KR102571967B1 - Biosensor for Alkaline phosphatase measurement - Google Patents

Abstract

The present invention relates to a biosensor for measuring ALP, and it is possible to quantify ALP in a biological sample through electrochemical analysis, and thus has an effect of performing a liver function test simply and quickly. Since it is configured to electrically connect the biosensor and an external device through a connection terminal prepared in the form of USB or pin, it can be applied to various devices to reduce costs, and it can be easily used in real life, increasing convenience and product utilization. There are advantages to this increase.
In addition, since the biosensor of the present invention is composed of a connection module electrically connected to an external device and a sensing module detachable from the connection module, various types of inspections can be performed by replacing the sensing module, and the connection module is connected to an external device. There is an advantage in that only the sensing module can be replaced in the state of being coupled to, so that rapid inspection can be performed continuously.

Description

Biosensor for ALP measurement {Biosensor for Alkaline phosphatase measurement}

The present invention relates to a biosensor for measuring ALP.

Alkaline phosphatase (ALP) is an enzyme found in tissues of the whole body, such as liver, bone, kidney, intestine, and placenta of pregnant women. However, the concentration of alkaline phosphatase is highest in the cells that make up bones and liver. In the liver, it is found at the edges of the cells that form the bile ducts, which are small tubes that drain bile from the liver into the intestine to aid in the digestion of fats in the diet. Bone alkaline phosphatase is produced by specific cells called 'osteoblasts' involved in bone formation. Each different tissue produces a characteristic type of alkaline phosphatase called an isoenzyme.

Elevated levels of alkaline phosphatase in the blood are most commonly observed with liver disease or bone lesions. Increases in enzyme concentrations are most likely to occur when the bile ducts are obstructed. Liver cancer, cirrhosis, taking drugs with hepatotoxicity, and taking hepatitis can increase the concentration of enzymes in the blood, although not severely. Anything that promotes excessive bone formation, such as Paget's disease, rheumatoid arthritis, or during the healing of a fracture, can increase alkaline phosphatase levels. Accordingly, liver function and the like can be diagnosed by measuring the level of alkaline phosphatase.

On the other hand, target substances such as hormones, proteins, and pathogens, which enable the prediction of symptoms and progression of human diseases and furthermore the condition of food, can be analyzed by immunoassay using an antigen-antibody reaction. On the other hand, analysis of target substances based on immunoassays has usually been performed in clinical laboratories equipped with special equipment. However, as the need for self-diagnosis at home and examinations at hospitals or emergency rooms has recently increased, the development of an immunoassay platform that does not require specialized knowledge or complicated processes and has a short analysis time has been continuously required. .

Therefore, as a solution for this, it is necessary to develop a biosensor capable of rapid and accurate analysis of alkaline phosphatase by electrochemical analysis.

One aspect is an external device and electrically connectable connection module; A sensing module configured to detect an Alkaline phosphatase (ALP) material from the analysis sample introduced into the interior and to transmit an electrical signal generated in response to the detected ALP material to the connection module; the sensing module includes , A sensor for detecting the ALP material from the analysis sample and reacting with the ALP material to generate the electrical signal is provided, and the ALP material is quantitatively measured by the generated electrical signal, and the sensor, A substrate; and a plurality of electrodes provided on one surface of the substrate and reacting with the ALP material to generate an electrochemical signal, and the sensing module is detachable from the connection module to provide a biosensor.

One aspect is an external device and electrically connectable connection module; A sensing module configured to detect an Alkaline phosphatase (ALP) material from the analysis sample introduced into the interior and to transmit an electrical signal generated in response to the detected ALP material to the connection module; the sensing module includes , A sensor for detecting the ALP material from the analysis sample and reacting with the ALP material to generate the electrical signal is provided, and the ALP material is quantitatively measured by the generated electrical signal, and the sensor, A substrate; and a plurality of electrodes provided on one surface of the substrate and reacting with the ALP material to generate an electrochemical signal, wherein the sensing module is detachable from the connection module, and provides a biosensor.

In one embodiment, the sensor may include a nanowell, microwell, or milliwell structure consisting of a plurality of grooves on the electrode.

In one embodiment, the nanowell, microwell, or milliwell may have a diameter of 50 nm to 50 mm.

In one embodiment, the analysis sample may be a biological sample isolated from an individual.

In one embodiment, the electrode may be a working electrode, a counter electrode, or a reference electrode.

In one specific example, the connection module and the sensing module may be detachably coupled to each other through magnetism and electrically connected to each other through surface contact.

In one specific example, the sensing module may be one or more, and the one or more sensing modules may be detachable from one connection module.

In one embodiment, the quantitative measurement of the ALP material may be measured by any one of Equations 1 to 3 below:

[Equation 1]

y(uA) = -257.00 + 256.96*exp(-6304.58x(g/mL))

[Equation 2]

y(uA) = -1.35 + 1.15*exp(-1.74*107x(g/mL))

[Equation 3]

y(uA) = -2.76 + 1.63*exp(-3.53*107x(g/mL))

The present invention relates to a biosensor for measuring ALP, and it is possible to quantify ALP in a biological sample through electrochemical analysis, and thus has an effect of performing a liver function test simply and quickly. Since it is configured to electrically connect the biosensor and an external device through a connection terminal prepared in the form of USB or pin, it can be applied to various devices to reduce costs, and it can be easily used in real life, increasing convenience and product utilization. There are advantages to this increase.

In addition, since the biosensor of the present invention is composed of a connection module electrically connected to an external device and a sensing module detachable from the connection module, various types of inspections can be performed by replacing the sensing module, and the connection module is connected to an external device. There is an advantage in that only the sensing module can be replaced in the state of being coupled to, so that rapid inspection can be performed continuously.

