KR20060018698A - Plastic chip for enzyme assay in microfluidic channels and methods for enzyme activity measurement using thereof - Google Patents

Abstract

The present invention relates to a chip for analyzing enzyme activity using microfluidic channel technology, specifically, a microfluidic channel region (1) for dispensing a sample, a microfluidic channel region (3) for dilution of a reagent, and a reaction between a sample and a reagent. And it relates to an enzyme activity analysis chip comprising a module consisting of the reaction chamber site (2) where the measurement is performed. Enzyme activity analysis chip using the microfluidic channel of the present invention is capable of enzyme-substrate reaction at different substrate concentrations, and by measuring the reaction result by ELISA, enzyme kinetic analysis can be performed simply, and enzyme activity It can be useful for biochemical experiments using the analysis of or prediction of diseases.

Enzyme Activity Analysis, Microfluidic Channels, Chips

Description

Chip for enzyme activity analysis using microfluidic channel and method for measuring enzyme activity using same {{Plastic chip for enzyme assay in microfluidic channels and methods for enzyme activity measurement using             

1 is a plan view of one enzyme activity analysis module constituting the chip for enzyme activity analysis using the microfluidic channel of the present invention;

2 is a perspective view of one enzyme activity analysis module constituting the enzyme activity analysis chip,

3 is remind The overall configuration of the chip for enzyme activity analysis,

Figure 4 is a side view of the enzyme activity analysis chip,

5 is a graph comparing the concentration prediction and the actual experimental results of the production of different concentration of substrate solution in the six reaction chambers of the enzyme activity analysis chip,

Figure 6 is a conversion graph that can predict the enzyme activity value (unit / L) through the V _max value by the enzyme kinetic.

* Description of the symbols for the main parts of the drawings *

(1): microfluidic channel for delivering the same amount of enzyme sample for the same time to the inlet and reaction chamber of the enzyme sample.

(2): A reaction chamber in which a substrate solution having six different concentrations and an enzyme sample are mixed at the same time so that an enzyme-substrate reaction occurs and is measured.

(3): Part of the microfluidic channel network to produce different concentrations with the inlet of the substrate solution.

(4): Two injection ports into which high and low concentration substrate solutions are injected.

(5): Injection hole into which a high concentration of substrate solution is injected.

(6): Injection hole into which a low concentration substrate solution is injected.

(7): Injection hole into which an enzyme sample is injected.

(10): A chamber for controlling the amount and time to reach each reaction chamber at the time of injection of an enzyme sample.

(11) to (16): A reaction chamber in which a substrate solution and an enzyme sample having a value from 0 to 5 in sequence as the w value of formula (1) are mixed to cause an enzyme-substrate reaction and to be measured.

(41): Cross sectional view of an injection port into which an enzyme sample is injected and a delivery route through which a sample is delivered in a side view.

(42): Cross-sectional view of the inlet through which the substrate solution is injected and the delivery path through which the solution is delivered in a side view.

(43): The upper layer of the board of three stacked chips in which a microfluidic channel pattern for delivering an enzyme sample to the reaction chamber is present.

(44): The middle layer of the board | substrate of the three laminated chip | grains which has a micro channel pattern for delivering a substrate solution to a reaction chamber and carrying out multi-step concentration dilution.

(45): Lower layer of the board | substrate of the three laminated chip | tips which make the bottom surface of a microfluidic channel.

The present invention relates to a chip for analyzing enzyme activity using microfluidic channel technology, specifically, a microfluidic channel region (1) for dispensing a sample, a microfluidic channel region (3) for dilution of a reagent, and a reaction between a sample and a reagent. And it relates to an enzyme activity analysis chip comprising a module consisting of the reaction chamber site (2) where the measurement is performed.

