KR20140143860A - Biosensor for detecting target materials using protease reaction - Google Patents
Biosensor for detecting target materials using protease reaction Download PDFInfo
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- KR20140143860A KR20140143860A KR1020130065247A KR20130065247A KR20140143860A KR 20140143860 A KR20140143860 A KR 20140143860A KR 1020130065247 A KR1020130065247 A KR 1020130065247A KR 20130065247 A KR20130065247 A KR 20130065247A KR 20140143860 A KR20140143860 A KR 20140143860A
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- G—PHYSICS
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/403—Cells and electrode assemblies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The present invention relates to a biosensor for detecting a target substance using a proteolytic enzyme reaction. More specifically, the present invention relates to a biosensor for detecting a target substance using proteolytic enzyme reaction, A biosensor capable of diagnosing cancer and capable of being downsized by using an electrochemical impedance spectroscopy (EIS) and being easy to use.
Description
The present invention relates to a biosensor for detecting a target substance using a proteolytic reaction, a method for producing the biosensor, and a method for quantitatively detecting a target substance using the biosensor.
Recently, people with prostate cancer have been increasing rapidly in recent decades due to aging population and westernization of diet. In general, cancer does not have any obvious symptoms at an early stage, and it often spreads to other organs when surgery is impossible. It is hard to expect a cure. However, early detection and treatment, it is said that the survival rate exceeds 82%. Therefore, the necessity of early diagnosis and development of point-of-care-testing (POCT) that can directly detect disease in real life is emphasized.
Protease is widely found in the tissues, cells and microorganisms of animals and plants. Most of them are simple proteins with a molecular weight of tens of thousands, which may be complex proteins with sugar or metal ions. It is a kind of enzyme that breaks down the protein chain made of amino acid and gives new physiological activity. (Exopeptidase, endopeptidase), and classified into 6 types according to the action mechanism (serine, threonine, cysteine, aspartic acid, glutamic acid proteases and metalloproteases). It is present in various organs and participates in important metabolic control and physiological responses in vivo.
The matrix metalloproteinases (MMPs) family is a zinc-dependent endopeptidase that hydrolyzes all types of extracellular matrix proteins and is involved in a variety of in vivo metabolism . In addition, MMP-2 and MMP-7 are known to play an important role in cancer development and metastasis. Especially, MMP-2 and MMP-7 can be useful as effective biomarkers of ovarian cancer and colon cancer, respectively.
Bio-chip technology is a core fusion technology that constitutes an axis of nano-biotechnology in addition to biosensor and micro electro-mechanical system (MEMS) technology. have. Since the field of biosensor was born, researches on biosensors using detection method of cancer biomarkers have been actively carried out. Cancer markers in cancer diagnosis are essential for precise, rapid and inexpensive detection of specific cancers. Detection of Cancer Markers Various detection methods such as an electrochemical method, an optical method and a mass spectrometric method can be applied to the biosensor, and many research results on the respective detection methods have been reported.
Diagnosis of cancer by conventional biomarkers was mostly done with proteins. Most of these protein biomarkers use proteins that are either newly emerging or rapidly increase in specific disease outbreaks. However, problems with reference points may arise in these proteins.
Recent researches have been conducted on biosensors that perform protease assay using the fact that a protease that plays an important role in the development or metastasis of cancer can be precisely detected . There is another significance in that it takes advantage of features that play an important role in determining protein amino acid sequence in protein chemistry. Proteolytic enzymes exist in various organs and are involved in important metabolic control and physiological responses in vivo. As such, the proteolytic enzyme has a merit as a biomarker for diagnosis of diseases because a change in the amount of the proteolytic enzyme occurs according to the biological rhythm and the activity is also changed.
