KR20190000851A - Lap on a chip, method for manufacturing the same and method for testing using the same - Google Patents

Abstract

Provided is a lab-on-a-chip comprising: a platform having a channel through which a fluid is delivered; a supplying unit for supplying a sample to the channel of the platform; at least one analyzing unit disposed along the channel to detect a biomarker contained in the sample; a reagent receiving unit connected to the channel for receiving a reagent to be supplied to the analyzing unit; and a driving unit for transferring the reagent and the sample to the analyzing unit along the channel.

Description

TECHNICAL FIELD [0001] The present invention relates to a lab-on-a-chip, a lab-on-a-chip, and a diagnostic method using a lab-

A lab-on-a-chip for detecting various biomarkers contained in a biological sample, a manufacturing method thereof, and a diagnostic method using a lab-on-a-chip.

For example, in the case of disease diagnosis using enzyme immunoassay (ELISA), a lot of time and expensive equipment were used and skilled engineers were needed. Therefore, the diagnosis of the disease can be made only in the laboratory.

Recently, the concept of Point Of Care Testing (POCT) has been demanded for diagnosing a patient's condition in a short time. Accordingly, researches on the development of a lab-on-a-chip based disease diagnosis chip capable of microfluidic control have been conducted. These diagnostic chips use blood, urine, etc. as representative samples to detect and quantify biomarkers for specific diseases.

However, conventional diagnostic chips have a difficulty in quantitatively analyzing biomarkers when using a small amount of samples, and only qualitative results can be obtained.

In order to detect two or more biomarkers, antibody particles for each biomarker must be immobilized in the chip. However, conventional chips can not fix a plurality of antibody particles properly in a chip, and it is difficult to obtain a desired diagnostic effect.

Provided is a lab-on-a-chip capable of quickly and accurately detecting a plurality of biomarkers through a small sample, a method of manufacturing the same, and a diagnostic method using the lab-on-a-chip.

The present invention provides a lab-on-a-chip, a method for manufacturing the lab-on-a-chip, and a diagnostic method using the lab-on-a-chip.

Provided is a lab-on-a-chip, a method of manufacturing the same, and a diagnostic method using the lab-on-a-chip, which can accurately diagnose even a small amount of samples by increasing the reactivity of antibody particles and increase the reliability of analysis results.

The lab-on-a-chip of this embodiment includes a platform having a channel through which a fluid is delivered, a supply for supplying a sample to a channel of the platform, at least one analyzer disposed along the channel for detecting a biomarker contained in the sample, A reagent accommodating unit connected to the channel to accommodate the reagent to be supplied to the analyzing unit, and a driving unit for transferring the reagent and the sample to the analyzing unit along the channel.

And a waste part formed on the platform and connected to the analysis part out side to receive the sample passed through the analysis part.

The supply unit may further include a filter for filtering and supplying the sample.

The supply unit may further include a micro flow path formed on a filter outlet side of the platform and connected to the channel for transferring the sample through the filter.

The supply unit may be separately provided from the platform and may be detachably coupled to the groove formed in the platform.

Wherein the supply unit includes a substrate having a fine flow path formed on its surface at an interval and an outlet through which a sample is discharged by being communicated with a fine flow path at a tip thereof, a filter installed in contact with the substrate surface to filter a sample, And a cover plate which is attached to the surface and has a hole for sample supply formed at a position corresponding to the filter.

The microfluidic channel may be a capillary tube through which a sample is transferred due to a capillary phenomenon.

The hydrophilic coating film may be formed on the surface of the micro channel.

The micro channel may have a structure in which a buffer solution for reducing the surface tension between the filter outlet side and the micro channel is accommodated as a wetting agent.

And a wax layer disposed on the filter outlet side or the micro flow passage to selectively block the buffer solution and the filter outlet side.

The substrate and / or the cover plate may be formed of an elastic material or an elastomeric material including an elastomer.

The analysis unit may have a structure in which a plurality of analyzers are sequentially arranged along a channel and connected in series.

The analysis unit may include an analysis line through which the fluid flows, a plurality of fine grooves arranged on the bottom surface of the analysis line, and antibody particles accommodated in the fine grooves to detect a biomarker of the sample.

The fine grooves may have a size corresponding to the size of the antibody particles, and one antibody particle may be inserted and fixed in each of the fine grooves.

The depth of the fine groove may be d < h < 2d. Where h = the depth of the fine groove, and d = the diameter of the antibody particle.

The fine grooves may be formed in a direction perpendicular to the fluid transport direction at the bottom of the analysis line.

Each of the analysis units may have a structure in which different antibody particles are accommodated.

The antibody particle may be a structure in which a capture antibody is attached to the surface of beads having magnetic properties.

The reagent receiving portion may include an accepting line connected to the channel and containing a reagent, a plurality of reagents sequentially accommodated in the accepting line, and an intermediate layer formed between the reagents to prevent interference between the reagents.

The driving portion may include a driving chamber connected to the receiving line of the reagent receiving portion, and a thermal expansion material accommodated in the driving chamber and expanding by heat.

The thermal expansion material may be a gas or a liquid containing air.

The driving unit may further include a heating unit installed on the platform for applying heat to the driving chamber.

The driving chamber may be formed in a zigzag shape extending in one line and spaced apart in the setting area or in a wound shape toward the center.

The method of manufacturing a lab-on-a-chip according to this embodiment includes a step of fixing an antibody particle having a capture antibody attached to a bead surface having magnetic properties to an analysis unit, wherein the fixing process is performed on the bottom surface of the analysis line Preparing a lab-on-a-chip having an analysis unit in which a plurality of fine grooves are arrayed, and inserting and fixing the antibody particles in the respective fine grooves.

The step of inserting and immobilizing the antibody particles in the respective fine grooves may include the steps of accommodating the antibody particles in the analysis line, applying magnetic force to the antibody particles outside the bottom surface of the analysis line, and moving the magnetic force along the analysis line, And inserting the antibody particles into the fine grooves on the bottom of the analysis line while moving the antibody particles.

And a washing step of transferring the fluid to the analysis line to remove the antibody particles immobilized in the fine grooves and removing the remaining antibody particles from the analysis line.

