TW201326813A - Detecting system and detecting method - Google Patents

Abstract

A detecting system comprises a microfluidic element and a complex. The microfluidic element includes an inlet, an outlet and a microfluidic channel structure which has a first microfluidic channel and a second microfluidic channel interconnected with each other. The complex includes chemical substances and carriers. Moreover, a detecting method is disclosed, in which the complex is filled into the microfluidic element so as to be arranged in the microfluidic channel structure, and a sample is then into the microfluidic element and contacts with the complex.

Description

Detection system and detection method

The present invention relates to a detection system, and more particularly to a detection system having a microfluidic component.

Traditional biochemical testing is limited by equipment and technology, and has high cost, inefficiency, and man-made operational pollution. Micro-electromechanical system technology can be used to minimize the detection instrument on the wafer, which not only reduces costs, but also improves its performance. And added value. A microfluidic device is a device that introduces a trace amount (microliter/nanoliter) of liquid into an element having a microchannel to carry out a reaction. Through the combination of MEMS technology and microfluidics technology, sampling, mixing, transmission, pre-processing, separation, reaction, detection and other functions can be integrated on the microfluidic chip for automated analysis. It not only has the advantages of small size, low cost, high detection efficiency, high sensitivity and specificity, low consumption of samples and reagents, low energy consumption, etc., but also avoids problems such as pollution and errors of human operation. Taiwan Patent No. 200911375 discloses a microfluidic wafer in which fluid droplets can be transported and analyzed.

Through the integration of biomedical technology, MEMS technology and microfluidic technology, biomolecules can be applied to microfluidic devices to perform various biochemical tests. Such microfluidic devices (eg, microfluidic biomedical wafers) can be widely used in clinical testing, genetic engineering, environmental testing, food testing, drug development, military detection and the like. For example, an enzyme can be immobilized on a sensor (electrode surface) in a wafer to detect by a specific reaction of an enzyme with a specific substance. Conventional techniques use a physical adsorption method, an enzyme embedding method, a molecular covalent bond method, and the like to immobilize an enzyme on a sensor (electrode surface) without losing its activity to be integrated in a wafer. Taiwan Patent No. I328115, Taiwan Patent No. I318107, and Taiwan Patent No. I247113 disclose the use of poly(vinyl alcohol) styrylpyridinium groups (PVA-SbQ) to embed an enzyme on an ion sensing electrode. A strong enzyme film is formed, or the enzyme is immobilized on the ion sensing electrode using 3-glycidoxypropyltrimethoxysilane (GPTS).

When making an enzyme-encapsulated device, the enzyme can be immobilized on the sensor and placed in a microfluidic device (such as a microfluidic wafer). However, in this manufacturing process, subsequent packaging processes can affect or even destroy the activity of the enzyme. Alternatively, when fabricating a device encapsulating an enzyme, the sensor may be first embedded in a microfluidic device (such as a microfluidic wafer), and then physically adsorbed, enzymatically embedded, and molecularly covalently bonded. The enzyme is strong in the sensor. However, since the sensor is confined to a tiny device (wafer), it is quite technically difficult to immobilize the enzyme on the sensor. In addition, the enzyme encapsulated in the device may affect/deprive its activity due to long-term use or poor preservation, and at the same time, it may be easily contaminated by repeated use, and such problems may affect the sensing quality. Furthermore, since the enzyme is firmly immobilized on the sensor by the above method, the enzyme cannot be exchanged depending on the detection. Therefore, there is still a need to develop a detection system and method that can solve the above-mentioned shortcomings and have industrial applicability.

In view of the deficiencies of the prior art, the present invention provides a detection system comprising: a microfluidic element comprising an inlet, an outlet and a microchannel structure, wherein the microchannel structure is in communication with the inlet and the outlet, the micro The flow path structure includes a first micro flow channel and a second micro flow channel, wherein the first micro flow channel is in communication with the second micro flow channel, and an inner diameter of the first micro flow channel is greater than the second micro flow channel An inner diameter; and a composite comprising a carrier and a chemical for detecting, wherein the carrier is combined with a chemical to form the composite, and the composite enters the microchannel structure to be placed in the first micro Flow path.

According to a particular embodiment of the invention, the composite is inaccessible to the second microchannel. According to an embodiment of the invention, the composite is sized such that the composite cannot enter the second microchannel. According to a particular embodiment of the invention, the composite passes through the inlet to enter the microchannel structure.

According to an embodiment of the invention, the microfluidic element further comprises a sensing member disposed adjacent to the microchannel structure. In some aspects, the sensing component is an electrochemical sensing component.

In accordance with an embodiment of the invention, the detection system includes positioning elements for positioning the composite entering the microfluidic structure. In some aspects, the positioning element is a magnetic substance.

According to a specific embodiment of the invention, the carrier is a colloid, and the carrier coats the biomolecule to form a microbead. According to a particular embodiment of the invention, the carrier is a magnetic substance and the chemical is bonded to the surface of the carrier.

According to a specific embodiment of the invention, the composite comprises the steps of: mixing the chemical and the material used to form the carrier to form a mixture; and emulsifying the mixture to form the chemical coated by the carrier Made of micro beads.

According to a specific embodiment of the invention, the chemical substance comprises a substance for reacting with the analyte in the sample. In some aspects, the inspection system enters the microchannel structure to contact the composite that has entered the microchannel structure.

The present invention provides a detection method for detecting a test object in a sample using the detection system of the present invention, the detection method comprising: introducing the composite into the microfluidic element to be placed in the micro flow channel structure; And, the specimen is brought into the microfluidic element to contact the composite.

According to an embodiment of the invention, the detecting method may comprise removing the composite from the microfluidic element.

According to a specific embodiment of the invention, the object to be tested comprises a substance for reacting with the chemical substance.

The detection system and method of the invention not only improves the freedom of use of the detection system, but also increases the use value thereof. Through the invention, not only the lack of the prior art can be solved, but also the quality of the detection can be improved, and the industrial utilization is extremely high.

The embodiments of the present invention are described by way of specific examples, and those skilled in the art can understand the advantages and advantages of the present invention. The present invention may be embodied or applied in various other specific embodiments. The details of the present invention can be variously modified and changed without departing from the spirit and scope of the invention.

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The term "or" as used in the specification and the scope of the appended claims generally includes the meaning of "and/or" unless otherwise indicated.

The present invention provides a detection system comprising: a microfluidic element comprising an inlet, an outlet and a microchannel structure, wherein the microchannel structure is in communication with the inlet and the outlet; and the composite, including the carrier and the chemical for detection A substance, wherein the carrier is combined with a chemical to form the composite, and the composite enters the microchannel structure for detection. Microfluidic components are used to provide a place to perform the test. According to the present invention, the detection system is configured to react with a sample entering the microfluidic structure of the microfluidic element and react with the analyte in the sample of the microfluidic structure entering the microfluidic element to perform detection ( Including: sensing, analysis (such as qualitative analysis, quantitative analysis, etc.), etc.). The specimen can enter the microfluidic element through the inlet of the microfluidic element and exit the microfluidic element through the outlet of the microfluidic element.

A microfluidic element refers to an element having a microchannel structure. Microfluidic structures can be formed in the substrate using a variety of conventional techniques to fabricate microfluidic components. Examples of the substrate include, but are not limited to, a germanium substrate, a glass substrate, a quartz substrate, a high molecular polymer (for example, polycarbonate, polymethyl methacrylate, cyclic olefin copolymers, polystyrene, Polydimethylsiloxane (PDMS) substrate. Microfluidic components can be fabricated using one or more substrates. Microfluidic components can be fabricated using one or more substrates. The microfluidic element can be of one or more layers. Further, a protective layer may be provided on the substrate. The protective layer can be made using materials well known in the art, such as a resin such as an epoxy resin. In accordance with an embodiment of the invention, the inlet and outlet of the microfluidic element can be defined using openings in the substrate and/or protective layer.

According to the present invention, the microchannel structure in the microfluidic element may have a plurality of microchannels, and the cross-sectional shape of the microchannels may be any shape. The plurality of microchannels in the microchannel structure can have different inner diameters (including: height, width) and/or different cross-sectional shapes. When the composite enters the microchannel structure, it can be positioned in the microchannel structure due to changes in inner diameter or cross-sectional shape. For example, when the composite enters the microchannel structure, it may not advance due to changes in the inner diameter (including: height, width), and thus may be positioned or fixed at a position before the above change occurs. In accordance with an embodiment of the invention, the change in the inner diameter and/or cross-sectional shape of the microchannels allows the composite to be positioned or secured in a desired position. According to an embodiment of the invention, the microchannel structure comprises a plurality of microchannels having different inner diameters (including height and width), since the composite cannot enter the microchannel with a larger inner diameter into the smaller inner passage. The micro flow path of the path, thus achieving the effect of positioning or fixing. In accordance with an embodiment of the present invention, the composite is sized such that it does not have a microchannel having a larger inner diameter into the microchannel having a smaller inner diameter. In addition, the microchannel structure may also be provided with microchannels that can be respectively passed into different specimens.

