KR200440969Y1 - Quartz crystal microbalance biosensor apparatus - Google Patents

Abstract

The present invention relates to a quartz crystal microbalance (QCM) biosensor device, and more particularly, it is possible to fix the receptor molecules on the surface of the sensor chip in a simple manner and to easily contact the test object in the gas phase or liquid phase with the sensor. A crystal crystal microbalance (QCM) biosensor device capable of obtaining a stable signal.

According to the present invention, a biosensor chip using a QCM is connected to a circuit, and a batch type cell and a flow type cell can be interchanged with each other. Enables measurement of all test objects in a condition.

In addition, according to the present invention, the oscillator circuit and the cell containing the biosensor chip, which are sensitive to the surrounding environment, such as temperature, are adopted to separate the chambers into independent chambers, thereby improving signal stability.

Quartz Crystal Microbalance (QCM), Flow Type, Well Type, Biosensor

Description

Quartz Crystal Microbalance (UCM) Biosensor Device {QUARTZ CRYSTAL MICROBALANCE BIOSENSOR APPARATUS}

1 is a block diagram of a quartz crystal microbalance (QCM) biosensor device.

2A is a view showing a cell bottom part.

Figure 2b is a view showing the actual appearance of the bottom of the cell.

Figure 2c is a view showing the actual state of a state equipped with a biosensor chip (hereinafter referred to as biosensor chip) using a QCM at the bottom of the cell.

Figure 2d is a view showing a state connected to the oscillator with a biosensor chip mounted on the lower end of the cell.

3A shows a well cell top end;

Figure 3b is a view showing the actual state with the guide attached to the upper end of the well-shaped cell.

Figure 3c is a view showing a state coupled to the top of the well-type cell in a state in which the biosensor chip is mounted on the lower end of the cell.

4A shows the flow cell upper end;

Figure 4b is a view showing the actual state of the state attached the guide, the sample inlet tube and the sample outlet tube is inserted into the upper end of the flow cell.

Figure 4c is a view showing a state combined with the upper end of the flow-type cell with a biosensor chip mounted on the lower end of the cell.

5A is a view illustrating an electrode connection part and a cell support of an oscillator.

5B is a view illustrating a state in which a biosensor chip is mounted at a lower end of a cell and a connection line connected to the biosensor chip is connected to an electrode connection unit of an oscillator.

5C is a diagram illustrating a state in which a lower end of a cell and an upper end of a well cell are connected to an oscillator in a state of being coupled;

FIG. 5D is a view illustrating a state in which a lower end of a cell and an upper end of a flow cell are connected to an oscillator.

6 shows two chambers for isolating and receiving an oscillator and a biosensor chip.

FIG. 7 shows a chamber door with three sides open;

8 is a view showing a connection structure between an injection valve and a cell.

9A illustrates the stability of a QCM biosensor device when the biosensor chip is in air.

9b shows the stability of the QCM biosensor device in contact with a buffer solution flowing at a rate of 50 μl / min at the biosensor chip in the flow cell.

10 shows a change in frequency after sample injection.

11 is a view showing a change in frequency according to the concentration of a sample.

The present invention relates to a quartz crystal microbalance (QCM) biosensor device, and more particularly, it is possible to fix the receptor molecules on the surface of the sensor chip in a simple manner and to easily contact the test object in the gas phase or liquid phase with the sensor. A crystal crystal microbalance (QCM) biosensor device capable of obtaining a stable signal. QCM uses the phenomenon that the frequency of crystal crystals fluctuates when molecules react with the surface of the sensor. The vibration of the crystal is converted into an electrical signal by an oscillator, and the frequency meter uses the electrical signal to measure the frequency of the crystal. QCM can measure both gaseous and liquid objects and generates signals that are generally proportional to the concentration of the reacting molecules. Current QCM systems, however, are designed to create a well-shaped space on the sensor when the test specimen comes in contact with the sensor, or to choose one of two ways in which the solution specimen flows over the sensor. It was not possible to alternate between the two methods.

The present invention was devised to solve the above problems, and it is possible to interchange the batch type cell and the flow type cell with the biosensor chip using QCM connected to the circuit. It is an object of the present invention to provide a QCM biosensor device capable of easily measuring both the stationary test specimen and the test specimen in the flow state.

In addition, the purpose of the present invention is to provide a quartz crystal microbalance (QCM) biosensor device in which an oscillator circuit sensitive to temperature and surrounding environment and a cell containing a biosensor chip are isolated in separate chambers to enhance signal stability. .

