KR100921237B1 - High Sensitivity Measurement Method of C-Reactive Protein Using Crystal Oscillator Immunosensitivity Sensor and C-Reactive Immune Sensor - Google Patents

Abstract

The present invention provides a crystal oscillator immunosensor measuring device and a crystal oscillator immunosensor for measuring C-reactive protein which can measure C-reactive protein which is one of biomarkers. It relates to a high sensitivity measurement method.

Biomarkers, C-Reactive Proteins, Crystal Oscillators, Immune Sensors

Description

Cart-reactive protein detection and high-sensitivity detection method for C-reactive protein using crystal oscillator immunosensor measuring device for C-reactive protein measurement and C-reactive protein using crystal oscillator immunosensor using quartz crystal microbalance immunosensor}

The present invention provides a crystal oscillator immunosensor measuring device and a crystal oscillator immunosensor for measuring C-reactive protein which can measure C-reactive protein which is one of biomarkers. It relates to a high sensitivity measurement method.

With the recent entry of the Health Functional Food Act, the door to manufacture food ingredients and processed foods based on natural products in Korea and to be recognized as health functional foods has been opened. However, to this end, it is necessary to present data that scientifically demonstrate the functional functionality of health functional foods, such as cardiovascular disease prevention, anti-aging, cancer prevention, obesity prevention, immune regulation, and gastrointestinal disease prevention. Therefore, the importance of food functional evaluation is increasing, and as a result, the possibility of commercialization of functional evaluation field is very high.

The functional evaluation of food includes in vitro and in vivo functional evaluation and clinical trials centering on the above six functional types.

The pattern of disease is changing from infectious disease to chronic degenerative disease all over the world due to the improvement of living standard, diversification of lifestyle, decrease of activity and overnutrition. The Science has reported that the main causes of modern death are cancer and cardiovascular diseases caused by smoking and obesity, and The Nature cited that about 50% of adult deaths are due to cardiovascular disease. have. Therefore, from the perspective of the food industry, the necessity of food development to prevent cardiovascular diseases is gradually increasing. For this purpose, a search method for efficiently and quickly performing cardiovascular functional evaluation of food materials and functional foods, especially in vivo search The need for law development is increasing.

One new way to detect cardiovascular functionality in food and functional foods in vivo is to measure the effects of food intake on blood changes in cardiovascular biomarkers in animal models. At this time, food functionalities related to cardiovascular disease prevention can be classified into four categories: coronary artery disease improvement, blood pressure drop, blood clot, and arteriosclerosis improvement, and related biomarkers are C-reactive protein. In addition, low density lipoprotein (LDL), fibrinogen (fibrinogen), angiotensin II (angiotensin II), cardiac troponin (cardiac troponin) and the like are attracting attention.

Among the biomarkers, C-reactive protein is known as one of the major indicators for coronary artery disease, hypertension, and the like by interleukin-6 and interleukin-1beta in mammalian liver. Pentamer protein with molecular weight of 118 kilodaltons (kDa) synthesized by induced stimulation ( Biosensors and Bioelectronics , Vol. 19, p. 1193, 2004; Clinical Chemistry , Vol. 47, p. 403, 2001; New England Journal of Medicine , Vol. 340, p. 448, 1999; Biochemical Journal , Vol. 327, p. 425, 1997).

In the past, enzyme-linked immunosorbent assay (ELISA) has been mainly used to measure C-reactive protein. The method is well established and the sensitivity of the protein is known to be very good ( American Journal of Cardiology). , Vol. 1, p. 155, 2005; American Journal of Veterinary Research , Vol. 1, p. 62, 2005; Journal of Clinical Laboratory Analysis , Vol. 18, p. 280, 2004).

However, as in all other photometric methods, the reaction signals obtained by enzyme-immunoassays may be inhibited by colorants present in various samples, such as reaction solutions, blood, and saliva. ( Sensors and Actuators B: Chemical , Vol. 121, p. 606, 2007; Biosensors and Bioelectronics , Vol. 11, p. 881, 1996) By addressing this, the C-reactive protein can be There is a need for the development of new measurement methods that can be easily and sensitively analyzed.

As described above, the conventional method for measuring C-reactive protein may show measurement inhibition and complex analysis process, resulting in low efficiency and economical analysis kit for C-reactive protein per kit. It is expensive, costing hundreds of thousands of won, and consequently making it difficult to routinely measure C-reactive proteins, one of the biomarkers that assess food functionality. Therefore, it is possible to improve the high sensitivity of C-reactive protein which can be measured easily and quickly and minimize the measurement inhibition by the effect of food and functional food supplementation on the concentration change of C-reactive protein in blood of laboratory animals. The development of new field-adaptive measurement technology is critical, and the need for this has been constantly raised.

The present invention provides a crystal oscillator immunosensor measuring device and a crystal oscillator immunosensor for measuring C-reactive protein which can measure C-reactive protein which is one of biomarkers. It is intended to provide a high sensitivity measurement method.

The crystal oscillator immunosensor measuring device for measuring a biomarker of the present invention immobilizes an antibody against C-reactive protein on a quartz crystal microbalance sensor chip, which is one of biosensor transducers, to measure biomarkers with high sensitivity. Instead, immobilized C-reactive protein, which is an antigen and a target of measurement, is added to a sample containing a predetermined concentration of antibodies to the biomarker and C-reactive protein by concentration, thereby causing a competitive immune response. The sensitivity of the crystal oscillator immunosensor to measure reactive protein dramatically improves the crystal oscillator immunosensor measuring device and crystal oscillator immunity for C-reactive protein measurement with limit of detection (LOD) of picomolar (pM) concentration level. To provide a high sensitivity measurement method of C-reactive protein using a sensor.

The present invention is a C-reactive protein known as one of the major biomarkers for coronary artery disease, hypertension and inflammation can be measured at a world-class sensitivity using a crystal oscillator immunosensor, one of the unlabeled immune sensors In vivo food functionality can be quickly assessed in real time using the corresponding biomarkers.

