TWI411779B - Microfluidic bio-chip and automatic reaction detection system thereof - Google Patents

Abstract

A microfluidic bio-chip and an automatic reaction detection system thereof are provided. A plurality of microfluidic bio-chips are installed on a turntable, and an extraction chamber of each of the microfluidic bio-chips is radially outward connected to a main distribution channel in relation to a rotation shaft of the turntable, and then the main distribution channel is laterally connected to a pair of side distribution channels, each of which is further radially inward connected to a plurality of reaction chambers. Because the side distribution channels are relatively away from the rotation shaft of the turntable in relation to the reaction chambers, fluids can firstly flow into the side distribution channels due to the centrifugal force and then can gradually overflow into each of the reaction chambers and averagely distribute therein due to the centrifugal force and the communicating pipe principle. Thus, the consistency, accuracy and reproducibility of the microfluidic experiments can be relatively enhanced.

Description

Microfluidic biochip and its automated reaction detection system

The present invention relates to a microfluidic biochip and an automated reaction detection system thereof, and more particularly to a microfluidic biochip and an automated reaction detection system for uniformly distributing fluids according to centrifugal force design of special microchannels.

At present, there are many biochemical detection requirements for various biopsies in the fields of medical treatment, biotechnology, chemistry, food and environment. For example, polymerase chain reaction (PCR) instruments are often used to increase the number of specimens. The total amount of genetic material such as DNA, in order to conduct more accurate and more biochemical analysis and detection of the specimen. However, due to problems such as equipment, technology, and reagent consumption, these biochemical detection instruments still have detection limits in terms of sensitivity. At the same time, detection time is too long, operation is complicated, equipment costs are high, and transportation is inconvenient. And other technical issues, thus affecting the popularity of its applications.

In order to solve the above problems, the first task is to miniaturize biochemical detection instruments to increase the speed of analysis and simplify the operation procedures. In recent years, the increasingly mature micro-electro-mechanical system (MEMS) process technology has the advantage of miniaturizing and manufacturing large-scale inspection instruments into wafer sizes. Therefore, MEMS technology is gradually being widely applied to analytical chemistry and In the field of biomedicine, biochips are produced to achieve significant reductions in inspection costs, speed of inspection and analysis, increased detection sensitivity, and simplified operational procedures.

In the case of existing biochips, they usually have functions such as sampling, mixing, sample transport, reaction, separation, and detection, that is, Integrating the instrument functions of the entire laboratory onto a single wafer, it is also known as Lab-on-a-chip, where laboratory wafers not only reduce reagents and human contamination, but also achieve The purpose of fully automated analysis. Furthermore, since the laboratory wafer is analyzed by a fluid flowing through several different functional regions in the microchannel of the wafer to complete the entire biochemical experiment process, it can also be called a microfluidic chip. .

For example, referring to FIG. 1, the invention patent of US Pat. No. 7,558,810 discloses a disc-type laboratory wafer 10 which is formed with a fixing hole 12 and a plurality of micro-channels on a disc substrate 11. Group 13. The disc substrate 11 can be placed in an optical disc drive and passed through the fixing hole 12 by the drive shaft of the optical disc drive to drive the disc substrate 11 to rotate at a high speed. Each of the microchannel groups 13 includes the same tank 131, a reagent tank 132, a buffer tank 133, a plurality of first valves 134, a mixing tank 135, a plurality of second valves 136, and a plurality of reaction tanks 137. . In the experiment, the disc substrate 11 is first rotated at a high speed, and then the first valve 134 is sequentially opened by the laser beam to make the sample tank 131, the reagent tank 132, the buffer tank 133, the sample and the reagent. The buffer flows into the mixing tank 135 through the respective pipes to be mixed with each other. Subsequently, each of the second valves 136 is sequentially opened by the laser beam, and the mixed liquid in the mixing tank 135 flows into the respective reaction tanks 137 through the respective distribution channels, and the respective reaction tanks 137 are heated by infrared rays to carry out the reaction. After the reaction is completed, an external optical detecting device detects the reaction result in each reaction tank 137. Such a disc-type laboratory wafer 10 can also be referred to as a disc laboratory (Lab-CD, Lab-on-a-CD).

However, on the disc substrate 11, the fluid flows radially outward from the sample tank 131, the reagent tank 132, and the buffer tank 133 near the fixing hole 12 in accordance with the centrifugal force, and is distributed to each of the tubes and the mixing tank 135. Reaction tank 137. Only during the distribution of the fluid from the mixing tank 135 to each of the reaction tanks 137, usually the fluid must fill the reaction tank 137 closest to the mixing tank 135, and can be filled to the left and right in order to be relatively far from the mixing tank 135. The reaction tank 137 cannot distribute the limited fluid evenly to each of the reaction tanks 137 when the fluid is insufficient to fill all the reaction tanks 137, or causes the reaction tanks 137 at the proximal end and the distal end to have different fluid amounts, thereby causing each The reaction tanks 137 may have different reaction and detection results, thereby affecting the consistency, accuracy and reproducibility of the microfluidic experiments.