Crystal crystal microbalance (QCM) biosensor device according to the present invention in order to achieve the above object, a disk-like crystal crystal microbalance (QCM), a metal thin film layer provided on each side of the disk-shaped crystal crystal, the A biosensor chip having a self-assembly monomolecular layer formed on any one of the metal thin film layers provided on both sides of the quartz crystal microbalance, and a receptor fixed on the self-assembled monomolecular layer; A cell having a space for accommodating the biosensor chip therein and allowing a gaseous or liquid sample to contact the receptor of the biosensor chip; A connection line electrically connected to the metal thin film layers provided on each side of the biosensor chip and extending out of the cell; An oscillator connected to a connection line of the biosensor chip and converting vibration information of the biosensor chip into an electrical signal and outputting the electrical signal; And a frequency measuring device for measuring the frequency of the biosensor chip from the electrical signal output from the oscillator, wherein the cell has a lower end of a cell accommodating and fixing the biosensor chip, and a lower end of the cell A silicon pedestal for fixing a sensor chip is attached, and at the bottom of the cell, a cell upper end (hereinafter referred to as a flow cell upper end) or a stationary sample for allowing a flowing sample to contact the receptor of the biosensor chip. A cell upper end (hereinafter, referred to as a well cell upper end) for selectively contacting with a receptor of a sensor chip is selectively coupled, and the biosensor chip accommodated at the lower end of the flow cell and the well cell upper end may be In a fixed state, the cell is removable at the lower end.

The upper end of the flow cell includes a hole for inserting a sample inlet tube and a sample outlet tube, and one end of the sample inlet tube and one end of the sample outlet tube are respectively inserted into the hole.

Preferably, the upper end of the flow cell may include a space (hereinafter referred to as a flow sample contact space) in which the sample introduced through the sample inlet tube and the sample inlet hole contacts the biosensor chip.

The upper portion of the flow cell has a circular groove around the flow sample contact space, and the circular groove has a circular shape that prevents the sample from leaking when the lower end of the cell and the upper portion of the flow cell are coupled to each other. It is recommended that the ring be attached.

Preferably, the lower end of the cell has a circular groove that prevents the bottom electrode of the received biosensor chip from directly contacting the lower surface of the cell when the biosensor chip is accommodated, and supports the biosensor chip in the circular groove. The ring may be attached to.

The upper end of the well-type cell may include a space (hereinafter, referred to as a well-type sample contact space) in which the injected sample contacts the biosensor chip.

The well-type cell upper end portion has a circular groove around the well-type sample contact space, and the circular groove has a circular shape that prevents the sample from leaking when the cell lower portion and the flow-type cell upper portion are coupled together. It is recommended that the ring be attached.

The oscillator is preferably connected to a connection line of the biosensor chip, and is located in a chamber separate from the cell containing the biosensor chip.

An opposite end of one end of the sample inlet tube may be connected to a syringe pump for injecting a solvent to be delivered to the cell.

One point of the sample inlet tube is preferably provided with an injection valve for controlling the injection of the sample.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. Prior to this, the terms or words used in this specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventors will properly describe the concept of terms in order to best explain their own design. Based on the principle that it can be defined, it should be interpreted as meaning and concept corresponding to the technical idea of the present invention. Therefore, the embodiments described in the specification and the drawings shown in the drawings are only the most preferred embodiment of the present invention and do not represent all of the technical ideas of the present invention, various modifications that can be replaced at the time of the present application It should be understood that there may be equivalents and variations.

1 is a block diagram of a quartz crystal microbalance (QCM) biosensor device 100. Referring to the drawings, the quartz crystal microbalance (QCM) biosensor device 100, a biosensor chip (hereinafter referred to as a biosensor chip) 105 using the QCM, a cell 110, and a biosensor chip (receiving the same) Oscillator 120 to supply electricity to the 105 and sends vibration information of the biosensor chip 105 to the frequency measuring instrument, a connection line 115 connecting the biosensor chip 105 and the oscillator 120, the oscillator 120 Frequency counter 125 to measure the frequency of the signal output from the computer, computer 130 to view the change in frequency in real time, the syringe pump to transfer the solvent 135, controlling the injection of the sample Is composed of an injection valve 140, a collection cup 145 for collecting the sample solution passed through the biosensor chip 105 in the flow cell 110, and a power supply unit 150 for supplying power to the oscillator 120. . Although the measuring apparatus for the flowing sample is shown in the drawing, the measuring of the stationary sample is also possible, and the well-type cell for the measurement of the stationary sample and the flow-type cell for the measurement of the flowing sample are illustrated in FIGS. 3 to 4. It will be described later with reference to.