In addition to the C-reactive protein, the present invention can establish a basic technology related to food functional evaluation required for high-speed simultaneous measurement of biomarkers existing in various concentration ranges in the blood of experimental animals such as ELDL, fibrinogen, and angiotensin II in the future. have.

The present invention shows a crystal oscillator immunosensor measuring device for measuring C-reactive protein.

The crystal oscillator immunosensor measuring device for measuring the C-reactive protein of the present invention is shown in FIG.

The crystal oscillator immunosensor measuring device for measuring the C-reactive protein of the present invention will be described with reference to FIG.

Crystal oscillator immunosensor measuring device for measuring C-reactive protein of the present invention is a reaction buffer solution tank (1),

A supply means (2) connected to the reaction buffer solution tank (1) for supplying the reaction buffer solution in the reaction buffer solution tank to the C-reactive protein immobilized on the crystal oscillator sensor chip inside the reaction cell (4) ),

A sample containing a reaction buffer solution, a C-reactive protein antibody, and a C-reactive protein, which is connected to the supply means 2 and supplied from the supply means 2, to a crystal oscillator sensor chip inside the reaction cell 4. Sample inlet for supplying immobilized C-reactive protein (3),

A sample containing a reaction buffer solution, a C-reactive protein antibody and a C-reactive protein is supplied through the sample inlet (3), and a crystal oscillator (7) including a sensor chip of an electrode on which an antigen, C-reactive protein, is immobilized. A reaction cell 4 provided with an O-ring 6 for protecting the crystal oscillator sensor chip and a holder 5 connected to the joint 8 while surrounding the crystal oscillator 7 and the O-ring 6;

An oscillation module 10 connected to the crystal oscillator 7 for vibrating the crystal oscillator inside the reaction cell 4 and transmitting the vibration of the crystal oscillator to the vibration measuring device 11,

Vibration measuring device 11 for measuring the vibration of the crystal oscillator (7) in the reaction cell (4) through the oscillation module 10,

A computer 12 connected to the vibrometer 11 and indicating a frequency change generated when the C-reactive protein immobilized on the sensor chip electrode of the crystal vibrator 7 binds to the C-reactive protein antibody;

And a waste solution tank 9 for collecting the reaction buffer solution supplied into the reaction cell 4 and the waste solution of a sample including the C-reactive protein antibody and the C-reactive protein.

The reaction buffer solution is used to obtain the steady-state frequency for measuring the C-reactive protein using the crystal oscillator immunosensor measuring device for C-reactive protein measurement of the present invention.

The reaction buffer solution may be used a phosphate buffer solution of 0.05 to 0.15 molarity (M).

The reaction buffer solution may be used a phosphate buffer solution having a pH of 7.0.

The reaction buffer solution is 0.05 to 0.15 molarity (M), pH phosphate buffer solution may be used.

In the above, the supply means 2 may use a pump.

The supply means 2 may be a micro-dispensing pump (micro-dispensing pump).

The reaction buffer solution tank, the supply means, the sample injection port, the reaction cell, the waste solution tank may be connected to each other through a tube through which the reaction buffer solution flows.

The reaction buffer solution tank, the supply means, the sample injection port, the reaction cell, the waste tank may be connected to the tube (tube) through which the reaction buffer solution flows.

The reaction buffer solution tank, the supply means, the sample injection port, the reaction cell, the waste solution tank may be connected to a capillary tube through which the reaction buffer solution flows.

In the sample inlet (3) is a reaction solution capable of reacting with the C-reactive protein, which is an antigen immobilized on the crystal oscillator inside the reaction cell (4) is supplied with a sample containing a C-reactive protein antibody and C-reactive protein do.

The loop capacity of the sample inlet 3 may be 20 to 500 microliters (μl).

Reaction cell (4) is provided with a holder (5) connected by a joint (8), the crystal oscillator (7) sensor chip is immobilized in the center of the holder (5) the antigen C-reactive protein is immobilized In addition, the crystal oscillator sensor chip has an electrode to which the C-reactive protein is immobilized, and an O-ring 6 for preventing leakage of the reaction liquid around the electrode.

The crystal oscillator sensor chip may use an AT-cut, and a fundamental resonant frequency is 8 to 10 MHz.

The nominal capacity inside the reaction cell may be 100 to 200 microliters (μl).

The present invention shows a high sensitivity measurement method of C-reactive protein using a crystal oscillator immunosensor.

The present invention can provide a high sensitivity measurement method of C-reactive protein using a measuring device comprising a crystal oscillator immune sensor.

The present invention can provide a high sensitivity measurement method of C-reactive protein using a measuring device including a crystal oscillator immune sensor as shown in FIG.

In the present invention, in the high sensitivity measurement of C-reactive protein using a crystal oscillator immunosensor, (1) the frequency at steady state by adding a reaction buffer solution to the crystal oscillator sensor chip to which the C-reactive protein as an antigen is immobilized (F1) Getting it,

(2) After the step (1), a mixture of C-reactive protein antibody and a sample containing C-reactive protein is added to the crystal oscillator sensor chip to which the antigen C-reactive protein is immobilized, and then, in a steady state after a competitive immunity reaction occurs. Obtaining the frequency F2 of

(3) Hydrostatic oscillator immunity, characterized in that it comprises the step of measuring the frequency change (ΔF = F1-F2) from the frequency (F1) obtained in step (1) and the frequency (F2) obtained in step (2) A high sensitivity measurement method of C-reactive protein using a sensor is shown.

Immobilization of the C-reactive protein as an antigen in the crystal oscillator sensor chip can be carried out using the following steps (a) to (d).