In some aspects of the invention, the microchannel structure includes a first microchannel and a second microchannel, the first microchannel and the second microchannel having different inner diameters (including: height, width) ). According to an embodiment of the invention, the first microchannel is in communication with the second microchannel. According to an embodiment of the invention, the microchannel structure comprises a first microchannel and a second microchannel that are continuously disposed.

According to a particular embodiment of the invention, the composite enters the first microchannel and the composite cannot enter the second microchannel. In some aspects of this embodiment, the inner diameter of the first microchannel is greater than the second microchannel. In some aspects of this embodiment, the composite is placed in the first microfluidic channel because the size of the composite is such that it cannot enter the second microfluidic channel having a smaller inner diameter from the first microfluidic channel. And achieve the effect of positioning or fixing.

The composite can be introduced into or out of the microfluidic element in any manner. According to one embodiment of the invention, the composite enters the microfluidic structure through the inlet of the microfluidic element. In some aspects of these embodiments, various assisting means can be used to cause the composite to enter or exit the microfluidic component. According to an embodiment of the present invention, the composite entering the microchannel structure cannot enter the first microchannel having a larger inner diameter into the second microchannel having a smaller inner diameter, thereby achieving the effect of positioning or fixing. . In some aspects of this embodiment, the composite is sized such that it does not have a first microchannel having a larger inner diameter into the second microchannel having a smaller inner diameter. In accordance with an embodiment of the invention, the composite enters and exits the microchannel structure through the inlet of the microfluidic element.

In addition, it is also possible to modify the surface of the microchannel (for example, using a specific substance or a specific functional group for modification). Furthermore, the material of the microchannel structure, the inner diameter of the microchannel, and/or the shape of the cross section may be used to adjust the flow rate or flow pattern of the fluid sample (such as the specimen) in the microfluidic element. Alternatively, a fluid sample (e.g., sample) volume control unit can be provided in the microfluidic element to control the amount of fluid sample (e.g., sample) entering the microfluidic element.

According to a particular embodiment of the invention, the detection system can have positioning elements. The positioning member is not particularly limited as long as the effect of positioning the composite entering the microchannel structure can be achieved. The positioning element can be configured to position or secure the composite at a desired location. For example, the positioning element can be a magnetic substance. The magnetic substance is not particularly limited as long as it has magnetic properties (for example, but not limited to, a group B element of the periodic table of elements, a compound thereof, a mixture thereof, and the like). According to a particular embodiment of the invention, the positioning element is arranged to position or secure the composite entering the microchannel structure. For example, by utilizing the attraction of the magnetic material, the composite entering the microfluidic structure can be positioned and more evenly aligned. According to an embodiment of the present invention, the positioning or fixing effect of the composite can be achieved by a plurality of microchannel structures including microchannels having different inner diameters; and at the same time, the positioning of the composite can be enhanced by the positioning of the positioning elements. Or fixed effect. According to an embodiment of the present invention, the micro flow channel structure includes a first micro flow channel and a second micro flow channel, the first micro flow channel is in communication with the second micro flow channel, and the first micro flow channel and the first The two microchannels have different inner diameters, the composite enters the first microchannel, and the composite cannot enter the second microchannel. In some aspects of these embodiments, the positioning element can be configured to position or secure the composite to the first microfluidic channel. In addition, the positioning element can also be configured to have other desired effects on the fluid sample (eg, the sample) entering the microchannel structure.

According to the detection system of the present invention, the composite can be removed into or removed from the microfluidic element, which not only improves the freedom of use of the detection system, but also increases its use value. According to one embodiment of the invention, the composite enters the microfluidic structure through the inlet of the microfluidic element. In accordance with an embodiment of the invention, the composite exits the microfluidic structure through the inlet of the microfluidic element. Through the present invention, the complex can be used or replaced as needed (for example, the type of the analyte, the state of the chemical in the composite) by simply passing in or removing the composite. In accordance with an embodiment of the present invention, the composite is positioned or secured to the microchannel structure through a plurality of microchannel structures including microchannels having different inner diameters and/or different cross-sectional shapes. The composite thus positioned or fixed can be easily removed from the microfluidic element. According to an embodiment of the invention, the composite is positioned or fixed in the microfluidic element through a plurality of microchannel structures comprising microchannels having different inner diameters and/or different cross-sectional shapes, and further, micro Positioning elements (eg, magnetic materials) in the fluid element further enhance the effect of positioning or securing the composite. The composite thus positioned or fixed can be easily removed from the microfluidic element. Therefore, according to the present invention, the desired composite can be selected and replaced as needed simply by simply inserting or removing the composite. Moreover, with the present invention, the detection system can be inspected as needed (e.g., detecting different analytes) without the need to replace or discard the microfluidic components (replacement of only the composite). Further, according to the present invention, it is possible to solve the problem of reduction/deactivation of activity due to long-term use or poor storage of chemical substances, contamination due to repeated use, and the like in the prior art by simply replacing the composite. In addition, according to the present invention, it is possible to place the composite into the microfluidic component as needed when performing the detection, which not only improves the convenience, but also improves the quality of the detection system (for example, avoids the reduction/deactivation of the activity of the chemical substance, and reuses The lack of pollution, etc.).

The microfluidic element can include a sensing member. The sensing member is not particularly limited. Examples of sensing members for microfluidic components include, but are not limited to, thermal sensing members, optical sensing members, electrochemical sensing members, acoustic sensing members, and the like. According to an embodiment of the invention, the sensing member in the microfluidic element can use an electrochemical sensing member. The sensing component can have units such as signal transduction, signal processing, and the like. The sensing member is disposed adjacent to the microchannel structure. In accordance with an embodiment of the invention, the composite entering the microfluidic component is positioned or fixed to correspond to the sensing member. After the composite reacts with the analyte to be reacted, the physical (heat, pressure, mass, magnetic field) amount or chemical amount change generated by the reaction can be converted into, for example, an electrical signal, for detection by the sensing member.

The sensing material as is known in the art can be used as the sensing member, and the sensing member can be fabricated and integrated into the detection system of the present invention using techniques known in the art. For example, the sensing member can be, for example, a transistor (such as, but not limited to, a field effect transistor (such as an ion sensitive field effect transistor). In these examples, for example, but not limited to: cerium oxide, cerium nitride, cerium oxide, aluminum oxide, tin dioxide, indium tin oxide, titanium nitride, cerium oxide, cerium-titanium compound, etc. Sensing film. In these examples, the composite entering the microfluidic component is positioned or fixed to correspond to the transistor for sensing in the sensing member; or, corresponding to the sensing electrode (the sensing electrode system Connecting the transistor with a wire. According to an embodiment of the invention, the sensing component comprises a sensing electrode, the sensing electrode is connected to the transistor by a wire, and the sensing electrode is disposed adjacent to the micro Flow path structure. In these embodiments, the composite entering the microfluidic component is positioned or fixed as a sensing electrode corresponding to the sensing member. Further, conventional techniques (eg, microelectromechanical technology) can be utilized to micro Further integration in the fluid element, Such as, but not limited to, reference electrode (or transistor), comparison electrode (or transistor), measurement circuit / measurement system (for measurement, for example: optical (absorbed light of specific wavelength, fluorescent, luminescent) , electricity (voltage, current, potential, resistance), magnetic, thermal, mechanical and other signals, color changes, etc.) and / or related circuits / devices (for example, for signal conversion, signal filtering, signal amplification), etc. In some examples, reference electrodes (or transistors), comparator electrodes (or transistors), and/or associated circuitry may be integrated into the microfluidic component using conventional techniques, such as microelectromechanical techniques, and further connected to the measurement. In addition, in addition to the complexing for sensing, the sensing member can also be designed to sense one or more parameters. For example, the sensing member can be set in addition to the composite through sensing. There is a sensing unit for performing additional sensing. For example, the sensing member can pass through the composite for detecting a specific object to be tested, and at the same time, other sensing units can be disposed in the sensing member for performing the sample. Testing of other analytes or The detection parameters.