Further, during the reaction in which each reaction tank 137 is heated by infrared rays, the temperature of each reaction tank 137 gradually increases, and at this time, the surface tension of the fluid in the reaction tank 137 is lowered, and may be generated in the reaction tank 137. Tiny bubbles. However, the decrease in surface tension or the generation of minute bubbles will cause the fluid to be pushed into the distribution channel outside the reaction tank 137, causing each reaction tank 137 to have a different total amount of fluid, thus also causing each reaction tank 137. There may be different reactions and detection results, which in turn affect the consistency and reliability of the microfluidic experiments.

In addition, although the disc-type laboratory wafer 10 is provided with a plurality of micro-channel groups 13 on the same disc substrate 11, the plurality of specimens can be independently detected at the same time, but only a single specimen is required. When the test is performed, the test piece still needs to use one of the disc type laboratory wafers 10, thereby wasting other unused micro flow path groups 13 and reducing the usable system if the disc is to be used continuously. Quantity. Therefore, the use efficiency of the disc-type laboratory wafer 10 is lowered, and the material cost of the inspection is relatively increased.

In addition, in practice, many biological reaction, extraction and detection processes have the need for centrifugation or even high-speed centrifugation, and the above-mentioned disc-type laboratory wafer 10 is limited by a small radius, so that it is often required to achieve the target centrifugal force. The high rotational speed (for example, greater than 10,000 rpm), but the material of the disc substrate 11 itself cannot withstand excessively high rotational speed. As a result, for bioassays, it is equivalent to the lack of a very common and important centrifugation procedure, which causes the entire test procedure to be discontinuous.

Therefore, it is necessary to provide a microfluidic biochip and its automated reaction detection system to solve the problems of the conventional technology.

The main object of the present invention is to provide a microfluidic biochip and an automated reaction detecting system thereof, which mounts a microfluidic biochip on a turntable and radially directs an extraction bath of the microfluidic biochip relative to a rotating shaft of the turntable. The main distribution flow channel is connected to the main distribution flow channel, and the pair of side distribution flow channels are connected to the left and right sides by the main distribution flow channel, and the plurality of reaction cells are connected radially inward by the distribution channels of the respective sides, because the side distribution flow channel is compared with The reaction tank is farther away from the rotating shaft of the turntable, so the fluid can first flow into the side distribution flow channel due to the centrifugal force, and then gradually overflows and evenly distributes into the reaction tanks due to the centrifugal force and the principle of the communication tube, thereby solving the problem of the prior art fluid. The problem of being evenly distributed to each reaction tank, in turn, enhances the consistency, accuracy, and reproducibility of the microfluidic experiment.

A secondary object of the present invention is to provide a microfluidic biochip and an automated reaction detecting system, wherein each reaction tank is provided with a plurality of microcolumns for adsorbing fluid when the fluid is heated in each reaction tank to reduce surface tension. In the microcolumn and the inner wall of the reaction tank, the total contact area between the fluid and the inner wall of the reaction tank is increased, thereby solving the problem of external heat overflow of the fluid in the prior art, thereby improving the consistency and reliability of the microfluidic experiment.

Another object of the present invention is to provide a microfluidic biochip and an automated reaction detection system thereof, wherein a plurality of microfluidic biochips are detachably mounted symmetrically on a plurality of wafer positions of a turntable, and can be experimentally required. The experiment is performed using one, two, several or all microfluidic biochips. After the experiment is completed, the microfluidic biochips that have been tested can be replaced with new ones, while the microfluidic biochips that are not experimental can still be used. Retained for the next experiment, thereby solving the problem that the same disc-type laboratory wafer can only perform a single inspection in the prior art, thereby relatively improving the use efficiency of the microfluidic biochip and reducing the material cost of the inspection, and The maximum number of systems available for each experiment was available.

It is still another object of the present invention to provide a microfluidic biochip and an automated reaction detecting system thereof, which use a turntable made of a solid material such as metal to carry a plurality of microfluidic biochips, and the effective radius of the turntable is greater than or Equal to 20 cm, to use a larger centrifugal radius to relatively reduce the rotational speed and achieve a preset and sufficient high centrifugal force value, thereby solving the problem of the prior art limited disc substrate material and failing to provide high centrifugal force, thereby ensuring long-term rotation of the turntable Perform safety and durability in centrifugation.

It is still another object of the present invention to provide a microfluidic biochip and an automated reaction detecting system thereof, which integrate all the microchannels required for the reaction step into the same microfluidic biochip, and simultaneously use all the units used. They are integrated into the same automated reaction detection system to provide automated, consistent operations such as sample, cleaning, centrifugation, extraction, distribution, mixing reagents, heating temperature control and reaction result detection, without the need for manpower or other The additional assistance of the device can relatively simplify the structure of the wafer and system, save manpower and time required for operation, and improve the convenience and practicability of the wafer and system.