Conventional proteolytic enzyme detection studies used optical methods using fluorescence, methods using molecular materials, and methods of detecting proteins using cells. However, the study of proteolytic enzyme analysis in the biosensor is in the early stage and has not been studied yet. More specifically, proteolytic enzymes such as MMP-2 and MMP-7 can be detected using energy transfer phenomena (BRET) between a quantum dot and bioluminescence in a well. A system was reported. In addition, a biochip capable of detecting proteolytic enzymes such as MMP-7, thrombin, and caspase-3 using a fluorescence resonance energy transfer (FRET) Was reported. In addition, MMP-1 was detected on a piezoelectric immunosensor chip.
However, these methods have complicated experimental procedures, require expensive equipment and cost, and take a long time. In particular, since cancer therapy requires long-term and steady diagnosis in the early diagnosis of cancer, the above methods are time consuming and costly, and there is a disadvantage that many samples and reagents are required. Therefore, Not suitable for POCT.
The label-free method generally uses a labeling substance that binds a labeling substance such as absorption, fluorescence, phosphorescence, color development, or radiation to a recognition substance that binds strongly to the analyte in order to determine the presence or absence of the analyte The non-labeling method refers to a method of measuring without such a labeling substance. Non-labeling methods are preferred by researchers because the labeling method can cause structural changes of biopolymers or proteins due to the attachment of labeling materials and lose their activity. The measurement of the sample without labeling is intended to detect the characteristics of the sample without changing the characteristics of the sample itself. If the sample is subjected to a separate task, the characteristics of the sample itself may change, or it may be troublesome and disadvantageous to study the conditions of the fusion with another research or invention. The advantage of the unlabeled method is that it can be detected and analyzed in real time without any additional analysis time because the sample is not treated separately. The shortening of the pretreatment time of the sample means that the target substance can be detected within a short time, so that it is advantageous in that it is possible to reduce the time constraint when the detection time and other additional tests are required. In this method, unnecessary waste can be prevented because the number of samples required is small, and the cost saving effect can also be said. Because it is a non-destructive substance detection method, it is applicable to various fusion researches.
Accordingly, the inventors have made efforts to develop a biosensor having a wide application range applicable to various types of cancer, and as a result, it has been possible to easily diagnose and diagnose various cancers in a short time by simply establishing conditions optimization and detection methods while maintaining a basic sensor framework. We have developed a biosensor capable of multiple detection as much as possible.
Specifically, the biosensor of the present invention is capable of performing precise detection by using a protease, which plays an important role in cancer development or metastasis, It is possible to diagnose cancer through continuous activity confirmation in the middle and long term. In addition, the biosensor of the present invention can be miniaturized and easily used by applying an electrochemical impedance spectroscopy (EIS), and is capable of precisely detecting a trace amount of biochemical reaction by precisely measuring various physicochemical characteristics .
It is an object of the present invention to provide a biosensor for detecting a proteolytic enzyme in a sample using a proteolytic reaction.
Another object of the present invention is to provide a method of manufacturing the biosensor.
It is still another object of the present invention to provide a method for quantitatively detecting a protease in a sample using the biosensor or a method for diagnosing a disease associated with the activity of the protease.
In order to achieve the above object,
The present invention
A channel is formed on the lower surface of the substrate. The channel includes an inlet for injecting an electrocyte, an inlet for injecting a substrate of a target protease to be fixed to the electrode, an inlet for injecting a sample containing a target protease, An upper substrate including an outlet through which a residue and an electrolyte are discharged;
The electrode unit is arranged on the upper surface of the substrate, and the working electrode unit is arranged at a right angle to the flow direction of the sample, and the reference electrode unit and the counter electrode unit are arranged so as to be parallel to the working electrode unit A lower substrate on which a substrate of a target protease is immobilized; And
An adhesive plate on an edge of the upper substrate and a lower substrate such that a channel of the upper substrate and an electrode portion of the lower substrate intersect to form a reaction space;
A biosensor for detecting a target protease in a sample is provided.