The diagnostic method of this embodiment includes a sample supply process for preparing a lab-on-a-chip and supplying a sample to a channel formed on a platform of the lab-on-a-chip, a movement process for moving the sample to an analysis unit provided in the platform, And detecting the biomarker of the sample with the antibody particles contained in the fine grooves arranged along the analysis part and labeling them with a reagent.

The sample supply step may include filtering the sample with a filter.

The sample supply step may further include wetting the filter outlet with the buffer solution before filtering the filter.

The moving process may include pressing the sample by pressing the channel to move the sample.

The movement process may include a step of applying heat to the driving chamber of the platform on which the reagent is received.

According to this embodiment, a plurality of biomarkers can be detected more quickly and accurately using a small amount of sample.

The plurality of antibody particles are uniformly and stably fixed to the respective analysis units, so that it is possible to perform quantitative diagnosis while using fewer samples, and to improve the reliability of the analysis results.

Fixing to the antibody particles is made easier and easier, so that the production is simple and the productivity is increased, and the production cost can be lowered.

The plasma can be separated from the whole blood and diagnosed immediately, so that the diagnosis procedure can be simplified, and the time and cost required for the diagnosis can be minimized.

1 is a schematic view showing a configuration of a lab-on-a-chip according to the present embodiment.
FIGS. 2 to 4 are views showing the structure of a supply portion of the lab-on-a-chip according to the present embodiment.
5 and 6 are views showing the structure of a driving unit of the lab-on-a-chip according to the present embodiment.
7 is a diagram showing an analysis unit structure of the lab-on-a-chip according to the present embodiment.
8 is a schematic diagram of a lab-on-a-chip integrated with a supply unit and an analysis unit according to the present embodiment.
FIG. 9 is a schematic view showing a process of arranging antibody particles in the analysis unit of the lab-on-a-chip according to the present embodiment.
10 is a photograph showing a state in which antibody particles are arrayed and fixed in the analyzer according to the present embodiment.
11 (a) and 11 (b) illustrate a manufacturing process of a lab-on-a-chip integrated with a supply part and an analysis part according to the present embodiment, (B) shows a state in which a substrate, a cover plate, and a filter are adhered to each other, and (c) shows a photograph when dye is flowed in a lab-on-a-chip.
12 is a photograph showing an experimental result of the lab-on-a-chip according to the present embodiment.
13 is a human total PSA ELISA standard curve obtained by quantitatively analyzing the amount of human total PSA using a conventional well plate method as reference data.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Wherever possible, the same or similar parts are denoted using the same reference numerals in the drawings.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms as used herein include plural forms as long as the phrases do not expressly express the opposite meaning thereto. Means that a particular feature, region, integer, step, operation, element and / or component is specified, and that other specific features, regions, integers, steps, operations, elements, components, and / And the like.

All terms including technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Predefined terms are further interpreted as having a meaning consistent with the relevant technical literature and the present disclosure, and are not to be construed as ideal or very formal meanings unless defined otherwise.

Hereinafter, the lab-on-a-chip of the present embodiment will be described by taking as an example the case of using plasma separated from whole blood as a sample for diagnosing a vaginal flap. Various samples such as a lymph fluid, a tissue fluid, a biological fluid including urine, or a fluid including cells such as somatic cells, bacteria, and viruses may be applied in addition to plasma separated from whole blood as a sample.

Fig. 1 schematically shows a configuration of a lab-on-a-chip according to the present embodiment.

1, the lab-on-a-chip 100 of the present embodiment is provided with a supply unit 20, an analysis unit 50 for detecting a biomarker, a reagent The housing portion 30 and the driving portion 40 are arranged in an array. The platform 10 may further include a discarding unit 70 connected to the analyzer 50 through the analyzer 50 to receive the discarded sample.

The platform 10 may be a plate structure. The shape of the platform 10 may be variously modified and is not particularly limited. The platform 10 is formed with a channel 12 through which fluid is transported. For example, the platform 10 may consist of a first substrate and a second substrate which is attached to the first substrate. A space formed between the first substrate and the second substrate forms a channel (12) for fluid transport. The channel 12 is formed as a cross-sectional area of a predetermined size, and can be understood as a passage through which the fluid is transferred. The internal cross-sectional size of the channel 12 may be varied. Each constituent part is arranged along the channel 12 formed in the platform 10. The liquid sample containing the plasma component may be transferred from the supply unit 20 through the analysis unit 50 to the waste unit 70 through the channel 12 which is a space between the first substrate and the second substrate .

The supply part 20 supplies the sample to the analysis part 50 along the channel 12. [ Figs. 2 to 4 show a supply unit 20 according to the present embodiment. Hereinafter, the structure of the supply unit 20 will be described with reference to Figs. 2 to 4. Fig.

The supply unit 20 may include a filter 21 for separating and supplying plasma from whole blood. The filter 21 filters the blood cells in the whole blood and passes the fluid sample containing the plasma component. The filter 21 may be formed of various sizes of pores or various materials so as to filter living cells, inorganic particles, or organic particles. The filter 21 of the present embodiment can be formed into pores capable of filtering out hemocyte cells having a size of about 5 to 10 mu m. In addition, the filter 21 may be formed of a biologically inert material so as to be applicable to a biological sample.

On the outlet side of the filter (21) of the supply part (20), the micro flow path (22) through which the filtered plasma is transferred via the filter (21) is located. The micro channel 22 contacts the outlet side of the filter 21 and extends toward the channel 12 and is connected to the channel 12. [ A plurality of fine flow paths 22 are formed at intervals so as to be in contact with the outlet side of the filter 21. The plasma filtered through the filter 21 flows through the micro channel 22 in contact with the outlet side of the filter 21 and is transported to the channel 12.

In this embodiment, the supply unit 20 may be constructed separately from the platform 10 and detachably coupled to the groove 14 formed in the platform 10. Accordingly, since the supply unit 20 is separated from the platform 10, it is possible to perform a necessary operation such as a sample supply more conveniently.

In addition to the above structure, the supply part may be formed integrally with the platform. In such a structure, a fine channel and an outlet are formed in a first substrate constituting a platform, a hole is formed in a second substrate, a first substrate and a second substrate are attached, and a filter is mounted through a hole of the second substrate, Can be achieved.

Hereinafter, a structure in which the supply unit 20 is attached to and detached from the platform 10 will be described as an example.