1 is a use state diagram showing a state in which a biosensor according to an embodiment of the present invention is connected to an external device.
2 is a perspective view illustrating a biosensor in which a milliwell or microwell structure is formed according to an embodiment of the present invention.
3 is a plan view showing a disassembled biosensor according to an embodiment of the present invention.
Figure 4 is a VI-VI line cross-sectional view of Figure 4;
5 is a side view illustrating part “A” of FIG. 4 .
6 is a plan view schematically illustrating a disassembled state of a biosensor according to another embodiment of the present invention.
FIG. 7 is an enlarged view of part “B” of FIG. 6 .
8 is a plan view schematically illustrating a disassembled state of a biosensor according to another embodiment of the present invention.
9 is a diagram schematically illustrating a process in which part “C” of FIG. 8 is combined.
10 is a side view schematically showing a disassembled state of a biosensor according to another embodiment of the present invention.
11 is a graph showing current values measured for each concentration of ALP using a nanowell electrode.
12 is a graph showing current values measured for each concentration of ALP using a microwell electrode.
13 is a graph showing current values measured for each concentration of ALP using a milliwell electrode.
14 is a graph showing the current value measured for each ALP concentration converted into a ratio divided by the negative control of each electrode in order to compare the sensitivity of each electrode.
15 is a graph showing the relationship between the current value in the nanowell electrode and the ALP concentration.
16 is a graph in which the relationship between the current value in the microwell electrode and the ALP concentration was derived.
17 is a graph in which the relationship between the current value and the ALP concentration at the milliwell electrode was derived.

This specification clarifies the scope of the present invention, explains the principles of the present invention, and discloses embodiments so that those skilled in the art can practice the present invention. The disclosed embodiments may be implemented in various forms.

Expressions such as “include” or “may include” that can be used in various embodiments of the present invention indicate the existence of a function, operation, or component that has been disclosed, and may include one or more additional functions, operations, or components. components, etc. are not limited. In addition, in various embodiments of the present invention, terms such as "comprise" or "having" are intended to designate that there are features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, It should be understood that it does not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

Terms including ordinal numbers, such as first and second, may be used to describe various components, but these components are not limited by the above terms. The terminology described above is only used for the purpose of distinguishing one component from another.

When an element is referred to as being "connected or coupled" to another element, the element may be directly connected or coupled to the other element, but there is a gap between the element and the other element. It should be understood that other new components may exist in On the other hand, when an element is referred to as being “directly connected” or “directly coupled” to another element, it will be understood that no new element exists between the element and the other element. should be able to

Meanwhile, a “module” or “unit” for a component used in this specification performs at least one function or operation. Also, a “module” or “unit” may perform a function or operation by hardware, software, or a combination of hardware and software. In addition, a plurality of “modules” or “units” other than “modules” or “units” to be executed in specific hardware or to be executed in at least one processor may be integrated into at least one module. Singular expressions include plural expressions unless the context clearly dictates otherwise.

In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be abbreviated or omitted.

The present invention relates to a biosensor for ALP measurement, and relates to a biosensor in which a sensing part can be replaced by using a connection module electrically connectable to an external device and a sensing module detachable from the connection module. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Referring to FIG. 1 , a biosensor 10 according to an embodiment of the present invention uses a nanowell, microwell or milliwell structure 220 to improve resolution and detects electrical signals generated by the biosensor 10. It is configured to be electrically connected to an external device 20 in which measurement and analysis capable software is installed.

Specifically, the biosensor 10 is electrically connected to an external device 20 in the form of a smartphone or tablet as shown in FIG. 1 (a), or connected to an external device 20 in the form of a PC as shown in FIG. can be electrically connected. However, the external device 20 is not limited thereto, and may be changed and used in various forms electrically connectable to the biosensor 10 .

The external device 20 includes a terminal unit (not shown) that can be coupled to the first connection terminal 110 provided in the connection module 100 so as to be electrically connected to the connection module 100 of the biosensor 10 to be described later. may be provided. In addition, the external device 20, when connected to the biosensor 10, measures and analyzes the potential according to the oxidation-reduction reaction of the biosensor 10, thereby enabling qualitative and quantitative analysis of the ALP material; or Applications and the like may be installed. Through this, the external device 20 collects and analyzes signals generated by the biosensor 10, and through this, various diseases or disorders can be detected, or changes thereof can be measured and displayed.

In addition, the external device 20 may be connected to the Internet through wired or wireless communication and transmit data measured by the biosensor 10 to a user terminal or a server of a medical institution. Through the transmitted data, the health condition of the test subject or medical institution can be monitored in real time.

For example, exemplarily, the external device 20 includes an input unit (not shown) electrically connected to the biosensor 10 to which the biosensor 10 is coupled, a signal (data) transmitted through the input unit, A control unit (not shown) that analyzes and diagnoses, a display unit (not shown) that outputs the data received from the control unit in an externally identifiable form, and a communication unit (not shown) that transmits the data received from the control unit to a selected user terminal and medical institution server. ) and a power supply unit (not shown) supplying power to an input unit, a control unit, a display unit, and a communication unit.

A biosensor according to an embodiment of the present invention includes a connection module 100 , a sensing module 200 , and a sensor 210 . Hereinafter, preferred embodiments of the present invention will be described in detail.

Referring to FIGS. 2 and 3 , the connection module 100 is electrically connectable to the external device 20 . To this end, the connection module 100 includes a first connection terminal 110, a second connection terminal 120, a controller 130, and a body 140.