In addition,
1) printing a reference or counter electrode portion for forming a potential difference between the working electrode portion and the working electrode in the electrode print region on the lower substrate, respectively, so as to be separated from each other between the electrode portions;
2) A channel is formed in the channel forming region on the upper substrate, the channel including an inlet for injecting an electrolyte, an inlet for introducing a substrate of a target protease to be fixed to the electrode, an inlet for injecting a sample containing a target protease , A residue after the reaction and an outlet through which the electrolyte is discharged;
3) attaching an adhesive plate to an edge position on the lower substrate;
4) attaching an upper substrate to the adhesive board; And
5) injecting a substrate of a target protease into an injection port into which the substrate of the target protease is injected to fix the substrate of the target protease;
The present invention provides a method for producing a biosensor for detecting a target protease in a sample.
In addition,
1) In the biosensor according to the present invention, injecting a test sample containing a target proteinase into a sample injection port and injecting an electrolyte into the electrolyte injection port;
2) contacting the sample injected in the reaction space in the biosensor with a substrate of a target protein protease immobilized on the electrode unit;
3) measuring an electrochemical signal of the working electrode and the reference electrode by the reaction; And
4) measuring the impedance through the electrochemical signal;
A method for quantitatively detecting a target protease in a sample.
In the early diagnosis of cancer using conventional proteolytic enzymes, cancer treatment requires a long-term and steady diagnosis. Therefore, it is time consuming and costly, requires a lot of samples and reagents, and the detection equipment is relatively large and complicated Therefore, it is not suitable for POCT.
The present invention can improve patient results through accurate diagnosis by using proteolytic enzymes, using differentiated detection methods through impedance detection methods, and using microfluid control technology, And developed a biosensor that can save cost and create a cost-effective effect.
The biosensor according to the present invention can be applied to research on the development of a biosensor capable of mass production at a low cost by applying various kinds of materials and early diagnosis studies of various kinds of cancer.
1 is a view illustrating a process of manufacturing an electrode unit on a glass substrate.
2 is a view illustrating a process of fabricating a channel on a PDMS substrate.
FIG. 3 is a schematic diagram showing an electrochemical detection method between a target material and a reactive material. FIG.
4 is a view of the completed biosensor. PDMS having a large channel and a glass substrate having an electrode part can be distinguished. The PDMS in which the channels are constructed has a total of five inlets and two outlets, all of which are perforated. A description of each inlet and outlet is as follows:
The first inlet is where the electrolyte for impedance measurement is injected into the channel of the biosensor;
The
The
The
5 is an actual photograph of the completed biosensor.
Hereinafter, the present invention will be described in detail.
The present invention
A channel is formed on the lower surface of the substrate. The channel includes an inlet for injecting an electrocyte, an inlet for injecting a substrate of a target protease to be fixed to the electrode, an inlet for injecting a sample containing a target protease, An upper substrate including an outlet through which a residue and an electrolyte are discharged;
The electrode unit is arranged on the upper surface of the substrate, and the working electrode unit is arranged at a right angle to the flow direction of the sample, and the reference electrode unit and the counter electrode unit are arranged so as to be parallel to the working electrode unit A lower substrate on which a substrate of a target protease is immobilized;
An adhesive plate on an edge of the upper substrate and a lower substrate so that a channel of the upper substrate and an electrode of the lower substrate intersect to form a reaction space;
A biosensor for detecting a target protease in a sample is provided.
In the biosensor, the protease is preferably matrix metalloproteinases (MMPs), more preferably MMP-2 or MMP-7, which is known as an effective biomarker of ovarian cancer and colon cancer But is not limited to the above protease.
The working electrode part is coated with gold (Au), and the reference electrode part is coated with platinum (Pt), but is not limited to the metal.
The working electrode part and the reference electrode part may arrange the working electrode part so as to be perpendicular to the flow direction of the sample, and arrange the reference electrode part and the counter electrode part so as to be parallel to the working electrode part.
Preferably, the upper substrate is a polydimethylsiloxane (PDMS) substrate and the lower substrate is a glass substrate, but the material of the substrate is not limited thereto.
