the control of the computer. Switching, so that the heat exchange medium in the corresponding liquid storage tank circulates through the heat exchanger, and the surface temperature of the heat exchanger quickly reaches the required value.

A method for controlling a polymerase chain reaction chip array, the method comprising:

Place a number of PCR chips with microcavities but without heaters and sensors on the upper surface of the heat exchanger to form an array; place a flexible printed circuit board with an array of heaters and sensor units on top of the PCR chip array And in contact with it, each heater and sensor unit corresponds to each PCR chip one by one;

Under the action of the computer control system, each PCR chip obtains a temperature close to the ideal temperature value of the polymerase chain reaction through the upper surface of the heat exchanger; the computer control system collects the temperature of the heat exchange medium at the entrance of the heat exchanger in each temperature zone, the The surface temperature and the temperature of the liquid inside the prototype chip implement a feedforward-cascade composite control strategy to control the corresponding heater on the flexible printed circuit board and heat the corresponding PCR chip to obtain an accurate temperature value;

At the same time, the difference between the upper surface temperature of each chip and the upper surface temperature of the model chip is collected, and statistical process control is implemented to monitor whether the contact thermal resistance of each chip is in a normal state.

The feed-forward-cascade control method is as follows: the temperature detection signal of the heat exchange medium at the entrance of each temperature zone in the heat exchanger is input to the computer control system, and the signal is transformed by a given signal generator formed by software on the one hand inside the computer system. As the setting signal of the main controller also formed by software; on the other hand, the dynamic feedforward link formed by software is integrated with the output signal of the main controller; at the same time, the temperature signal on the surface of each sample chip is detected as the corresponding temperature zone The feedback signal of the sub-controller forms an inner loop, and the sub-controller is also formed by software; the temperature of the liquid inside each sample chip is used as the feedback signal of the main controller in the corresponding temperature zone to form an outer loop; the output signal of the sub-controller is amplified by power and simultaneously Applied to each heater on the flexible printed circuit board corresponding to the group of PCR chips, so as to complete the precise control of the temperature of each PCR chip.

The statistical process control is to use the difference between the upper surface temperature of the chip and the upper surface temperature of the sample chip in the temperature zone where the chip is located as a monitoring parameter to perform single-value-moving range control, that is, to control the difference between the upper surface of the chip and the flexible printed circuit board. Statistical process control is carried out on the dispersion of the indirect contact thermal resistance, and the statistical process control is carried out indirectly on the dispersion of the contact pressure on the upper and lower surfaces of the chip, which is equivalent to the dispersion of the contact thermal resistance between the lower surface of the chip and the heat exchanger. Statistical process control is achieved; when the system is working under statistical control, it can be considered that the dispersion of the contact thermal resistance of each chip is within the allowable range, and the sample chip is fully representative; when a chip is affected by abnormal factors When the monitoring parameters are out of the statistically controlled state, it is considered that the contact thermal resistance of the chip has deviated significantly from the normal value, and the sample chip is no longer representative of the chip, and the computer system will give a corresponding warning.

The beneficial results of the present invention are as follows: a number of PCR chips are placed on the upper surface of a specially designed heat exchanger to form an array; some suitable liquid (such as pure water) is used as the heat exchange medium, controlled by a circulation pump and a solenoid valve, The heat exchange medium of the corresponding temperature is circulated in the heat exchanger according to the needs; the system contains four liquid storage containers, and the liquid temperature in the three containers is respectively controlled at the three characteristic temperatures of the polymerase chain reaction (55°C, 72°C, 95°C); the cooling liquid is stored in the fourth container, which is used to speed up the cooling process from high temperature denaturation to low temperature annealing. A heating and sensing unit is provided for each micro-reaction tank PCR chip to provide accurate and fast temperature control for each PCR chip, and each heating and sensing unit is fabricated on the same flexible printed board; according to the heat exchanger The uniformity and accuracy of the surface temperature require that the upper surface of the heat exchanger be reasonably partitioned, and the temperature in each small area is considered to be the same, which is called an isothermal area. In each isothermal zone, select the PCR chip located in the middle as the measurement and control sample chip, place a miniature temperature sensor (such as a miniature thermocouple) in its microgroove and fill it with a liquid that has similar thermal properties to the PCR reaction solution ( Such as distilled water), it is considered that the temperature of the liquid in each chip in the isothermal zone is the same under the condition that the input electric power of the heater and the sensor heating unit is the same. The polymerase chain reaction is not carried out in the measurement and control sample chip, and the polymerase chain reaction is completed in the remaining chips, but no miniature temperature sensors are placed in these chips. When carrying out different types or different batches of polymerase chain reaction, only the rest of the chips need to be replaced, and there is no need to replace the measurement and control sample chip. The measurement and control sample chip can be regarded as an inherent configuration of the measurement and control system. The concept of the measurement and control sample chip not only simplifies the configuration of the chip temperature sensor and some corresponding operations, but also enables people to accurately calibrate it because it is a fixed configuration of the measurement and control system; the computer automatically collects each measurement and control sample The temperature of the chip forms each control output according to a predetermined control strategy, and the output is not only applied to the corresponding measurement and control sample chip, but also applied to other chips in the same isothermal zone as the sample chip. The main features of the system are "macroscopic temperature control, microscopic temperature correction, use of model chips, and formation of composite control".