According to an embodiment of the invention, the positioning element is arranged to position or fix the composite to correspond to the sensing element. In accordance with an embodiment of the invention, the change in the inner diameter and/or cross-sectional shape of the microchannels causes the composite to be positioned or fixed to correspond to the sensing member. According to an embodiment of the invention, the sensing member is disposed adjacent to the microchannel structure. In some aspects of the embodiments, the sensing member is configured to take into account changes in the inner diameter and/or cross-sectional shape of the microchannel, and/or the positioning of the positioning elements, thereby allowing the composite into the microchannel structure. Can correspond to the sensing piece. In some aspects of these embodiments, the positioning of the positioning elements takes into account the arrangement of the sensing members and/or changes in the inner diameter and/or cross-sectional shape of the microchannels. According to an embodiment of the present invention, the micro flow channel structure includes a first micro flow channel and a second micro flow channel, the first micro flow channel is in communication with the second micro flow channel, and the first micro flow channel and the first The two microchannels have different inner diameters, the composite enters the first microchannel, and the composite cannot enter the second microchannel. In some aspects of these embodiments, the sensing member can be configured to correspond to the first microfluidic channel. In some aspects of this embodiment, the size of the composite is such that the composite cannot enter the second microchannel.

In addition, in the microfluidic component, other functional units may be provided as needed. For example, for performing, for example, but not limited to, washing, dilution, separation, decomposition, drying, concentration, mixing, temperature adjustment, pH adjustment, modification, labeling (such as: enzyme, antigen, antibody, fluorescent, luminescence) , radioactive elements, chromogenic substances, nanoparticles, magnetic beads, etc.), anticoagulation, anti-degradation, anti-degradation, gas emissions (for example, discharge of gases contained in fluid samples, to avoid the impact of bubbles on detection) Functional units such as. The functional unit described above can be used to process, for example, a sample before or after sensing/analysing (in contact with the composite). In addition, the microfluidic component may also be disposed as needed, such as, but not limited to, a driving device (eg, a mechanical pump, an electric pump, etc.), a flow rate control device (eg, a control pump), a spoiler generating device, and an electrical device. Etc., to produce the desired adjustment to the sample into the microfluidic element.

According to an embodiment of the invention, the microfluidic component is a microfluidic wafer. In some aspects of these embodiments, the microfluidic component is a microfluidic wafer fabricated using microelectromechanical technology. The microfluidic structure can be formed in the substrate using various conventional techniques (eg, etching, direct molding, PDMS demolding, LIGA-like processes, etc.), and techniques such as wafer bonding can be used to fabricate the microfluidic wafer. Examples of the substrate include, but are not limited to, a ruthenium substrate, a glass substrate, a quartz substrate, a high molecular polymer (for example, polycarbonate, polymethyl methacrylate, cycloolefin polymer, polystyrene, polydimethyl hydrazine). Oxytomane (PDMS) substrate. Microfluidic components can be fabricated using one or more substrates. Microfluidic components can be fabricated using one or more substrates. Microfluidic components can be fabricated using one or more substrate materials. The microfluidic element can be of one or more layers. For example, a multilayer substrate can be bonded to make a microfluidic component. As another example, portions made on different substrates can be integrated to make microfluidic components. Further, a protective layer may be provided on the substrate. The protective layer can be made using materials well known in the art, such as a resin such as an epoxy resin. In accordance with an embodiment of the invention, the inlet and outlet of the microfluidic element can be defined using openings in the substrate and/or protective layer. In some aspects of these embodiments, the sensing member can be placed by MEMS technology. In some aspects of the embodiments, the transistors, circuits, instruments, devices, functional units, and the like as described above may be provided by MEMS technology as needed. In some aspects of the embodiments, a microchannel structure can be formed in the substrate and bonded to the substrate provided with the electrodes to fabricate a microfluidic component, wherein the sensing component corresponds to the electrode and the microchannel The structure is set. According to an embodiment of the invention, the microfluidic element can be one or more microfluidic wafers. The fabrication of microfluidic wafers using MEMS technology is well known in the art and will not be described again. Microfluidic components can be fabricated using methods known in the art for making microfluidic wafers. In addition, digital microfluidic wafers or continuous microfluidic wafers can also be used.

The composite of the detection system of the present invention comprises a carrier and a chemical for detection. The carrier used for the complex of the detection system of the present invention is not particularly limited. The carrier can be combined with chemicals in a variety of ways. For example, the carrier can be permeable to various physical or chemical means (such as, but not limited to, physical absorption, ion bonding, covalent coupling, cross-linking, embedding). (entrapment), copolymerization, etc.) Fix the chemical substance used for detection. In accordance with the present invention, the chemical species and the carrier can be interactively formed to form a composite using various conventional means in the art. For example, the carrier can be coated/embedded with a variety of conventional methods to form a composite. Alternatively, various physical and/or chemical means can be utilized to create a complex in which the chemical species is combined with the carrier, such as by binding the chemical to the surface of the carrier.

According to a particular embodiment of the invention, the carrier can be coated (including: embedded) with the chemical, for example, a colloid can be used as the carrier. Examples of colloids include, but are not limited to, algin, furcellaran, pectin, agar, agarose, chitosan, glucomannan (konjac glucomannan), curdlan, gellan, etc., derivatives thereof, analogs thereof, and mixtures thereof and the like. According to one embodiment of the invention, a colloid can be used as a carrier to cause the carrier to coat the chemical and form microbeads (or nanobeads). The chemical substance and the carrier can be formed into a composite of microbeads using various means known in the art. In some aspects of these examples, a composite of microbeads is produced by means of emulsification or the like.

According to a specific embodiment of the invention, the manufacture of the composite comprises the steps of: mixing the chemical and the material used to form the carrier to form a mixture; and emulsifying the mixture to form the chemical coated by the carrier Made of micro beads. For example, in the case of using a colloid as a carrier, a chemical substance may be mixed with a material for forming the colloid, and the mixture may be treated by emulsification to form microbeads. The use of an emulsified method for producing a composite is not only convenient, but also inexpensive, and the desired bead size can be selected according to requirements. Emulsification can be carried out using various conventional methods and materials in the art. An embodiment in which a composite of microbeads is produced by emulsification is as described in the following examples, but is not limited thereto. According to a particular embodiment of the invention, the manufacture of the composite comprises curing after emulsification of the mixture. Various methods and materials known in the art can be used (such as, but not limited to, various sources of cations (such as calcium ions, potassium ions, etc.) (such as, but not limited to, calcium chloride, potassium chloride, Etc.)) to cure. The curing may be carried out in the manner as described in the examples below, but is not limited thereto.

According to an embodiment of the invention, the particle size of the microbeads can be designed according to the inner diameter of the microchannels in the microchannel structure. According to an embodiment of the invention, the inner diameter of the microchannel in the microchannel structure can be designed according to the particle size of the microbeads. Preferably, the particle size of the microbeads allows the microbeads to pass through the microchannels having a relatively large inner diameter and not through the microchannels having a relatively small inner diameter. According to an embodiment of the present invention, the micro flow channel structure includes a first micro flow channel and a second micro flow channel, the first micro flow channel is in communication with the second micro flow channel, and the first micro flow channel and the first The two microchannels have different inner diameters, the composite enters the first microchannel, and the composite cannot enter the second microchannel. In some aspects of this embodiment, the size of the composite is such that the composite cannot enter the second microchannel. In some aspects of this embodiment, the bead size is such that the bead is permeable to the inlet of the microfluidic element to enter the first microchannel and is inaccessible to the second microchannel. The microbeads may have a particle size of from 100 to 2000 microns, preferably from 90 to 170 microns.

According to an embodiment of the invention, in addition to the chemical, the carrier may be coated (including: embedded) with a magnetic substance (such as, but not limited to, a magnetic bead). The magnetic substance is not particularly limited as long as it has magnetic properties (for example, but not limited to, a group B element of the periodic table of elements, a compound thereof, a mixture thereof, and the like). In some aspects of these embodiments, the magnetic material in the composite can be coupled to a positioning element (eg, a magnetic substance) of the microfluidic element to enhance the positioning or fixation of the composite. For example, the magnetic material in the composite and the positioning elements of the microfluidic element (eg, magnetic material) can position or secure the composite entering the microfluidic structure and be more evenly aligned. According to an embodiment of the present invention, the micro flow channel structure includes a first micro flow channel and a second micro flow channel, the first micro flow channel is in communication with the second micro flow channel, and the first micro flow channel and the first The two microchannels have different inner diameters, the composite enters the first microchannel, and the composite cannot enter the second microchannel. In some aspects of this embodiment, the size of the composite is such that the composite cannot enter the second microchannel. In some aspects of these embodiments, the magnetic material in the composite can be coupled to a positioning element (eg, a magnetic substance) of the microfluidic element to position or immobilize the composite to the first microfluidic channel. The carrier may be coated with a chemical substance and a magnetic substance to form a composite using a method known in the art. The carrier may be coated with a chemical substance and a magnetic substance by a method as described above to prepare a composite of microbeads.