(a) adding a phosphate buffer solution to the C-reactive protein to obtain a phosphate buffer solution containing the C-reactive protein, (b) sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido ] Reacting an aqueous solution of sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido] hexanoate with the phosphate buffer solution of step (a), (c) dithio to the solution obtained in step (b) Reacting by adding a dithiothreitol solution, (d) applying the solution obtained in step (c) to the electrode surface of the crystal oscillator sensor chip and dried.

Immobilization of the C-reactive protein as an antigen in the crystal oscillator sensor chip can be carried out using the following steps (a) to (d).

(A) adding a phosphate buffer solution to the C-reactive protein to obtain a phosphate buffer solution with a concentration of 0.5-2.5 mg / ml C-reactive protein, (b) 10 ~ 30mM sulfosuccinimidyl 6- [3 10 to 50 µl of {2-pyridyldithio} propionamido] hexanoate aqueous solution with 10 to 50 µl of the phosphate buffer solution of step (a) for 30 to 90 minutes, (c) the (b) 10-30 μl of dithiothreitol solution was added to the solution obtained in the step, and reacted for 10 to 50 minutes. (D) 10-15 μl of the solution obtained in step (C) was applied to the electrode surface of the crystal oscillator sensor chip. Drying at room temperature for 30 to 90 minutes and washing with distilled water and 0.05 to 0.15 molar phosphate buffer solution 3 to 7 times.

In the above reaction buffer solution, a phosphate buffer solution of 0.05 to 0.15 molarity (M) may be used.

The reaction buffer solution may be used a phosphate buffer solution having a pH of 7.0.

In the above reaction buffer solution is 0.05 ~ 0.15 molarity (M), the pH may be used a phosphate buffer solution of 7.0.

The frequency (F1) of the step (1) in the above can be measured by the addition of the reaction buffer solution to the crystal oscillator sensor chip C-reactive protein is immobilized after 5-20 minutes to measure the frequency (F1) in the steady state.

In the frequency (F2) measurement of step (2) above, a competitive immunization reaction occurs by adding a mixture of a sample containing C-reactive protein and C-reactive protein to a crystal vibrator sensor chip on which C-reactive protein is immobilized. After 20 minutes, the frequency F2 at the steady state can be measured.

As the mixture of the sample containing the C-reactive protein and the C-reactive protein, a sample containing the C-reactive protein and the C-reactive protein may be used in a volume ratio of 1: 9 to 9: 1. .

The C-reactive protein antibody may be any one selected from antibodies prepared using C-reactive protein of rats, mice, rabbits, monkeys or human mammals. At this time, the preparation of the C-reactive protein antibody using C-reactive protein of rat, mouse, rabbit, monkey or human mammal is carried out by a person of ordinary skill in the art. As it can be done, the detailed description thereof will be omitted.

The C-reactive protein antibody in the above can be used as a commercially available product, as an example of such a commercialized C-reactive protein antibody monoclonal C-reactive protein antibody of R & D Systems (Minneapolis, MN, USA) Can be used.

The sample for measuring C-reactive protein is selected from tissue extracts such as standard solution, C-reactive protein dissolved in the reaction buffer solution, blood, blood serum, blood plasma, saliva, body fluid, liver, etc. Either one can be used.

The present invention represents a crystal oscillator immunosensor measuring apparatus including a reaction buffer solution and a waste solution tank, a microdispensing pump, a sample inlet, a reaction cell, an oscillation module, a frequency meter, and a personal computer as a device for measuring C-reactive protein. .

In addition, the present invention shows a method for measuring C-reactive protein using a crystal oscillator immunosensor measuring device, for example, a crystal oscillator immunosensor measuring device that can be shown in FIG.

C-reactive protein measurement method using the crystal oscillator immunosensor measuring device of the present invention comprises the steps of immobilizing the C-reactive protein as an antigen to the sensor chip electrode of the crystal oscillator inside the reaction cell; Significantly enhance the sensitivity of the fertilizer's immune sensor by inducing a competitive immune response by adding a sample containing an optimized C-reactive protein antibody and concentration-specific C-reactive protein to an immobilized crystal vibrator sensor chip with an optimized concentration of antigen It provides a method for high sensitivity measurement of C-reactive protein, one of the major biomarker proteins required for food functional evaluation, including the step of making.

After immobilizing the C-reactive protein as an antigen on the sensor chip electrode of the crystal oscillator inside the reaction cell, the C-reactive protein as the antigen is immobilized on the crystal oscillator sensor chip mounted on the reaction cell by concentration and the C-reactivity The method may further include optimizing the concentration of the C-reactive protein used for immobilization and the C-reactive protein antibody added to the reaction cell by measuring the sensor signal according to the binding of the antibody while adding the protein antibody by concentration.

The inventors of the present invention have examined the method of measuring high sensitivity of C-reactive protein, which is a biomarker protein related to cardiovascular food functional evaluation, using the piezoelectric properties of a crystal oscillator. Using a crystal oscillator immunosensor measuring device that can be displayed, and immobilized C-reactive protein as an antigen as an optimized concentration for a crystal oscillator sensor chip, mounted in a reaction cell, and optimized concentration of C-reactive protein antibody and concentration The fact that C-reactive protein can be measured at picomol level without labeling differently from enzyme-immunoassay using labeling enzyme by adding a mixture of specimens containing C-reactive protein to produce a competitive immunity. Found. As a result of intensive research from these facts, we use a continuous crystal oscillator immunosensor measurement system that can measure C-reactive protein with high sensitivity and is widely applicable to high sensitivity measurement of other food functional biomarkers. Immobilized C-reactive protein as an antigen at an optimized concentration in the crystal oscillator sensor chip and non-labeling competitive immune response by adding a mixture of an optimized concentration of C-reactive protein antibody and a sample containing concentration-specific C-reactive protein The present invention has been completed, which is the main feature of causing.

Other food functional biomarkers above are related to cardiovascular biomarkers such as LDL, fibrinogen, angiotensin II, cardiac troponin, aging inhibitors, cancer prevention, obesity prevention, immunomodulation, gastrointestinal disease prevention, etc. Biomarker proteins can be targeted.