The first connection terminal 110 is coupled to the external device 20 and can be electrically connected to the external device 20 . The first connection terminal 110 may have various configurations as long as it can be electrically connected to the external device 20 .

The first connection terminal 110 is coupled to the terminal portion provided in the external device 20 to be electrically connected to the external device 20 in a form corresponding to the terminal portion of the external device 20. It can be done. Specifically, the first connection terminal 110 may be provided in the form of a USB, micro USB, or pin, but is not limited thereto, and various configurations may be provided if it can be electrically connected to the external device 20. can be used

The second connection terminal 120 is coupled to the sensing module 200 to be described later and can be electrically connected to the sensing module 200 . The second connection terminal 120 may be provided in the form of a micro USB or PIN, but is not limited thereto, and various configurations may be used as long as they can be electrically connected to the sensing module 200.

The controller 130 is electrically connected to the first connection terminal 110 and the second connection terminal 120, and between the first connection terminal 110 and the second connection terminal 120, a signal ( data) may be configured to control transmission. The controller 130 may be electrically connected to the first connection terminal 110 and the second connection terminal 120 through wires, but is not limited thereto, and the first connection terminal 110 and the second connection terminal 120 may be electrically connected. Various configurations may be used as long as the two connection terminals 120 can be electrically connected.

The main body 140 includes a lower body in which the first connection terminal 110, the second connection terminal 120, and the controller 130 are installed, and coupled to the lower body, the first connection terminal 110 , It may include an upper body that protects the upper portion of the second connection terminal 120 and the controller 130.

Referring to FIGS. 2 and 3 , the sensing module 200 detects an ALP material from an analysis sample introduced into the interior, and transmits an electrical signal generated by reacting with the detected ALP material to the connection module 100. configured to transmit, it is equipped with a nanowell, microwell or milliwell structure 220 .

The analysis sample may refer to a biological sample separated from the subject, and may include blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, intestinal fluid, genitourinary tract fluid , breast milk, lymphatic fluid, semen, cerebrospinal fluid, intratracheal fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof. However, the analysis sample is not limited thereto, and may be a variety of materials if it is a biological sample. In addition, the biological sample may include an intact protein. An intact protein may be a protein isolated from a biological sample without modification of the protein. An intact protein may be a protein that has been isolated from a biological sample, for example without proteolytic degradation by proteolytic enzymes.

The sensing module 200 may be provided with a sensor 210 that senses the ALP material from the analysis sample and reacts with the ALP material to generate the electrical signal. The sensor 210 may include a substrate 211, a plurality of electrodes 212 and a nanowell, microwell or milliwell structure 220.

Specifically, the sensor 210 may be provided with a sensing material that detects the ALP material by reacting with the ALP material included in the analysis sample. When the sensor 210 comes into contact with the analysis sample, it generates an electrical signal by interacting with the ALP material included in the analysis sample. The ALP material may be quantitatively measured by the electrical signal generated in this way. Quantitative measurement of the ALP material may be measured by any one of Equations 1 to 3 below.

[Equation 1]

y(uA) = -257.00 + 256.96*exp(-6304.58x(g/mL))

[Equation 2]

y(uA) = -1.35 + 1.15*exp(-1.74*107x(g/mL))

[Equation 3]

y(uA) = -2.76 + 1.63*exp(-3.53*107x(g/mL))

Accordingly, the external device 20 connected to the biosensor 10 analyzes the electrical signal generated by the biosensor 10 to detect the presence or concentration of the ALP material, and analyzes only the electrical signal. Since the presence or concentration of ALP can be known quickly, diseases such as liver function can be quickly and conveniently diagnosed. However, the sensor 210 is not limited thereto, and may be configured to move, stop, filter, purify, react, and mix the analysis sample.

The substrate 211 provided in the sensor 210 may have a predetermined size, and the substrate 211 may have a size of 2 x 2 mm. However, the size of the substrate 211 is not necessarily limited thereto and may be changed to various sizes.

In addition, the substrate 211 may be made of a flexible, bendable material. For example, the substrate 211 may be a polyurethane (Poly urethane, PU)-based, polydimethylsiloxane (PDMS)-based, NOA (Noland Optical Adhesive)-based, epoxy-based, polyethylene terephthalate (Polyethylene terephthalate, At least one of PET), polymethyl methacrylate (PMMA), polyimide (PI), polystyrene (PS), polyethylene naphthalate (PEN), and polycarbonate (PC) It can be made of the material of

However, the material of the substrate 211 is not necessarily limited thereto, and electrodes generating a potential difference according to reaction with a target material may be disposed, and various materials having flexibility may be changed and applied.

The electrode 212 may be provided in plurality, and the electrode 212 may be provided on one surface of the substrate 211 and react with a target material included in the analysis sample to generate an electrochemical signal. .

The electrode may be a working electrode, a counter electrode, or a reference electrode.

The plurality of electrodes 212 include a first electrode 212a for oxidation or reduction reaction with ALP material, a second electrode 212b for oxidation or reduction reaction with ALP material in opposition to the reaction of the first electrode 212a, and a third electrode 212c configured to maintain a constant operating voltage between the first electrode 212a and the second electrode 212b.

The plurality of electrodes 212 may be formed on the insulating substrate 211 through a screen printing method. The second electrode 212b and the third electrode 212c may be disposed to surround the periphery of the first electrode 212a.

The first electrode 212a and the second electrode 212b may be disposed on the substrate 211 while being spaced apart from each other by a predetermined distance. The first electrode 212a may have a circular shape, and the second electrode 212b may have a semicircular shape to surround a portion of the first electrode 212a. However, the arrangement of the first electrode 212a and the second electrode 212b and the shape of each of these electrodes are not limited thereto and may be changed as needed.