Further, other materials may be used as the carrier. According to a particular embodiment of the invention, for example, a magnetic substance can be used as the carrier. In some aspects of these embodiments, the chemical is bonded to the surface of the support to form a composite. The chemical can be directly or indirectly bound to the surface of the carrier using a variety of methods known in the art. The magnetic substance is not particularly limited as long as it has magnetic properties (for example, but not limited to, a group B element of the periodic table of elements, a compound thereof, a mixture thereof, and the like). In some aspects of these embodiments, the magnetic material as the carrier may be coupled to a positioning element (eg, a magnetic substance) of the microfluidic element to enhance the positioning or fixation of the composite. For example, a magnetic substance as a carrier and a positioning element of a microfluidic element (eg, a magnetic substance) can position the composite entering the microchannel structure and be more evenly aligned. According to an embodiment of the present invention, the micro flow channel structure includes a first micro flow channel and a second micro flow channel, the first micro flow channel is in communication with the second micro flow channel, and the first micro flow channel and the first The two microchannels have different inner diameters, the composite enters the first microchannel, and the composite cannot enter the second microchannel. In some aspects of this embodiment, the size of the composite is such that the composite cannot enter the second microchannel. In some aspects of these embodiments, the magnetic material as the carrier may be coupled to a positioning element (eg, a magnetic substance) of the microfluidic element to position or fix the composite to the first microfluidic channel.

As the chemical substance used in the composite of the present invention, any chemical substance which can be used for detecting the analyte can be used. According to a particular embodiment of the invention, the chemical is coated (including: embedded) with a carrier. According to a particular embodiment of the invention, the chemical is bonded to the surface of the carrier. According to a particular embodiment of the invention, the chemistry of the composite of the invention comprises a substance for reacting with the analyte in the sample. In some aspects of these embodiments, the chemical of the complex includes a substance that is specifically reactive with the analyte in the sample. Examples of the substance to be reacted with the analyte in the specimen include, but are not limited to, nucleic acids, peptides, proteins, enzymes, antigens, antibodies, ligands, receptors, cells, viruses, bacteria, derivatives thereof, Analogs thereof, or mixtures thereof and the like. The chemical substance used can be selected according to the object to be tested. According to one embodiment of the invention, the chemical is used for detection. Specifically, the chemical is used to react with the analyte in the sample for detection. After the sample enters the microchannel structure, it contacts the complex that has entered the microchannel structure, and the chemical substance of the complex is reacted with the analyte (which can react with the chemical substance) in the sample to perform sensing. For example, as described above, when an electrochemical sensing member is used, a chemical quantity change generated by a reaction of a chemical substance with a test object can be converted into, for example, an electrical signal for sensing.

According to the detection system of the present invention, the composite can be removed into or removed from the microfluidic element, which not only improves the freedom of use of the detection system, but also increases its use value. Through the present invention, the complex can be used or replaced as needed (for example, the type of the analyte, the state of the chemical in the composite) by simply passing in or removing the composite. In accordance with an embodiment of the present invention, the composite is positioned or secured to the microchannel structure through a plurality of microchannel structures including microchannels having different inner diameters and/or different cross-sectional shapes. The thus positioned or fixed composite can be easily removed from the microfluidic element. According to an embodiment of the invention, the composite is positioned or fixed in the microfluidic element through a plurality of microchannel structures comprising microchannels having different inner diameters and/or different cross-sectional shapes, and further, micro Positioning elements (eg, magnetic materials) in the fluid element further enhance the positioning or fixation effect. The thus positioned or fixed composite can be easily removed from the microfluidic element. Therefore, according to the present invention, the desired composite can be selected and replaced as needed simply by simply inserting or removing the composite. Moreover, with the present invention, the detection system can be inspected as needed (e.g., detecting different analytes) without the need to replace or discard the microfluidic components (replacement of only the composite). Further, according to the present invention, it is possible to solve the problem of reduction/deactivation of activity due to long-term use or poor storage of chemical substances, contamination due to repeated use, and the like in the prior art by simply replacing the composite. In addition, according to the present invention, it is possible to place the composite into the microfluidic component as needed when performing the detection, which not only improves the convenience, but also improves the quality of the detection system (for example, avoids the reduction/deactivation of the activity of the chemical substance, and reuses The lack of pollution, etc.).

The substance to be tested is a substance that can react with the chemical substance of the complex. For example, the test object in the sample may be a substance that can specifically react with the chemical substance of the complex. The applicable test substance depends on the chemical substance used in the composite. Examples of the analyte include, but are not limited to, glucose, amino acid, urea, uric acid, creatinine, nucleic acid, peptide, protein, enzyme, antigen, antibody, ligand, receptor, cell, virus, bacteria, Derivatives, analogs thereof, or mixtures thereof, and the like. According to an embodiment of the present invention, the sample enters the microchannel structure and is in contact with the composite that has entered the microchannel structure, and the chemical substance of the complex is reacted with the analyte in the sample (can react with the chemical substance) Reaction) for sensing. For example, as described above, when an electrochemical sensing member is used, sensing can be performed by electrochemical changes produced by the reaction of the chemical with the analyte. The sample can be a variety of fluid samples. Fluid samples include gases, liquids, plasmas, mixtures thereof, etc., which may contain solids to some extent, as well as biochemical substances (eg, glucose, amino acids, urea, uric acid, creatinine, nucleic acids, peptides, proteins, Enzymes, antigens, antibodies, ligands, receptors, cells, viruses, bacteria, derivatives thereof, analogs thereof, or mixtures thereof, etc.). For example, the fluid sample can be, for example, but not limited to, a biological sample (eg, body fluid (blood, interstitial fluid, urine, etc.), tissue, cells, biochemical samples containing the above-described analytes, etc.), Chemical samples, artificially supplied fluid samples, etc.

The detection system according to the present invention will be exemplified below with reference to Figures 1 to 3. It is to be understood that the drawings are only schematic, and are not intended to limit the scope of the invention.

Please refer to Figures 1A through 1B, which are schematic illustrations of a detection system in accordance with an embodiment of the present invention. As shown, the detection system 1 includes a microfluidic element 11 and a composite 13. The microfluidic element 11 includes an inlet 111, an outlet 113, and a microchannel structure 115. The microfluidic element 11 is used to provide a place for detection. The specimen can enter the microfluidic element 11 through the inlet 111 of the microfluidic element 11 and exit the microfluidic element 11 through the outlet 113 of the microfluidic element 11. Microfluidic components can be fabricated using conventional techniques. Microfluidic components can be fabricated using one or more substrate materials. The microfluidic element can be of one or more layers.

The microchannel structure 115 is in communication with the inlet 111 and the outlet 113. The microfluidic structure 115 can be formed in a substrate (an example of a substrate as described above) by various conventional techniques to fabricate the microfluidic element 11. Microfluidic components can be fabricated using one or more substrates. Microfluidic components can be fabricated using one or more substrates. Further, a protective layer may be provided on the substrate. As shown in Fig. 1A', the microfluidic element 11 is provided with a protective layer 19. According to an embodiment of the invention, the inlet 111 and the outlet 113 of the microfluidic element 11 can be defined using openings of the substrate and/or the protective layer. According to a particular embodiment of the invention, the microfluidic element 11 is a microfluidic wafer. In some aspects of these embodiments, the microfluidic component 11 is a microfluidic wafer fabricated using microelectromechanical technology.

The microfluidic structure 115 in the microfluidic element 11 is used to provide a location for detection. The microchannel structure 115 can have a plurality of microchannels that can have different inner diameters (including: height, width) and/or different cross-sectional shapes. According to an embodiment of the invention, the microchannel structure 115 includes a first microchannel 1151 and a second microchannel 1153, and the first microchannel 1151 is in communication with the second microchannel 1153. The first microchannel 1151 and the second microchannel 1153 have different inner diameters (including: height, width). According to an embodiment of the invention, the microchannel structure 115 includes a first microchannel 1151 and a second microchannel 1153 that are continuously disposed. As shown in FIG. 1B, if the inner diameter of the first microchannel 1151 is larger than the second microchannel 1153, the composite 13 cannot enter the second microchannel having a smaller inner diameter from the first microchannel 1151. 1153, thus placing the composite 13 in the first microchannel 1151 to achieve the effect of positioning or fixation. According to an embodiment of the invention, the composite 13 is placed in the first microchannel due to the size of the composite 13 such that it cannot enter the second microchannel 1153 having a smaller inner diameter from the first microchannel 1151. 1151, and achieve the effect of positioning or fixing. In accordance with an embodiment of the invention, the composite 13 enters the microchannel structure 115 through the inlet 111 of the microfluidic element 11. In some aspects of the embodiments, the composite 13 entering the microchannel structure 115 cannot enter the second microchannel 1153 having a smaller inner diameter from the first microchannel 1151 having a larger inner diameter. Thus achieving the effect of positioning or fixing. In accordance with an embodiment of the present invention, the composite 13 is permeable to the microfluidic structure 115 through the inlet 111 of the microfluidic element 11.

