TWI386253B - Heater-type tilting device - Google Patents

Description

Heatable bead device

The heating type liquid bead device of the invention can be widely used in an inspection device requiring rapid temperature control, such as an instant quantitative PCR device, an RNA reverse transcription PCR (RT-PCR) device, etc., to improve the reaction efficiency, and indeed achieve instant quantitative detection. The purpose.

With the rapid development of micro-electromechanical technology (MEMS) and biomedical technology, many technologies have been used in the field of medical care in recent years, and medical care equipment is also becoming miniaturized, personalized, nano-customized, customized and wireless. In the direction of research and development, it is necessary to achieve green energy-saving materials that can save energy and save carbon.

Microelectromechanical technology is a technology that can miniaturize biochips. Therefore, in conjunction with biomedical applications, bio-MEMS technology has been developed with the aim of developing miniaturized portable rapid detection instruments. In the field of biomedicine, temperature is often required as a catalytic effect of biochemistry, such as PCR technology, RT-PCR, Digest, and the like.

A prior art technique for accomplishing microheaters using microelectromechanical process technology [Journal of Thermal Sciences, 46, 580-588, 2007], which is characterized in that gold (Au) and titanium (Ti) are deposited on a glass substrate. As a means of heating, and giving a fixed voltage can produce a comparable heat source. Therefore, in terms of power (P) conversion, it can be expressed by equation (1): P = i ² R ... (1)

In the formula (1), i is a current, and R is a metal barrier.

PCR technology

The polymerase chain reaction (PCR) was invented by Kary Mullis in 1985, and Mullis received the Nobel Prize and patent rights [US 4,683,195] [US 4,683,202]. PCR requires three steps to refer to (1) double stranding: heating to 94 ° C, open the double-strand structure of the DNA template; (2) primer hybridization (induction): cooling to 50-65 ° C, at this time a pair The primer enters the double-stranded DNA molecule, and searches for the base sequence complementary to itself and binds to this position; (3) nucleic acid synthesis (extension): the temperature is raised to 65-75 ° C, thereby activating the polymerization and binding to the 3 end of the primer, and grabbing the corresponding dNTPs according to the base sequence on the template to form a new nucleic acid molecular chain; two polymerases The nucleic acid molecular chain is simultaneously grown in a face-to-face direction until both models are finished. However, the traditional PCR procedure, due to the need to control the temperature rise and fall, leads to an increase in the time of the entire procedure, as well as a non-specific DNA reaction, so in order to improve the reaction time of the overall detection, it is necessary to increase the heat transfer efficiency and reduce the reagent volume.

Conventional PCR equipment often needs to use a rapid temperature rise and fall module to heat the metal plate in the cavity, and the temperature of the hot plate is transferred to the plastic tube placed on the metal plate, which has a volume of at least 15 ul, at three different temperatures. The cycle was carried out at 65 ° C, 95 ° C, and 75 ° C. At this time, the biological signal will be amplified by 2 ⁿ , and the n value is 1 when the temperature is cycled once. When n reaches 25 to 30 times, a very small amount of the medical signal can be amplified. In recent years, with the detection of fluorescent systems, real-time detection technology can be achieved.

In recent years, in order to reduce the overall reaction time, there is a two-stage temperature PCR reaction procedure, which is carried out by combining the temperatures of Annealing/Extension, while the part of Denature is maintained, so the entire reaction The program requires only two temperature points and a reaction procedure that can complete the PCR. However, the use of conventional heating systems still requires procedures for temperature rise and fall, thus still increasing the overall time of the reaction.

In addition to the refinement of reagents, micro-heaters developed using microelectromechanical process technology are used in the field of biomedical applications, which have the advantages of rapid temperature rise and decrease of reagent volume.

In recent years, since the technology of PCR has been developed, it has gradually been applied to the field of real-time detection, and currently more used techniques are optical detection. In the fluorescent real-time detection system, the detection by SYBR Green is often used, and the characteristics of the fluorescent dye are embedded in the double-stranded DNA, so when the PCR process is performed, and when the double-strand The greater the amount of DNA, the stronger the fluorescence intensity. Equipment traditionally used requires precise optical components, excitation sources such as lasers, and precision optics.