Hereinafter, the present invention will be described in more detail step by step.

The present invention comprises a continuous crystal oscillator immunosensor measuring system having the configuration of FIG. 1 to be mounted on the reaction cell to measure C-reactive protein, which is one of the major biomarkers required for evaluating cardiovascular food functionalities, with high sensitivity. Instead of immobilizing the C-reactive protein antibody on the crystal oscillator sensor chip, the degree of immune response between the antigen and the antibody can be greatly increased by immobilizing the C-reactive protein as an antigen. Next, C is prepared by adding a mixture of optimized concentrations of C-reactive protein antibodies and samples containing concentration-specific C-reactive proteins to the immobilized crystal oscillator sensor chip. Reactive protein can be measured with high sensitivity.

(i) Immobilization of C-Reactive Protein as Antigen

Immobilization of the C-reactive protein as an antigen to the crystal oscillator sensor chip mounted in the reaction cell of the crystal oscillator immunosensor measuring device as shown in FIG. 1 can be carried out as follows (1) to (3).

(1) Be careful not to touch the surface of the gold electrode, the sensitive area of the crystal oscillator sensor chip inside the reaction cell, and soak it in caustic soda, wash with distilled water, immerse in hydrochloric acid, wash with distilled water, treat with hydrochloric acid and wash with distilled water. After drying. For example, be careful not to touch the electrode wire on the surface of the gold electrode, which is the sensitive region of the crystal oscillator sensor chip inside the reaction cell, and use 3 to 7 moles of hydrochloric acid at 0.8 to 1.5 molar concentration (M) and 0.8 to 1.5 molar concentration (M). Dip sequentially for a minute and wash with distilled water in between. Thereafter, concentrated hydrochloric acid having a pH of 0.5-1.0 may be treated for 0.5-1.5 minutes, washed again with distilled water, and dried in a convection oven for 10-30 minutes.

(2) To recover the C-reactive protein in the freeze-dried state, the solution was added with 0.05 to 0.15 molar phosphate buffer solution (phosphate buffer saline, PBS, containing 0.05 to 0.15 molar sodium chloride) and recovered as a solution. Obtain a phosphate buffer solution with a concentration of 0.5-2.5 mg / ml. Then, 10 to 30 mmol of sulfosuccinimidyl 6- [3- {2- dissolved in distilled water in 10 to 50 µl of a phosphate buffer solution containing 0.5 to 2.5 mg / ml of the C-reactive protein. 10-50 μl of pyridyldithio} propionamido] hexanoate (sulfosuccinimidyl 6- [3- {2-pyridyldithio} -propionamido] hexanoate solution was added with a thiolation cross-linker for 30-90 minutes. React. Thereafter, 10 to 30 µl of a dithiothreitol solution is treated for 10 to 50 minutes in order to reduce the disulfide bond of the C-reactive protein in the reaction solution to expose the thiol group.

(3) 5 to 15 µl of the solution obtained in (2) are evenly applied to the electrode surface of the crystal oscillator sensor chip of (1), dried at room temperature for 30 to 90 minutes, and distilled water and reaction buffer solution 0.05 to 0.15 Rinse with a molar concentration of phosphate buffer solution (pH 7.0) three to seven times in succession.

(ii) Evaluation of signal characteristics of crystal oscillator immunosensor measuring device

In order for the crystal oscillator immune sensor of the present invention to be developed as a field and workstation device for a food functional biomarker, it is necessary to secure the stability of the sensor signal and minimize the measurement time. To configure the immune sensor measurement system and to evaluate its signal characteristics, the specific method is as follows.

Operation of the crystal oscillator immunosensor measuring device as shown in FIG. 1 is a reaction buffer solution tank (1) connected to a capillary tube by a microdispensing pump (2) supplying a reaction buffer solution stored in the reaction buffer solution tank (1) And flow through the fine dispensing pump (2), the sample inlet (3), and the reaction cell (4) to the waste tank (9). At this time, the flow rate of the reaction buffer solution is 20-230 μl / min. The loop volume of the sample inlet 3 is 20 to 500 µl. When electromagnetic force is applied through the oscillation module 10 during the operation of the crystal oscillator immunosensor measuring device, the crystal oscillator sensor chip in which the C-reactive protein is immobilized by the process (i) is 2-10 minutes later. The oscillation is caused by the resonance frequency (F ₁ ) in the steady-state according to the flow, and when the sample containing the C-reactive protein antibody is injected, the mass accumulation of the surface of the sensor chip due to the immune reaction occurs. As the frequency of the chip decreases, the frequency change starts to appear, and after 2 to 20 minutes, it reaches the steady state resonance frequency (F ₂ ), from which the frequency shift (ΔF) corresponding to the sensor response can be calculated (ΔF = F ₁ -F ₂ ). The sensor signal according to time during the operation of the crystal oscillator immunosensor measurement system is measured in real time by the frequency measuring device 11 and then displayed on the personal computer 12 as an image.

In order to determine the specificity, stability, and measurement time of the sensor signal when the crystal oscillator immunosensor measuring device is operated, a small amount of C-reactive protein is added to the crystal oscillator sensor chip inside the reaction cell of the crystal oscillator immunosensor measuring device of FIG. 1. The serum albumin was immobilized at a concentration of 2 mg / ml and the monoclonal C-reactive protein antibody of R & D Systems (Minneapolis, MN, USA) was immobilized on the crystal oscillator sensor chip by adding 250 μg / ml of the antibody through the sample inlet. As a result of measuring the binding degree of the above-mentioned C-reactive protein antibody to bovine serum albumin, as shown by reference numeral 13 of FIG. . In addition, C-reactive protein of 2 mg / ml concentration instead of bovine serum albumin is immobilized on the crystal vibrator sensor chip of the crystal oscillator immunosensor measuring device of FIG. When the antibody was added through the sample inlet at a concentration of 250 µg / ml, the resonance frequency change pattern was very stable over time as a result of the immune response, and the time required for obtaining a steady state sensor signal was 3 to 5 It can be confirmed that the continuous crystal oscillator immunosensor measuring device configured in the present invention is an efficient measuring device for high sensitivity measurement of C-reactive protein using the piezoelectric properties of the crystal oscillator which is the ultimate target of the present invention.