Preferably, the upper substrate has a width of 25 to 35 mm and a length of 20 to 30 mm, a channel width of 600 to 800 μm, a height of 50 to 150 μm, and an angle of 40 to 50 °.
Preferably, the lower substrate has a width of 33 to 45 mm and a length of 20 to 30 mm, and the electrode portion has a width of 300 to 600 μm, but is not limited thereto.
The upper substrate
One inlet through which the electrolyte is injected is located at the farthest center of the outlet;
One inlet through which the substrate of the target protein protease is injected is located in the middle in each channel divided into two from the inlet of the electrolyte;
One inlet through which a sample is injected is located in each channel leading from the inlet of the substrate of the target protease;
It is preferable that one outlet is located at the center farthest from the injection portion of the electrolyte in each of the channels leading from the injection port of the sample.
In a specific embodiment of the present invention, the biosensor is designed as follows.
Since five proteinase enzymes such as MMP-2 and 7 use the same electrolyte, peptides and enzymes such as one injection port, MMP-2 and 7 commonly used in the center are different materials, Because it is not possible, it is necessary to enter each channel divided into two, and it is designed with four each of two, that is, a total of five inlets.
The position of the injection port was selected in consideration of the amount and role of the material to be injected and the distance between the injection port and the discharge port. The inlet port for the electrolyte, which also serves to push the existing materials present in the channel to the outlet, is located at the farthest center and the second at the outlet, And the injection port for the proteolytic enzyme with the least amount of injected was located closest to the outlet.
The size of the channel was selected to be 700 μm wide and 100 μm high showing the best fluid flow on the electrode part deposited on a glass wafer through simulation. The width of the electrode portion was selected at a ratio of 1: 2 with Au of 300 μm and Pt of 600 μm. The distance between the electrode parts was 1 mm, and the interval at which the noise was smallest was selected as the test result. At both ends of the biosensor, the distance between the electrodes at the part connected to the measuring device is 2 mm, so that they do not interfere with each other.
In order to compensate for the small signal received by only one reaction part in the conventional linear channel and the electrode part, the channel and the electrode part are designed as shown in FIG. 4 to increase the reaction part to six The reaction area is also increased six times, and theoretically, it is designed to receive six times the signal from the existing design. The electrode part can be measured in two places at the same time through the 'U' shaped Au (working) electrode part on both sides and the Pt (reference and counter) electrode part. The shape of the electrode and the shape of the channel are designed to enlarge the reaction area to receive more accurate and larger signals.
In order to increase the reaction area while occupying a small area, both the channel and the electrode part are designed to be bent. In addition, when connected to the main channel from the injection port, the angle of the point where the channels meet each other is designed to be 45 degrees to induce fluid flow in the direction of the discharge port.
The biosensor according to the present invention can be performed as follows. First, a sample is injected, and a substrate of a target protease is injected into a biosensor to induce a specific reaction, and an impedance is measured by injecting an electrolyte solution to obtain a reference impedance signal. Then, the target proteinase is injected to induce the reaction with the immobilized peptide, and then the non-specific binding or unnecessary reaction by-products are removed using a buffer solution. Finally, the impedance is measured by injecting an electrolytic solution, and the difference between the measured impedance and the reference impedance is compared to obtain the result of the concentration of the peptide or the concentration of the proteolytic enzyme, and the quantitative detection can be performed based on the result. [Fig. 3] is a schematic diagram showing an electrochemical detection method between a target material and a reactant. By using a target substance having a property of specifically decomposing and cleaving a reaction substance, the polypeptide is fixed to the biosensor using the polypeptide sequence, and the size of each electric signal obtained by decomposing or cutting the target substance is measured A quantitative signal analysis according to the amount of the reactant can be performed.
The biosensor according to the present invention can inject a plurality of peptide samples through a sample injection port. The reaction chambers are arranged in a series connection, and a plurality of proteolytic enzymes can be mixed and injected into one injection port. At this time, a check valve or a passive valve may be installed in order to solve the reverse flow phenomenon of the proteolytic enzyme sample and the mixing phenomenon of the respective peptide samples.