2A is a diagram illustrating the cell lower end 20. The cell bottom portion 20 has a square groove 28 that fits the size of the biosensor chip 25 (shown in FIG. 2C) and has a circular groove 21 receiving the silicon circular ring 22 in the square groove 28 again. There is. The inner diameter of the silicon circular ring 22 is slightly larger than the diameter of the electrode of the biosensor chip 25. As a result, the two surfaces of the biosensor chip 25 toward the lower end of the cell 20 are in contact with air, and the biosensor chip 25 may vibrate even when the opposite surface is in contact with the liquid. In front of the square groove 28 is a groove 29 for attaching the silicon support 23 for fixing the biosensor chip 25. On the opposite side to which the biosensor chip 25 is mounted, there is a groove 24 into which the guide at the top of the cell is fitted. A groove 23.1 is provided above the pedestal 23 to accommodate the connection line 26 (shown in FIG. 2C) of the biosensor chip 25. Due to the elasticity and friction of the silicone pedestal 23, the biosensor chip 25 may be fixed in a compressed state. As described above, since the biosensor chip 25 is fixed to the cell lower end 20 even in the absence of the upper end of the cell, the biosensor chip 25 may be connected to the oscillator circuit 27 as shown in FIG. 2D.

2B is a view showing the actual appearance of the cell lower end 20.

2C is a diagram illustrating an actual state of a state in which the biosensor chip 25 is mounted on the cell lower end 20. The state in which the connecting line 26 of the biosensor chip 25 is accommodated in the groove 23.1 above the silicon pedestal 23 is shown.

2D is a diagram illustrating a state in which the biosensor chip 25 is mounted on the cell lower end 20 and connected to the oscillator 27.

3A shows a well cell upper end 30. The well-shaped cell upper end 30 has a hole 34 that is slightly larger than the electrode of the biosensor chip 25 and on one side of the hole accommodates the same silicon circular ring 32 as used in the cell lower end 20. There is a groove 31 that can be. Guide 35 (shown in FIG. 3B) is attached to opposite groove 33.

3B is a view showing the actual state of the guide 35 is attached to the well-type cell upper end 30.

FIG. 3C is a view illustrating a state in which the biosensor chip 25 is mounted on the cell lower end 20 and coupled to the well-type cell upper end 30. When the guide 35 covers the well cell upper end 30 over the cell lower end 20 so that the guide 35 fits snugly with the lower groove 24, the biosensor chip 25 is connected to the cell lower end 20 and the well cell upper end 30. It is positioned between two silicon circular rings 22 and 32 which are present and a circular well 36 is made on the upper electrode of the biosensor chip 25. Therefore, when the liquid sample is put into the well 36, the upper electrode of the biosensor chip 25 may come into contact with the liquid sample.

4A is a diagram illustrating a flow cell upper end 40. At the top end 40 of the flow cell there is a groove 41 which can receive the same circular ring 42 used in the cell bottom 20 and there are two small holes 43 for the insertion of the tube. have. Guide 48 (shown in FIG. 4B) is attached to opposite groove 44.

4B shows the actual state of attaching the guide 48 to the flow cell upper end 40 and inserting the sample inlet tube 45 and the sample outlet tube 46 into the two small holes 43. It is a figure which shows. The position of the hole is in contact with the inner surface of the silicon circular ring 42 when the silicon circular ring 42 is attached, so that the liquid flowing into the sample inlet tube 45 covers the entire surface, and then the sample outlet tube ( 46). When connecting the sample inlet tube 45 connected to the flow cell upper part 40 and the tube from the inlet valve 140, a luer lock connector 47 without dead volume is used for easy connection and experiment. It can withstand the required pressure and prevent dilution of the sample due to increased volume at the connection. The flow cell top 40 also has a guide 48 so that the bio-sensor chip 25 is the cell bottom 20 when the guide 48 covers the flow cell top 40 so that it fits snugly into the cell bottom groove 24. And between the two silicon circular rings 22 and 42 on the flow cell top 40 and between the upper electrode of the biosensor chip 25 and the flow cell top 40. Small space is created. The thickness of this space is adjusted to minimize the volume while ensuring a smooth flow of the liquid for precise measurement.

4C is a view illustrating a state in which the biosensor chip 25 is mounted on the cell lower end 20 and coupled to the flow cell upper end 40.