It is still another object of the present invention to provide a microfluidic biochip and an automated reaction detecting system thereof, which are particularly suitable for extracting micro-RNA biological samples in plasma or serum, and performing reverse transcription (Reverse transcription). Real-time detection of polymerase chain reaction (Real Time-PCR) and detection analysis, and only a very small amount of sample, reverse transcription and Real Time-PCR related reagents, can complete several identical reactions simultaneously, thereby being relatively Reduce sampling requirements, reduce inspection costs, increase detection sensitivity, and help expand the popularity of its wide range of applications.

To achieve the above object, the present invention provides a microfluidic biochip comprising: a wafer substrate mounted on a turntable of an automated reaction detection system: and a micro flow channel group formed on the wafer substrate, and The microchannel group comprises: an extraction tank relatively close to a rotating shaft of the turntable, the extraction tank is for injecting the same body or a fluid; and a main distribution flow channel, the extraction tank is radially outward with respect to the rotating shaft of the turntable a pair of side distribution flow passages respectively extending from the main distribution flow passage to both sides; a plurality of reaction tanks respectively extending from the side distribution flow passages radially inwardly relative to the rotation shaft of the rotary disc And a plurality of reagent temporary storage tanks, wherein each of the reaction tanks is connected radially inward to a reagent temporary storage tank, and the reagent temporary storage tank is configured to provide at least one reagent to the reaction tank; wherein the side distribution flow channel is opposite The reaction tank is located farther away from the rotating shaft of the turntable, and the sample and the fluid are evenly distributed into each of the reaction tanks via the side distribution flow passage.

In one embodiment of the invention, each of the reaction tanks has a plurality of microcylinders therein.

In one embodiment of the present invention, the sample injected into the extraction tank is a plasma or serum sample, and the extraction tank uses a colloid to extract a micro-RNA fragment having a nucleotide number of less than or equal to 40. .

In one embodiment of the invention, the reagent in the reagent storage tank is a reagent required for performing a reverse transcription reaction and/or detecting a polymerase chain reaction (Real Time PCR).

In an embodiment of the invention, each of the reaction tanks has a first exhaust port.

In an embodiment of the present invention, the micro flow channel group further includes a single closing valve and a waste liquid storage tank, wherein the main distribution flow channel is sequentially radially outwardly connected to the side distribution flow channel, and is closed once. Valve and waste storage tank.

In an embodiment of the invention, the waste liquid storage tank has at least one second exhaust port.

To achieve the above object, the present invention further provides an automated reaction detection system for a microfluidic biochip, comprising: a turntable having a rotating shaft and symmetrically disposed a plurality of wafer positions around the rotating shaft; and several microfluidic organisms The wafers are respectively detachably mounted symmetrically at respective wafer positions of the turntable, wherein the microfluidic biological wafers each comprise: a wafer substrate mounted on a turntable of an automated reaction detection system: and a micro flow channel group Formed on the wafer substrate, and the micro-flow channel group comprises: an extraction tank relatively close to a rotating shaft of the rotating disc, the extraction tank is used to inject the same or a fluid; a main distribution flow channel, the extraction tank A pair of side distribution flow passages are respectively extended from the main distribution flow passage to the two sides; a plurality of reaction tanks are respectively arranged by the respective side distribution flow passages relative to the turntable The rotating shaft extends radially inward; and a plurality of reagent temporary storage tanks, wherein each of the reaction tanks is connected radially inward to a reagent temporary storage tank, and the reagent temporary storage tank is configured to provide at least one reagent The reaction tank; and the side distribution flow channel of the side is relatively far from the rotation axis of the turntable, the sample and the fluid are evenly distributed to each of the reaction tanks through the side distribution flow channel; an automatic spotting unit is used for Injecting the sample or fluid into the extraction tank, or injecting the reagent into the reagent storage tank; and an infrared heating unit for generating an infrared light beam to illuminate the reaction tank to increase the temperature of the reaction tank.

In one embodiment of the invention, the effective centrifugal radius from one of the axes to the wafer position is greater than or equal to 20 centimeters.

In an embodiment of the invention, the position of the side distribution channel and the reaction channel is curved in an arc shape, and the curvature of the side distribution channel is the same as the circle drawn on the basis of the effective centrifugal radius. Curvature.

In an embodiment of the present invention, the automated reaction detection system further includes: a fluorescence excitation unit for generating an excitation beam to illuminate the sample in the reaction tank and reacting with the reagent to generate a fluorescent light; And a fluorescent detection system for detecting the intensity of the fluorescent signal.

In an embodiment of the invention, the fluorescence detection system includes an optical filter unit for filtering the detected fluorescence wavelength.

The above and other objects, features and advantages of the present invention will become more <RTIgt; Furthermore, the directional terms mentioned in the present invention, such as "radial", "axial", "upper", "lower", "previous", "rear", "left", "right", "inside" , "outside" or "side", etc., only refer to the direction of the additional schema. Therefore, the directional terminology used is for the purpose of illustration and understanding of the invention.