The microfluidic structure 115 can be modified as desired (eg, modified with a particular substance or specific functional group). It is also possible to influence the flow rate or flow pattern of the fluid sample (e.g., sample) that has passed through the microchannel structure 115 by utilizing changes in the material, inner diameter, and/or cross-sectional shape of the microchannel structure 115. Additionally, a fluid sample (e.g., sample) volume control unit may be provided in the microfluidic element 11 to control the amount of fluid sample (e.g., sample) entering the microfluidic element 11.

The composite 13 can be brought into or out of the microfluidic element 11 in any manner. As previously mentioned, the composite 13 can be passed into or removed from the microfluidic element 11, thereby increasing the freedom of use of the detection system 1 and increasing the value of the detection system 1. For example, the composite can be replaced with a rinse or the like or any other means. With the present invention, the composite 13 can be used or replaced as needed simply by inserting or removing the composite 13. As shown in FIG. 1B, the composite 13 is positioned or fixed to the microchannel structure 115 through the microchannel structure 115 including the first microchannel 1151 and the second microchannel 1153. The composite 13 thus positioned or fixed can be easily removed from the microfluidic element 11 (e.g., by passing a liquid or gas or the like to remove the composite 13). Furthermore, with the present invention, the detection system 1 can be inspected as needed (e.g., detecting different analytes) without the need to replace or discard the microfluidic element 11 (which can be replaced only with the composite 13). Further, according to the present invention, it is possible to solve the problem of reduction/deactivation of activity due to long-term use or poor storage of chemical substances, contamination due to repeated use, and the like in the prior art by simply replacing the composite 13. Further, according to the present invention, the composite 13 can be placed in the microfluidic element 11 as needed when performing the detection, which not only improves the convenience, but also improves the quality of the detection system 1.

The detection system 1 can include a sensing member 15. The sensing member 15 may have a signal conversion unit (converting the physical (heat, pressure, mass, magnetic field) amount or chemical quantity change of the composite 13 to the object to be tested into a signal), a signal processing unit, and the like. In addition, other sensing units may be disposed in the sensing component 15 to detect the detection of other objects in the sample or other parameters. According to an embodiment of the invention, an electrochemical sensing member is used as the sensing member 15. According to a particular embodiment of the invention, the microfluidic element 11 is a microfluidic wafer. In some aspects of these embodiments, the microfluidic component 11 is a microfluidic wafer fabricated using microelectromechanical technology. In some aspects of the embodiments, the sensing member 15 can be disposed in the detection system 1 by a technique such as microelectromechanical.

The sensing material 15 as known in the art can be used as the sensing member 15, and the sensing member 15 can also be fabricated and integrated into the detection system 1 of the present invention using techniques known in the art. According to an embodiment of the invention, the detection system 1 is detected by the composite 13 and the sensing member 15. As described above, the sensing member 15 can be fabricated using, for example, a transistor. In these examples, the composite 13 entering the microfluidic element 11 is positioned or fixed to correspond to the transistor used in the sensing member 15 for sensing. In accordance with an embodiment of the present invention, the sensing member 15 includes electrodes, such as a bottom plate electrode 151 formed in the microfluidic element 11 formed of a multilayer substrate, disposed adjacent to the microchannel structure 115. For example, as shown in FIGS. 1A and 1A', the bottom plate electrode 151 is formed on the glass substrate 11a under the microfluidic element 11, and the function of the bottom plate electrode 151 is to pull out the electrical signal of the sensing member 15 as a signal. For measurement purposes. In these embodiments, the composite 13 entering the microfluidic element 11 is positioned or fixed to correspond to the electrodes of the sensing member 15. In accordance with an embodiment of the present invention, further integration may be utilized in microfluidic devices using conventional techniques, such as microelectromechanical techniques, such as, but not limited to, a reference electrode (or transistor), a comparison electrode (or transistor), Measuring circuit / measuring instrument (for measuring, for example: optical (absorbed light of specific wavelength, fluorescent, luminescent), electric (voltage, current, potential, resistance), magnetic, thermal, mechanical and other signals, color change And/or related circuits/devices (for example, for signal conversion, signal amplification).

In accordance with an embodiment of the present invention, the microchannel structure 115 is designed such that the composite 13 is positioned or fixed to correspond to the sensing member 15. In accordance with an embodiment of the invention, the sensing member 15 is disposed adjacent to the microfluidic structure 115. In some aspects of these embodiments, the sensing member 15 is configured to take into account the design of the microfluidic structure 115 such that the composite 13 entering the microfluidic structure 115 can correspond to the sensing member 15. As shown in FIG. 1B, the composite 13 entering the microfluidic element 11 is positioned or fixed to correspond to the sensing member 15. The microchannel structure 115 includes a first microchannel 1151 and a second microchannel 1153. The first microchannel 1151 is in communication with the second microchannel 1153, and the first microchannel 1151 and the second microchannel The 1153 has a different inner diameter, and the composite 13 is able to enter the first microchannel 1151 but cannot enter the second microchannel 1153. In this example, the sensing member 15 is disposed to correspond to the first microfluidic channel 1151.

The sample can enter the microfluidic element 11 through the inlet 111 of the microfluidic element 11 and contact with the composite 13 that has entered the microchannel structure 115, and the chemical of the complex 13 can react with the analyte in the sample to Test. For example, through the sensing member 15, the change caused by the reaction of the chemical substance with the analyte (eg, electrochemical change) can be utilized for sensing.

Further, as described above, various functional units, transistors, circuits, instruments, devices, and the like can be disposed in the microfluidic element 11 as needed using techniques known in the art.

Next, please refer to Figures 2A and 2B, which are schematic views of a detection system in accordance with an embodiment of the present invention. The difference between the 2A/2B diagram and the 1A/1B diagram is that the detection system 1 is provided with the positioning element 17 and the reference electrode 20.

The reference electrode 20 is formed in the upper half of the microfluidic element 11, and the inlet 111 or the outlet 113 is connected to the reference electrode 20.

The positioning member 17 is not particularly limited as long as it can exert a function of positioning or fixing the composite 13 entering the microchannel structure 115 (as shown in FIG. 2B). The positioning element 17 can be arranged to position or secure the composite 13 in the desired position. For example, the positioning element 17 can be a magnetic substance. The magnetic substance is not particularly limited as long as it has magnetic properties. The attraction of the magnetic material can be utilized to position the composite 13 entering the microchannel structure 115 and the alignment is more uniform. As described above, the effect of positioning or fixing the composite 13 can be achieved by the microchannel structure 115 including the first microchannel 1151 and the second microchannel 1153. At the same time, the positioning or fixing effect can be further enhanced by the setting of the positioning member 17.

In accordance with an embodiment of the present invention, the microchannel structure 115 is designed such that the composite 13 is positioned or fixed to correspond to the sensing member 15. In accordance with an embodiment of the invention, the sensing member 15 is disposed adjacent to the microfluidic structure 115. In some aspects of these embodiments, the positioning element 17 is configured to take into account the design of the microchannel structure 115 such that the composite 13 entering the microchannel structure 115 can correspond to the sensing member 15. In some aspects of these embodiments, the positioning element 17 is configured to take into account the design of the microfluidic structure 115 and/or the placement of the sensing member 15 such that the composite 13 entering the microfluidic structure 115 can correspond to Sensing member 15. As shown in FIG. 2B, the composite 13 entering the microfluidic element 11 is positioned or fixed to correspond to the sensing member 15. The composite 13 is positioned or fixed to correspond to the sensing member 15 by the design of the microchannel structure 115 and/or the positioning of the positioning member 17. The microchannel structure 115 includes a first microchannel 1151 and a second microchannel 1153, and the first microchannel 1151 and the second microchannel 1153 are in communication. According to an embodiment of the invention, the microchannel structure 115 includes a first microchannel 1151 and a second microchannel 1153 that are continuously disposed. According to an embodiment of the invention, the first microchannel 1151 and the second microchannel 1153 have different inner diameters, and the composite 13 can enter the first microchannel 1151 but cannot enter the second micro. Flow path 1153. In this example, the positioning member 17 is disposed to correspond to the first microfluidic channel 1151. The composite 13 is positioned or fixed to the first microchannel 1151 and corresponds to the sensing member 15 by the design of the microchannel structure 115 and/or the positioning of the positioning member 17.