(iii) Optimization of C-reactive protein antibody concentrations added to C-reactive protein and reaction cells used for immobilization

Established a high sensitivity measurement method of C-reactive protein using the crystal oscillator immunosensor measuring device of Figure 1 comprising a crystal oscillator sensor chip immobilized C-reactive protein prepared by the step (i) and a reaction cell equipped with the same To this end, in the present step, the binding degree of the antibody that appears when the C-reactive protein concentration as an antigen is immobilized to the crystal oscillator sensor chip and applied through the sample inlet while changing the concentration of the C-reactive protein antibody bound by the immune response thereto By measuring the C-reactive protein used for immobilization and C-reactive protein antibody concentration added to the reaction cell to optimize the specific method is as follows.

C-reactive protein as an antigen was prepared using a phosphate buffer solution (pH 7.0) of 0.05 to 0.15 molar concentration as a reaction buffer solution to prepare a phosphate buffer solution having a concentration of 0.5 to 2.5 mg / ml of C-reactive protein. By i) the C-reactive protein is immobilized on the crystal oscillator sensor chip electrode inside the reaction cell of the crystal oscillator immunosensor measuring device of FIG. Afterwards, the microdispensing pump is operated to flow the reaction buffer solution stored in the reaction buffer tank at a flow rate of 20 to 230 µl / min through the wire. This system stabilization is performed by the crystal oscillator immunosensor chip in which the reaction buffer solution flows. Run for 5 to 20 minutes until the resonance frequency reaches steady state. When the system is stabilized, C-reactive protein antibody is dissolved in the reaction buffer solution to prepare a reaction buffer solution having a C-reactive protein antibody concentration of 1 to 1000 µg / ml, and a sample inlet having a loop of 20 to 500 µl is attached. C-reactive protein used for immobilization and C-reactive protein antibody applied to immobilized cell by measuring the frequency change due to the accumulation of mass resulting from the immune response in the crystal oscillator sensor chip mounted on the reaction cell As a result of optimizing the concentration, as shown in Example 2 of the present invention described below, the concentration of C-reactive protein immobilized on the electrode of the crystal oscillator sensor chip of the reaction cell was 2 mg / ml, the electrode of the crystal oscillator sensor chip of the reaction cell. The concentration of C-reactive protein antibody added to the C-reactive protein immobilized to 250 μg / ml was found to be appropriate.

(iv) Enhance the sensitivity of the sensor by competing immune response using optimized concentrations of immobilized antigen and C-reactive protein antibody

In order to establish a high sensitivity measurement method of C-reactive protein using the piezoelectric properties of the crystal oscillator which is the ultimate object of the present invention under the optimization conditions derived from step (iii), the internal modification of the reaction cell of the crystal oscillator immunosensor measuring apparatus of FIG. After immobilizing the C-reactive protein as an antigen on the vibrator sensor chip, a mixture of C-reactive protein antibody and C-reactive protein as a measurement material is injected into the reaction cell through a sample inlet, and then the sensor chip for the C-reactive protein antibody. The change in the frequency as a sensor response resulting from the competitive immunity reaction between the C-reactive protein immobilized on and the C-reactive protein by concentration in the sample is measured. Through this, the detection limit of the C-reactive protein measurement by the crystal oscillator immunosensor of the present invention, which is a non-labeled property, was confirmed to complete the high sensitivity measurement of the C-reactive protein.

The C-reactive protein as an antigen was used as a buffer buffer solution (pH 7.0) of 0.05 to 0.15 molar concentration, and the phosphate buffer solution having a concentration of 2 mg / ml C-reactive protein was added to the crystal oscillator sensor chip. It is immobilized by i) and mounted in the reaction cell of the crystal vibrator immunosensor measuring device of FIG. 1, and then the reaction buffer solution is flowed to stabilize the system. Immediately thereafter, the C-reactive protein antibody (monoclonal C-reactive protein antibody from R & D Systems (Minneapolis, MN, USA)) was added to the solution having a concentration of 250 μg / ml of the C-reactive protein antibody using a reaction buffer solution. 20 to 500 microliters (μl) of loops are attached after 1 to 10 minutes of mixing the sample (saliva) with C-reactive protein for each concentration of picomolar concentration (pM) to 300 nanomolar concentration (nM). Through the sample inlet. The C-reactive protein immobilized on the sensor chip with respect to the C-reactive protein antibody and the C-reactive protein of each concentration in the sample are measured as frequency change as a sensor response resulting from the competition immune reaction. The detection limit for the measurement of C-reactive protein by the crystal oscillator's immunosensor is set. As a result, as in the embodiment of the present invention, the detection limit for the C-reactive protein was shown as a high sensitivity of 1.1 picomol concentration.

Hereinafter, the content of the present invention will be described in detail through examples and test examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

<Example 1>

Immobilize C-reactive protein antibodies (monoclonal C-reactive protein antibodies from R & D Systems, Inc. (Minneapolis, MN, USA)) instead of immobilizing C-reactive proteins as antigens on the crystal oscillator sensor chip as in step (i) above. 1 is mounted on the reaction cell of the crystal vibrator immunosensor measuring device of FIG. 1 and a direct binding immunity reaction with the C-reactive protein antibody immobilized on the sensor chip is performed while applying a certain concentration of C-reactive protein through the sample inlet. The sensitivity of the crystal oscillator's immune sensor was measured as follows.