In 1958, Palecek discovered that DNA has a redox behavioral response at the electrochemical electrode. The subsequent use of electrochemistry for DNA-related detection is also initiated. A prior art technique for performing real-time PCR using electrochemical methods [Biosensors and Bioelectronics, 24, 2131-2136, 2009], which is characterized by the use of a polymer material such as PDMS as a transmission The flow path of the microfluid is passed and the fluid is passed through three different heating blocks at the bottom to complete the PCR reaction procedure. In order to instantly detect the amplification effect of DNA, a reagent that reacts with DNA, such as methyl blue, is added to the reagent, and the reagent is embedded in the DNA of the double helix, and when the reaction is generated, the current signal is generated. Will decrease. Therefore, the PCR reaction for instant detection is completed in this way. A device utilizing electrochemical detection [US 7, 135, 294], [US 7,393,644] which uses DNA immobilization technology to immobilize DNA on a substrate surface, then to give DNA reagents, and to perform PCR reaction procedures, and finally to measure Its Impedance signal.

There are many ways to perform DNA measurement, and the sensor of the DNA is directly used on the surface of the electrode, but if the surface is detected on a surface that is nanocrystalline, the surface of the nanostructure has a high contact surface area, and the electrode has direct measurement. The ability of DNA, therefore, on the surface electrode of nanocrystallization, will contribute to the sensing of DNA.

The invention utilizes the micro-electromechanical process to produce a tiny heater required for providing a biochemical process, in addition to providing thermal energy during biochemical detection, and also providing a detecting component on the surface of the wafer as an instant detection during biochemical reaction. . Via the design of the wafer, the biomolecule is driven to move in a specific direction. However, when the sensor is designed in a specific area, the biomolecule is sensed in a specific area.

In thermodynamics, the effect of Free Convection is caused by temperature changes and density changes. Therefore, when the temperature changes (when the temperature is not equal), an unstable cycle will occur, which will result in a change in the velocity field inside the liquid. . When the inside of the liquid is heated to cause the particles to buoyant, the density changes, and after the liquid is heated, the inside of the liquid will form a first-class field due to the change of buoyancy, in such a manner. It is thus also possible to drive the molecules of the liquid through a specific area, such as an electrode, an optical detection zone. The device of the invention uses the prepared microelectrode to heat, and drives the bottom of the liquid bead to reach 95 ° C. At this time, the molecules generate buoyancy due to heat, which drives the molecules to move upward, and the molecular movement path is limited by the appearance of the liquid bead. Therefore, reaching the top of the bead will cause the biomolecule to move around the bead due to the geometry. After the biomolecule reaches the bottom, it will drive the biomolecule upwards due to the heating factor. By circulating in this way, the thermal cycle of the polymerase chain reaction can be completed.

During the heating process, the evaporation of liquid will be an important issue, and mineral oil is often A liquid used in a PCR reaction to avoid volatilization. Therefore, the patent of the present invention is designed to have two liquids that can be stored on the surface of the device, and two liquids are defined by using SU-8 photoresist in combination with standard yellow light photolithography (Photolithography). In the storage area, the reagents for the PCR reaction can be volatilized by the protection of mineral oil during the heating process.

Temperature control method (the first temperature is fixed, and the second temperature can be adjusted)

In order to accurately control the temperature to reach a certain temperature, the voltage of the feedback is used to monitor the actual generated thermal power. According to the formula ( ), when the metal resistance (R) is determined, the value can be determined. As a result, the power consumption (P) is fixed, so that the current change (i) is constantly monitored as a precise control of the temperature in the biochemical experiment. Therefore, after the substrate is fixedly cooled, the state of the heater is adjusted and monitored to achieve a temperature regulation mechanism. In another part, the disturbance caused by this flow field will force the object to be tested to pass through the surface of the detector, and then the signal will be taken out. Through the control of the software, it is possible to have different temperatures, for example, using Plus to give signals.