The first electrode 212a may mean a working electrode that performs an oxidation or reduction reaction by reacting with an ALP material or an analysis sample including the ALP material on the substrate 211 .

A bioreceptor (not shown) that specifically binds to an ALP material may be disposed on the first electrode 212a. Pillars on which a conductive layer is deposited are disposed on the first electrode 212a, and bioreceptors such as antibodies, antigens, and aptamers may be disposed on the pillars. A material reacting with the ALP material to induce an electrochemical signal may be further disposed on the first electrode 212a.

Through the pillars and bio-receptors disposed on the first electrode 212a and the second electrode 212b, the reaction area with the ALP material is widened, and through this, the biosensor 10 reacts to a small amount of ALP material. It can also provide highly sensitive qualitative and quantitative analysis results. Here, the pillars may be nano-sized polymer structures.

The pillar may be made of at least one of polyurethane, polydimethylsiloxane, NOA (Norland Optical Adhesives), epoxy, polyethylene terephthalate, polymethyl methacrylate, polyimide, polystyrene, polyethylene naphtharate, polycarbonate, and combinations thereof. there is.

In addition, the pillars may be made of a combination of polyurethane and NOA (eg, NOA 68). However, the pillar is not limited thereto, and may be made of more diverse polymers as long as it has flexibility.

And, the conductive layer deposited on the pillar may refer to a layer made of a conductive material. The conductive layer may be made of at least one of Ni, Zn, Pd, Ag, Cd, Pt, Ga, In, and Au, but is not limited thereto.

The second electrode 212b may mean a counter electrode facing the first electrode 212a on the substrate 211 . Accordingly, if an oxidation reaction occurs in the first electrode 212a due to a reaction with the ALP material, a reduction reaction may occur in the second electrode 212b. The above-described pillar on which a conductive layer is deposited may be disposed on the second electrode 212b.

The third electrode 212c may mean a reference electrode whose potential is stably maintained even when in contact with an ALP material. A reaction layer capable of maintaining a constant potential even when in contact with an ALP material may be provided on the third electrode 212c.

The reaction layer was Ag/AgCl, Ag, Hg2SO4, Ag/Ag+, Hg/Hg2SO4, RE-6H, Hg/HgO, Hg/Hg2Cl2, Ag/Ag2SO4, Cu/CuSO4, KCl saturated calomel half cell (SCE) and salt bridge. It can be made of a material whose potential difference between platinum is known in advance.

Referring to FIG. 2 , the nanowell, microwell, or milliwell structure 220 is provided on the electrode 212 and consists of a plurality of grooves. The nanowell, microwell or milliwell structure 220 may be provided in an inlet portion into which the analysis sample is inserted into the sensing module 200, and the nanowell, microwell or milliwell structure 220 It has the advantage of improving the resolution as it is used.

Specifically, the nanowell, microwell or milliwell structure 220 may be provided above a working electrode, and the nanowell, microwell or milliwell structure 220 is composed of a working electrode. It may be provided above the first electrode 212a.

In addition, the nanowell, microwell, or milliwell may have a diameter of 50 nm to 50 mm. Specifically, 50 nm to 30 mm, 50 nm to 10 mm, 50 nm to 900 μm, 50 nm to 600 μm, 50 nm to 300 μm, 50 nm to 100 μm, 100 nm to 50 mm, 100 nm to 30 mm , 100 nm to 10 mm, 100 nm to 900 μm, 100 nm to 600 μm, 100 nm to 300 μm, 300 nm to 50 mm, 300 nm to 30 mm, 300 nm to 10 mm, 300 nm to 900 μm, 300 nm to 600 μm, 500 nm to 50 mm, 500 nm to 30 mm, 500 nm to 10 mm, 500 nm to 900 μm, 700 nm to 50 mm, 700 nm to 30 mm 700 nm to 10 mm 700 nm to 900 μm can be

The sensing module 200 according to an embodiment of the present invention may further include a connection pad 230, a connection port 240, a housing 250, and a bonding wire 260.

The connection pad 230 may be provided in single or plural numbers, and may be electrically connected to the sensor 210 through the bonding wire 260 . 3 and 5, on the lower surface of the sensor 210, a plurality of connection pads 230 and a plurality of electrodes 212 provided in the sensor 210 are electrically connected to each other. ) may be further provided.

One surface of the connection pad 213 attached to the bottom surface of the sensor 210 is electrically connected to a plurality of electrodes 212 provided in the sensor 210, and the other surface of the connection pad 213 is It may be electrically connected to the plurality of connection pads 230 through the bonding wire 260 . Specifically, the plurality of connection pads 230 and the connection pad 213 are provided in the form of copper foil, and gold foil may be further coated on surfaces of the plurality of connection pads 230 .

In addition, the connection pad 213 and the sensor 210 may be electrically connected to each other through a conductive material (not shown), and a double-sided adhesive carbon tape or the like may be used as the conductive material. However, the conductive material is not limited thereto, and various materials may be used as long as the connection pad 213 and the sensor 210 can be electrically connected.

The bonding wire 260 is made of gold (Au), and one end of the bonding wire 260 is bonded to the connection pad 213 attached to the sensor 210 to form a plurality of pluralities provided in the sensor 210. It may be electrically connected to the electrode 212 of the dog, and the other end of the bonding wire 260 may be bonded to the plurality of connection pads 230 to be electrically connected to the plurality of connection pads 230 .

2 and 3, the connection port 240 may be electrically connected to a plurality of connection pads 230 and coupled to the connection module 100 to be electrically connected to the connection module 100. There is.