Referring to FIG. 2, the automatic reaction detection system of the microfluidic biochip according to the preferred embodiment of the present invention mainly comprises: a turntable 20, a plurality of microfluidic biochips 30, an automatic spotting unit 40, and an infrared heating system. The unit 50, a fluorescent excitation unit 60 and a fluorescent detection system 70, the detailed construction of the above components will be further described in detail below with reference to Figures 1 and 2.

Referring to FIG. 2, the turntable 20 of the preferred embodiment of the present invention is designed according to the specifications of the microfluidic biochip 30 of the present invention, wherein the turntable 20 is made of a solid material such as metal and has a sufficient thickness. The disk member may be in the shape of a circle, a rectangle, a triangle, a square or a regular polygon, and the shape preferably corresponds to the number and shape of the microfluidic biochip 30 to be carried by the turntable 20. In the present invention, the turntable 20 has a rotating shaft 21 for driving the turntable 20 to rotate at a high speed, wherein a plurality of wafer positions 22 are preferably equiangularly and axially symmetric around the rotating shaft 21, and the rotating shaft 21 is provided. One of the effective centrifugal radii R to the wafer position 22 is greater than or equal to 20 cm, for example 20, 25, 30 cm or more. When the effective centrifugal radius R of the turntable 20 is greater than or equal to 20 cm, the present invention can utilize a larger centrifugal radius to relatively reduce the rotational speed to achieve a preset and sufficient high centrifugal force value, thereby ensuring that the turntable 20 performs centrifugation for a long period of time. Safety and durability of the work.

Referring to Figures 2 and 3, the microfluidic biochip 30 of the preferred embodiment of the present invention can be removably mounted to the turntable by means of suitable fixtures (e.g., clamps, slots, screws or adhesives). The shape of the microfluidic biochip 30 is preferably rectangular in shape at each wafer position 22 of 20, but is not limited thereto. The microfluidic biochips 30 each have a wafer substrate 31 and a microchannel group 32. The wafer substrate 31 is preferably made of one, two or more transparent transparent plastic substrates, such as poly(methyl methacrylate), PMMA, and polydimethyl methoxy oxane. (polydimethylsiloxane, PDMS) or polycarbonate (PC), etc., but is not limited thereto. The interior of the wafer substrate 31 can be prefabricated to form the microchannel group 32 by prior art techniques such as etching, molding, or stamping. The microchannel group 32 includes an extraction tank 321, a main distribution channel 322, a pair of side distribution channels 323, a plurality of reaction tanks 324, a plurality of reagent temporary storage tanks 325, a single closing valve 326, and a waste. Liquid storage tank 327.

In more detail, the extraction groove 321 is formed in the wafer substrate 31 and is located on a side of the wafer substrate 31 relatively close to the rotating shaft 21 of the turntable 20. The extraction groove 321 is a relatively wide spindle-shaped long groove. And an upstream end of the extraction tank 321 (ie, one end relatively close to the rotating shaft 21) communicates with the same injection port 321a. The sample injection port 321a extends through the wafer substrate 31 to selectively inject the same or a fluid in different steps, wherein the sample is preferably a plasma or serum sample; the type of the fluid is selected according to the sample. For example, a cleaning solution or a buffer. Furthermore, one downstream end of the extraction tank 321 (ie, one end relatively away from the rotating shaft 21) is radially outwardly connected to the main distribution flow channel 322, and the main distribution flow channel 322 is formed in the wafer substrate 31, and A relatively narrow slot, the main distribution channel 322 is further radially outwardly coupled to the two side distribution channels 323, the single closing valve 326, and the waste reservoir 327. Hereinafter, "radially inward" refers to a direction toward the rotating shaft 21 of the turntable 20, and "radially outward" refers to a direction away from the rotating shaft 21 of the turntable 20, as will be described first.

The two side distribution channels 323 are formed in the wafer substrate 31, and are respectively extended from the main distribution channel 322 to the left and right sides, and the side distribution channel 323 is a relatively narrow slot. The width of the side distribution channel 323 and the reaction channel are slightly curved, wherein the curvature of the side distribution channel 323 is substantially the same as the width of the main distribution channel 322. The effective centrifugal radius R is the curvature of the circle drawn by the reference. Furthermore, the plurality of reaction tanks 324 are formed in the wafer substrate 31 and extend radially inwardly from the side distribution channels 323, that is, the plurality of reaction tanks 324 are adjacent to the rotation shaft 21 The direction is extended. The plurality of reaction tanks 324 are symmetrically arranged on both sides of the two side distribution flow passages 323 relatively close to one side of the rotation shaft 21, and each of the reaction tanks 324 is a relatively wide elliptical long groove, and the width thereof is significantly larger than the side distribution flow. Road 323. Since the side distribution flow path 323 is farther away from the rotation axis 21 of the turntable 20 than the reaction flow path 324, in the centrifugal step described below, the sample and the fluid will be evenly distributed to each of the reaction channels via the side distribution flow path 323. 324.