The composite 13 can be brought into or out of the microfluidic element 11 in any manner. As previously mentioned, the composite 13 can be passed into or removed from the microfluidic element 11, thereby increasing the freedom of use of the detection system 1 and increasing the value of the detection system 1. With the present invention, the composite 13 can be used or replaced as needed simply by inserting or removing the composite 13. As shown in FIG. 2B, the composite 13 is positioned or fixed to the microchannel structure 115 through the first microchannel 1151 and the second microchannel 1153. Furthermore, the positioning of the positioning element 17 enhances the effect of this positioning or fixing. The thus positioned/fixed composite 13 can be easily removed from the microfluidic element 11. With the present invention, the microfluidic element 11 can be replaced or discarded (the composite 13 can be replaced only), so that the detection system 1 can be detected as needed (for example, detecting different analytes). Further, according to the present invention, it is possible to solve the problem of reduction/deactivation of activity due to long-term use or poor storage of chemical substances, contamination due to repeated use, and the like in the prior art by simply replacing the composite 13. Further, according to the present invention, the composite 13 can be placed in the microfluidic element 11 as needed when performing the detection, which not only improves the convenience, but also improves the quality of the detection system 1.

The sample can enter the microfluidic element 11 through the inlet 111 of the microfluidic element 11, and contact with the composite 13 that has entered the microchannel structure 115, and the chemical of the complex 13 can react with the analyte in the sample to perform Detection. For example, through the sensing member 15, the change caused by the reaction of the chemical substance with the analyte (eg, electrochemical change) can be utilized for sensing.

The present invention provides a detection method comprising detecting using a detection system as described above. According to a specific embodiment, the detection method of the present invention uses a detection system as described above to detect a test object in a sample, the detection method comprising: introducing a composite into a microfluidic element for placement in a microfluidic structure And placing the sample into the microfluidic element to contact the composite. The sample can be used to enter or leave the microfluidic element in any manner. As described in the foregoing, the purpose of the test can be achieved by the reaction of the analyte in the sample with the chemical substance in the complex. For example, when an electrochemical sensing member is used, sensing can be performed by electrochemical changes produced by the reaction of the chemical substance in the composite with the analyte in the sample. According to a particular embodiment of the invention, the composite can be removed from the microfluidic element.

According to the detection system and method of the present invention, the composite can be removed into or removed from the microfluidic element, which not only improves the freedom of use of the detection system, but also increases its use value. With the detection system and method of the present invention, it is only necessary to simply pass through or remove the complex, and the composite can be used or replaced as needed (for example, the type of the analyte, the state of the chemical in the composite). In accordance with an embodiment of the present invention, the composite is positioned or secured to the microchannel structure through a plurality of microchannel structures including microchannels having different inner diameters and/or different cross-sectional shapes. The thus positioned or fixed composite can be easily removed from the microfluidic element. According to an embodiment of the invention, the composite is positioned or fixed in the microfluidic element through a plurality of microchannel structures comprising microchannels having different inner diameters and/or different cross-sectional shapes, and further, micro The positioning elements of the fluid element (eg, magnetic materials) further enhance the positioning or fixation effect. The thus positioned or fixed composite can be easily removed from the microfluidic element. Therefore, according to the present invention, the desired composite can be selected and replaced as needed simply by simply inserting or removing the composite. Moreover, with the present invention, the detection system can be inspected as needed (e.g., detecting different analytes) without the need to replace or discard the microfluidic components (replacement of only the composite). Further, according to the present invention, it is possible to solve the problem of reduction/deactivation of activity due to long-term use or poor storage of chemical substances, contamination due to repeated use, and the like in the prior art by simply replacing the composite. In addition, according to the present invention, it is possible to place the composite into the microfluidic component as needed when performing the detection, which not only improves the convenience, but also improves the quality of the detection system (for example, avoids the reduction/deactivation of the activity of the chemical substance, and reuses The lack of pollution, etc.).

The detection system and method of the present invention can be applied to, for example, but not limited to, biomedical (such as gene sequencing, gene expression analysis, protein analysis, drug screening, clinical detection), environmental testing, food testing, and the like.

The invention will be more specifically described by the examples, but these examples are not intended to limit the scope of the invention. Unless otherwise specified, the "%" and "parts by weight" used in the following examples and comparative examples to indicate the content of any component and the amount of any substance are based on the weight.

Preparation of complex Preparation Example 1: Preparation of Enzyme-encapsulated Alginate Microbeads

Take 10 mg (mg) of Glucose Oxidase type II (SIGMA, USA), 50 mg of urease (Urease type III, SIGMA, USA) and 10 mg of Creatinine deiminase (SIGMA, USA). After uniformly mixing with 100 μl of μgin sodium (Alginic acid sodium salt, SIGMA, USA, 2% by weight (wt%)), corn oil (Corn oil C8267, SIGMA, USA) was added dropwise. Because the seaweed gum is in the water phase, the seaweed gel droplets and the corn oil are in a water-in-oil state by the incompatibility of the oil and water, so as to form the emulsified particles by utilizing the principle of emulsification. The emulsified droplets were then dropped into a calcium chloride solution at a concentration of 0.4 millimolar (mM) to solidify the algae beads to obtain algae beads embedded in the enzyme.

Preparation Example 2: Preparation of seaweed microbeads encapsulating enzymes and magnetic beads

Take 10 mg of Glucose Oxidase type II (SIGMA, USA), 50 mg of urease (Urease type III, SIGMA, USA) and 10 mg of Creatinine deiminase (SIGMA, USA) and 100 Μl of Alginic acid sodium salt (USA) (2wt%), a concentration of 2 × 10 ⁹ beads / ml (bead / ml) of magnetic beads (Myone, Invitrogen, USA) solution 100 μl evenly mixed, drip Corn oil (Corn oil C8267, SIGMA, USA). Because the seaweed gum is in the water phase, the seaweed gel droplets and the corn oil are in a water-in-oil state by the incompatibility of the oil and water, so as to form the emulsified particles by utilizing the principle of emulsification. The emulsified droplets were then dropped into a 0.4 mM calcium chloride solution to solidify the algae beads to obtain algae beads embedded in the enzyme and magnetic beads.

Using the methods of Preparation Examples 1 and 2 above, the microfluidic device utilizing pneumatic micro-vibrators to generate alginate microbeads for microencapsulation of cells, Sensors and Actuators B 147 (2010) 755-764 can be prepared by adjusting different flow rates and frequencies. Complexes of different sizes (particle sizes). As shown in Fig. 3, algae microbeads having a diameter of 90, 110, 130, 150, and 170 micrometers (μm) can be separately prepared for quantitative analysis of various particle sizes, as shown in Fig. 4.

Preparation Example 3: Making Magnetic Beads of Bonding Enzymes

The enzyme is bonded to the magnetic beads by a biochemical reaction. A magnetic bead having a functional group such as a magnetic bead having a carboxyl group or a magnetic bead having an amino group (M270, Invitrogen, USA) may be used for the binding enzyme.

In this embodiment, magnetic beads with carboxyl groups (MyOne, Invitrogen, USA) are used to prepare magnetic beads bound to glucose enzyme, and carbodiimide is used (eg, (1-ethyl-3-() 3-ethyl-3-(3-dimethylaminopropyl-carbodiimide (EDC)) is used as a bonding bridge between carboxyl and glucose enzymes on magnetic beads (EDC is available) The carboxyl group on the activated magnetic beads further binds the carboxyl group to the glucose enzyme).

Further, in the present embodiment, magnetic beads with an amine group (M270, Invitrogen, USA) were used to prepare magnetic beads bound to creatinine zymase, and glutaraldehyde was used as an amine-based magnetic bead and creatinine zymase. Connection bridge.

The aforementioned carboxyl magnetic bead bond glucose method is divided into several steps:

(1) Dynabeads MyOne Carboxylic Acid was vortexed for 30 minutes to uniformly mix the magnetic beads in the solution to maintain a fixed concentration.

(2) Next, 1 ml (10 mg/ml, 2 × 10 ⁹ beads/ml) of magnetic beads was taken from the original solution using a micropipette and placed in a test tube.

(3) Fix the tube with the magnetic holder for 3 to 5 minutes, and the magnetic beads in the solution are magnetically fixed to the tube wall, and the suspension in the tube is taken.

(4) Wash the magnetic beads twice with 1 ml of MES (2-(N-morpholino)ethanesulfonic acid) for the purpose of removing residual magnetic bead preservation solution and increasing The ability to bond with an enzyme.

(5) The washed magnetic beads were concentrated to 100 μl of MES, and uniformly mixed by shaking.

(6) Add 100 μl of EDC (10 mg/ml in D.I.) to the test tube and mix gently with shaking. It was then mixed at room temperature for 30 minutes with a rotary mixer.