For this, the supply unit 20 includes a substrate 23 having a micro-channel 22 formed on its surface at an interval and an outlet 26 communicating with the micro-channel 22 to discharge the sample, A filter 21 attached to the surface of the substrate 23 to filter the sample and a cover plate 25 attached to the surface of the substrate 23 with the filter 21 interposed therebetween and having a hole 25 corresponding to the filter 21 24).

A cover plate 24 attached to the substrate 23 and the substrate 23 is formed in a size and shape corresponding to the grooves 14 of the platform 10. The cover plate 24 may be attached to the substrate 23 to form a body. A space formed between the substrate 23 and the cover plate 24 forms the micro flow path 22. The substrate 23 is exposed through the hole 25 formed in the cover plate 24 and the filter 21 is inserted and installed on the substrate 23 through the hole 25. The filter 21 may be formed in a shape and a size corresponding to the holes 25.

The cover plate 24 or both the substrate 23 and the cover plate 24 may be formed of an elastic material or a plastic material including an elastomer. Accordingly, when pressure is applied to the lid plate 24, the micro channel 22 is pressed to narrow the cross-sectional area, so that the fluid transferred to the micro channel 22 can be more easily transferred to the tip outlet 26. Specifically, the plastic material may be made of a polyester material.

The platform 10 is formed with a groove 14 having a size corresponding to the supply unit 20. The channel 12 is formed in an opened state on the inner side surface of the groove 14 at a position corresponding to the outlet 26 formed on the substrate 23. [ When the supply portion 20 is coupled to the groove 14 of the platform 10, the outlet 26 of the supply portion 20 is connected to the channel 12 of the platform 10. Thus, fluid flowing through the outlet 26 of the feeder 20 can be conveyed to the channel 12.

On the surface of the substrate 23, a plurality of micro channels 22 are formed with a space therebetween. A plurality of micro flow paths 22 extend linearly on the substrate 23 and communicate with the outlet 26 on the tip end side of the substrate 23.

In this embodiment, the micro channel 22 may be formed of a capillary to which a sample is transferred due to a capillary phenomenon.

Therefore, a flow of the fluid by the capillary phenomenon is generated on the outlet side of the filter 21. Therefore, when the whole blood is supplied to the mouth side of the filter 21, plasma is sucked into the micro flow path 22 contacting the outlet side of the filter 21 by the capillary phenomenon. Thus, the plasma can be filtered through the filter 21 more quickly in the whole blood. The plasma that has passed through the filter 21 quickly flows out along the micro flow path 22 in contact with the outlet side of the filter 21 by the capillary phenomenon.

A hydrophilic coating film may be further formed on the surface of the micro channel 22 so that the fluid passing through the filter 21 can be smoothly transported along the micro channel 22. [ For example, the substrate 23 and the cover plate 24 can be subjected to an oxygen plasma treatment, followed by oxygen plasma treatment, or a hydrophilic polymer coating. Hydrophilic coating treatment of the micro channel 22 reduces the surface tension of the fluid and air so that the plasma can flow at a higher rate along the micro channel 22.

In addition, a blood-friendly buffer solution such as a PBS solution can be previously accommodated in the micro channel 22 so as to further lower the fluid surface resistance of the micro channel 22. That is, the supply unit 20 may be a structure in which the buffer solution is accommodated as a wetting agent between the outlet side of the filter 21 and the micro flow path 22. The buffer solution comes into contact with the filter outlet side and makes the filter outlet side wetted.

The buffer solution may be the same solution as the fluid through the filter 21. Alternatively, the buffer solution may be a solution free of particles to be separated and not affecting the biological sample. The buffer solution is applicable to any solution that can reduce the capillary pressure of the pores of the filter 21 on the outlet side of the filter 21 and reduce the surface resistance of the microchannel 22. [

The buffer liquid can be previously accommodated in the microchannel 22 of the substrate 23 when the supply section 20 is manufactured by attaching the substrate 23 and the lid plate 24, for example. Alternatively, when the supply unit 20 is used, the buffer solution may be first dropped to the filter 21 and supplied to the outlet side of the filter 21 and the microchannel 22 before the whole blood is dropped on the filter 21.

Due to the buffer solution, the space between the outlet 21 of the filter 21 and the microchannel 22 comes into a state of being in advance contact with the buffer solution. When the whole blood is supplied to the mouth side of the filter 21 in this state, the plasma of the whole blood passes through the pores of the filter 21 without surface resistance by the cohesive force of the buffer solution contacting the outlet side of the filter 21, So that it flows smoothly.

Therefore, the resistance of the fluid flow is reduced, so that the whole blood can be sufficiently filtered by the filter 21 only by the capillary phenomenon without exerting any force from the outside.

In this embodiment, the buffer solution acting as a wetting agent can be brought into contact with the filter outlet side only when the sample is supplied to the filter while being accommodated in the micro flow path.

To this end, a wax layer may be provided between the outlet side of the filter and the buffer solution contained in the micro flow path 22 to selectively block contact between the filter outlet side and the buffer solution. The wax layer may be formed on the exit side of the filter 21 or in the micro flow path 22. The wax layer cuts off between the filter outlet side and the buffer solution, and is removed when necessary to bring the buffer solution into contact with the filter outlet side. The wax layer may be a thin film structure of a wax material which is melted by heat. The wax layer may be melted and removed by, for example, a user's body temperature or a light source or a heat source irradiated from the outside.

Therefore, when this apparatus is not used, the filter outlet side is blocked by the wax layer and is not brought into contact with the buffer solution. When using the device, if the user applies body heat or a heat source to the filter side, the wax layer is melted and removed. When the wax layer is removed, the buffer solution contained in the micro flow path is brought into contact with the filter outlet side, thereby making the filter outlet side wet.

The plasma filtered through the filter 21 flows toward the outlet 26 along the microchannel 22 due to the capillary phenomenon and is moved to the outlet 26 by the pressure pressing the cover plate 24, And is transported to the channel 12 of the connected platform 10. As mentioned above, the supply unit 20 is made of an elastic material and can transfer the fluid by applying an external force and narrowing the internal cross sectional area of the micro flow path 22 narrowly.

The platform 10 is provided with a reagent supply unit 20 and a driving unit 40 on the inlet side of the channel 12 connected to the supply unit 20.