As shown in FIG. 3A, in one embodiment of the present invention, each of the reaction tanks 324 has a first exhaust port 324a, and further a plurality of micro-cylinders 324b. The first exhaust port 324a penetrates the wafer substrate 31 and is used to immediately discharge the original internal gas when the sample, fluid or reagent enters the reaction tank 324, so that the sample, the fluid or the reagent can smoothly enter the reaction tank 324. The microcylinder 324b functions to increase the contact area between the fluid and the inner wall of the reaction vessel 324 by the microcylinder 324b when the fluid is heated to reduce the surface tension, so that the fluid is adsorbed on the microcylinder 324b and the reaction vessel 324. The inner wall avoids the problem of fluid being externally flooded to the side distribution channel 323, thereby increasing the consistency and reliability of the microfluidic experiment.

Moreover, each of the reaction tanks 324 is connected inwardly to the reagent temporary storage tank 325. The reagent temporary storage tank 325 is formed in the wafer substrate 31 and is relatively close to the rotating shaft 21 of the turntable 20. The reagent storage tank 325 is configured to provide a reagent to the reaction tank 324. The reagent temporary storage tank 325 has a reagent injection port 325a extending through the wafer substrate 31 for injecting related reagents. In the present invention, when the sample is a microRNA fragment, the reagents injected into the reagent storage tank 325 are preferably related reagents of reverse transcription and immediate detection of a polymerase chain reaction. In addition, the single-time closing valve 326 is formed in the wafer substrate 31 and located on the main distribution flow channel 322, and at a boundary point between the main distribution flow channel 322 and the side distribution flow channel 323 and the waste liquid storage tank. Between 327. The single closing valve 326 is normally open, but can be permanently closed due to the illumination of the infrared beam or the laser beam, that is, it can only be turned off once and cannot be turned on again. The single shut-off valve 326 can be selected from a suitable existing valve body, such as a capillary valve, a hydrophobic valve, a mechanical valve, or a phase change valve. The single closing valve 326 is further radially outwardly connected to the waste liquid storage tank 327. The waste liquid storage tank 327 is formed in the wafer substrate 31, and functions to clean the sample after the cleaning step is performed in the following cleaning step. Waste liquid. The waste liquid storage tank 327 generally has at least one second exhaust port 327a extending through the wafer substrate 31 and used to immediately discharge the original internal gas when the cleaning waste liquid enters the waste liquid storage tank 327, so as to clean the waste liquid. The waste liquid storage tank 327 is smoothly entered.

Referring to FIG. 2 again, the automatic spotting unit 40, the infrared heating unit 50, the fluorescent excitation unit 60 and the fluorescent detection system 70 of the preferred embodiment of the present invention are located on the turntable 20 and the microfluidic biochip 30. At the appropriate position above. The automatic spotting unit 40 can be moved relative to the microfluidic biochip 30 to selectively inject the sample (eg, plasma or serum) or a fluid (eg, a cleaning solution or buffer) into the extraction tank 321 or the reagent (For example, a reverse transcription reaction and a reagent for immediately detecting a polymerase chain reaction) are injected into the reagent storage tank 325. The infrared heating unit 50 generates an infrared light beam to illuminate the reaction tank 324 to increase the temperature of the reaction tank 324. The fluorescent excitation unit 60 is configured to generate an excitation beam (for example, UV ultraviolet light) to illuminate the sample in the reaction tank 324 and react with the reagent to generate a fluorescent light. Thereby, the fluorescence detection system 70 can be used to detect the signal intensity of the fluorescent light to detect the experimental reaction result.

Referring to Figures 2, 4A and 4B, when using the automated reaction detection system of the microfluidic biochip of the preferred embodiment of the present invention, the automated reaction detection system can be one, two, several or all The microfluidic biochip 30 provides automated operations such as sample feeding, washing, centrifuging, extracting, dispensing, mixing reagents, heating temperature control, and reaction result detection. The automated reaction detection system is used to perform the following steps in sequence. As shown in Figures 2 and 4A, in a feeding step, a plurality of microfluidic biochips 30 are first fixed on each wafer position 22 of the turntable 20, and the sample is injected from the sample by the automatic spotting unit 40. The inlet 321a injects the same (e.g., serum) solution into the extraction tank 321, which contains sample molecules or particles and a suitable fluid (e.g., sterilized Phosphate buffered saline, PBS). Since the extraction tank 321 is previously filled with a suitable gel, such as agarose, the sample molecules or particles in the sample solution temporarily adhere to the gel. Then, the rotating shaft 21 is used to drive the rotating wheel 20 to rotate at a high speed, and the fluid portion (ie, waste liquid) of the sample liquid in the extraction tank 321 is radially outwardly flowed to the waste liquid storage tank via the main distribution flow path 322 by centrifugal force. Stored in 327. After the waste collection is completed, the rotation is suspended.

Then, in a cleaning step, a cleaning liquid (for example, a liquid containing isopropanol/alcohol isopropanol/alcohol) is injected into the extraction tank 321 from the sample injection port 321a by the automatic spotting unit 40. And staying in the extraction tank 321 for a predetermined time, in order to carry out the waste liquid in the gel.