As the human genome project is completed and the post-genome era arrives, a large amount of bioinformation is difficult to process quickly with existing laboratory analysis systems. In response to these trends, biological detection systems for the identification of life phenomena, drug development and diagnostics include micro-total analysis systems and lab-on-a-chip to analyze samples quickly, accurately and conveniently in smaller quantities. It is evolving in the form of a lab-on-a-chip. Lab-on-a-chip means 'the lab is placed on one chip' and is referred to as 'lab in chip' or 'lab on chip'. This usually involves the use of materials such as plastics, glass, etc. to create microchannels of less than a nanoliter (one billionth) liters through which the sample can move, resulting in small liquid samples (most biochemical samples). It exists in solution so that the existing experiment or research process can be performed quickly.

Research on how to implement a lab-on-a-chip has been around for a while. In order to analyze biochemical samples, most of the biochemical samples exist in a solution state, and thus, a technique for delivering a liquid sample is the most important factor. The research field for controlling the flow of microfluidics includes microfluidics ( microfluidics).

So far, microfluidic control techniques have been approached in a number of ways, including electroosmotic flow, membrane pumps, and syringe pumps, which electrically transfer small amounts of liquid samples. Etc.

The method using electroosmoric flow controls the microscopic sample flow without a pump or valve because it induces the flow of the solution by capillary electroosmotic by applying voltage across the microfluidic channel filled with the liquid sample and the buffer solution. However, the liquid sample delivery method using the electric field has a limitation in the design of a chip in which one or more microfluidic channels are complicated because the electric field uses the electric field to induce the flow of the fluid. Since it is affected by ionic strength and viscosity, it is difficult to apply universally to various samples.

On the other hand, liquid sample delivery using on-chip pumps such as membrane pumps is an essential technology for the ultimate lab-on-a-chip technology, but the manufacturing method is complicated, there is a limitation in mass production, and It is not suitable to use it as a fluid delivery method in the current lab-on-a-chip development stage because the reliability of the pump is inferior to the conventional pump (William L. Benard, et al., Journal of Microelectromechanical Systems, 7 (2); 251, 1998)

Syringe pump refers to a device that puts a sample in a syringe and pushes the syringe from behind using a motor to flow the sample at an appropriate flow rate. Connecting the syringe and the microchip is a teflon or silicon tube, which has different outer and inner diameters depending on the intended use. Among the fluid control technologies, the syringe pump has the advantage of being able to control the widest range of flow rates and having a relatively high reliability.

In addition, in order to apply the enzyme kinetics analysis method, the experimenter needs to separately prepare and inject a substrate solution having different concentration into the chip, which causes a problem of measurement error and cumbersome process ( Robert Scopes, Protein Purification; Principles and Practice, Springer-Verlag, 1982). In the present invention, a series of biochemical experiment preparations performed by an existing experimenter is replaced with a reaction in a microfluidic channel to provide a high reproducibility, a shorter time, and a convenient analysis method.

On the other hand, in developing a lab-on-a-chip, a part to be considered along with the chip design is a detection system that analyzes the result of the chip in which the reaction occurred.

In the case of chips developed by related companies such as Caliper, Tecan, and Gyros, the lab-on-a-chip has not been standardized, and most of them have a limitation in that a measurement system must be separately developed according to the chip design developed by the company. In addition, in the case of lab-on-a-chip measurement, an expensive optical measurement system is required, which limits the spread and utilization of the lab-on-a-chip. Recently, Nanostream is developing a microfluidic device that is compatible with existing measurement systems (Ken Rubenstein, "Microfluidics Second Generation Technologies Driving Commercial Applications", D & MD report, 2003).

Therefore, by combining the lab-on-a-chip with the enzyme kinetic analysis method, it will be possible to analyze the enzyme activity quickly and accurately. Including a module consisting of a channel part (3) and a reaction chamber part (2) where reaction and measurement of a sample and a reagent are carried out, an enzyme activity analysis chip capable of easily forming a substrate solution of different concentrations and reacting with an enzyme is provided. By developing and measuring the reaction result by ELISA, which is a standardized measurement system, it was confirmed that the enzyme activity analysis can be performed quickly and simply and completed the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a chip in which microfluidic channels are integrated, which enables reaction measurement in an existing measurement system in order to easily perform enzyme activity analysis required for biochemical experiments.

In order to achieve the above object, the present invention provides a chip for analyzing enzyme activity using a microfluidic channel.