The reagent storage unit 30 is connected to the channel 12 to receive the reagent 32 to be supplied to the analysis unit 50. The reagent container 30 is connected to the channel 12 and includes a receiving line 31 containing reagents 32 required for analysis, a plurality of reagents 32 sequentially accommodated in the receiving line 31, 32 to prevent interference between the reagents 32. In the embodiment shown in FIG. The receiving line 31 can be understood as a passage formed by a cross-sectional area of a predetermined size, such as the channel 12, to which the fluid is transferred. The internal cross-sectional size of the receiving line 31 is variable in various ways. The receiving line 31 may be formed on the first substrate of the platform 10.

In this embodiment, the receiving line 31 is a long elongated single passage, and a plurality of reagents 32 are sequentially contained therein. The plurality of reagents 32 can be sequentially arranged in accordance with the order of use for disease analysis. The reagent 32 may be reagents for biomarker measurement provided to each analyzer 50. The reagent 32 may be a primary detection antibody according to the biomarker, or a secondary detection antibody with fluorescence attached to the primary detection antibody. The reagent 32 can be applied to any substance that can react with the biomarker to analyze and quantify the biomarker by using different optical signals such as fluorescence and luminescence. The reagent disposed at the end of the reagent 32 sequentially disposed along the receiving line 31 may be a buffer solution 34 for washing the analyzing section 50. The buffer solution 34 is moved along the channel 12 and finally passes through the analysis unit 50 to clean the analysis unit 50.

In this embodiment, the reagent includes a washing buffer solution, a primary detection antibody according to the biomarker, a washing buffer solution, a secondary detection antibody with fluorescence attached to the primary detection antibody, and a washing buffer solution in sequence . The wash buffer solution may be, for example, PBST or TBST. The primary detection antibody used for detection may be a polyclonal antibody. The primary detection antibody may be, for example, a polyclonal anti-PSA antibody for prostate cancer. The secondary detection antibody is a fluorescently labeled detection antibody that binds to the primary detection antibody. The secondary detection antibody may be, for example, a fluorophore-labeled anti-rabbit antibody if the primary detection antibody is an antibody made in rabbits. Then, the reagent is moved to the analyzing section along the channel in the order of the washing buffer solution, the primary detecting antibody, the washing buffer solution, the secondary detecting antibody, and the washing buffer solution.

An intermediate layer 33 is formed between the plurality of reagents 32 and the reagent 32 in the receiving line 31 to prevent the reagents 32 from intermixing with each other. The intermediate layer 33 may be a gas such as air or a liquid that is not mixed with a reagent. For example, the intermediate layer 33 can be an organic solution which is not mixed with water.

The receiving line 31 is connected to the channel 12 through the inlet side of the channel 12 formed in the platform 10. When the reagent 32 accommodated in the receiving line 31 moves to the channel 12, the sample flowing into the channel 12 is pushed so that the sample and the reagent 32 sequentially move along the channel 12 And is transferred to the analysis unit 50.

The driving unit 40 applies a transfer pressure to the transfer line and transfers the reagent 32 and the sample to the analysis unit 50.

5 and 6 show embodiments of the driving unit 40. Fig. 5 and FIG. 6 are different from each other only in the arrangement of the driving unit 40, and the structure thereof is the same. Therefore, the embodiment shown in FIG. 5 will be described below.

5, the driving unit 40 includes a driving chamber 41 connected to the receiving line 31 of the reagent receiving unit 30, a thermal expansion material accommodated in the driving chamber 41, . &Lt; / RTI &gt;

The driving chamber 41 can be understood as an inner space having an area of a predetermined size. The inner space size of the driving chamber 41 can be variously modified. The drive chamber 41 may be formed on the first substrate of the platform 10.

The driving chamber may be a space in which the thermal expansion material can be expanded by receiving the thermal expansion material therein, and the shape thereof may be variously modified. For example, the drive chamber may be in the form of a long extended line.

The leading end of the driving chamber 41 extends to the receiving line 31 and communicates with the receiving line 31. [ The thermal expansion material discharged from the tip end of the driving chamber 41 is pushed to the receiving line 31 so that the reagent 32 accommodated in the receiving line 31 can be pushed and moved.

In this embodiment, the drive chambers 41 may be formed in a shape extending from one line and being wound toward the center at intervals in the setting region. The drive chamber 41 may be formed in a zigzag shape in spaced apart relation as shown in Fig. 6, in addition to the wound structure.

Therefore, the entire length of the drive chamber 41 can be made longer even in a limited narrow region. Thus, the drive chamber 41 receives a sufficient amount of the thermal expansion material, and the thermal expansion material can be expanded along the drive chamber 41 to be ejected into the intake line 31. The thermal expansion material is ejected through the exit of the driving chamber 41 and the sample and the reagent 32 are pushed and moved to the analysis unit 50 through the channel 12. [

The expanding thermal expansion material is moved to the channel 12 and pushes the sample and the reagent toward the analysis part. The outlet 26 of the supply part or the hole 25 of the supply part may be blocked so that the thermal expansion material moved to the channel does not escape to the supply part through the outlet part 26 of the supply part connected to the channel. For example, after the sample has been transported to the channel, the hole in the supply can be blocked off before the actuation of the drive. Thus, the expanded thermal expansion material does not move to the channel and does not escape to the supply part, but the sample and the reagent can be continuously pushed toward the analysis part.

The thermal expansion material may be a gas or a liquid containing air. The thermal expansion material may be a material which expands in volume by heat, and which is expanded in volume by heat of various temperatures including a user's body temperature. In addition, the thermal expansion material may be a material that does not react with the sample or reagent 32.

For example, if the thermal expansion material is inflated to the user's body temperature, the device can be driven without a separate heat source. In such a structure, the user can supply the sample and the reagent to the analysis unit by expanding the thermal expansion material by applying the body temperature by holding the one side of the drive chamber or the supply unit of the apparatus by hand.

In another embodiment, the driving unit 40 of the present apparatus may further include a heating unit (not shown) installed in the platform 10 to apply heat to the driving chamber 41.

The heating unit may be an electrothermal heater that receives external power and emits heat. For example, the heat transfer heat can be installed on the first substrate or the second substrate at a position corresponding to the drive chamber 41 with an area heater having a relatively thin thickness and a large area.