At this time, 0.10 molar phosphate buffer solution (pH 7.0) was used as the reaction buffer solution, and the immobilization of the C-reactive protein antibody was performed as follows. In other words, care was taken not to touch the surface of the gold electrode, which is the sensitive area of the sensor chip, and soaked sequentially for 5 minutes with 1.2 moles of caustic soda and the same concentration of hydrochloric acid, and then washed with distilled water. Thereafter, the mixture was treated with concentrated hydrochloric acid having a pH of 0.5 for 1 minute, washed again with distilled water, and dried in a convection oven for 20 minutes. To a lyophilized C-reactive protein antibody, 0.10 molar concentration of phosphate buffer solution (PBS) was added to recover the solution, and then to 30 µl of an antibody solution diluted with PBS to a concentration of 50 µg / ml, which was previously optimized. An equivalent amount of 20 mmol of sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido] hexanoate dissolved in distilled water was added to a thiolated crosslinking agent and allowed to react for 60 minutes. Thereafter, 20 µl of dithiothreitol solution was treated for 30 minutes in order to reduce the disulfide bond of the C-reactive protein antibody in the reaction solution to expose the thiol group. 10 μl of the thiolated antibody prepared as described above was evenly applied to the surface of the gold electrode, which is a sensitive part of the crystal oscillator, dried at room temperature for 60 minutes, and washed with distilled water and reaction buffer solution five times in succession.

Through the crystal oscillator immunosensor measuring apparatus of FIG. 1, the reaction buffer solution was flowed at a flow rate of 199 μl / min, and the resonance frequency F1 at the steady state was measured. In addition, a sample containing C-reactive protein as a measurement target at 106 nanomolar concentrations and 424 nanomolar concentrations, respectively, was added to the sample inlet with a 200 microliter loop, and the surface of the sensor chip as a result of the immune reaction was detected. By measuring the frequency reduction according to the mass accumulation occurring, the resonance frequency F2 of the steady state was measured, and the frequency change ΔF (F1-F2) was measured. From this, the C-reactive protein concentration was 106 nanomolar concentration and 424 nanomolar, respectively. In case of concentration, the frequency change as sensor response was only 16.87Hz and 29.63Hz, respectively, and there was no significant sensor response when C-reactive protein concentration was lower than this.

As can be seen in Example 1, the C-reactive protein immobilized antibody to the crystal oscillator sensor chip to operate the crystal oscillator immunosensor measurement system of FIG. In order to make high-sensitivity measurements of reactive proteins, it is urgent to establish new measurement methods to improve them.

<Example 2>

In case of measuring C-reactive protein by continuous crystal oscillator immunosensor measuring device operated by direct coupling method, it is possible to improve the sensitivity of C-reactive protein by improving the problem that sensor sensitivity is very low as in Example 1. C-reactive protein as an antigen used for immobilization and the crystal oscillator immunosensor measuring device of FIG. 1, which are necessary for conducting the sensitivity of the sensor by the competition reaction as shown in the above steps (iii) and (iv). The concentration optimization of the C-reactive protein antibody added to the reaction cell through the sample inlet of was performed as follows.

At this time, 0.10 molar concentration of phosphate buffer solution (pH 7.0) was used as the reaction buffer solution, and C-reactive protein was immobilized as follows. In other words, care was taken not to touch the surface of the gold electrode, which is the sensitive area of the sensor chip, and soaked sequentially for 5 minutes with 1.2 moles of caustic soda and the same concentration of hydrochloric acid, and washed with distilled water in between. Thereafter, the mixture was treated with concentrated hydrochloric acid having a pH of 0.5 for 1 minute, washed again with distilled water, and dried in a convection oven for 20 minutes. 0.10 molar concentration of PBS was added to the lyophilized C-reactive protein to recover the solution, and the same 20 mmol molar concentration dissolved in distilled water in 30 µl of PBS diluted solution to a concentration of 1 mg / ml and 2 mg / ml ( mM) sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido] hexanoate was added to a thiolated crosslinking agent and allowed to react for 60 minutes. Thereafter, 20 microliters (μl) of dithiothritol solution was treated for 30 minutes in order to reduce the disulfide bond of the C-reactive protein in the reaction solution to expose the thiol group. Ten microliters (μl) of thiolated C-reactive protein prepared as described above was evenly applied to the surface of the gold electrode, which is the sensitive part of the crystal oscillator, dried at room temperature for 60 minutes, and washed with distilled water and reaction buffer solution five times in succession. .

The resonance vibration F1 of the steady state was measured while flowing the reaction buffer solution at a flow rate of 199 microliters / min (μl / min) through the crystal oscillator immunosensor measuring apparatus of FIG. 1. Herein, C-reactive protein antibodies (monoclonal C-reactive protein antibodies from R & D Systems (Minneapolis, MN, USA)) were respectively 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 250 Samples containing ㎍ / ml and 500 ㎍ / ml concentration were added to a sample inlet with a loop of 200 microliters (μl) to track the frequency reduction caused by mass accumulation on the surface of the sensor chip as a result of the immune reaction. The resonance frequency F2 was measured and the frequency change ΔF (F1-F2) was measured.

As a result, the concentration of C-reactive protein used for immobilization was high because the sensor signal was high at 2 mg / ml as shown in Table 1, and the concentration of C-reactive protein antibody applied to the reaction cell was determined by the size of the sensor signal and the amount of antibody used. In consideration of this, it was found that 250 micrograms / milliliter (µg / ml) was appropriate and based on this, high sensitivity measurement for C-reactive protein could be performed as in Example 3.

Table 1.

 Antibody to C-reactive protein ((micrograms / milliliters (μg / ml)) Frequency change (Hz) ^{1 )} C-reactive protein (milligrams / milliliters (mg / ml)) One 2 10  13.83 ± 0.12  6.42 ± 0.11 25  19.15 ± 0.17 12.46 ± 0.22 50  24.23 ± 0.89 24.71 ± 0.15 100  32.49 ± 0.39 60.26 ± 0.12 250  56.18 ± 0.35 71.78 ± 0.27 500 182.67 ± 0.22 195.29 ± 0.11

¹⁾ Average the five values of adjacent measuring time and display standard deviation.