Next, in an extraction step, the centrifugation is then re-rotated, so that the cleaning liquid (also referred to as waste liquid) in the extraction tank 321 flows into the waste liquid storage tank 327 through the main distribution flow path 322. After the waste liquid collection is completed, only the sample molecules or particles are left in the gel of the extraction tank 321 .

Next, as shown in FIGS. 2 and 4B, in the step of closing the valve, when the turntable 20 is rotated (or stationary), the infrared light beam of the infrared heating unit 50 or a laser unit (not shown) is used. Irradiation of the beam causes the previously opened single shut-off valve 326 to be permanently closed.

Then, in a dispensing step, when the turntable 20 is stationary, a discharge liquid (for example, a salt-containing liquid) is injected into the extraction tank 321 from the sample injection port 321a by the automatic spotting unit 40, and then utilized. The rotating shaft 21 drives the turntable 20 to rotate at a high speed, and the sample molecules or particles in the gel of the extraction tank 321 are taken away by the releasing liquid by centrifugal force, and flow radially outward and to the left and right sides through the main distributing flow path 322. It is distributed into the flow channel 323 to this side. After the release liquid fills the side distribution flow path 323, the release liquid gradually overflows radially inwardly due to the centrifugal force and the communication tube principle and is evenly distributed into each reaction tank 324, wherein when the release liquid is not filled yet In each reaction tank 324, the release liquid and the sample molecules or particles carried therein are substantially relatively close to the side of the side distribution flow path 323.

Next, in a mixing reagent step, when the turntable 20 is at rest, the reagent (such as a reverse transcription reaction and a reagent for detecting a chain reaction of a polymerase chain reaction) is injected from the reagent injection port 325a by the automatic spotting unit 40. Into the reagent storage tank 325, the reagent then flows radially outward into each reaction tank 324 by the centrifugal force of the rotation of the turntable 20.

Then, in a heating temperature control step, when the turntable 20 rotates, the infrared heating unit 50 generates an infrared light beam to illuminate each of the reaction tanks 324 to increase the temperature of the reaction tank 324 for reverse transcription reaction and instant detection. Polymerase chain reaction. If not every microfluidic biochip 30 is tested, the rotational position of the turntable 20 can be sensed by a position sensor to determine the instantaneous position of the microfluidic biochip 30 to be tested, and the microfluidic biochip to be tested. When the reaction tank 324 of 30 is aligned to the infrared heating unit 50, the infrared heating unit 50 generates an infrared light beam to illuminate the reaction tank 324 to be tested.

It is worth noting that the reverse transcription reaction requires several different temperature control steps to reverse-transcribe the microRNA sample into complementary DNA (cDNA), and for subsequent PCR reactions, it is usually necessary to repeat dozens of mixed reagents. The step and the cyclic reaction of the heating temperature control step can amplify the total amount of cDNA. Furthermore, each heating temperature control cycle may be subdivided into several different heating temperature periods to sequentially perform denaturation, annealing, and extension reactions.

Finally, in a reaction result detecting step, when the turntable 20 is stationary, a fluorescent labeling reagent (for example, SYBR Green I) is injected into the reagent temporary storage from the reagent injection port 325a by the automatic spotting unit 40. In the tank 325, the fluorescent labeling reagent then flows radially outward into each reaction tank 324 by the centrifugal force of the rotation of the turntable 20 to be combined with the sample molecules or particles that have completed the RT-PCR (in this case, the synthesis should be mainly On the cDNA product). When the turntable 20 rotates, the fluorescent excitation unit 60 generates an excitation light beam (for example, UV ultraviolet light) to illuminate the sample in the reaction tank 324 combined with the fluorescent labeling reagent to generate a fluorescent light. In this way, the fluorescence detection system 70 can be used to detect the signal intensity of the fluorescent light. The fluorescent detection system 70 must include an optical filter unit for pre-filtering the detected fluorescent light. Light wavelength. In this way, the total amount of cDNA can be detected by PCR reaction results, and the qualitative or quantitative data of the original microRNA can be reversed. Since the data of multiple sets of reaction results can be obtained by single or multiple microfluidic biochips 30 at a time, it will be advantageous to substantially reduce the need for repeated experiments and relatively reduce the complexity of the experimental operation.

As described above, the present invention of FIGS. 2 to 4B mounts the microfluidic biochip 30 on the turntable 20 in comparison with many technical problems existing in the conventional disc type laboratory wafer 10 of FIG. The extraction groove 321 of the microfluidic biochip 30 is first radially connected to the main distribution flow channel 322 with respect to the rotating shaft 21 of the turntable 20, and the main distribution flow channel 322 is connected to the left and right sides to connect a pair of side distribution flow channels. 323, and a plurality of reaction tanks 324 are radially inwardly connected by the respective side distribution flow passages 323. Since the side distribution flow passages 323 are farther away from the rotation shaft 21 of the turntable 20 than the reaction tanks 324, the fluid may be firstly subjected to centrifugal force. The action flows into the side distribution flow channel 323, and then the centrifugal force is used to gradually overflow and evenly distribute the liquid in the pipe to each reaction tank 324 due to the principle of the communication pipe, thereby solving the problem that the fluid in the prior art cannot be evenly distributed to several The problem of the reaction tank, in turn, relatively improves the consistency, accuracy and reproducibility of the microfluidic experiment.