The connection port 240 may be electrically connected to the plurality of connection pads 230 through wires 261, and when coupled to the connection module 100, the sensor ( 210) may be supplied with power, and electrical signals generated by the sensor 210 may be transmitted to the connection module 100. The connection port 240 may be provided in a form corresponding to the second connection terminal 120 provided in the connection module 100 .

The housing 250 is installed with the sensor 210, a plurality of the connection pads 230, and the connection port 240, and the housing 250 includes the sensor 210, a plurality of the connection pads ( 230), the circumference of the connection port 240 may be wrapped to protect the sensor 210, the plurality of connection pads 230, and the connection port 240 from the outside.

The housing 250 faces a first member 251 provided with a channel 253 for guiding an analysis sample provided from the outside to the electrode 212 of the sensor 210 and the first member 251. and is coupled to the first member 251, and includes a second member 252 on which the sensor 210, the plurality of connection pads 230, and the connection port 240 are seated.

The channel 253 is a hole formed through the first member 251, and the channel 253 is an inclined surface 254 for guiding an analysis sample provided from the outside to the electrode 212 of the sensor 210. ). The inclined surface 254 may be provided along the circumference of the channel 253, and the analysis sample provided from the outside moves along the inclined surface 254 to stably reach the electrode 212 of the sensor 210. be able to reach

Here, referring to FIG. 4 , the nanowell, microwell, or milliwell structure 220 may be provided in the channel 253 . The nanowell, microwell or milliwell structure 220 may be provided on top of the first electrode 212a-working electrode, and the channel 253 is the first electrode 212a -Can be provided on top of the working electrode.

Therefore, the nanowell, microwell or milliwell structure 220 is preferably provided in the channel 253, while the nanowell, microwell or milliwell structure 220 is coupled to the channel 253 can be supported. As the nanowell, microwell or milliwell structure 220 is provided in the channel 253, the resolution can be improved when the analyzed sample is moved to the electrode 212 through the channel 253 do.

The first member 251 is coupled to the second member 252 and covers the sensor 210 and the connection port 240 disposed on the second member 252 to protect them from the outside. . In addition, the first member 251 may firmly support the sensor 210 and the connection port 240 by pressing them with a predetermined force. In addition, the first member 251 prevents the analysis sample provided to the sensor 210 through the channel 253 from leaking to the outside of the housing 250 through a combination with the second member 252. can do.

3 and 5, between the first member 251 and the second member 252, the sensor 210, the plurality of connection pads 230, and the connection port 240 are accommodated. And, an accommodation space 250a protecting the bonding wire 260 may be provided. The accommodating space 250a may be provided in an intaglio pattern on the inside of the first member 251 and the second member 252, respectively.

Referring to FIG. 4, the second member 252 is coupled to the first member 251, and at least one coupling protrusion 252a supporting the first member 251 is provided. One member 251 may have at least one coupling groove 251a corresponding to the coupling protrusion 252a.

When the coupling protrusion 252a and the coupling groove 251a are coupled, the center of the channel 253 and the center of the sensor 210 can be naturally aligned at a predetermined position. Inside the coupling groove 251a, a sealer made of rubber or silicon that seals between the coupling protrusion 252a and the coupling groove 251a when the coupling protrusion 252a is inserted into the coupling groove 251a. may be further provided.

Referring to FIG. 4 , the second member 252 includes a seating groove 252b in which the sensor 210 is seated and supported, and a collecting groove 252c in which analysis samples flowing out of the sensor 210 are stored. This may be further provided. A sample guide surface 252d may be further provided between the sensor 210 and the collection groove 252c to guide the analysis sample flowing out of the sensor 210 toward the collection groove 252c. (252d) may be made in an inclined shape.

Referring to FIGS. 2 and 3 , the housing 250 is a flow state of the analysis sample, whether or not there is leakage of the analysis sample, the alignment state of the channel 253 and the sensor 210, and the bonding wire 260. A transparent material is provided so that the connection state of the sensor 210 and the plurality of connection pads 230 and the electrical connection state between the plurality of connection pads 230 and the connection port 240 can be easily observed from the outside. It can be.

For example, the housing 250 is made of at least one of polymethyl methacrylate, polycarbonate, cyclic olefin copolymer, polyethylene sulfone and polystyrene, or at least two or more of them. This combination of materials can be provided. However, the material of the housing 250 is not necessarily limited thereto, and may be made of polydimethylsiloxane, which is a silicon-based organic polymer.

A biosensor using a nanowell, microwell or milliwell structure according to an embodiment of the present invention may be modified and used as follows.

Referring to FIG. 6 , a plurality of sensing modules 200 of the biosensor according to an embodiment of the present invention may be provided, and the plurality of sensing modules 200 are detachably coupled to the connection module 100. It can be. That is, the connection module 100 may be electrically connected to the plurality of sensing modules 200 .

To this end, the connection module 100 includes the first connection terminal 110 coupleable to the external device 20 and the second connection terminal 120 coupleable to the sensing module 200, and A plurality of two connection terminals 120 may be provided. The plurality of sensing modules 200 may be selectively coupled to the plurality of second connection terminals 120 .

In this way, when a plurality of the sensing modules 200 are provided and a plurality of second connection terminals 120 are provided, there is an advantage in that a plurality of tests can be performed at once by detecting ALP materials of different objects. .

Here, when the plurality of sensing modules 200 are all connected to the plurality of second connection terminals 120 to generate electrical signals, the controller 130 operates through the plurality of second connection terminals 120. After storing the order in which electrical signals are transmitted, electrical signals of the sensing module 200 connected to the corresponding second connection terminals 120 may be sequentially received and transmitted to the first connection terminals 110 .