The heating unit may be a structure using chemical exothermic energy in addition to electrical energy. The heating part can generate heat by mixing a liquid, a solid, or a substance causing an exothermic reaction when the gas is mixed. For example, the heating portion may be a structure using thermal energy generated by a curing reaction.

As the driving unit 40 is subjected to heat, the thermal expansion material accommodated in the driving unit 40 expands. The expansion force of the thermal expansion material pushes the reagent 32 received in the receiving line 31 to the channel 12 to be transported. Therefore, the sample flowing into the channel 12 is pushed by the reagent 32 and moved to the analysis unit 50.

The analyzer 50 is disposed along the channel 12 to detect biomarkers included in the sample.

The analysis unit 50 includes an analysis line 55 through which the fluid flows, a plurality of fine grooves 56 arranged on the bottom surface of the analysis line 55, And antibody particles 57 for detecting biomarkers.

In the present embodiment, a plurality of analyzers 50 may be serially arranged along the channel 12 and sequentially connected thereto. Although FIG. 1 illustrates a structure in which four analyzers 50 are sequentially arranged along the channel 12, the present invention is not limited thereto, and the analyzer 50 may be formed in three or less or more than five different numbers. For convenience of explanation, each analysis unit 50 connected to the channel 12 along the fluid transfer direction is referred to as a first analysis unit 51, a second analysis unit 52, a third analysis unit 53, 4 analyzing unit 54, respectively. Simply the analysis unit 50 may refer to all of the analysis units.

Each of the analysis units 50 may have a structure in which different antibody particles 57 are received and detected for different biomarkers. Accordingly, multiple detection of various disease factors is possible using one lab-on-a-chip 100.

For example, the first analyzer 51 receives antibody particles 57 bound to PSA (prostate cancer marker), and the second analyzer 52 receives antibody particles (CEA) (gastrointestinal cancer tumor marker) And the third analyzer 53 receives the antibody particles 57 that are bound to AFP (liver cancer tumor marker).

The fourth analyzer 54 can receive a control group. The control group was used to determine whether the biomarker was attached to the antibody particle nonspecifically. In normal ELISA analysis, a positive test is performed to determine whether it is a result of a specific binding or a false positive due to nonspecific binding. In this embodiment, the fourth analyzing unit 54 is provided as a control group, so that it can be compared with the experimental results of the other analyzing units. The fourth analyzing unit 54 may contain, for example, an antibody particle surface-treated with a surface group (such as PEG) that does not react with any biomarker that is not an antibody that retains PSA on the antibody particle. Thus, in the fourth analyzing unit, the primary detection antibody sequentially injected and the secondary detection antibody labeled with fluorescence do not bind to the antibody particles contained in the fourth analysis unit. As a result, no fluorescence signal appears in the fourth analyzing unit. Therefore, when signals are output from the first analyzing unit to the third analyzing unit, it is possible to use the fourth analyzing unit as a result comparison control group to compare the results.

The analysis line 55 can be understood as a passage formed by a cross-sectional area of a predetermined size, such as the channel 12, to which the fluid is transferred. The internal cross-sectional size of the analysis line 55 may be varied. The analysis line 55 may be formed on the first substrate of the platform 10.

The analysis line 55 may consist of a single line. In this embodiment, the analysis line 55 may extend in one line and be formed in a zigzag shape in the setting area. Thus, the analysis line 55 can be arranged closely in a limited narrow area. Therefore, the antibody particles 57 arranged along the analysis line 55 are densely gathered in the confined area of the analysis part 50, so that the diagnostic effect can be enhanced.

The fine grooves 56 fix each of the antibody particles 57 in an even distribution in the analysis line 55. The antibody particles 57 are uniformly arrayed and fixed on the bottom surface of the analysis line 55 by the fine grooves 56. [

Fig. 7 shows the fixing structure of the analysis line according to the present embodiment.

As shown, the fine grooves 56 are arranged along the analysis line 55 at intervals on the bottom surface. The fine grooves 56 are designed to be arranged in a uniform distribution on the bottom surface of the analysis line 55. The arrangement of the fine grooves 56 can be variously modified as long as a uniform distribution can be obtained.

The fine grooves 56 are formed in a groove shape on the bottom surface of the analysis line 55 to capture and fix the antibody particles 57. The fine grooves 56 are formed in a size corresponding to the size of the antibody particles 57. In this embodiment, the fine grooves 56 may be formed in a hemispherical shape corresponding to the spherical antibody particles 57. One antibody particle 57 is inserted and fixed in each fine groove 56.

In this embodiment, the depth of the fine grooves 56 may be d <h <2d. Here, h = the depth of the fine groove 56, and d = the diameter of the antibody particle 57. The depth h of the fine grooves is the distance from the bottom surface of the analysis line to the inner edge of the fine grooves.

When the depth h of the fine grooves 56 is smaller than the diameter d of the antibody particles 57, the antibody particles are unstable and the antibody particles escape from the fine grooves due to the flow of the fluid flowing along the analysis line. Is generated. If the depth h of the fine grooves 56 exceeds twice the diameter d of the antibody particles 57, the thickness of the device becomes thick and the problem of prolonged diffusion of the cleaning liquid for washing the antibody particles and the assay line .

Further, in this embodiment, the fine grooves 56 may be formed in the direction perpendicular to the fluid flow direction at the bottom of the analysis line 55. [ Thus, the flow of the fine grooves and the fluid is arranged at right angles. Therefore, the antibody particles inserted into the fine grooves can not easily escape from the fine grooves along the fluid flow.

Thus, the antibody particles 57 can be stably inserted and fixed in the fine grooves to maintain the uniform alignment state on the analysis line regardless of the fluid flow.

The fine grooves 56 are arranged along the analysis line 55 and the antibody particles 57 are inserted and fixed in the respective fine grooves 56 so that the antibody particles 57 are uniformly distributed along the analysis line 55 Arrays can be fixed. As described above, since the antibody particles 57 are uniformly arranged on the analysis line 55, the quantitative measurement effect can be enhanced.

The antibody particles 57 may have a structure in which a capture antibody is attached to the surface of a magnetic bead. Capture antibody is an antibody that binds to a biomarker to capture and fix the biomarker.