In order to automatically control the temperature and loading, the design of the mechanism, the automatic loading of the wafer and the precise contact with the probe card, and the precise fixing of the probe card can make the human error and pollution during the test, so the wafer The signal transmission can be transmitted by means of a probe card or a metal wire.

Biomedical science often requires precise temperature control, and micro-heaters have the advantage of fast response and reduced energy loss. Since rapid temperature changes can be generated, it can be used in the fields of RT-PCR (reverse transcription), Real-Time PCR and Digest. After the polymerase chain reaction has been proposed, it has been widely used in biochemical, agricultural and other related industries. Since the conventional PCR instrument performs the PCR process by temperature, the temperature stability of the instrument will increase the reaction time of the PCR process in addition to the reaction time of the biological sample itself. In order to respond quickly and in a small volume, a device with a small volume and fast response has been proposed to provide an instant detection technique.

Real-time detection Principle of electrochemistry

Generally, the electrochemical detection test utilizes the mechanism of redox to perform sample detection. The present invention drives the molecules in the liquid to circulate through the surface of the sensor via a temperature difference to perform real-time monitoring of the biological sample. In the sensor part, a nano structure is added to increase the contact area, and not only the surface modification can be made to have a specific detection. In addition, the nanostructure of the surface of the sensor can also change the flow velocity of the liquid molecules, thereby regulating the velocity of the internal flow field of the liquid.

Fluorescent detection system

In the instant detection of the present invention, part of the detection is by optical detection, because the liquid will form a semicircular shape during the reaction, so when a signal source is generated, the liquid forms a lens shape, and the weak light source is focused to Above the sensor.

Traditional PCR often requires rapid temperature rise and fall, so it requires high power consumption. In addition, more biological reagent volume is required for reaction. In addition, when fluorescent detection is performed, the fluoroscope group will increase the bulk of the instrument system, but PCR The technology is mature and widely used, and with the improvement of the energy concept, the present invention will propose a novel technology to improve the traditional instrument system: First, reduce the power consumption of the instrument system, achieve energy-saving benefits, and second, reduce Volume of biological reagents: micro-volume detection is accomplished by novel miniaturized PCR wafers, because in many studies, many biological samples are less accessible and very expensive, so the use of micro-reagent samples is used. It will be a key point. Third, the miniaturization system: Since the traditional instrument system utilizes the detection of the optical system, the instrument system is huge, and the miniaturization system can save the material and avoid the waste of resources.

Therefore, the inventors of the present invention have various disadvantages derived from the above-mentioned instant quantitative PCR temperature control device, and the wide application of the heatable liquid bead device in a rapid temperature control inspection device, such as an instant quantitative PCR device, RNA reverse Transcription PCR (RT-PCR) equipment, etc., can improve the reaction efficiency, and indeed achieve the purpose of real-time quantitative detection. It is improved and innovated by 亟思, and after years of painstaking research, finally successfully developed this piece of heatable liquid bead device. .

The present invention provides a heatable bead apparatus.

The object of the present invention is to use the heatable liquid bead device to instantly control the temperature change during the reaction.

Another object of the present invention is to use an optical detection or electrochemical detection to instantly detect a PCR reaction performed by the heatable liquid droplet device.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

For a detailed description of the heatable bead device of the present invention, please refer to FIG. 1 , a reaction device 100 including 101 first liquid, 102 second liquid, 103 heater, 104 protective layer, 105 external lead, 106 substrate, 107 cooling device, 108 first group of sensing elements, 109 second group of sensing elements, 110 third group of sensing elements, 111 fourth group of sensing elements, 112 first signal source, 113 second signal source, 114 A ring layer, 115 second ring layer, 116 first cycle line, 117 second cycle line, 118 third cycle line.