Furthermore, in the present invention, as shown in FIG. 3A, a plurality of micro-cylinders 324b are preferably disposed in each of the reaction tanks 324 to adsorb the fluid when the fluid is heated in each of the reaction tanks 324 to reduce the surface tension. The microcolumn 324b and the inner wall of the reaction tank 324 are used to increase the total contact area between the fluid and the inner wall of the reaction tank 324, thereby solving the problem of external heat overflow of the fluid in the prior art, thereby improving the consistency and reliability of the microfluidic experiment. Sex.

In addition, in the present invention, a plurality of microfluidic biochips 30 are symmetrically mounted on the plurality of wafer positions 22 of the turntable 20 in a detachable manner, and one, two, several or ones may be selected according to experimental requirements. All of the microfluidic biochips 30 were tested. After the experiment was completed, the microfluidic biochip 30 that had been tested could be replaced with a new one, while the unexperimented microfluidic biochip 30 could still be used for the next experiment. Therefore, the problem that the same disc type laboratory wafer can only be single-detected in the prior art is solved, thereby improving the use efficiency of the microfluidic biochip 30 and reducing the material cost of the detection, and maximizing each experiment. The number of systems is available.

Further, in the present invention, the turntable 20 made of a solid material such as metal can be used to carry a plurality of microfluidic biochips 30, and the effective centrifugal radius of the turntable 20 is greater than or equal to 20 cm to utilize a large centrifugal radius R. To reduce the rotational speed and achieve a preset and sufficient high centrifugal force value, thereby solving the problem of the prior art limited disc substrate material and failing to provide high centrifugal force, thereby ensuring the safety and durability of the turntable 20 for performing long-term centrifugation work. .

Furthermore, in the present invention, all the microchannels required for the reaction step can be integrated into the same microfluidic biochip 30, and all the units used are integrated into the same automated reaction detection system. It provides automated, consistent operations such as sample, cleaning, centrifugation, extraction, distribution, mixing reagents, heating temperature control and reaction result detection, and requires no additional assistance from human or other equipment during the experiment, thus simplifying the structure of the wafer and system. Save manpower and time required for operation, and improve the ease of use and practicability of wafers and systems.

In addition, in the present invention, the microfluidic biochip and its automated reaction detection system are particularly suitable for extracting, reverse transcribed, and polymerase chain reaction of microRNA biological samples in plasma or serum (Real Time) -PCR) experiments and detection analysis, and only a very small number of samples and reverse transcription / polymerase chain reaction related reagents, can complete several identical reactions simultaneously, thereby reducing the sampling requirements, reducing the detection cost, and improving Sensitivity is detected and is conducive to expanding the popularity of its wide range of applications.

The present invention has been disclosed in its preferred embodiments, and is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

10. . . Disc type laboratory chip

11. . . Disc substrate

12. . . Fixed hole

13. . . Microchannel group

131. . . Sample slot

132. . . Reagent tank

133. . . Buffer tank

134. . . First valve

135. . . Mixing tank

136. . . Second valve

137. . . Reaction tank

20. . . Turntable

twenty one. . . Rotating shaft

twenty two. . . Wafer location

30. . . Microfluidic biochip

31. . . Wafer substrate

32. . . Microchannel group

321. . . Extraction tank

321a. . . Sample injection port

322. . . Main distribution runner

323‧‧‧ side distribution runner

324‧‧‧Reaction tank

324a‧‧‧first exhaust

324b‧‧‧Microcolumn

325‧‧‧Reagent temporary storage tank

325a‧‧‧Reagent inlet

326‧‧‧Single valve closure

327‧‧‧ Waste storage tank

327a‧‧‧Second vent

40‧‧‧Automatic spotting unit

50‧‧‧Infrared heating unit

60‧‧‧Fluorescence excitation unit

70‧‧‧Fluorescence detection system

Figure 1: View of the top view of a conventional disc-type laboratory wafer.

2 is a perspective view of an automated reaction detection system for a microfluidic biochip according to a preferred embodiment of the present invention.

Figure 3: An enlarged top view of a microfluidic biochip of a preferred embodiment of the invention.

Figure 3A is a partial enlarged view of a reaction vessel of a microfluidic biochip of a preferred embodiment of the present invention.

Fig. 4A is a schematic view showing the microfluidic biochip of the preferred embodiment of the present invention when the waste liquid is removed.

Figure 4B is a schematic illustration of a microfluidic biochip in accordance with a preferred embodiment of the present invention.