The connection module 100 is a power supply unit 150 that controls the connection between the plurality of second connection terminals 120 and the controller 130 between the plurality of second connection terminals 120 and the controller 130. may further include.

A plurality of power supply units 150 may be provided, and may be individually connected to a plurality of second connection terminals 120 . In addition, the power supply unit 150 may be connected to each of the controllers 130 .

The power supply unit 150 may be configured to be ON/OFF controlled through a user's operation, and only when the power supply unit 150 is controlled to be in an ON state through a user's operation, the second connection terminal 120 Electrical signals of the connected sensing module 200 may be transmitted to the controller 130 .

6 and 7, the connection module 100 is installed around the first connection terminal 110 and the angle adjusting unit for allowing the first connection terminal 110 to rotate by a preset angle ( 160) may be further included.

The angle adjuster 160 is coupled to the first connection terminal 110 and is installed on the rotating member 161 configured to rotate together with the first connection terminal 110 and the main body 140, and is installed on the inside. The rotating member 161 may include a guide member 162 for guiding the rotation of the rotating member 161 is accommodated.

A plurality of protrusions 161a spaced apart from each other are provided on the outer circumferential surface of the rotating member 161, and a plurality of grooves 162a corresponding to the plurality of protrusions 161a are provided on the inner circumferential surface of the guide member 162. can Therefore, the user can arrange the plurality of second connection terminals 120 in predetermined positions by rotating the main body 140 in a state where the first connection terminals 110 are coupled to the external device 20. there is.

Referring to FIG. 8 , according to an embodiment of the present invention, the connection module 100 and the sensing module 200 may be detachably coupled to each other through magnetism, and may be electrically connected to each other through surface contact. .

To this end, the connection module 100 may include a first coupling member 170, and the sensing module 200 may include a second coupling member 270 coupled to the first coupling member 170. .

Referring to FIG. 9 , the first coupling member 170 includes a coupling protrusion 171 protruding from one end of the connection module 100 and a first magnetic body 172 accommodated at an end of the coupling protrusion 171. can include

The coupling protrusion 171 may be formed in a wedge structure in which an outer diameter gradually decreases along a protruding direction. Through this, when the connection module 100 and the sensing module 200 are coupled, the inclined outer peripheral surface of the coupling protrusion 171 is the coupling groove of the second coupling member 270 provided in the sensing module 200. It can be easily coupled by being guided to the inner circumferential surface of (271).

The second coupling member 270 is the coupling groove 271 concavely formed at the other end of the sensing module 200 facing one end of the connection module 100 and the inside of the coupling groove 271. It may include a second magnetic body 272 accommodated in and capable of being coupled with the first magnetic body 172 through magnetic force.

The connection module 100 and the sensing module 200 are primarily coupled through structural coupling of the coupling protrusion 171 and the coupling groove 271, and the first magnetic body 172 and the second As they are secondarily coupled through the coupling using the magnetism of the magnetic body 272, they can be stably fixed to each other.

Referring to FIG. 8 , the sensing module 200 shields the magnetic force of the second magnetic body 272 so that the magnetic force of the second magnetic body 272 does not affect other parts of the sensing module 200 . A shielding structure 280 configured to do so may be provided.

The shielding structure 280 is provided inside the housing 250 and may be provided in a form surrounding the coupling groove 271 of the second coupling member 270 and the circumference of the second magnetic body 272. there is. The shielding structure 280 may be formed of the same material as the housing 250 and may be made of a material capable of shielding magnetic force.

According to an embodiment of the present invention, the connection module 100 and the sensing module 200 may be electrically connected to each other through surface contact coupling. Referring to FIG. 8 , the second connection terminals 120 of the connection module 100 connected to the connection port 240 of the sensing module 200 are plate-shaped electrically connectable to each other through surface contact. It may be provided in the form of a terminal.

When the first coupling member 170 of the connection module 100 and the second coupling member 270 of the sensing module 200 are coupled to each other, the connection port 240 and the second connection terminal 120 ) can be electrically connected to each other only by being in surface contact.

Through this, the coupling and separation of the connection module 100 and the sensing module 200 become easier, and it is possible to prevent the connection port 240 and the second connection terminal 120 from being damaged even in a detachable type. there will be

An identification film capable of confirming use of the sensing module 200 through damage may be attached to an end of the sensing module 200 according to an embodiment of the present invention. An accommodation groove (not shown) capable of engaging with a wedge-shaped pressing protrusion (not shown) provided at an end of the connection module 100 is provided at an end of the sensing module 200, and an entrance of the receiving groove is provided with the sensing module ( 200) may be attached to the identification film in the form of a thin film configured to block the entrance of the receiving groove.

The identification film may be configured to be damaged by being pressed with a predetermined force by a pressing protrusion of the connection module 100 inserted into the receiving groove when the sensing module 200 and the connection module 100 are coupled. Therefore, the user can determine whether the sensing module 200 is used by checking whether or not the identification film provided at the end of the sensing module 200 is damaged. However, the configuration capable of confirming whether the sensing module 200 is used is not necessarily limited thereto, and may be changed and applied in various forms.

Referring to FIG. 10 , the sensing module 200 according to an embodiment of the present invention may include a plurality of sensing units configured to detect a target material from an analysis sample. The plurality of sensing units are electrically connected to the connection port 240 and may be installed inside the housing 250 .

Each of the plurality of sensing units includes the sensor 210, and the plurality of sensing units are separated from each other through the partition wall 214 and through the connection port 240 electrically coupled to the connection module 100. They can be electrically connected to each other.