The electrode can be fabricated by using a micro-electromechanical method. In order to heat the intermediate portion, the heating metal 103 will be made of platinum metal, and the outer conductor 105 can be a metal wire (in this case, aluminum is used as the wire). . By means of resistance heating, when the wire metal 105 is connected to a voltage or a current, the heater 103 generates a heat source due to the relationship of current or voltage, thereby generating a first circulation line 116, a second circulation line 117, and a third cycle. The circular trajectory of line 118. And the instant response signal is detected through the first group of sensing elements 108, the second group of sensing elements 109, the third group of sensing elements 110, and the fourth group of sensing elements 111 inside the sensing area 125.

In the technique of PCR, the first loop line 116 performs the annealing and extension regions for the PCR, so that the sensing region is designed in this region, and the direct detection effect can be achieved. In addition, a temperature portion is required to generate a specific temperature through the 103, thereby generating a trajectory of the first circulation line 116, the second circulation line 117, and the third circulation line 118, and performing an instantaneous detection through the electrode group inside the sensing region 125.

The temperature profile shown in Figure 2 is the heater 103, through the first signal source 112, 113 The second signal source, and a voltage of 3 volts (30 mA) is applied to the external conductor via 105, and the result obtained by the 107 cooling device is stabilized. The result is data measured by an infrared thermal imager, wherein at 103 The zone can be stabilized at a first temperature of 307 (95 ° C) while the 107 cooling device is maintained at a second temperature (60 ° C).

As can be seen from FIG. 3, the temperature of the temperature rise and fall is very fast, the curve 301 is the temperature provided by the cooling substrate 107, and the curve of 302-305 is to give different voltages or currents to 112 and 113, and to make the heating element 103 Different temperature points are generated, in such a way that a direct temperature rise curve 306 can be obtained after the signal is given, and a stable effect 305 can be quickly achieved.

As shown in FIG. 4, the reaction device 200 includes 101 first liquid, 102 second liquid, 103 heater, 104 protective layer, 105 outer lead, 106 substrate, 107 cooling device, 108 first set of sensing elements, 109 a second set of sensing elements, 110 a third set of sensing elements, 111 a fourth set of sensing elements, 112 a first signal source, 113 a second signal source, 114 a first ring layer, 115 a second ring layer, 116 First cycle line, 117 second cycle line, 118 third cycle line, 201 first light source, 202 second light source, 203 first barrier layer, 204 signal receiver, 205 support frame. When the first light source is irradiated on the liquid of 102, the biomolecule generates a first light source 202 (fluorescent signal) due to the amplification result, and the signal is separated by the 203, and is detected by the signal receiver 204. As shown in Figure 4. In order to control the flow rate inside the second liquid 102 to achieve the required time and reaction, 102 the second liquid will add different proportions of liquid, the purpose of which is to increase the viscosity coefficient thereof, so that the viscosity of the second liquid of 102 can be different. In turn, the control of different flow rates is completed. The excitation of the laser can be detected above the side-excited fluorescence or excited and detected by the coaxial source.

Example 1 : Using two-stage temperature control to complete the biochemical reaction

Since the micro-scale heat dissipation has the characteristics of rapid temperature rise and fall, the heatable liquid droplet device of the present invention, one of which is completed by utilizing this characteristic. A two-stage temperature control is used to achieve amplification of the PCR. First, the 107 cooling device is first brought to the annealing temperature point (65 ° C), and then the amplification effect of the PCR can be started. Therefore, the heating electrode of 103 is used to heat the temperature to reach the denature temperature (95 ° C), and the time is controlled. To complete the amplification mechanism of PCR. This method can avoid heat loss during the temperature rise and fall process. At the same time, the temperature is raised and lowered and detected at the same time to achieve the purpose of instant detection.

With 103 heating electrode control, temperature control of three stages of 95 ° C, 65 ° C, and 70 ° C can be achieved.