Description of drawings

Fig. 1 is a structural representation of the present invention;

Fig. 2 is the schematic diagram of the micro-reaction tank PCR chip without heater and sensor inside;

Fig. 3 is a structural schematic diagram of a heater and a sensor unit based on a flexible PCB;

Fig. 4 is a kind of hardware configuration of PCR chip array temperature control system based on industrial computer;

Fig. 5 is a structural schematic diagram of a temperature zone on the special heat exchanger;

Figure 6 is a schematic cross-sectional view of Figure 5;

Fig. 7 is a feedforward-cascade compound control block diagram of the chip.

Among them, 1. Heat exchanger, 2. Flexible printed circuit board, 3. PCR chip, 4. Circulation pump, 5. Liquid storage tank, 6. Solenoid valve, 7. Heater, 8. Sensor.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

In Fig. 1, the PCR chips 3 are placed in an array on the upper surface of the heat exchanger 1, and the flexible printed circuit board 2 is covered above the chip array, and the heat exchanger 1 is connected with the circulating pump 4 and four liquid storage tanks 5 through pipelines. Also be provided with eight solenoid valves 6 on the pipeline.

The design of the PCR chip 3 is shown in FIG. 2 . Its substrate is made of silicon material. Silicon has good thermal conductivity, and its thermal conductivity is as high as 1.57W/(cm·℃). Two identical serpentine microgrooves were fabricated on the PCR chip 3 , with dimensions of 1200 μm in width, 200 μm in depth, and 105 mm in length, each with a volume of 25 μL. The serpentine microgroove facilitates the loading and unloading of the reaction solution and the cleaning of the chip. Above the substrate is a glass cover slip, on which four small holes with a diameter of 1 mm are made as the entrances and exits for loading and unloading the reaction solution. The capillary principle is used to load the reaction solution, and the compressed air is used to unload the reaction solution. During the reaction process, the inlet and outlet were sealed with paraffin oil. The microfabrication process of the above-mentioned chip is briefly described as follows: a 2-inch silicon wafer is thermally oxidized to form a 1 μm oxide layer, and then the pattern of the micro-reaction tank is formed on the oxide layer by ultraviolet light exposure photolithography and HF, and then anisotropic KOH wet A micro-reaction groove with a depth of 200 μm was etched on the silicon wafer by etching method; a YH-D4030 laser engraving machine was used to drill the entrance and exit holes at the corresponding positions on the 0.6mm thick PYREX glass cover; the graphics of the substrate and the cover were aligned. Anodic bonding is performed at 400°C and 800V to form a micro-reaction tank PCR chip. There are no heating elements, cooling elements, and sensing elements inside the chip, so the cost can be reduced.