The plurality of sensing units may include a first sensing unit 210a and a second sensing unit 210b. The first sensing unit 210a and the second sensing unit 210b may be disposed in a space independent of each other through the partition wall 214 provided in the housing 250 .

The first sensing unit 210a and the second sensing unit 210b detect a target material from a contacted analysis sample, and react with the target material to generate an electrical signal. The sensor 210 and the bonding wire The connection pad 230 electrically connected to the sensor 210 through 260 and electrically connected to the connection port 240 through the wire 261 may be included.

In addition, the first sensing unit 210a and the second sensing unit 210b may each have the channel 253, and each of the channels 253 has the nanowell, microwell or milliwell structure. (220) may be provided.

Further, in each of the first sensing unit 210a and the second sensing unit 210b, the connection pad 230 and the connection port 240 are connected between the connection pad 230 and the connection port 240. A power supply unit 290 controlling the connection may be further provided.

The power supply unit 290 may be electrically connected to the connection pad 230 and the connection port 240, and may be configured to be ON/OFF controlled through a user's manipulation. Therefore, only when the power supply unit 290 is controlled to be in an ON state through a user's manipulation, the electrical signal of the sensor 210 connected to the connection pad 230 can be transmitted to the connection port 240.

A biosensor using a nanowell, microwell or milliwell structure according to an embodiment of the present invention has been described as including the first sensing unit 210a and the second sensing unit 210b, but is not limited thereto No, two or more sensing units may be provided.

A biosensor using a nanowell, microwell or milliwell structure according to an embodiment of the present invention described above has the following effects.

Since the biosensor 10 using a nanowell, microwell or milliwell structure according to an embodiment of the present invention is configured to be electrically connected to the external device 20 through a connection terminal provided in the form of a USB or pin, various devices Since it can be applied to, cost can be reduced, and it can be easily used in real life, so convenience and usability of the product can be increased.

In addition, the biosensor 10 using a nanowell, microwell or milliwell structure according to an embodiment of the present invention includes a connection module 100 electrically connected to an external device 20 and a connection module 100. Since it consists of a detachable sensing module 200, it is possible to perform various types of inspections through replacement of the sensing module 200, and the sensing module 200 in a state where the connection module 100 is coupled to the external device 20 It is replaceable only so that continuous and rapid inspection can be performed.

In addition, in the biosensor 10 using a nanowell, microwell or milliwell structure according to an embodiment of the present invention, the sensing module 200 and the external device 20 are electrically connected to each other via the connection module 100. Since it is configured to be connected, even when the biosensor 10 is coupled to or separated from the external device 20, the sensing part is prevented from being damaged by the external device 20.

In addition, by improving the shape of the electrodes provided on the substrate 211 applied to the biosensor 10 using a nanowell, microwell or milliwell structure according to an embodiment of the present invention, a larger number of sensors than the prior art 210 can be manufactured, and manufacturing costs can be reduced.

Specifically, by improving the shape of the electrode provided on the substrate 211, it is possible to manufacture the sensor 210 in a size of 2 x 2 mm. Therefore, the number of sensors 210 that can be manufactured using an existing 8-inch wafer has greatly increased from as little as 1000 to as much as 20,000. Other parts except for the sensor 210 may further reduce manufacturing costs by using a PCB process having a low manufacturing cost.

In addition, the biosensor 10 using a milliwell or microwell structure according to an embodiment of the present invention can improve resolution by inserting an analysis sample through the nanowell, microwell or milliwell structure 220. There are advantages to being

Example 1. Quantitative analysis of alkaline phosphatase (ALP) using nanowell, microwell, and milliwell electrodes

The effectiveness of ALP quantitative analysis using nanowell, microwell, and milliwell electrodes was confirmed.

Specifically, first, nanowell NWA_U101 electrodes (manufactured by Mara Nanotech Co., Ltd.), microwell MWA_U201 (manufactured by Mara Nanotech Co., Ltd.), and Milli_U301 (manufactured by Mara Nanotech Co., Ltd.) were used as electrodes. ALP standard solution was prepared as shown in Table 1, and the cut-off value of ALP was formed at 1 to 3 ng/ml, and the concentrations of the positive and negative controls in this experiment were prepared as shown in Table 2. A negative control group did not contain ALP. ALP was purchased from Rand system, and sigmafast p-nitrophenyl phosphate, a substrate reaction solution, was purchased from sigma. ALP was measured using a 1030C Multi-potentiostat (purchased from CH instrument), and the specific conditions are shown in Table 3.

standard sample 30ul per test SIGMAFAST 90ul ALP 10ul Total 100ul

division density volume Additive Substrate Solution positive control 3 ng/ml 10ul sigmafast tablet phosphate (90ul) 10 ng/mL 30 ng/mL 100 ng/mL 200 ng/ml negative control 0

equipment parameters Detail CH1030C measurement method SWV (SQUARE WAVE VOLTAMMETRY) starting voltage -0.3V final voltage 0.7V step voltage 0.005V size 0.025V frequency 10Hz set sensitivity 1uA

Samples treated by concentration as described above were dropped on the prepared electrode, and after 10 minutes, electrical signals were measured under the conditions shown in Table 3. All measurements were repeated three times. Based on the value of the measured current, it was shown as an electrochemical measurement curve graph. As a result, current values measured for each concentration of ALP using nano-well or micro-well electrodes were shown in FIGS. 11 and 12 . As the ALP concentration increased, the current value between 0.1 and 0.5 V increased. Among them, the current value increased to the maximum at 0.4 V, so it was easy to detect the ALP concentration, and the measured value for each concentration was recorded at 0.4 V. Current values measured for each concentration of ALP using a milliwell electrode are shown in FIG. 13 . As the ALP concentration increased, the current value between 0.1 and 0.7 V increased, and the maximum current value increased at 0.5 to 0.6 V, so it was easy to detect the ALP concentration, and the measured value for each concentration was recorded at 0.5 V.