For example, streptavidin or avidin, or anti-biotin protein, is immobilized by covalent bonding using a EDC / NHS coupling method on a micro sized bead surface having magnetic properties. Then, the capture antibody with biotin attached thereto is reacted to fix the capture antibody on the surface of the magnetic beads, thereby preparing antibody particles. Streptavidin is coated on the bead surface. Biotin, which binds to streptavidin, is attached to the capture antibody. When the bead and the capture antibody are left at room temperature for 30 minutes, the capture antibody binds to the bead.

8 is a schematic diagram of a lab-on-a-chip in which the supply unit 20 and the analysis unit 50 are integrated according to the present embodiment. 8, the plasma is filtered through the filter 21 of the supply unit 20, and then supplied to the analysis unit 50 through the micro flow path 22.

FIG. 9 illustrates a process of fixing antibody particles to an analysis unit for manufacturing a lab-on-a-chip according to the present embodiment.

The lab-on-a-chip of the present embodiment undergoes a process of arraying and fixing the antibody particles 57 to the analyzer 50 during the manufacturing process.

To this end, a lab-on-a-chip having an analysis section 50 in which a plurality of fine grooves 56 are arranged on the bottom surface of the analysis line 55 through which the fluid flows is prepared as shown in Fig. 9, The antibody particles 57 are injected into the space inside the analysis line 55 of the analysis unit 50. At this time, a magnet 80 for applying magnetic force to the antibody particles 57 having magnetism is disposed outside the analysis line 55 of the lab-on-a-chip 100. The magnet 80 is disposed outside the bottom surface of the analysis line 55. The magnet may be a permanent magnet or an electromagnet. The magnet 80 may be positioned on one side of the analytical line 55 side.

The antibody particles 57 injected into the analysis line 55 flow along the analysis line 55 by the magnetic force of the magnet 80 and are gathered at the position where the magnet 80 is located. In this state, the magnet 80 disposed on one side of the analysis line 55 is moved along the analysis line 55 to the other side. The magnet 80 is moved toward the other end while applying a magnetic force to the analysis line 55. [ As the magnet 80 moves with magnetic force, the antibody particles 57 injected into the analysis line 55 are moved along the magnet 80 by the magnetic force.

The antibody particles 57 are brought close to the bottom side of the analysis line 55 where the magnet 80 is located by the magnetic force and are moved along the analysis line 55. In this process, the antibody particles 57 are trapped and fixed in the fine grooves 56 formed on the bottom surface of the analysis line 55, respectively.

After the magnet is completely moved to the other end of the analysis line 55, the residual antibody particles are removed from the analysis line by washing. The magnetic force can be removed from the analysis line 55 during washing. The washing line is transferred to the analysis line 55 to wash the analysis line 55. The washing solution may be a solution such as PBS (phosphate buffered saline) or pluronic solution. The remaining antibody particles 57 in addition to the antibody particles 57 inserted and fixed in the fine grooves 56 by the washing liquid are removed in the analysis line 55. During the washing process, the antibody particles are inserted into the fine grooves formed deep in the direction perpendicular to the flow direction of the washing liquid, so that the antibody particles can be maintained in a fixed state without escaping from the fine grooves by the flow of the washing liquid. Thus, only the remaining antibody particles that have not been immobilized in the assay well in the assay line flow out of the analysis line along the flow of the wash solution.

10 is a photograph showing a state in which the antibody particles 57 are arranged and fixed to the analysis part 50. Fig. As shown in Fig. 9, the antibody particles 57 can be evenly arranged and fixed on the analysis line 55 of the analysis unit 50 through the present embodiment.

The sample or reagent 32 or the like to be discarded via the analyzer 50 is transferred to the disposal unit 70 connected to the analyzer 50 and processed. The waste part 70 is connected to the outlet of the analyzer 50 to receive a fluid such as a sample or a reagent to be discarded. The waste portion 70 is formed to have a predetermined size and can be understood as an empty space. The shape and size of the waste portion 70 can be variously modified. The waste portion 70 may be formed on the first substrate of the platform 10. The waste portion 70 may further include an air hole communicating with the outside at one side thereof. The channel 12 formed in the platform 10 and the outside air flow through the air holes to minimize the resistance of the fluid transported along the channel 12 so that the fluid is smoothly transported from the supply portion 20 to the waste portion 70 .

11 illustrates a manufacturing process of a lab-on-a-chip in which the supply unit 20 and the analysis unit 50 are integrated according to the present embodiment.

The lab-on-a-chip of the present embodiment includes a substrate 23, a cover plate 24, and a hole 25 of a cover plate 24, which are formed with a channel 12 including a supply unit 20 and an analysis unit 50, The trimmed filter 21 is prepared and adhered using a double-sided tape. After trimethoxy silane is applied to the cover plate 24, the substrate 23 and the cover plate 24 are subjected to oxygen plasma treatment to form a filter 21 ).

Hereinafter, a diagnosis process using the lab-on-a-chip according to the present embodiment will be described.

The whole blood can be supplied to the lab-on-a-chip 100 according to the present embodiment to diagnose a plurality of diseases more easily and effectively.

The lab-on-a-chip 100 is divided into three analyzers 50 and one control group. Each analyzing unit 50 receives antibody particles 57 to which capture antibodies binding to different disease agent biomarkers are immobilized. The antibody particles 57 to which the capture antibody is immobilized are inserted and fixed in the fine grooves 56 uniformly arranged along the analysis line 55 in each analysis unit 50. The receiving line 31 is pre-filled with reagents 32 necessary for analysis. In the receiving line 31, the respective reagents 32 are separated by air or an organic solvent immiscible with water.

When the lab-on-a-chip is prepared, a sample is supplied to the lab-on-a-chip, filtered with a filter, and transferred to the analyzer.

When whole blood is dropped on the mouth side of the filter 21 mounted on the supply part 20 of the lab-on-a-chip 100, the whole blood cells are filtered by the filter 21, .

The sample containing the plasma in the microchannel 22 is moved to the exit side of the supply section 20 by the capillary phenomenon of the microchannel 22 and the external pressure pressing the cover plate 24 to be supplied to the channel 12 of the platform 10 do.