The present invention also provides a method for measuring enzyme activity using an enzyme activity analysis chip using the microfluidic channel.

In addition, the present invention provides a method for predicting disease by measuring enzyme activity using the enzyme activity analysis chip using the microfluidic channel.

Hereinafter, the present invention will be described in detail.

The present invention provides a chip for analyzing enzyme activity using a microfluidic channel.

The chip for enzyme activity analysis using the microfluidic channel of the present invention comprises a microfluidic channel region (1) for dispensing a sample, a microfluidic channel region (3) for dilution of a reagent, and a reaction chamber in which reaction and measurement of a sample and a reagent are performed. A module consisting of the site 2.

The microfluidic channel portion 1 for dispensing the measurement sample of the chip of the present invention comprises a sample inlet 7 and a microfluidic channel for equally dispensing the injected sample into a plurality of reaction chambers.

The microfluidic channel portion 3 for dilution of the reaction reagent is composed of inlets 5, 6 of the reaction reagent solution for analysis and a multistage microfluidic channel network for forming different reagent concentrations for the analysis in the reaction chamber.

In addition, the reaction chamber site 2 where the reaction and measurement of the sample and the reagent is performed is a site where the reaction between the sample and the diluted substrate takes place and the reaction is measured, and is aligned with the well position of a conventional standardized well-plate. 1 and 2 .

The enzyme activity analysis chip using the microfluidic channel may be composed of a plurality of analysis modules capable of measuring several samples ( FIG. 3 ), and four enzyme activity analysis modules having the same size as a conventional well plate. It is preferable that it consists of.

In addition, the chip for analyzing enzyme activity using the microfluidic channel has a structure stacked in layers of a lower layer 45, an intermediate layer 44, and an upper layer 43 ( FIG. 4 ) for microfluidic delivery. The lower layer 45 is composed of a flat plastic layer without a microfluidic channel pattern, and the lower surface of the middle layer 44 has a microfluidic channel pattern for different concentrations of the substrate solution, and an enzyme is disposed on the lower surface of the upper layer 43. It has a microfluidic channel pattern structure for injection of a solution.

The enzyme solution sample inlet 41 and the microfluidic channel portion 1 for sample distribution are microfluidic channel portions designed and manufactured to deliver the same amount of enzyme sample to each reaction chamber 2 at the same time. The substrate solution delivery section also consists of a dilution microfluidic channel section 3 consisting of a high and low concentration substrate solution inlet 42 and a multistage microfluidic channel for obtaining six different concentrations of the substrate solution. The part of the reaction chamber 2 in which the enzyme-substrate reaction takes place and is measured has a form in which the upper layer is open to the outside.

The substrate solution inlet of the reaction fluid dilution microfluidic channel region 3 consisting of multistage microfluidic channels consists of two inlets. One side (5) injects a high concentration substrate solution and the other (6) injects a low concentration substrate solution. Substrate solution passed through the dilution microfluidic channel section (3) consisting of multistage microfluidic channels for substrate solution delivery is finally stored in the six reaction chambers (11)-(16) for enzyme-substrate reactions. . The enzyme sample sample injected for enzyme activity analysis is injected through the enzyme sample inlet (7) and generates an enzyme-substrate reaction in the reaction chambers (11)-(16) and is optically measured by an ELISA meter. The chamber 10 here is a control for equal distribution of the sample. At this time, the concentration of the substrate solution gathered in the six reaction chambers must be known so that the enzyme activity can be analyzed by the Micheles-Menten relationship. Assuming that the concentration of the substrate solution injected into the substrate solution inlets (5) and (6) is C ₁ and C ₂ , the concentration in each reaction chamber (11)-(16) is determined by the following equation.

식(1)

Formula (1)

Where N represents the number of stages of the microfluidic channel from the substrate solution inlets (5), (6) to the reaction chamber (2) and has an integer. The chip model according to the present invention has an N value of 5 since the number of stages of the microfluidic channel required to set six substrate solutions of the reaction chamber 2 is five. i is a variable having a value starting at 0 and increasing by 1 toward the right of the leftmost chamber of the part of the dilution microfluidic channel portion 3 consisting of multistage microfluidic channels for substrate solution delivery.