Example 2 , using electrochemical mechanism to measure the product of PCR

Using the electrical signal to complete the real-time monitoring of the PCR product, the schematic diagram of the wafer shown in FIG. 9 is fabricated through the microelectromechanical process technology, which includes 108 first set of sensing elements (Work electrodes) and 109 second senses. Measuring element (reference electrode), 110 third group of sensing elements (counter electrode), 111 fourth group of sensing elements (Working Electrode). In this system, the fluid molecules will drive the liquid to flow through the surface of the electrode due to the influence of the temperature field, and then the signal is detected, and the measured value (which can be the current value) is increased or decreased. Therefore, the electrode on the surface of the heatable bead device of the present invention uses a nanostructure to enhance the detection sensitivity. It is also possible to make the flow field changeable. The combination of designs can be either symmetrical or sandwich.

Example 3 : Using optical means to complete detection of PCR products

By using a laser to excite the PCR product of the bead and through the optical detection system, the PCR product can be detected immediately. The state in which the liquid exists is in the form of a liquid bead. Therefore, after the fluorescent signal is generated, the fluorescent signal is transmitted to the detector by the focusing characteristic of the liquid itself, which can be a CCD, a PMT, and an effect of enhancing the signal. As shown in Figure 5, the results of the detection above the liquid using the side light source excitation, as can be seen from the figure, after two minutes, the DNA has begun to react and complete the reaction mechanism within 10 minutes.

Example 4 , micro-nano surface electrode

The electrode used in the heatable bead device of the present invention may also be a micro-nano surface electrode (Fig. 7). The first group of sensing growth objects 120 are added to the sensing area 125 of FIG. 6 and FIG. The second group senses the growth object 121, the third group of sensing growth objects 122, and the fourth group of sensing growth objects 123. The sensing growth object can not only detect the 116-driven detection object by the electrode in the sensing area 125. When it comes out, it can also make the movement of the flow field form a moving path of 124, which can change the flow field at the same time (Fig. 6) and increase the sensitivity. The first group of sensing growth objects 120, the second group of sensing growth objects 121, the third group of sensing growth objects 122, and the fourth group of sensing growth objects 123 can be more chemically and physically modified to achieve a more specific one. Sexual reaction.

In order to be able to determine the characteristics of the sensing region 125, the measurement of its characteristics is performed using an electrochemical principle. The first set of sensing elements 108 and the third set of sensing elements 110 inside the sensing region 125 are given positive The negative voltage is switched, and the measured result is shown in Figure 8.

Example 5 , RNA reverse transcription PCR (RT-PCR)

Following reverse transcription PCR, followed by RT-PCR, the heatable bead device of the present invention can also be applied to a temperature control device in an RT-PCR instrument to rapidly regulate temperature changes in the instrument to improve the efficiency of the RT-PCR reaction.

Example 6 Application of Enzyme Digeation

The heatable liquid bead device of the invention can also be applied to the temperature regulating instrument required for Enzyme Digeation in the biochemical reaction, thereby improving the temperature rise and fall, reducing the heat loss, and improving the decomposition ability of the enzyme.

The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention. Equivalent embodiments of the liquid bead device applied to devices with rapid temperature control requirements, such as real-time quantitative PCR, RNA reverse transcription PCR (RT-PCR), etc., are all included in the patent scope of the present application.

In summary, this case is not only innovative in terms of method and form, but also can enhance the above-mentioned multiple functions compared with conventional articles. It should fully comply with the statutory invention patent requirements of novelty and progressiveness, and apply for it according to law. This invention patent application, in order to invent invention, to the sense of virtue.