In the sample supply process, the filter outlet side may be wetted with the buffer solution by wetting the filter outlet side before filter filtration. When the user applies heat such as body temperature to the micro channel of the filter or the supply part, the wax layer blocking the gap between the filter outlet side and the buffer solution contained in the micro channel is melted and removed. As a result, the filter outlet side is exposed and comes into contact with the buffer solution previously contained in the micro flow path to become wet while being wetted. Therefore, plasma of the whole blood supplied to the mouth side of the filter can be smoothly filtered through the filter pores without surface resistance to the filter outlet side.

When the sample is supplied from the plasma separation and supply unit 20 to the channel 12, the driving unit 40 is operated to transfer the sample and the reagent 32 to the analysis unit 50.

The driving unit 40 may be driven by a user's body temperature or a separate heat source provided by the user. Alternatively, the driving unit 40 may be driven by a heat source generated in the heating unit provided on the platform 10. [

The volume of the thermal expansion material filled in the driving chamber 41 expands as heat is applied to the driving unit 40. The expansion pressure of the thermal expansion material is applied to the receiving line 31 connected to the driving chamber 41 so that the reagent 32 accommodated in the receiving line 31 is pushed toward the channel 12. That is, the volume of the thermal expansion material inside the driving chamber 41 is increased by the heat energy applied to the driving unit 40, and the thermal expansion material is discharged to the exit side of the driving chamber 41 by the increased volume. Then, the thermal expansion material pushes the reagent 32 in the receiving line 31, so that the fluid is moved. The reagents 32 are moved in the filling line 31 in the filling order.

The reagent 32 pushed out from the receiving line 31 pushes the sample flowing into the channel 12 to move. Thus, the sample and the reagent 32 are moved along the channel 12 to the analysis unit 50.

When the sample and the reagent are supplied to the analyzing unit, the biomarker of the sample is detected by the antibody particles contained in the fine grooves of the analyzing unit and is labeled with a reagent, so that the sample can be diagnosed.

In the analysis unit 50, the antibody particles 57 are regularly arranged along the analysis line 55 and fixed in a uniform distribution. The sample passes through antibody particles (57) fixed to each analyzing unit (50) while passing through each analyzing unit (50) arranged in series. A specific biomarker contained in the sample passes through the analyzing unit 50 and is captured by the antibody particle 57 in response to a specific capture antibody attached to the antibody particle 57 of each analyzing unit 50.

The antibody particles 57 having different specific capture antibodies are immobilized on the respective analyzers 50 so that different biomarkers pass through the analyzers 50 to the analyte 50 The particles 57 are caught.

The sample is flowed by the driving unit 40 and passes through the analysis unit 50. In this process, the sample is brought into contact with the antibody particles 57 of the analysis unit 50 in a dynamic state. Therefore, the antigen-antibody reaction occurs between the sample and the antibody particle 57 in the fluid flowing state by the driving unit 40, and thus the reaction can be induced faster than in the static state.

The biomarker is attached to the antibody particle 57 as the sample passes sequentially through the antibody particles 57 uniformly arranged along the analysis line 55. The reagent 32 moved along with the sample passes through each analysis unit 50. In this process, the primary detection antibody and the secondary detection antibody with fluorescence are sequentially bound to the biomarker held on the surface of the antibody particle (57).

For example, if the biomarker is PSA, the primary detection antibody may be a polyclonal anti-PSA antibody. The secondary detection antibody with fluorescence may be an anti-PSA FITC labeled antibody that binds to the biomarker. For example, the secondary detection antibody is an anti-rabbit antibody capable of attaching to the primary detection antibody when the host is a rabbit, or an enzyme such as fluorescent or peroxidase (HRP) or alkaline phosphatase (AP) Can be used. The reagent 32 is attached to the biomarker captured on the surface of the antibody particle 57, and finally the biomarker can be detected as a fluorescence signal.

Biomarkers can be quantitatively analyzed using enzyme-linked immunosorbent assay (ELISA) based on fluorescence signals.

In this manner, the sample and the reagent 32 are transferred to the analysis unit 50, the biomarker can be attached to the antibody particles 57 arranged in the analysis unit 50, and fluorescence treatment can be performed with the reagent 32. Each analyzer 50 provided in the lab-on-a-chip 100 detects biomarkers in response to different biomarkers. The sample and the reagent 32 that have passed through the analysis unit 50 are moved to the waste unit 70 connected to the analysis unit 50 and discarded.

The buffer solution 34 positioned at the rear end of the reagent 32 finally moved through the channel 12 passes through the analyzer 50 to clean the analyzer 50. The surfaces of the antibody particles 57 fixed to the inner surface of the analysis line 55 and the fine grooves 56 and the fine grooves 56 are washed by the buffer solution 34 to remove the biomarkers and reagents All the residue is removed. Thus, the concentration of the biomarker captured on the surface of the antibody particle 57 can be more accurately detected.

The concentration of the biomarker is detected in proportion to the intensity of the fluorescence signal. As mentioned above, the analyzer 50 has a uniform distribution of the antibody particles 57 regularly arranged along the analysis line 55 within a certain space. Therefore, it is possible to more accurately detect and analyze the fluorescence signal with respect to each analyzer 50. Therefore, more reliable biomarker quantitative analysis can be performed.

[Experimental Example]

12 is a photograph showing an experimental result of the lab-on-a-chip according to the present embodiment.

On the lab-on-a-chip prepared in this example, the antibody particles with the capture antibody were arrayed and immobilized in the analyzer 50, and then the sample and the primary detection antibody and the secondary detection antibody, which were reagents, were sequentially transferred and treated. In this experiment, the disease marker biomarkers were PSA, 10 ng / mL, 20 ng / mL and 40 ng / mL, respectively.

In FIG. 12, 10 ng / mL, 20 ng / mL and 40 ng / mL of each sample were subjected to the above-described treatment to transfer the sample containing the biomarker. The PSA binds to the antibody particles and finally the secondary antibody It is a photograph measured after processing. The PSA of 0 ng / mL in FIG. 12 is a photograph of a state in which PSA is not bound to the antibody particle after transferring the sample without the biomarker.

As shown in FIG. 12, in the case where PSA is present in plasma, when fluorescence is observed as in the case of PSA 10 ng / mL, PSA 20 ng / mL and PSA 40 ng / mL in FIG. 12, Fluorescence is not observed with 0 ng / mL of PSA. Thus, it can be confirmed that the biomarker can be accurately detected through the lab-on-a-chip manufactured according to the present embodiment.