11 is a graph showing current values measured for each concentration of ALP using a nanowell electrode.

12 is a graph showing current values measured for each concentration of ALP using a microwell electrode.

13 is a graph showing current values measured for each concentration of ALP using a milliwell electrode.

In order to confirm the sensitivity of each electrode, the current value measured for each ALP concentration was converted into a ratio divided by the negative control of each electrode and shown in a graph as shown in FIG. 14. As a result, the microwell and nanowell electrodes showed higher current/negative control values than the milliwell electrode, and the nanowell electrode showed higher values than the microwell electrode at high ALP concentration. These results mean that when using a microwell electrode or nanowell electrode, ALP analysis with higher sensitivity than a milliwell electrode is possible.

14 is a graph showing the current value measured for each ALP concentration converted into a ratio divided by the negative control of each electrode in order to compare the sensitivity of each electrode.

Using the above results, the relationship between current and ALP concentration was derived as a function for each electrode as shown in FIGS. 15 to 17. The function of the current value and ALP concentration at the nanowell electrode can be expressed as y (uA) = -257.00 + 256.96 * exp (-6304.58x (g / mL)), and the current value and ALP concentration at the microwell electrode The function for can be expressed as y(uA) = -1.35 + 1.15*exp(-1.74*10 ⁷ x(g/mL)), and the function for the current value and ALP concentration at the milliwell electrode is y(uA ) = -2.76 + 1.63*exp(-3.53*10 ⁷ x (g/mL)). The ALP concentration can be quantified according to the current value using such a function for each electrode.

15 is a graph showing the relationship between the current value in the nanowell electrode and the ALP concentration.

16 is a graph in which the relationship between the current value in the microwell electrode and the ALP concentration was derived.

17 is a graph in which the relationship between the current value and the ALP concentration at the milliwell electrode was derived.

In this way, the present invention has been described with reference to the embodiments shown in the drawings, but this is only exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. . Therefore, the true technical scope of protection of the present invention should be determined by the technical spirit of the appended claims.

10 ... Biosensor 20 ... External device
100 ... connection module 110 ... first connection terminal
120 ... second connection terminal 130 ... controller
140 ... main body 150 ... power supply unit
160 ... angle adjustment unit 161 ... rotating unit
162 ... guide member 170 ... first coupling member
171 ... coupling protrusion 172 ... first magnetic body
200 ... sensing module 210 ... sensor
211 ... substrate 212 ... electrode
212a ... first electrode 212b ... second electrode
212c ... third electrode 213 ... connection pad
214... bulkhead 220... milliwell or microwell structure
230 ... connection pad 240 ... connection port
250 ... housing 250a ... accommodating space
251 ... first member 251a ... coupling groove
252 ... second member 252a ... coupling protrusion
252b ... seating groove 252c ... collection groove
252d ... sample guide surface 253 ... channel
254 ... slope 260 ... bonding wire
261 ... wire 270 ... second coupling member
271 ... coupling groove 272 ... second magnetic body
280... Shielding structure 290... Power unit

Claims (9)

Connection module electrically connectable with external devices;
A sensing module configured to detect an alkaline phosphatase (ALP) material from the analysis sample introduced into the interior and to transmit an electrical signal generated in response to the detected ALP material to the connection module; includes,
The sensing module is provided with a sensor that detects the ALP material from the analysis sample and reacts with the ALP material to generate the electrical signal, and the ALP material is quantitatively measured by the generated electrical signal,
The sensor,
A substrate; and a plurality of electrodes provided on one surface of the substrate and reacting with the ALP material to generate an electrochemical signal,
The electrode is a working electrode, a counter electrode or a reference electrode,
A pillar on which a conductive layer is deposited is disposed on the working electrode,
The sensing module is detachable from the connection module, a connection pad electrically connected to the sensor through a bonding wire, a connection port electrically connected to the connection pad, and connectable to the connection module, the sensor, and the connection pad And further comprising a housing in which the connection port is installed, the biosensor for ALP measurement. The biosensor for measuring ALP according to claim 1, wherein the sensor comprises a nanowell, microwell or milliwell structure consisting of a plurality of grooves on the electrode. The biosensor of claim 2, wherein the nanowell, microwell or milliwell has a diameter of 50 nm to 50 mm. The biosensor for measuring ALP according to claim 1, wherein the analysis sample is a biological sample separated from the subject. delete The biosensor for measuring ALP according to claim 1, wherein the connection module and the sensing module are detachably coupled to each other through magnetism and electrically connected to each other through surface contact. The biosensor for measuring ALP according to claim 1, wherein there is one or more sensing modules, and one or more sensing modules are detachable from one connection module. The method according to claim 1, wherein the sensing module is provided with a plurality of sensing units including the sensor, and the plurality of sensing units are separated from each other through a barrier rib, but are electrically connected to each other through a connection port that can be electrically coupled to the connection module Characterized in that, a biosensor for measuring ALP. The biosensor for measuring ALP according to claim 1, wherein the quantitative measurement of the ALP material is measured by any one of Equations 1 to 3 below.
[Equation 1]
y(uA) = -257.00 + 256.96*exp(-6304.58x(g/mL))
[Equation 2]
y(uA) = -1.35 + 1.15*exp(-1.74*107x(g/mL))
[Equation 3]
y(uA) = -2.76 + 1.63*exp(-3.53*107x(g/mL))