<Example 3>

The high-sensitivity measurement of C-reactive protein using the piezoelectric characteristics of the crystal oscillator, which is the ultimate object of the present invention, is immobilized on the crystal oscillator sensor chip and C-reactive protein and C as antigens mounted on the reaction cell of the crystal oscillator immunosensor measuring device of FIG. At the optimized concentration of the reactive protein antibody, the C-reactive protein, which is the target substance, is mixed with the C-reactive protein antibody and added through the sample inlet, and the C-reactive protein immobilized on the sensor chip against the C-reactive protein antibody. C-reactive protein in the sample was measured as follows to measure the frequency change as a sensor response that appears as a result of the competitive immune response. At this time, 0.10 molar concentration of phosphate buffer solution (pH 7.0) was used as the reaction buffer solution, and C-reactive protein was immobilized as follows. In other words, care was taken not to touch the surface of the gold electrode, which is the sensitive area of the sensor chip, and soaked sequentially for 5 minutes with 1.2 moles of caustic soda and the same concentration of hydrochloric acid, and washed with distilled water in between. Thereafter, the mixture was treated with concentrated hydrochloric acid having a pH of 0.5 for 1 minute, washed again with distilled water, and dried in a convection oven for 20 minutes. The equivalent amount dissolved in distilled water in 30 microliters (μl) of a solution diluted with PBS to recover the solution by adding 0.10 molar concentration of PBS to the C-reactive protein in the lyophilized state to a concentration of 2 mg / ml C-reactive protein Sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido] hexanoate at 20 mol molar concentration of was added to a thiolated crosslinking agent and allowed to react for 60 minutes. Thereafter, 20 microliters (μl) of dithiothritol solution was treated for 30 minutes in order to reduce the disulfide bond of the C-reactive protein in the reaction solution to expose the thiol group. Ten microliters (μl) of the thiolated C-reactive protein prepared as described above was evenly applied to the surface of the gold electrode, which is the sensitive part of the crystal oscillator, dried at room temperature for 60 minutes, and washed with distilled water and reaction buffer solution for 5 consecutive times. .

The resonance vibration F1 of the steady state was measured while flowing the reaction buffer solution at a flow rate of 199 microliters / min (μl / min) through the crystal oscillator immunosensor measuring apparatus of FIG. 1. In addition, the C-reactive protein concentration (0.0011 nanomolar concentration of C-reactive protein in a reaction buffer solution containing 250 μg / ml of C-reactive protein antibody (monoclonal C-reactive protein antibody of R & D Systems (Minneapolis, MN, USA)) 0.0053 nanomolar concentration, 0.011 nanomolar concentration, 0.053 nanomolar concentration, 0.11 nanomolar concentration, 0.53 nanomolar concentration, 1.06 nanomolar concentration, 5.3 nanomolar concentration, 10.6 nanomolar concentration, 53 nanomolar concentration, 106 nanomolar concentration and After 5 minutes of mixing the sample (saliva) containing 212 nanomolar concentration, it is injected into the sample inlet with a 200 microliter loop to conduct a competitive immunity reaction and as a result, the frequency according to the mass accumulation occurring on the surface of the sensor chip. By tracking the decrease, the resonance frequency F2 at steady state was measured and the frequency change ΔF (F1-F2) was measured.

As a result, as shown in Table 2, even at the 0.0011 nanomole (1.1 picomol) concentration, the Fr value, which is the rate of change of frequency, is only 0.8283, and the change in frequency is about 9Hz lower than that of the antibody-only injection, so the detection limit is 1.1 pico. It was found to reach molarity. The sensor sensitivity was evaluated to be superior to the biomarker detection sensitivity measured by the autoimmune sensor and the surface plasmon resonance immunosensor ( Biosensors and Bioelectronics , Vol. 22, p. 973, 2007; Biosensors and Bioelectronics , Vol. 21, p. 1141, 2006; Biosensors and Bioelectronics , Vol. 21, p. 1631, 2006), through the present invention to C-reactive protein, one of the major biomarkers required to evaluate cardiovascular food functionality It was shown that high sensitivity measurements were taken for real-time.

Table 2.

C-reactive protein (nM) Fr ¹⁾ 0 ^-2) 0.0011 0.8283 0.0053 0.6236 0.0110 0.5177 0.0530 0.4727 0.1100 0.4507 0.5300 0.3574 1.0600 0.3080 5.3000 0.2499 10.6000 0.2049 53.0000 0.1581 106.0000 0.1354 212.0000 0.1023

¹⁾ Frequency change ratio = ΔF _{antibody + target substance} / ΔF _antibody .

²⁾ The frequency change when only antibody is injected into sensor system is 90.17 ± 0.35Hz.

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.

C-reactive protein, which is known as one of the major biomarkers for coronary artery disease, hypertension and inflammation by the present invention, can be evaluated in real time quickly, and in addition to C-reactive protein in the future, in addition to ELD, fibrinogen, and angiotensin It is possible to establish the basic technology related to food functional evaluation necessary for high-speed simultaneous measurement of biomarkers that exist in various concentration ranges in blood of experimental animals such as II.

1 is a schematic diagram of a crystal oscillator immunosensor measuring device for measuring C-reactive protein.

2 is a graph showing the degree of binding of C-reactive protein antibody to the crystal oscillator sensor chip is immobilized C-reactive protein and bovine serum albumin, respectively.