A unit of the heater and sensor array on the flexible printed circuit board 2 is shown in FIG. 3 . The flexible printed circuit board 2 is made of a soft base material, and its main feature is that it can be bent and folded, can be conveniently installed in a three-dimensional space, and has good wiring consistency. A single-sided flexible printed circuit board corresponding to the geometric dimensions of the upper surface of the heat exchanger 1 is used, and the principle that the copper foil on it has a certain resistance value and the resistance value has an exact corresponding relationship with the temperature is used to connect the board and each chip. The corresponding position is used to make the heating and sensing unit. Because the heater 7 needs to draw corresponding electric power from the power supply according to the requirements of temperature control, there is inevitably a self-heating effect, and the size of the self-heating effect is fluctuating. If the heater 7 is used as a temperature sensor at the same time, its temperature measurement accuracy will inevitably be subject to self-heating effects. Therefore, the heater 7 and the sensor 8 are designed separately, the sensor 8 only needs to be excited by a 5mA constant current source, and the maximum working current of the heater 7 can reach 300mA. The cross-sectional dimensions of the flexible printed circuit board metal foil lines are all 55um in width and 35um in thickness. At 20° C., the resistance of each heater 7 is about 3.3 ohms, and the resistance of each sensor 8 is about 6.5 ohms. A flexible printed circuit board 2 is placed over and in contact with the array of PCR chips 3 to provide temperature correction for each chip. Elastic elements and heat-conducting insulating glue are used to ensure good thermal contact between each heater 7 and sensor 8 on the flexible printed circuit board and each PCR chip 3, and the dispersion of contact thermal resistance is limited within an acceptable range. Due to the current mature flexible PCB board production technology, the consistency of each heating and sensing unit can be guaranteed. The thermal conductivity of the base material of the flexible printed circuit board is low, and the mutual coupling between the heaters and the chips brought about by it is negligible.

The hardware configuration of the computer control system based on the industrial computer is shown in Figure 4. The macroscopic centralized temperature control device consists of a PC bus industrial control computer, several corresponding interface boards, 8 direct-acting solenoid valves 6, a circulation pump 4, 4 2L liquid storage tanks 5, and a high-efficiency heat exchanger 1 composition. The heat exchange medium in the liquid storage tank is pure water. Each of the three liquid storage tanks has a built-in 500W electric heating wire and a K-type thermocouple temperature sensor. The temperature is controlled by the PC bus industrial control computer to correspond to the three characteristic temperatures of high-temperature denaturation, low-temperature annealing, and medium-temperature extension of the PCR reaction process. temperature. The fourth liquid storage tank 5 stores cooling water to ensure that the heat exchanger 1 can cool down rapidly. The ice-water mixture can be formed by adding ice manually, or by using a small compressor. According to the needs of the reaction process of the PCR chip 3, the industrial control computer continuously controls 8 solenoid valves 6 through the interface board, so that the water in the corresponding water tank circulates through the heat exchanger 1, and the surface temperature of the heat exchanger 1 quickly reaches the corresponding value . By controlling the temperature of the liquid storage tank 5, the steady-state temperature on the upper surface of the heat exchanger 1 is 2°C-3°C lower than the temperature required by the chip, and the difference is made up by the heater 7 above each chip. Since each heater 7 only needs to provide a temperature rise of 2° C. to 3° C., it is relatively easy to realize the accuracy and speed of chip temperature control. Each heater 7 is also controlled by the same computer. The PC bus industrial control machine system consists of a host, CRT, two 2-group 8-way intelligent thermocouple input modules IPC5455, four photoelectrically isolated 32-way 8-bit A/D boards IPC5482, a 32-way photoelectrically isolated switch output module IPC5373 and The self-made resistive temperature sensor signal conditioning board and solid state relay board are composed.

In Fig. 5 and Fig. 6, the heat transfer rate of the heat exchanger 1 should be compatible with the heat conduction time constant of the PCR chip 3, and should have better temperature uniformity. The upper surface of the heat exchanger 1 is an effective heat exchange surface for placing the PCR chip 3. The copper material with high thermal conductivity is used, and several through grooves are opened on the copper material to expand the heat exchange area, reduce the mass of the copper, and improve its heat exchange. rate. In order to reduce heat loss and ensure heat exchange rate, the rest of the heat exchanger (bottom, inlet and outlet, etc.) is made of plastic material with low thermal conductivity. The effective path of the fluid in the heat exchanger is about 2 meters long and 60 mm wide. According to the infrared thermal imaging camera, the temperature rise rate of the heat exchanger surface can reach 12°C/s, and the temperature drop rate can reach 10°C/s. When the fluid at the same temperature circulates stably, the temperature difference of the heat exchanger surface is within 2.0°C. However, in the dynamic process of the alternation of two temperature fluids, due to the lag of fluid transmission, the temperature difference on the surface of the heat exchanger can be as high as 9.3°C. Therefore, the surface of the heat exchanger is divided into 12 temperature zones according to their dynamic temperature distribution, as shown in Fig. 6 is a schematic diagram of the structure of a temperature zone. After repeated tests, the temperature extreme difference in each temperature zone during the dynamic process can be guaranteed to be less than 0.7°C. A K-type thermocouple temperature sensor is set at the fluid inlet of each temperature zone, which measures the fluid outlet temperature of the previous temperature zone while measuring the fluid inlet temperature of the temperature zone. 10 PCR chips can be installed on the upper surface of each temperature zone heat exchanger, and a total of 120 PCR chips can be installed, of which 12 are measurement and control sample chips, which are set at the positions shown in the shaded part in Figure 5, and 108 are for actual testing. Chips for PCR reactions.