(7) The magnetic beads are used to fix the magnetic beads on the test tube wall, and then the suspension is taken.

(8) Pre-dissolved in 200 μl of MES buffer solution using 400 μg of glucose enzyme.

(9) Mixing at room temperature overnight using a rotary mixer, since the glucose enzyme contains an amine group, it can undergo a substitution reaction with EDC to form a covalent bond.

(10) The magnetic beads are used to fix the magnetic beads on the test tube wall, and then the suspension is taken.

(11) Using 1 ml of PBS (1X PBS, 0.1% Tween 20), the magnetic beads were washed with a rotary mixer for 10 minutes. The magnetic beads of the modified glucose enzyme were fixed by magnets to the sensing area of the sensing part to measure different concentrations. The glucose solution produced by the electrochemical difference, the remaining magnetic beads can be stored in a 4 ° C environment and stored in 1 ml of PBS.

The method of immobilizing creatinine enzyme with amine-based magnetic beads is as follows:

(1) The magnetic bead stock solution (Dynabeads M-270 Amine) was shaken for 2 minutes to allow the magnetic beads to be uniformly mixed in the solution to maintain a fixed concentration.

(2) Next, 50 μl of magnetic beads were taken from the original solution using a micropipette and placed in a test tube.

(3) Fix the tube with the magnetic holder for 3 to 5 minutes. The magnetic beads in the solution are magnetically fixed to the tube wall and the suspension in the tube is taken.

(4) Add 100 μl of PBS to the test tube, shake for 2 to 3 minutes, then fix the magnetic beads on the tube wall with a magnetic holder. Finally, repeat this step three times in order to remove the residual magnetic bead preservation solution and increase The ability to bond with an enzyme.

(5) Fix the magnetic beads on the magnetic base, use a micropipette to extract the suspension in the test tube, and then add 100 μl of the cross-linking agent glutaraldehyde (10% glutaraldehyde in PBS, pH=7) at room temperature. Mix for two hours using a rotary mixer.

(6) Fix the magnetic beads on the test tube wall with a magnetic holder, use a micropipette to remove the cross-linking agent glutaraldehyde, add 100 μl of PBS to the test tube, shake for another 2 to 3 minutes, and then fix the magnetic beads to the test tube with the magnetic holder. On the wall, this step is repeated three times to remove residual glutaraldehyde, ensuring that the enzyme is immobilized on the magnetic beads by cross-linking.

(7) The magnetic holder was used and the suspension was withdrawn, followed by dissolving 30 μl of creatinine in 100 μl of PBS, added to a test tube, and mixed overnight at room temperature using a rotary mixer.

(8) Using a magnetic holder to fix the magnetic beads on the wall of the test tube and extract the suspension, add 100 μl of PBS to the test tube, shake for another 2 to 3 minutes, then fix the magnetic beads on the tube wall with a magnetic holder, and repeat this step three times. The creatinine enzyme, which has not been fixed in the suspension, is washed to make the measurement results more accurate.

(9) The magnetic beads of the modified creatinine enzyme can be fixed in the sensing area of the sensing part by using a magnet, and the electrochemical difference caused by the different concentrations of the creatinine solution can be measured, and the remaining magnetic beads can be stored in an environment of 4 ° C and used. Save in PBS.

Example

In the present embodiment, the detection system as shown in Figs. 2A and 2B is used, and the manufacturing method is not limited to the following. The microfluidic element has a microchannel structure and is integrated with an electrode, a sensing member, a positioning member, and the like. Each component can be fabricated and integrated through semiconductor manufacturing and MEMS technology.

First, the bottom plate electrode is formed on the glass substrate as the lower layer of the microfluidic element to form an aluminum metal wire, that is, a layer of positive photoresist is coated on the glass substrate, and then after soft baking, exposure and development are performed, and then aluminum is evaporated. Then remove the photoresist, that is, complete the fabrication of the bottom plate electrode.

The sensing member in the microfluidic component is fabricated by using an electrolyte-insulating layer-germanium semiconductor capacitor, and a p-type germanium wafer having a (100) direction and a resistance of 8-12 Ω·cm is used as a substrate. Initially, the native oxide layer was washed with hydrofluoric acid (HF), then the surface organic matter was washed away with acetone and methanol, and the residual acetone and methanol were rinsed with DI water, and then the temperature was adjusted to 120 ° C in an oven. The p-type substrate was baked for 30 minutes to remove moisture from the surface. In the low-vacuum cavity, the sputter is used to deposit the yttrium titanium compound with the target of 99.9% pure metal ruthenium and titanium, and the resulting plasma bombardment of Dysprosium (Dy) and Titanium (Ti). Insulation layer. After depositing the insulating layer of the bismuth titanium compound, the amorphous insulating layer is rearranged and bonded by rapid annealing at a temperature of 900 ° C. Finally, the back surface of the p-type substrate is plated by evaporation. 300 nm aluminum is used as the ohmic contact layer. Then, the prepared sensing member is joined with the bottom plate electrode by silver glue.

The microfluidic component is fabricated in a microfluidic structure by using a precision micro-engraving device of 0.5 cm (mm) to engrave the desired model on a PMMA (polymethylmethacrylate) substrate. Reperfusion PDMS (polydimethyloxane, Sil-More Industrial Ltd., USA) was formed on the PMMA model and baked at 70 ° C. Finally, the dried PDMS was peeled off to form the PDMS microchannel layer. .

The reference electrode is provided in the microfluidic component, and the reference electrode is prepared by coating a layer of AZ4620 positive photoresist on the glass substrate, then soft baking and then exposing and developing, then sequentially depositing aluminum and silver, and then removing the light. Resistance, and then modified with ferric chloride to Ag / AgCl.

Finally, the process of oxygen plasma bonding is performed to fabricate a microfluidic component, which is cleaned and dried with alcohol and water on the surface of the PDMS microchannel layer, placed in an oxygen plasma machine, evacuated, and passed through oxygen. After the plasma is generated and the plasma treatment is continued for two minutes, the PDMS micro-channel layer is aligned under the microscope with the glass substrate having the bottom plate electrode and the sensing member, and then placed on the heating plate to be heated to strengthen the joint. effect. Similarly, the reference electrode is then bonded to the PDMS microchannel layer, typically the inlet or outlet of the microfluidic element is in communication with the reference electrode to contact the solution of the composite. According to the above, a microfluidic member having a microchannel structure (having a first microchannel of 200 μm and a second microchannel of 70 μm) and a sensing member as shown in Figs. 2A and 2B can be obtained. The sensing unit is connected to the measurement system for analysis at the time of detection.

Example 1

The seaweed microbeads having a particle diameter of 170 μm obtained in Preparation Example 1 (in the PBS preservation solution, the solution containing the same concentration of the enzyme as in the microbeads) were introduced into the microfluidic element through the inlet of the microfluidic element. The number of entries is about 200. Since the height of the microchannel structure above the sensing member is reduced from 200 μm (first microchannel) to 70 μm (second microchannel), the 170 μm seaweed microbead cannot be the first height of 200 μm. The microchannel enters a second microchannel having a height of 70 μm and is thus blocked from being positioned on the surface of the sensing member.

Example 2

The seaweed microbead having a particle diameter of 170 μm obtained in Preparation Example 2 (in the PBS preservation solution, the preservation solution contains the same concentration of the enzyme as in the microbead), and the microfluidic element is introduced into the microfluidic element through the inlet of the microfluidic element. The number of entries is about 200. Since the height of the microchannel structure above the sensing member is reduced from 200 μm (first microchannel) to 70 μm (and the second microchannel), the 170 μm seaweed microbead cannot be replaced by a height of 200 μm. A microchannel enters a second microchannel having a height of 70 μm and is thus blocked from being positioned on the surface of the sensing member. In addition, a positioning member made of a magnetic material is disposed under the sensing member, and the material of the magnetic material is a sintered NdFeB super-strong magnet. The attraction of the magnetic device can make the algae microbeads above the sensing member more aligned. Evenly.

Example 3

The magnetic beads of the binding enzyme obtained in Preparation Example 3 were introduced into the microfluidic element from the inlet of the microfluidic element, and the number of introductions was about 200. A positioning member made of a sintered NdFeB super-strong magnet is disposed under the sensing member, and the magnetic bead having magnetic properties can be attracted by an applied magnetic field from the positioning member, and thus positioned on the surface of the sensing member.