In the chip design according to the present invention, the reaction chamber 11 has a value of 0, the reaction chamber 12 has a value of 1, 13 is 2, and in this manner, the reaction chamber 16 has a value of 5. In this way, all necessary parameters are defined and substituted in Equation (1) to obtain the concentration of the substrate solution in each reaction chamber. By comparing the experimentally predicted and actual experimental values, the reliability of the microfluidic channel system for different concentrations of the substrate solution can be determined. 5 is a graph comparing the predicted concentration mentioned above with actual experimentally measured values.

The number of different concentrations of the substrate solution generated at the microfluidic channel site for dilution of the reaction reagent is determined as N + 1 by the number (N) of the microfluidic channel network from the substrate solution inlet to the reaction chamber. The number of and reaction chambers can be changed as needed.

The material for preparing the chip of the present invention is polydimethylsiloxane, polymethylmethacrylate, polyacrylates, polycarbonates, polycyclic olefins, poly Various polymer materials such as polyimides and polyurethanes may be used.

The present invention also provides a method for measuring enzyme activity using an enzyme activity analysis chip using the microfluidic channel.

Enzyme activity measurement methods that have been performed in conventional biochemical experiments include a stop method and an enzyme kinetics analysis method.

Among them, the stop method is a method of determining the enzyme activity by measuring the amount of enzymatic conversion to the final product using a substrate for a given time. The advantage of simplicity of experiment and data processing is the time when the enzyme-substrate reaction occurs. Because of the color of the long, pre-reacted enzyme sample itself, the absorbance values before the reaction are included in the absorbance values of the final product, which is disadvantageous in terms of cost and complexity, requiring a large amount of reagents for analysis.

Enzyme kinetics, on the other hand, has the advantage of complex analysis, but takes less time and does not require absorbance correction because it measures the initial reaction rate. However, even when using the enzyme kinetic method, it is necessary to measure the initial reaction rate (V _o ) of the enzyme reaction in substrate solutions having different concentrations. There is a hassle to prepare yourself, the error that occurs according to the skill of the experimenter in this process has a problem that can affect the final enzyme activity analysis results.

Therefore, by using the enzyme activity chip of the present invention can solve the problems of the enzyme kinetic analysis method described above it is possible to measure the enzyme activity more simply and quickly.

In the embodiment of the present invention, in order to measure the enzyme activity by using the enzyme activity analysis chip of the present invention, the enzyme solution is injected into the inlet (7) of the microfluidic channel portion for dispensing the sample and the substrate solution is multistage microfluidic. It was injected into the inlets (5, 6) of the microfluidic channel portion for dilution of the reaction reagent consisting of the channel to cause the enzyme-substrate reaction in the reaction chamber (2). Using at the same time as the reaction of the absorbance values measured in ELISA to obtain the initial reaction velocity V _o Hans-draw activity graph (Fig. 6) for the next V _max obtained for V _max by Wolf expression found out the activity of the enzyme solution of unknown . As a result of comparing the predicted value with the experimental value found by the conventional stop method, it showed a small error value within 10.5%, and thus the usefulness of the enzyme activity analysis method using the chip of the present invention was confirmed.

In addition, the present invention provides a method for predicting disease by measuring enzyme activity using the enzyme activity analysis chip using the microfluidic channel.

Alkaline phosphatase is an enzyme present in the liver, bile ducts, intestines, bones, kidneys, placenta, and leukocytes, which produce slightly different isoenzymes for each organ. An increase in the concentration of the enzyme is observed. Therefore, in this case, by analyzing the homozygous enzyme of the enzyme, to determine which organ the amount of alkaline dephosphorase produced from it is increased and the test for the organ.

In the embodiment of the present invention, the enzyme activity analysis chip was used to observe that the activity of the enzyme was significantly increased in the blood of cancer patients, and the chip was found to be useful for predicting diseases such as cancer. .