<Explanation of symbols for the main parts of the drawings>

 1: reaction buffer solution 2: supply means (fine dispensing pump)

 3: sample injection port 4: flow-through cell

 5: holder 6: O-ring

 7: crystal oscillator 8: joint

 9: waste liquid tank 10: oscillation module

11: frequency meter 12: personal computer

13: Degree of binding of C-reactive protein antibody to crystal oscillator sensor chip immobilized with 2 milligrams of bovine serum albumin per milliliter

14: The degree of binding of C-reactive protein antibody to the crystal oscillator sensor chip immobilized with 2 mg of C-reactive protein per milliliter

Claims (9)

Reaction buffer bath (1), A supply means (2) connected to the reaction buffer solution tank (1) for supplying the reaction buffer solution in the reaction buffer solution tank to the C-reactive protein immobilized on the crystal oscillator sensor chip inside the reaction cell (4) ), A sample containing a reaction buffer solution, a C-reactive protein antibody, and a C-reactive protein, which is connected to the supply means 2 and supplied from the supply means 2, to a crystal oscillator sensor chip inside the reaction cell 4. Sample inlet for supplying immobilized C-reactive protein (3), A sample containing a reaction buffer solution, a C-reactive protein antibody and a C-reactive protein is supplied through the sample inlet (3), and a crystal oscillator (7) including a sensor chip of an electrode on which an antigen, C-reactive protein, is immobilized. A reaction cell 4 provided with an O-ring 6 for protecting the crystal oscillator sensor chip and a holder 5 connected to the joint 8 while surrounding the crystal oscillator 7 and the O-ring 6; An oscillation module 10 connected to the crystal oscillator 7 for vibrating the crystal oscillator inside the reaction cell 4 and transmitting the vibration of the crystal oscillator to the vibration measuring device 11, Vibration measuring device 11 for measuring the vibration of the crystal oscillator (7) in the reaction cell (4) through the oscillation module 10, A computer 12 connected with the vibration measuring device 11 to show the frequency change of the C-reactive protein immobilized on the sensor chip electrode of the crystal vibrator 7 and C-reactive protein measurement, characterized in that it comprises a waste tank (9) for collecting the waste buffer of the sample containing the reaction buffer solution and the C-reactive protein antibody and C-reactive protein supplied into the reaction cell (4) Crystal oscillator immunosensor measuring device. In the high sensitivity measurement of C-reactive protein using a crystal oscillator immunosensor, (1) obtaining a frequency (F1) at steady state by adding a reaction buffer solution to the crystal oscillator sensor chip to which the C-reactive protein as an antigen is immobilized, (2) After the step (1), a mixture of C-reactive protein antibody and a sample containing C-reactive protein is added to the crystal oscillator sensor chip to which the antigen C-reactive protein is immobilized, and then, in a steady state after a competitive immunity reaction occurs. Obtaining the frequency F2 of (3) a crystal oscillator immune sensor comprising measuring a frequency change (ΔF = F1-F2) from the frequency F1 obtained in step (1) and the frequency F2 obtained in step (2) High sensitivity measurement method of C-reactive protein using. The method of claim 2, wherein the immobilization of C-reactive protein as an antigen to the crystal oscillator sensor chip is (a) adding a phosphate buffer solution to the C-reactive protein to obtain a phosphate buffer solution containing the C-reactive protein, (b) step (a) using a solution of sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido] hexanoate (sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido] hexanoate Reacting with a phosphate buffer solution, (c) adding and reacting a dithiothreitol solution to the solution obtained in step (b), (d) a high sensitivity measurement method of C-reactive protein using a crystal oscillator immunosensor comprising applying and drying the solution obtained in step (c) to the electrode surface of the crystal oscillator sensor chip. The method of claim 2, wherein the immobilization of C-reactive protein as an antigen to the crystal oscillator sensor chip is (A) adding a phosphate buffer solution to the C-reactive protein to obtain a phosphate buffer solution having a concentration of 0.5-2.5 mg / ml C-reactive protein, (B) 10 to 50 µl of 10-30 mM sulfosuccinimidyl 6- [3- {2-pyridyldithio} propionamido] hexanoate aqueous solution, and 10 to 50 µl of the phosphate buffer solution of step (a). And reacting for 30 to 90 minutes, (C) adding 10-30 μl of dithiothritol solution to the solution obtained in step (b) and reacting for 10 to 50 minutes, (D) 10-15 μl of the solution obtained in step (C) is applied to the electrode surface of the crystal oscillator sensor chip, dried at room temperature for 30-90 minutes, washed 3-7 times with distilled water and 0.05-0.15 molar phosphate buffer solution. High sensitivity measurement method of C-reactive protein using a crystal oscillator immune sensor, characterized in that the step is carried out. The method according to claim 2, wherein the reaction buffer solution is 0.05 to 0.15 molar concentration (M) and the pH is 7.0 phosphate buffer solution, characterized in that the high sensitivity of the C-reactive protein using a crystal oscillator immunosensor. The method of claim 2, wherein the C-reactive protein antibody is any one selected from antibodies prepared using C-reactive protein of rat, mouse, rabbit, monkey or human mammal. High Sensitivity Measurement of C-Reactive Protein Using Crystal Oscillator Immunosensor. The frequency (F1) of step (1) is measured by adding the reaction buffer solution to the crystal oscillator sensor chip on which the C-reactive protein is immobilized, and measuring the frequency (F1) at a steady state after 5 to 20 minutes. High sensitivity measurement method of C-reactive protein using a crystal oscillator immune sensor, characterized in that. The frequency (F2) measurement of step (2) is a competitive immune response by adding a mixture of C-reactive protein antibody and a sample containing C-reactive protein to the crystal oscillator sensor chip to which the C-reactive protein is immobilized. High sensitivity measurement method of C-reactive protein using a crystal oscillator immune sensor, characterized in that the frequency (F2) in the steady state after 2-20 minutes after this occurs. The method of claim 2, wherein the mixture of the sample containing the C-reactive protein antibody and the C-reactive protein is mixed with the sample containing the C-reactive protein antibody and the C-reactive protein in a volume ratio of 1: 9-9: 1. High sensitivity measurement method of C-reactive protein using a crystal oscillator immune sensor, characterized in that.

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