100‧‧‧ polymerase chain reaction device

101‧‧‧First liquid

102‧‧‧Second liquid

103‧‧‧heater

104‧‧‧Protective layer

105‧‧‧External wire

106‧‧‧Substrate

107‧‧‧Cooling the substrate

108‧‧‧First set of sensing elements

109‧‧‧Second set of sensing elements

110‧‧‧The third set of sensing elements

111‧‧‧Fourth set of sensing elements

112‧‧‧first signal source

113‧‧‧Second signal source

114‧‧‧First ring layer

115‧‧‧second ring layer

116‧‧‧First cycle line

117‧‧‧second cycle line

118‧‧‧ third cycle line

120‧‧‧First Sensing Growth

121‧‧‧Second Sensing Growth

122‧‧‧ Third Sensing Growth

123‧‧‧ Fourth Sensing Growth

124‧‧‧ fourth cycle line

125‧‧‧Response sensing area

200‧‧‧ polymerase chain reaction device

201‧‧‧First light source

202‧‧‧second light source

203‧‧‧ first barrier

204‧‧‧Signal Receiver

205‧‧‧Support frame

206‧‧‧Fluorescent area

301‧‧‧The temperature provided by the cooling device 107

302-305‧‧‧ Temperature curves for different voltages or currents

306‧‧‧ Heating rate

307‧‧‧First temperature

308‧‧‧second temperature

Figure 1. Schematic diagram of the instant detection device of the heatable liquid bead device combined with the electrochemical detection device

Figure 2. Temperature distribution interval curve of the heating electrode

Figure 3, temperature curve

Figure 4. Schematic diagram of the instant detection device of the heatable liquid bead device combined with the optical detection device

Figure 5. Signal measurement curve for real-time measurement

Figure 6. Schematic diagram of the flow field change caused by the nanostructure

Figure 7. Schematic diagram of the carbon nanotubes on the surface of the test electrode

Figure 8. Curve of test results in the sensing area

Figure 9. Schematic diagram of the wafer

100‧‧‧ polymerase chain reaction device

101‧‧‧First liquid

102‧‧‧Second liquid

103‧‧‧heater

104‧‧‧Protective layer

105‧‧‧External wire

106‧‧‧Substrate

107‧‧‧Cooling the substrate

108‧‧‧First set of sensing elements

109‧‧‧Second set of sensing elements

110‧‧‧The third set of sensing elements

111‧‧‧Fourth set of sensing elements

112‧‧‧first signal source

113‧‧‧Second signal source

114‧‧‧First ring layer

115‧‧‧second ring layer

116‧‧‧First cycle line

117‧‧‧second cycle line

118‧‧‧ third cycle line

125‧‧‧Response sensing area

200‧‧‧ polymerase chain reaction device

201‧‧‧First light source

202‧‧‧second light source

203‧‧‧ first barrier

204‧‧‧Signal Receiver

205‧‧‧Support frame

206‧‧‧Fluorescent area

Claims (9)

A heatable liquid bead device comprising: a substrate; a liquid storage area disposed above the substrate; a composite liquid placed in the liquid storage area and confined by the liquid storage area without random flow a heating element disposed at a bottom of the center of the liquid storage area, the heating element further having an external wire for externally introducing a voltage and a current to heat the heating element, the heating element heating the composite liquid and generating an internal circulation flow a plurality of sensing elements disposed at the bottom of the liquid storage area, the sensing elements detecting a reaction signal of the internal circulation flow field of the composite liquid in the liquid storage area due to a temperature change; and a cooling a substrate disposed below the substrate, the cooling substrate is configured to cool the composite liquid in the liquid storage region; wherein the temperature difference between the heating element and the cooling substrate is used to drive the composite liquid to generate the internal circulation flow field And the reaction signal is detected by the sensing elements in real time. The heatable bead device of claim 1, wherein the sensing elements are CCD, PMT, alloy or metal electrodes. The heatable liquid bead device of claim 1, wherein the sensing element surface is a material of a nanostructure. The heatable liquid bead device of claim 3, wherein the nanostructure is surface modified to increase the sensitivity of the reaction. The heatable liquid bead apparatus according to claim 3, wherein the speed of the internal circulation flow field of the composite liquid is adjusted by a nanostructure. A method for immediate detection by using an inflatable bead device as described in claim 1 of the patent application in combination with an optical or electrochemical detection system for immediate detection. The method of detecting an instant detection as described in claim 6 wherein the electrical signal detected by the electrochemical detection system is a signal of a change in current value, and the optical signal detected by the optical detection system is It is a fluorescent signal. The method of instant detection as described in claim 7 wherein the composite liquid system is A liquid droplet in the form of a semi-circular lens that focuses the fluorescent signal to the sensing elements. The method of instant detection as described in claim 6 wherein the composite liquid comprises a reagent and a liquid which avoids volatilization.