In the present invention, by implementing different substrate concentrations on the plastic chip, the experimenter can directly reduce the sequence of preparing samples of different concentrations and reduce errors that may occur in the process. Enzyme activity analysis was carried out by kinetics to reduce the overall analysis time, and the same starting point was measured by allowing the enzyme-substrate reaction to be measured simultaneously in six measurement wells. It was designed to increase the reliability of the measurement results. In addition, in the present invention, the size of the plastic chip is normalized to be the same as a well plate, and the position of the reaction chamber in the plastic chip in which the enzymatic reaction occurs is arranged to match the existing detection system.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1> Fabrication of enzyme activity analysis chip using microfluidic channel

The enzyme activity analysis chip of the present invention is composed of four enzyme activity analysis modules (module), as shown in Figure 1 , each module is a substrate dilution consisting of enzyme sample injection unit (1), multi-stage microfluidic channel It consists of the reaction microfluidic channel region 3 and the reaction chamber region 2 in which the reaction between the enzyme sample and the diluted substrate takes place and measured simultaneously, and has a structure in which three layers are stacked.

The chip of the present invention was produced by a molding method using a transparent and elastic polydimethylsiloxane (hereinafter referred to as "PDMS") material. The mold for molding uses a method of manufacturing a micro pattern having a width of 100 μm and a height of 50 μm by using SU-8 as a photoresist on a silicon wafer substrate. It was. The mold was poured with pre-PDMS mixed with a curing agent in a ratio of 10: 1 and cured at 80 ° C. for 2 hours. Punching the PDMS substrate through the curing step to prepare a substrate and the enzyme sample injection port and each reaction chamber, and oxidized each PDMS substrate using an air plasma (laminated by lamination) to stack the enzyme activity analysis chip consisting of three layers Completed.

< Example  2> Enzyme Activity Analysis Using Chip Using Microfluidic Channel

Enzyme activity of alkaline phosphatase was analyzed by the following method using the enzyme activity analysis chip prepared in <Example 1> .

Substrate solutions C1 (3.0 mM p-nitrophenyl phosphate) and C2 (56.8 mM p-nitrophenyl phosphate) solutions were injected into the inlets (5) and (6) of the microfluidic channel region for substrate dilution, respectively. In Equation (1), N is the number of stages of the microfluidic channel network from the substrate solution inlet to the reaction chamber, so that the concentration [S] of the substrate solution stored in each reaction chamber is 3.00 mM ( i = 0), Chamber (9) is 14.76 mM ( i = 1), (10) is 24.52 mM ( i = 2), (11) is 35.28 mM ( i = 3), (12) is 46.04 mM ( i = 4) and ( 13) is 56.80 mM ( i = 5).

식(1)

Formula (1)

Enzyme-substrate reaction occurs in the reaction chamber by injecting the enzyme sample solution into the sample inlet (7) of the microfluidic channel for sample distribution. By measuring the reaction rate (V _o ) for 1 minute of the initial reaction, V _max was determined by the Michelis-Menton relation. The Michelis-Menton relationship is

식(2)

Formula (2)

At this time, since the V _max value is directly obtained from the Michelis-Menton relation rather than the linear function, there is a problem of generating a large error value. Therefore, in order to obtain the V _max of the smaller error range, Lineweaver-Burk formula, Edie-Hofstee formula and Hans-Woolf formula) can be used. The inverse of the slope by substituting the initial concentration (V _o ) and the substrate concentration value (S) obtained experimentally by the Hans-Wolf equation (Eq. (3)) reported as the safest equation through computer simulation. The V _max value was obtained by taking.

식(3)

Formula (3)

On the other hand, after obtaining the activity (unit, L) value of each enzyme sample using the conventional stop method and performing enzyme kinetic analysis using the chip of the present invention to obtain the V _max (μmol / min) value, V _max (μmol / min) values for each activity (unit / L) were graphed to obtain a conversion plot between enzyme activity and V _max ( FIG. 5 ). When V depletion of alkaline dephosphorase enzyme with unknown concentration is performed on a chip to obtain V _max by enzyme kinetic analysis, the conversion graph can finally determine the unknown enzyme activity value (unit / L). Was within 10.5%.