Meanwhile, the biosensor according to the present invention has a problem in that the reaction efficiency is lowered due to the different protease reaction conditions during the injection of a plurality of protease mixture samples, and when there is a problem that interference occurs in each protease reaction , We analyze the correlation between the two conditions through the preliminary experiment results and then determine the reaction conditions that can show the best efficiency within the range where the interference does not occur using the analyzed results, Type proteinase can be used for diagnosis.
In addition,
1) printing a reference or counter electrode portion for forming a potential difference between the working electrode portion and the working electrode in the electrode print region on the lower substrate, respectively, so as to be separated from each other between the electrode portions;
2) A channel is formed in the channel forming region on the upper substrate, the channel including an inlet for injecting an electrolyte, an inlet for introducing a substrate of a target protease to be fixed to the electrode, an inlet for injecting a sample containing a target protease , A residue after the reaction and an outlet through which the electrolyte is discharged;
3) attaching an adhesive plate to an edge position on the lower substrate;
4) attaching an upper substrate to the adhesive board; And
5) injecting a substrate of a target protease into an injection port into which the substrate of the target protease is injected to fix the substrate of the target protease;
The present invention provides a method for producing a biosensor for detecting a target protease in a sample.
In the above method, the electrode sub-printing of the lower substrate of the step 1) includes: 1) coating a glass wafer with a photoresist; 2) development of an electrode part in the shape of an electrode to be made; 3) depositing a metal film on the developed electrode using thermal evaporation; And 4) removing the photoresist using acetone.
Specifically, FIG. 1 is a view showing a procedure for manufacturing an electrode portion on a glass substrate. A thin layer of Ti / Au thin film was deposited on the glass wafer by coating the PR of
In the above method, the channel formation of the upper substrate in the step 2) may include: 1) coating a photoresist on a silicon wafer; 2) patterning the channel with a channel shape to be formed; 3) pouring the PDMS in liquid form onto the shaped channel, then baking in an oven, and then separating the cured PDMS from the silicon wafer.
Specifically, FIG. 2 is a diagram illustrating a procedure of manufacturing a channel in a PDMS. The silicon wafer was coated with PR (SU-8), shaped in the shape of a channel to be made, then PDMS liquid was poured on it, baked in an oven at 65 DEG C for 4 hours and then cured PDMS The separated PDMS is completed. This is bonded to the glass substrate on which the electrode part is formed by using
In addition,
1) In a biosensor according to the present invention, a sample containing a target proteinase is injected into a sample injection port, and an electrolyte is injected into an electrolyte injection port.
2) contacting the sample injected in the reaction space in the biosensor with a substrate of a target protein protease immobilized on the electrode unit;
3) measuring an electrochemical signal of the working electrode and the reference electrode by the reaction; And
4) measuring the impedance through the electrochemical signal;
A method for detecting a target protein protease in a sample.
In the above method, the electrochemical signal of step 3) is measured using a potential meter, and the impedance of step 4) can be measured using an impedance meter. Sensors and Actuators B129 (2008) 372-379; and Biosensors and Bioelectronics 22 (2007) 373-379; Sensors and Actuators B114 ) 2525-2531).
In the present invention, the basic principle of the impedance measurement method was used, and the invention was conducted using a comparative analysis method of reactivity with the reference electrode in the analysis. Specifically, the degree of activation of an enzyme can be detected using an electrochemical measurement method, and the impedance can be measured by converting it into an analytical signal.
Using the detection method according to the present invention, ovarian cancer and colon cancer can be diagnosed by measuring the target protease by applying MMP-2 and MMP-7. On the other hand, signals can be measured and analyzed using two or more electrochemical measurement methods.