Fig. 7 is a schematic diagram of the feedforward-cascade composite control strategy of the chip. For the reaction solution in each PCR chip, there are two control inputs, the first is water passing through the heat exchanger, silicon substrate and then to the reaction solution, which is automatically formed by the computer according to the PCR process to control the pump and valve; the second The two are the heater on the flexible PCB board, the glass cover and the reaction solution. During the heating process, most of the energy required by the PCR chip comes from the heat exchanger, and the heater 7 only provides a very small part, which plays the role of "correction" and "replenishment". The device is then distributed by water. For each PCR chip itself, the first input is actually measurable but not controllable, only the second input is controllable, but the second input must be adapted and coordinated with the first input. For this reason, in the processing of the control strategy, the double-input system is artificially regarded as a single-input system, and the first input is regarded as "interference", but the temperature given signal of the chip is controlled by the first control input and the PCR process. Co-generation, the cascade-feedforward composite control strategy is applied to control the PCR temperature of the chip to form a temperature follower system. For example, when the computer detects that the water temperature of the heat exchanger in a certain isothermal zone starts to rise from 72°C, it means that the medium temperature extension stage of the chip has ended, and it should enter the high temperature denaturation stage as soon as possible, so immediately set the given value of the chip temperature in the isothermal zone to is 95°C. The reason for not changing the temperature setpoints of all chips in the system at the same time is because of the transport lag in the flow of water through the heat exchanger. The given signal generator, main loop controller, secondary loop controller, dynamic feedforward link, PWM generator, etc. in Fig. 7 are all realized by computer through software. The control goal of the system is that the chip temperature in each isothermal zone should track its given temperature "quickly, accurately and stably". Since the given temperature is cyclically changing, this is a follow-up system. The inner ring of the cascade control system is the temperature control ring on the upper surface of the chip, and the outer ring is the temperature control ring of the liquid inside the chip. The transfer function structure output from the secondary loop controller to the upper surface temperature of the chip 3 is taken as:

${\mathrm{<span\; class="notranslate">\; G</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; K</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="1"\; label="T"\; filenames="A20061004324300103.png,A20061004324300104.png"\; state="\{\{state\}\}">T</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; e</span>}}^{<span\; class="notranslate">\; -</span>{{\mathrm{<span\; class="notranslate">\; \&tau;</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}^{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>$

Among them, T ₁ is a comprehensive reflection of the thermal resistance and heat capacity of the interface between the heater and the chip, τ ₁ is the heat transfer lag at the interface between the heater and the chip, and K ₁ is the static amplification factor between the electric power of the heater and the interface temperature . The transfer function structure from the upper surface temperature of the chip 3 to the chip sample temperature is taken as:

${\mathrm{<span\; class="notranslate">\; G</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; K</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="2"\; label="T"\; filenames="A20061004324300101.png,A20061004324300103.png"\; state="\{\{state\}\}">T</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; e</span>}}^{<span\; class="notranslate">\; -</span>{{\mathrm{<span\; class="notranslate">\; \&tau;</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}^{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 2</span>}<span\; class="notranslate">\; )</span>$

Among them, T ₂ is a comprehensive reflection of the thermal inertia of the chip itself and the thermal inertia of the sample; the transmission delay of the silicon structure part of the chip is at the millisecond level, which can be ignored, so τ ₂ includes the thermal transmission delay of the chip glass cover, the thermal transmission delay of the sample and the sample The detection of temperature is hysteresis, and _K2 is the static amplification factor. The transfer function structure from the temperature of the heat exchange medium at the inlet of each isothermal zone of the heat exchanger to the temperature of the liquid in the chip is taken as:

${\mathrm{<span\; class="notranslate">\; G</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; K</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}}{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="2"\; label="T"\; filenames="A20061004324300101.png,A20061004324300103.png"\; state="\{\{state\}\}">T</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="3"\; label="T"\; filenames="A20061004324300101.png"\; state="\{\{state\}\}">T</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>}{\mathrm{<span\; class="notranslate">\; e</span>}}^{<span\; class="notranslate">\; -</span>{{\mathrm{<span\; class="notranslate">\; \&tau;</span>}}_{\mathrm{<span\; class="notranslate">\; 3</span>}}}^{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 3</span>}<span\; class="notranslate">\; )</span>$

where _T3 is the heat exchanger time constant. The transmission lag of the silicon structure part of the chip is at the millisecond level, which can be ignored; the detection lag of the water temperature of the heat exchanger and the detection lag of the sample temperature cancel each other out, so it can be considered that _τ3 only includes the transmission lag of water in the heat exchanger, the heat exchanger The heat transfer lag of itself, the transfer lag of the interface between the heat exchanger and the chip, and the heat transfer lag of the chip sample. Tested by the step response method, T ₁ =0.5s, T ₂ =3.1s, T ₃ =1.1s, τ ₁ =0.1s, τ ₂ =1.1s, τ ₃ =1.36s. Based on this, the transfer function of the dynamic feed-forward link and the structure and approximate parameters of the main and auxiliary controllers can be determined. The specific parameters of the main and auxiliary controllers are finally completed by on-site setting. The time constant and time lag of the inner loop are very small, and the working speed is fast, forming a fast follow-up system for the output signal of the main controller, so that the temperature of the upper surface of the chip can quickly reach the required value, and can quickly eliminate various Disturbance has strong robustness to the contact thermal resistance and heat capacity fluctuations that inevitably exist on the upper surface of the chip; the lower surface of the chip is the main transmission interface of the chip's thermal energy, and its impact on the chip is finally reflected in the change of the liquid temperature inside the chip , this change is consistent with the change trend of the given signal, and objectively shares most of the tasks of the outer loop of the cascade control system. The final accurate control of chip temperature is done by the outer loop of the cascade control system.

Each PCR chip is affected by both the heat exchanger below it and the heater above it. The above analysis process considers that the heat transfer process of all chips in the isothermal zone of each heat exchanger is exactly the same, ignoring the objective existence of each chip. kind of dispersion. Among the many dispersions, the dispersion of the contact thermal resistance on the upper and lower surfaces of the chip has the greatest influence, and the dispersion of the contact thermal resistance on the upper and lower surfaces of the chip is directly affected by the dispersion of the contact pressure. The contact pressure is generated by the mechanical elastic structure, and its uniformity is guaranteed by the scientific design and precision manufacturing of the mechanical structure. However, in the actual use process, the contact pressure of each chip inevitably has small random fluctuations. This small random fluctuation has no obvious impact on the polymerase chain reaction process of the chip, which is within the allowable range and can be regarded as background noise. Don't bother. What needs to be prevented is the obvious uneven contact pressure of each chip caused by improper operation or equipment mechanical structure failure, which will destroy the representativeness of the sample chip and cause the failure of the polymerase chain reaction. For this purpose, indirect parameters are used to form the supervisory control of the contact pressure of each chip. Incorporating the dispersion of the temperature at the interface between each chip in the isothermal zone of the heat exchanger and the flexible printed circuit board (that is, the surface temperature on the chip) is included in the statistical process control, which can detect possible abnormal fluctuations in contact pressure at any time. Statistical process control is an important method of product quality control. Its basic point of view is: product quality is affected by two types of random factors and abnormal factors. Random factors exist objectively and are inevitable, but they have little impact on product quality, and obey a certain statistical distribution law. Abnormal factors do not always exist, but have a greater impact on product quality. When the abnormal factors do not exist, the product quality is only affected by random factors, and the quality parameters obey the uniform distribution. Within the acceptable range, the process is said to be in a statistically controlled state; and when the abnormal factors appear, the product quality fluctuates greatly. It no longer obeys the original statistical distribution law, saying that the product quality is not in a statistically controlled state. Once this situation occurs, it should be dealt with immediately to restore the product quality to a statistically controlled state. The function of statistical control is to conduct quantitative statistical analysis on the continuously collected process parameter data according to the theory of mathematical statistics, so as to judge whether the process is in a state of statistical control. When the process capability declines, the process is out of control or tends to be out of control, an alarm will be issued immediately, so that abnormal factors can be found and eliminated in time, so that the process can always be in a statistically controlled state. Statistical process control technology has played the role of "preventing" a large number of substandard products. According to the specific situation of this system, the difference between the upper surface temperature of each chip and the upper surface temperature of the model chip is used as the monitoring parameter, and the single value-moving range control chart is selected to perform statistical process control on the above 108 temperature difference parameters, which is essentially It is to carry out statistical process control on the dispersion of the contact pressure of each chip inside each isothermal zone, which is approximately equivalent to carrying out statistical process control on the dispersion of the contact thermal resistance of the upper and lower surfaces of the chip.