30. . . Microfluidic biochip

31. . . Wafer substrate

32. . . Microchannel group

321. . . Extraction tank

321a. . . Sample injection port

322. . . Main distribution runner

323. . . Side distribution runner

324. . . Reaction tank

324a. . . First exhaust port

325. . . Reagent temporary storage tank

325a. . . Reagent injection port

326. . . Single closing valve

327. . . Waste storage tank

327a. . . Second exhaust port

Claims (13)

A microfluidic biochip comprising: a wafer substrate mounted on a turntable of an automated reaction detection system; and a microchannel group formed on the wafer substrate, the microchannel group comprising: an extraction a groove, which is relatively close to a rotating shaft of the turntable, the extracting tank is used for injecting the same or a fluid; a main distributing flow channel is formed by the extracting groove extending radially outward relative to the rotating shaft of the rotating wheel; The passages are respectively extended from the main distribution flow passages to the two sides; the plurality of reaction tanks are respectively formed by the side distribution flow passages extending radially inward relative to the rotation shaft of the rotary disc; and a plurality of reagent temporary storage slots, Each of the reaction tanks is connected radially inwardly to a reagent temporary storage tank, and the reagent temporary storage tank is configured to provide at least one reagent to the reaction tank; wherein the side distribution flow channel is farther away from the turntable than the reaction tank a rotating shaft, the sample and the fluid are evenly distributed to each of the reaction tanks through the side distribution flow channel, the micro flow channel group further comprising a single closing valve and a waste liquid storage tank, wherein the main distribution flow channel is sequentially radial Connect the side to the side Channel, a single valve is closed and the waste reservoir. The microfluidic biochip of claim 1, wherein each of the reaction tanks has a plurality of microcylinders. The microfluidic biochip of claim 1, wherein the sample injected into the extraction tank is plasma or serum. The microfluidic biochip of claim 3, wherein the extraction tank extracts a microribonucleic acid fragment using a colloid. The microfluidic biochip of claim 4, wherein the microRNA fragment has a nucleotide number of less than or equal to 40. The microfluidic biochip of claim 4, wherein the reagent of the reagent storage tank is a reagent required for performing a reverse transcription reaction or a polymerase chain reaction. The microfluidic biochip of claim 1, wherein each of the reaction tanks has a first exhaust port. The microfluidic biochip of claim 1, wherein the waste liquid storage tank has at least one second exhaust port. An automated reaction detection system for a microfluidic biochip, comprising: a turntable having a rotating shaft, and symmetrically disposed a plurality of wafer positions around the rotating shaft; and a plurality of microfluidic biological wafers respectively detachably mounted on the symmetrical Each of the microfluidic biochips includes: a wafer substrate mounted on a turntable of an automated reaction detection system: and a micro flow channel group formed on the wafer substrate, and the wafer substrate The micro runner group contains: An extraction tank, which is relatively close to a rotating shaft of the turntable, the extracting tank is used for injecting the same body or a fluid; a main distributing flow channel is formed by the extracting groove extending radially outward relative to the rotating shaft of the rotating wheel; Distributing flow passages respectively extending from the main distribution flow passages to both sides; a plurality of reaction tanks respectively extending from the side distribution flow passages radially inwardly relative to the rotation shaft of the rotary disc; and a plurality of reagents temporarily stored a tank, wherein each of the reaction tanks is connected radially inwardly to a reagent temporary storage tank, and the reagent temporary storage tank is configured to provide at least one reagent to the reaction tank; and wherein the side distribution flow channel is farther away from the reaction tank system a rotating shaft of the turntable, the sample and the fluid are evenly distributed into each of the reaction tanks through the side distribution flow channel; an automatic spotting unit for injecting the sample or fluid into the extraction tank, or injecting the reagent into the The reagent temporary storage tank; and an infrared heating unit for generating an infrared light beam to illuminate the reaction tank to increase the temperature of the reaction tank, wherein the micro flow channel group further comprises a single closing valve and a waste liquid storage tank ,among them Primary distribution flow channels are sequentially connected radially outwardly to the side distributing flow path, a single valve is closed and the waste reservoir. An automated reaction detection system for a microfluidic biochip according to claim 9, wherein an effective centrifugal radius from the rotation axis to the wafer position is greater than or equal to 20 cm. The automatic reaction detection system for a microfluidic biochip according to claim 10, wherein the side distribution flow path and the reaction groove are arranged in an arc shape, and the curvature of the side distribution flow channel is the same as The curvature of the circle drawn on the basis of the effective centrifugal radius. The automatic reaction detection system for a microfluidic biochip according to claim 9, wherein the automated reaction detection system further comprises: a fluorescence excitation unit for generating an excitation beam to illuminate the reaction chamber and the The reagent is reacted to generate a fluorescent light; and a fluorescent detection system is used to detect the intensity of the fluorescent signal. The automatic reaction detection system for a microfluidic biochip according to claim 12, wherein the fluorescence detection system comprises an optical filter unit for filtering the detected fluorescence wavelength.