The detection method according to the present invention can be used for confirming the activity while keeping the concentration of the fixed target substance constant and changing the concentration of the enzyme as the reaction substance. Therefore, it is possible to use the enzyme for the early diagnosis of cancer by comparing the maximum value of the impedance value by measuring the activity depending on the concentration of the cancer early diagnosis biomarker.
In a specific example of the present invention, MMP-2 and MMP-7 were detected using the biosensor manufactured according to the present invention under the following conditions.
(Lysine-Serine-Arginine-Tryptophan-Leucine-Alanine-Leucine-Proline-Arginine)
(Arginine-Proline-Leucine-Alanine-Leucine-Tryptophan-Arginine-Serine-Lyusine)
Claims (16)
The electrode unit is arranged on the upper surface of the substrate, and the working electrode unit is arranged at a right angle to the flow direction of the sample, and the reference electrode unit and the counter electrode unit are arranged so as to be parallel to the working electrode unit A lower substrate on which a substrate of a target protease is immobilized; And
An adhesive plate on an edge of the upper substrate and a lower substrate such that a channel of the upper substrate and an electrode portion of the lower substrate intersect to form a reaction space;
A biosensor for detecting a target protease in the sample.
Wherein the proteolytic enzyme is matrix metalloproteinases (MMP).
Wherein the substrate metalloproteinase is MMP-2 or MMP-7.
Wherein the working electrode part is coated with gold (Au), and the reference electrode part is coated with platinum (Pt).
Wherein the upper substrate is a polydimethylsiloxane (PDMS) substrate.
Wherein the lower substrate is a glass substrate.
Wherein the upper substrate has a width of 25 to 35 mm and a length of 20 to 30 mm and the channel has a width of 600 to 800 μm and a height of 50 to 150 μm and an angle of 40 to 50 °.
Wherein the lower substrate has a width of 33 to 45 mm and a length of 20 to 30 mm, and the electrode portion has a width of 300 to 600 μm.
One inlet through which the electrolyte is injected is located at the farthest center of the outlet;
One inlet through which the substrate of the target protein protease is injected is located in the middle in each channel divided into two from the inlet of the electrolyte;
One inlet through which a sample is injected is located in each channel leading from the inlet of the substrate of the target protease; And
And one outlet is located at the center farthest from the injecting portion of the electrolyte in each channel leading from the injection port of the sample.
Wherein six reaction spaces in which one channel and one electrode intersect each other are formed.
2) A channel is formed in the channel forming region on the upper substrate, the channel including an inlet for injecting an electrolyte, an inlet for introducing a substrate of a target protease to be fixed to the electrode, an inlet for injecting a sample containing a target protease , A residue after the reaction and an outlet through which the electrolyte is discharged;
3) attaching an adhesive plate to an edge position on the lower substrate;
4) attaching an upper substrate to the adhesive board; And
5) injecting a substrate of a target protease into an injection port into which the substrate of the target protease is injected to fix the substrate of the target protease;
Wherein the target protein protease is present in the sample.
The electrode sub-printing of the lower substrate of step 1)
1) coating a photoresist on a glass wafer;
2) development of an electrode part in the shape of an electrode to be made;
3) depositing a metal film on the developed electrode using thermal evaporation; And
4) removing photoresist using acetone;
Wherein the biosensor is produced by a method comprising the steps of:
The channel formation of the upper substrate of step 2)
1) coating a photoresist on a silicon wafer;
2) patterning the channel with a channel shape to be formed; And
3) pouring the PDMS in liquid form onto the shaped channel and then baking in an oven, then separating the cured PDMS from the silicon wafer;
Wherein the biosensor is produced by a method comprising the steps of:
2) contacting the sample injected in the reaction space in the biosensor with a substrate of a target protein protease immobilized on the electrode unit;
3) measuring an electrochemical signal of the working electrode and the reference electrode by the reaction; And
4) measuring the impedance through the electrochemical signal;
A method for detecting a target protease in a sample.
Wherein the electrochemical signal of step 3) is measured using a potential meter.
Wherein the impedance of step 4) is measured using an impedance meter.
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