Assuming that in the state of statistical control, the interface temperature x _ij (i=1, 2, ... 12; j = 1, 2, ... 9) of the j-th chip in the i-th isothermal zone and the interface temperature of the sample chip in the corresponding isothermal zone The difference x _{ij -xi} of x _i obeys the distribution N(μ _ij , σ _ij ² ). Through mechanism analysis and experimental verification, it is found that μ _ij ≈0 and σ _ij ≈σ ² . That is to say, the distribution law of each temperature difference parameter is approximately the same, which is N(0, σ ² ). Select the jth chip in the i-th isothermal zone, and extract n consecutive sample data x _ijk -xi _ik (k=1, 2,...n) of x _ij -xi _i , then the moving range is defined as:

R _sk =|(x _ijk -x _ik )-(x _ij(k+1) -x _i(k+1) )|, k=1, 2,...n-1 (4)

and the average moving range is

$\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; no</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 1</span>}}\underset{\mathrm{<span\; class="notranslate">\; k</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; no</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; sk</span>}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 5</span>}<span\; class="notranslate">\; )</span>$

Since the distribution law of the temperature difference parameters of each chip is approximately the same, the average moving range obtained from the jth chip in the i-th isothermal zone under statistical control Applies to all chips. When the system is working, it collects temperature data in real time and performs the above calculations, but it does not need to do real-time drawing on the CRT, and the single value-moving range control chart is implicit in the working process of the system. In the case where the data points of the control chart are randomly arranged, if one of the following conditions is met, it can be determined that the system is working under statistical control, the contact thermal resistance of each chip is uniform, and the sample chip is fully representative:

(1) 25 consecutive data points, the number of out-of-bounds points d=0;

(2) 35 consecutive data points, the number of out-of-bounds points d≤1;

(3) 100 consecutive data points, the number of out-of-bounds points d≤2;

When one of the following situations occurs, it can be determined that the system is affected by abnormal factors and is out of statistical control. The contact thermal resistance of the corresponding chip obviously deviates from the normal value. The sample chip is no longer representative of the chip. Give corresponding warnings:

(1) If the data point is out of bounds or under the bounds, it will be judged;

(2) If the arrangement of points within the bounds is not random, it will be judged differently. Including ideas repeatedly approaching the control limit, ideas forming a chain on one side of the control center line, ideas making periodic changes, etc.

Through the introduction of statistical process control, the representativeness of the sample chip is under the real-time monitoring of the system itself. Although the actual PCR chip itself does not contain a temperature sensor, the system can guarantee the accuracy of its temperature control.

Claims (6)