< Example  3> Disease prediction using chips using microfluidic channels

The enzyme activity analysis chip using the microfluidic channel of the present invention was used to measure the activity of alkaline phosphatase present in the blood and based on the fact that the activity of the enzyme is related to various diseases of the human body. Applied to the prediction.

The procedure of enzyme activity analysis using the chip of the present invention is as described in < Example 2> , except that the enzyme sample is a serum of the blood (serum), not an alkaline dephosphorase for biochemical experiments.

Specifically, alkaline dephosphatase activity (mUnit / mL) was measured from the blood of renal cell carcinoma, melanoma and other carcinoma patients and used to predict disease. As a result, alkaline dephosphorase activity (mUnit / mL) values in serum of renal cell carcinoma, melanoma and other carcinoma patients were 9.84 ± 5.44 (minimum value 2.13- maximum value 22.62; n = 7), respectively; 5.15 ± 2.38 (min 1.62-max 8.12; n = 5) and 3.52 ± 1.41 (min 1.62-max 6.75; n = 6).

Considering that the activity of alkaline dephosphorase in blood of normal people was in the range of 0-1.0 mUnit / mL, it can be seen that the activity of alkaline dephosphorase in blood of cancer patients was significantly increased. Therefore, by using the enzyme activity analysis chip of the present invention, it is possible to predict diseases such as cancer by measuring the activity of alkaline dephosphorylase.

The present invention relates to a chip for analyzing enzyme activity using microfluidic channel technology. The chip of the present invention adopts a standard compatible with a measurement system that has been standardized and used as in the case of a conventional 96-well microplate. It can be used without a separate optical measuring device and has the expandability to increase the number of analysis modules included in one chip to fit a 384 well, 1536 well plate measuring device. Bio-analysis technology using such a chip can be used not only for detecting enzyme reactions, but also for various applications such as predictive protein chips, immunoassay chips, and ELISA assay chips.

Claims (13)

Chip for enzyme activity analysis using microfluidic channel. 2. The chip of claim 1, wherein the chip comprises a microfluidic channel portion (1) for dispensing a sample, a microfluidic channel portion (3) for dilution of a reagent, and a reaction chamber portion where reaction and measurement of a sample and a reagent are performed. Chip comprising a module consisting of (2). 3. The microfluidic channel of claim 2, wherein the microfluidic channel portion 1 for dispensing the measurement sample of the chip distributes the same amount of the sample inlet 7 and the unknown sample into the plurality of reaction chambers. Chip, characterized in that consisting of. The method of claim 2, wherein the microfluidic channel portion 3 for dilution of the reaction reagent of the chip comprises a multistage microfluidic channel for forming different reaction reagent concentrations in the reaction chamber with the reaction reagent solution inlets 5 and 6. Featured chip. 3. The chip as claimed in claim 2, wherein the reaction chamber portion (2) of the chip is configured to align with the well position of an existing standardized well-plate. The chip of claim 2, wherein the module of the chip is a plurality of analysis modules capable of measuring a plurality of samples, respectively. The chip of claim 2, wherein the chip has a structure in which a layer is formed of a lower layer, an intermediate layer, and an upper layer. 8. The chip of claim 7, wherein the layer of the chip has a microfluidic channel pattern structure for microfluidic delivery. 5. The method according to claim 4, wherein the number of concentrations of different substrate solutions generated in the reaction fluid dilution microfluidic channel region (2) is determined by the number N + of the microfluidic channel network from the substrate solution inlet to the reaction chamber. A chip characterized by being determined by one. According to claim 1, The chip material is polydimethylsiloxane (polydimethylsiloxane), polymethylmethacrylate (polymethylmethacrylate), polyacrylates (polyacrylates), polycarbonates (polycarbonates), polycyclic olefins (polycyclic olefins), Chips, characterized in that selected from the group consisting of polyimides and polyurethanes (polyurethanes). Enzyme activity measurement method using the enzyme activity analysis chip using the microfluidic channel of claim 1. The method of claim 11, wherein the measuring system of the enzyme activity measuring method is an enzyme-linked immunosorbent assay (ELISA). The method of predicting disease by measuring the enzyme activity using the chip of claim 1.

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