13 is a human total PSA ELISA standard curve obtained by quantitatively analyzing the amount of human total PSA using a conventional well plate method as reference data.

The assay system used an ELISA system capable of linearly quantifying PSA concentrations from a few pg / mL to 2000 pg / mL.

Figure 13 shows the detection dynamic range exhibited by the antibody-antigen-detection antibody system used in the Examples. The dynamic range of detection ranges from 0.5 to 2 ng / mL, similar to the concentration of PSA detected in patients with prostate cancer.

Thus, when an ELISA system performed on a well plate is implemented in the lab-on-a-chip of this embodiment, it can be expected that similar detection ranges are exhibited.

While the illustrative embodiments of the present invention have been shown and described, various modifications and alternative embodiments may be made by those skilled in the art. Such variations and other embodiments will be considered and included in the appended claims, all without departing from the true spirit and scope of the invention.

10: Platform 12: Channel
14: Groove 20: Supply
21: filter 22: fine flow path
23: substrate 24: cover plate
25: Hall 26: exit
30: Reagent receiving section 31: Receiving line
32: reagent 33: middle layer
34: Buffer solution 40:
41: driving chamber 50:
55: analysis line 56: fine groove
57: antibody particle 70: waste part
80: Magnet 100: Lab-on-a-chip

Claims (25)

At least one analyzing part disposed along the channel to detect a biomarker included in the sample, and a controller connected to the channel and supplying the analyzing part to the analyzing part A reagent accommodating portion for accommodating the reagent, and a driving portion for transferring the reagent and the sample to the analyzing portion along the channel,
Wherein the analyzing section includes an analyzing line through which a fluid flows, a plurality of fine grooves arranged on a bottom surface of the analyzing line, and antibody particles accommodated in the fine grooves to detect a biomarker of the sample. The method according to claim 1,
On-the-fly chip is formed with a size corresponding to the size of the fine groove antibody particles, and one antibody particle is inserted and fixed in each fine groove. 3. The method of claim 2,
Wherein the analyzer comprises a plurality of probes connected in series and sequentially connected along a channel. The method of claim 3,
Wherein each of the analyzing units contains different antibody particles. The method according to claim 1,
Wherein the antibody particle has a capture antibody attached to a surface of a bead having magnetism. The method according to claim 1,
Wherein the reagent receiving portion includes an accepting line connected to a channel and containing a reagent, a plurality of reagents sequentially accommodated in the accepting line, and an intermediate layer formed between the reagents to prevent interference between the reagents. The method according to claim 1,
The depth of the fine groove is d < h < 2d.
Where h = the depth of the fine groove, and d = the diameter of the antibody particle. The method according to claim 1,
Wherein the fine grooves are formed in a direction perpendicular to the fluid transport direction at the bottom of the analysis line. 9. The method according to any one of claims 1 to 8,
Wherein the driving portion includes a driving chamber connected to a receiving line of the reagent receiving portion, and a thermal expansion material accommodated in the driving chamber and expanding by heat. 10. The method of claim 9,
Wherein the driving part further comprises a heating part installed on the platform and applying heat to the driving chamber. 10. The method of claim 9,
Wherein the supply section includes a filter for filtering and supplying the sample. 12. The method of claim 11,
Wherein the supply section further comprises a micro flow path formed in a filter outlet side of the platform and connected to the channel to transfer a sample through the filter. 10. The method of claim 9,
Wherein the supply portion is provided separately from the platform and is detachably coupled to the groove formed in the platform. 14. The method of claim 13,
Wherein the supply unit includes a substrate having a fine flow path formed on its surface at an interval and an outlet at an end thereof communicating with the micro flow path to discharge the sample, a filter provided in contact with the substrate surface to filter the sample, And a cover plate attached to the substrate surface and provided with a hole for sample supply at a position corresponding to the filter. 15. The method of claim 14,
Wherein the substrate and / or the cover plate are formed of an elastic material or plastic material including an elastomer. 12. The method of claim 11,
The lab-on-a-chip has a structure in which a buffer solution for reducing surface tension between a filter outlet side and a micro channel is accommodated as a wetting agent. 17. The method of claim 16,
Further comprising a wax layer provided on the filter outlet side or the micro flow passage for selectively blocking between the buffer solution and the filter outlet side. A method for manufacturing a lab-on-a-chip,
Immobilizing the antibody particle to which the capture antibody is attached on the surface of the bead having magnetism,
The fixing process comprises preparing a lab-on-a-chip having an analyzing portion in which a plurality of fine grooves are arranged on the bottom surface of an analysis line through which the fluid flows, and inserting and fixing the antibody particles in the respective fine grooves A method for manufacturing a lab-on-a-chip. 19. The method of claim 18,
The process of inserting and immobilizing each of the antibody particles in each of the fine grooves includes immersing the antibody particles in the analysis line, applying magnetic force to the antibody particles outside the bottom surface of the analysis line, moving the magnetic force along the analysis line, And inserting the antibody particles into the fine grooves of the bottom surface of the assay line while moving the antibody particles. 20. The method of claim 19,
Further comprising transferring the fluid to the analysis line to remove the remaining antibody particles from the analysis line in addition to the antibody particles immobilized in the micro-grooves. A sample supply process for preparing a lab-on-a-chip according to any one of claims 1 to 8 and supplying a sample to a channel formed on a platform of a lab-
A movement process of moving the sample to an analysis section provided in the platform,
A reagent supply process for supplying the reagent to the analysis unit, and
Detecting the biomarker of the sample with the antibody particles contained in the fine grooves arranged along the analysis part and labeling them with a reagent
On-a-chip &lt; / RTI &gt; 22. The method of claim 21,
Wherein the sample supply step further comprises filtering the sample with a filter. 23. The method of claim 22,
Wherein the sample supplying step further comprises wetting the filter outlet with a buffer solution before filtering the filter. 25. The method according to any one of claims 21 to 24,
Wherein the moving process includes pressing the sample by applying pressure to the channel to move the sample. 25. The method according to any one of claims 21 to 24,
Wherein the moving process comprises applying heat to a driving chamber of a platform on which the reagent is housed.

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