1. A control device for a polymerase chain reaction chip array, which includes a heat exchanger, and a polymerase chain reaction chip with at least one microcavity, i.e. a PCR chip, is placed on the upper surface of the heat exchanger, and is characterized in that: There are multiple PCR chips, and they form a PCR chip array on the upper surface of the heat exchanger; a flexible printed circuit board is arranged above the PCR chip array, and heaters and sensor arrays corresponding to the chip array are made on the flexible printed circuit board At the same time, the upper surface of the heat exchanger is divided into several temperature zones, each temperature zone corresponds to a group of PCR chips in the PCR chip array, and a PCR chip is selected as a measurement and control model chip in each temperature zone, and in the microcavity of the model chip Place a miniature temperature sensor and fill it with a liquid that has similar thermal properties to the PCR reaction solution; electrical components such as the temperature sensor in each sample chip, the heater on the flexible printed circuit board, and the sensor array are respectively connected to the computer control system through the interface circuit. 2. The control device of the polymerase chain reaction chip array according to claim 1, characterized in that: The flexible printed circuit board is formed with a plurality of units composed of heaters and sensors, each unit corresponds to a PCR chip, and these units form a heater and sensor array corresponding to the PCR chip array; the flexible printed circuit board is arranged Above the PCR chip array and in contact with each PCR chip. 3. The control device for polymerase chain reaction chip array according to claim 1, characterized in that: The inside of the heat exchanger is a flowing heat exchange medium, the heat exchanger is a combination of heat conduction material and heat insulation material, the bottom and the inlet and outlet of the heat exchange medium are heat insulation materials, and the rest are heat conduction materials, and the heat conduction material forms several grooves In contact with the heat exchange medium; the upper surface of the heat exchanger is divided into several temperature zones to ensure that the temperature uniformity of the upper surface of each temperature zone is within a certain allowable range; at least one temperature sensor is set at the fluid inlet of each temperature zone The upper surface of the heat exchanger in each temperature zone corresponds to a number of PCR chips, one of which is a sample chip for measurement and control and the rest are chips for actual PCR reactions; the heat exchanger is connected to four liquid storage tanks and circulation pumps through pipelines. There are eight solenoid valves on the pipeline, and the four liquid storage tanks are respectively equipped with temperature sensors, heating elements or cooling elements. These electrical elements are connected to the computer control system through the interface circuit, and each solenoid valve is turned on and off under the control of the computer. Switching, so that the heat exchange medium in the corresponding liquid storage tank circulates through the heat exchanger, and the surface temperature of the heat exchanger quickly reaches the required value. 4. A method for controlling a polymerase chain reaction chip array, the method comprising: Place a number of PCR chips with microcavities but without heaters and sensors on the upper surface of the heat exchanger to form an array; place a flexible printed circuit board with an array of heaters and sensor units on top of the PCR chip array And in contact with it, each heater and sensor unit corresponds to each PCR chip one by one; Under the action of the computer control system, each PCR chip obtains a temperature close to the ideal temperature value of the polymerase chain reaction through the upper surface of the heat exchanger; the computer control system collects the temperature of the heat exchange medium at the entrance of the heat exchanger in each temperature zone, the The surface temperature and the temperature of the liquid inside the sample chip are implemented with a feed-forward cascade composite control strategy to control the corresponding heater on the flexible printed circuit board and heat the corresponding PCR chip to obtain an accurate temperature value; At the same time, the difference between the upper surface temperature of each chip and the upper surface temperature of the model chip is collected, and statistical process control is implemented to monitor whether the contact thermal resistance of each chip is in a normal state. 5. The method for controlling the polymerase chain reaction chip array according to claim 4, characterized in that: The feed-forward cascade control method is as follows: the temperature detection signal of the heat exchange medium at the inlet of each temperature zone in the heat exchanger is input to the computer control system, and the signal is transformed by a given signal generator formed by software on the one hand inside the computer system. As the setting signal of the main controller also formed by software; on the other hand, the dynamic feedforward link formed by software is integrated with the output signal of the main controller; at the same time, the temperature signal on the surface of each sample chip is detected as the corresponding temperature zone The feedback signal of the sub-controller forms an inner loop, and the sub-controller is also formed by software; the temperature of the liquid inside each sample chip is used as the feedback signal of the main controller in the corresponding temperature zone to form an outer loop; the output signal of the sub-controller is amplified by power and simultaneously Applied to each heater on the flexible printed circuit board corresponding to the group of PCR chips, so as to complete the precise control of the temperature of each PCR chip. 6. The method for controlling the polymerase chain reaction chip array according to claim 4, characterized in that: The statistical process control is to use the difference between the upper surface temperature of the chip and the upper surface temperature of the sample chip in the temperature zone where the chip is located as a monitoring parameter to perform single-value-moving range control, that is, to control the difference between the upper surface of the chip and the flexible printed circuit board. Statistical process control is carried out on the dispersion of the indirect contact thermal resistance, and the statistical process control is carried out indirectly on the dispersion of the contact pressure on the upper and lower surfaces of the chip, which is equivalent to the dispersion of the contact thermal resistance between the lower surface of the chip and the heat exchanger. Statistical process control is achieved; when the system is working under statistical control, it can be considered that the dispersion of the contact thermal resistance of each chip is within the allowable range, and the sample chip is fully representative; when a chip is affected by abnormal factors When the monitoring parameters are out of the statistically controlled state, it is considered that the contact thermal resistance of the chip has deviated significantly from the normal value, and the sample chip is no longer representative of the chip, and the computer system will give a corresponding warning.

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