Detection of glucose, creatinine and urea

First, using the detection systems of Examples 1 to 3, respectively, the standard solution is detected to obtain a standard curve (the standard curve is measured before each measurement of the sample, and each concentration has a corresponding voltage, When the detection of the sample is performed, the concentration can be obtained by measuring the voltage): a glucose solution (Glucose, SIGMA, USA) having a concentration ranging from 2 mM to 8 mM is measured using a complex containing glucose enzyme; The complex of creatinine zymase is measured in a concentration range of 0.01 mM to 10 mM creatinine acid solution (Creatine, SIGMA, USA); and a urea solution containing a concentration of 1 mM to 16 mM is measured using a complex containing urease ( Urea, SIGMA, USA). Different composites can be replaced by flushing with water or the like. The measurement method is to use a syringe pump (Legato 180, KD Scientific, USA) to measure the standard solution (glucose solution, creatinine solution or urea solution) in the syringe at an injection rate of 480 μl (microliter) / min (minutes). The injection time was 5 seconds, and the detection system was injected into the detection system by the inlet of the microfluidic element to react with the enzyme of the complex (the reaction time was 20 seconds after the completion of the injection), and the sensing was performed through the sensing member. The HP4284 Impedance Analyzer (HP) was used as the measurement system for analysis: the measurement voltage was -2 V (volts) ~ 2 V, the fixed frequency (500 Hz (Hz)); the aluminum part of the back of the sensing part (used to reduce The contact surface resistance of the sensing member and the bottom plate electrode is connected to the High end of the measuring system, and the reference electrode is connected to the Low end of the measuring system.

Next, the detection of the concentration of glucose, creatinine, and urea in the human serum (sample) is performed by using the detection systems of Examples 1 to 3, respectively: when necessary, the microfluidic element can be washed away with clean water before the detection is performed. The used complex (the algae beads or the magnetic beads of the bond enzyme), and then the new complex (the algae beads or the magnetic beads of the bond enzyme; the particle size is 170 μm, the quantity is about 200 a). The serum-loaded syringe was then injected into the detection system from the inlet of the microfluidic element using an injection pump (Legato 180, KD Scientific, USA) at an injection rate of 480 μl/min for an injection time of 5 seconds. The enzyme reaction (measured 20 seconds after the injection was completed) was passed through the sensing element and the HP4284 Impedance Analyzer (HP) (measurement system) for sensing and analysis (as described above). The concentration of blood glucose (glucose), creatinine, and urea contained in the serum can be detected using the measured potential.

Test results: Using the detection system of Example 2, the measured value of glucose was measured to be 12.19 mV (microvolt) / mM, and the linearity was 0.9944; the measured value of creatinine was measured to be 105.3 mV/pC _creatinine (pC _creatinine represents The linearity of each 10 mM creatinine concentration was 0.9926; the measured value of urea was 12.76 mV/mM, and the linearity was 0.9929. The same results were obtained by measuring with the detection system of Example 1. Furthermore, using the detection system of Example 3, the measured value of glucose was 5.28 mV/mM, and the linearity was 0.9897; the sensing value of creatinine was 15.15 mV/mM, and the linearity was 0.9844.

In addition, glucose and creatinine were prepared using commercially available glucose, creatinine detection reagents (available from GLUCOSE liquicolor and CREATININE liquicolor: Human, Wiesbaden, Germany); and urea reagents (purchased from MeDiPro-BUN Formosa Biomedical Technology, Taipei, Taiwan). The detection of urea was compared with the detection system of the examples. As a result, it was found that the commercially available reagent detected a glucose concentration of 6.58 mM, a urea concentration of 4.5 mM, and a creatinine acid concentration of 60.39 μM (micromolar concentration). In the case of the detection systems of Examples 1 and 2, the glucose concentration was 6.24 mM (error percentage was 5.16%), the urea concentration was 4.22 mM (error percentage was 6.22%), and the muscle liver acid concentration was 52.22 μM (the error percentage was 13.52%), as shown in Figure 5. Furthermore, the detection system of Example 3 of the present invention detected a glucose concentration of 5.93 mM (10% error percentage) and a muscle liver acid concentration of 50.9 μM (15% error percentage). It can be seen from the above that the detection result obtained according to the embodiment of the present invention is very close to and consistent with the detection result obtained by the commercially available reagent, and has practical clinical application value.

According to the above, the detection system of the present invention exhibits good sensing values and linearity, and has clinical applicability. At the same time, through the detection system and method of the invention, the use degree and convenience of the detection system are not only improved, but also the use value of the detection system and the quality of the detection are improved. Compared with commercially available detection reagents, the detection system and method of the present invention have higher clinical application value. Therefore, the detection system and method of the present invention can not only solve the lack of the prior art, but also have great industrial applicability.

The above embodiments are merely illustrative of the invention and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the claims of the present invention should be as set forth in the appended claims.

1. . . Detection Systems

11. . . Microfluidic component

11a. . . glass substrate

111. . . Entrance

113. . . Export

115. . . Microchannel structure

1151. . . First microchannel

1153. . . Second microchannel

13. . . Complex

15. . . Sensing piece

151. . . Bottom plate electrode

17. . . Positioning element

19. . . The protective layer

20. . . Reference electrode

1A to 1B are schematic views illustrating a detection system of a specific embodiment of the present invention;

2A and 2B are schematic views illustrating a detection system of another embodiment of the present invention;

Figure 3 is a view showing microscopic observation of microbeads obtained according to an embodiment of the present invention;

4 is a view showing a microbead particle size distribution of microbeads obtained according to an embodiment of the present invention;

Figure 5 is a graphical representation of a comparison with commercially available reagent test results in accordance with an embodiment of the present invention.

1. . . Detection Systems

11. . . Microfluidic component

11a. . . glass substrate

111. . . Entrance

113. . . Export

115. . . Microchannel structure

1151. . . First microchannel

1153. . . Second microchannel

15. . . Sensing piece

151. . . Bottom plate electrode

Claims (20)

A detection system comprising: a microfluidic element comprising an inlet, an outlet, and a microchannel structure, wherein the microchannel structure is in communication with the inlet and the outlet, the microchannel structure comprising a first microchannel and a second micro a flow path, the first micro flow channel is in communication with the second micro flow channel, and an inner diameter of the first micro flow channel is greater than an inner diameter of the second micro flow channel; and the composite includes a carrier and is used for The chemical substance is detected, wherein the carrier is combined with the chemical to form the composite, and the composite enters the microchannel structure to be placed in the first microchannel. The detection system of claim 1, wherein the composite cannot enter the second microchannel. The detection system of claim 1, wherein the composite passes through the inlet to enter the microchannel structure. The detection system of claim 1, wherein the microfluidic component comprises a sensing component, and the sensing component is adjacent to the microchannel structure. The detection system of claim 4, wherein the sensing component is an electrochemical sensing component. The detection system of claim 1, wherein the microfluidic element comprises a positioning element for positioning the composite entering the microchannel structure. The detection system of claim 6, wherein the positioning element is a magnetic substance. The detection system of claim 1, wherein the microfluidic component is a microfluidic wafer. The detection system of claim 1, wherein the carrier is a colloid, and the carrier coats the chemical to form a microbead. The detection system of claim 9, wherein the composite comprises a magnetic substance coated in the carrier. The detection system of claim 1, wherein the carrier is a magnetic substance, and the chemical is bonded to the surface of the carrier. The detection system of claim 9, wherein the manufacturing of the composite comprises the steps of: mixing the chemical and the material used to form the carrier to form a mixture; and emulsifying the mixture to form The carrier coats the chemical material into microbeads. The detection system of claim 12, wherein the manufacturing of the composite comprises the steps of: curing after emulsification of the mixture. The detection system of claim 1, wherein the chemical substance comprises a substance for reacting with the analyte in the sample. The detection system according to claim 14, wherein the substance for reacting with the analyte in the sample comprises a nucleic acid, a peptide, a protein, an enzyme, an antigen, an antibody, a ligand, a receptor, A cell, a virus, a bacterium, a derivative thereof, an analog thereof, or a mixture thereof. The detection system of claim 1, wherein the chemical is used to react with the analyte in the sample, and wherein the inspection system enters the microchannel structure to enter the The composite of the microchannel structure is in contact. A detection method using the detection system according to any one of claims 1 to 16 for detecting a test object in a sample, the detection method comprising: causing the composite to enter the microfluidic element Placed in the microchannel structure; and subject the sample into the microfluidic element to contact the composite. The method of detecting according to claim 17, further comprising removing the composite from the microfluidic element. The test method of claim 17, wherein the test object comprises a substance for reacting with the chemical substance. The method according to claim 19, wherein the substance for reacting with the chemical substance comprises glucose, amino acid, urea, uric acid, creatinine, nucleic acid, peptide, protein, enzyme, antigen , an antibody, a ligand, a receptor, a cell, a bacterium, a derivative thereof, an analog thereof, or a mixture thereof.