5A is a diagram illustrating an electrode connection part 51 and a cell pedestal 52 of the oscillator 50. A flow cell or a well cell is placed on the cell support 52, and a connection line 26 from the biosensor chip 25 is inserted into and connected to the electrode connector 51.

FIG. 5B is a view illustrating a state in which the biosensor chip 25 is mounted on the lower end of the cell 20 and the connection line 26 connected to the biosensor chip 25 is connected to the electrode connection part 51 of the oscillator 50. .

5C is a view illustrating a state in which the lower end 20 of the cell and the well type cell upper end 30 are connected to the oscillator 50 in a coupled state. The clamp 53 serves to secure the coupling of the lower end 20 of the cell and the well type cell upper end 30.

FIG. 5D is a view illustrating a state in which the lower end 20 of the cell and the flow cell upper end 40 are connected to the oscillator 50 in a coupled state. The clamp 54 serves to secure the coupling of the lower end 20 and the flow cell upper end 40 of the cell.

FIG. 6 shows two chambers that isolate and accommodate the oscillator 64 and the biosensor chip 25. The inner space of the portion indicated by the left dotted line 61 is an oscillator chamber, and the inner space of the portion indicated by the right dotted line 62 includes the cell including the biosensor chip 25. It is a biosensor chip cell chamber. Only the cell pedestal 65 is shown in FIG. 6. In order to increase the signal stability, the oscillator circuit 64 and the biosensor chip 25 sensitive to temperature and the surrounding environment are separated from each other in an independent chamber. The two chambers are isolated by the chamber isolation wall 63, and only the electrode connection 51 of FIG. 5A is exposed toward the biosensor chip cell chamber so that the connecting line 26 can be inserted.

FIG. 7 is a view showing a chamber door 70 in which three surfaces are opened. The chamber door is designed to open the front, top and side as shown when the door is opened, making it very convenient for the user to insert or remove the biosensor chip and to insert or remove the sample into the well cell.

8 is a view showing a connection structure between the sample injection valve 81 and the cell. A sample inlet tube 84 to the flow cell top 40 and a tube 82 connected to the inlet valve 81, a luer lock connector 83 to ensure no dead volume when connecting these two tubes, and a sample outlet A tube 85 is shown. Although not shown in the figure, the syringe pump 135 shown in FIG. 1 supplies a buffer solution to the sample injection valve so that the sample is included in the buffer solution and delivered to the cell. By using the syringe pump 135 to transfer the buffer solution, the buffer solution is introduced into the cell without pulsation. A three-way stopcock (not shown) can also be attached to the end of the syringe pump to connect the vials of the syringe and buffer solution to the biosensor chip cells. Here, when the syringe is pushed while the valve is adjusted to connect the syringe and the cell, the buffer solution flows into the cell. However, when the syringe is pulled while the valve is adjusted to connect the syringe and the bottle, the buffer solution flows into the syringe. In this way it is easy to switch between filling the syringe with buffer and feeding the cell with buffer without having to remove the tube.

9A is a diagram showing the stability of the QCM biosensor device 100 when the QCM is in air. In order to increase the sensitivity of the biosensor chip, it is very important to minimize noise. Therefore, in this design, as described above with reference to FIGS. 1 to 8, a cell capable of accommodating a biosensor chip to which an electrode is attached to an electrode, an electrode connection part for tightly connecting an electrode terminal, and a power supply to the entire device It is designed to reduce noise by designing a pedestal structure that can be fixed in the chamber and a chamber designed to prevent the biosensor chip from being affected by the environment. As a result, as shown in FIG. 9A, when the biosensor chip without any treatment on the electrode surface is placed in the air, the noise is less than ± 0.3 Hz and there is almost no drift.

FIG. 9B is a diagram illustrating the stability of the QCM biosensor device 100 in a state in which the biosensor chip is in contact with a buffer solution flowing at a rate of 50 μl / min in a flow cell. When the biosensor chip is mounted in the flow cell and the buffer solution is flowed at a rate of 50 μl / min, the noise is less than ± 0.5 Hz and there is almost no drift. For a QCM with a natural frequency of 10 MHz, the frequency change of 1 Hz corresponds to a mass change of about 0.9 ng, so the QCM biosensor device is estimated to be able to detect mass changes on the order of nanograms.

10 is a view showing a change in frequency after sample injection. In order to confirm the actual operation of the biosensor device, an anti-bovine haptoglobin antibody was immobilized on the surface of the biosensor chip electrode as a receptor, and the heptoglobin protein was flowed and measured. to be. As a result, a signal began to appear about 1 minute after the sample was injected and the analysis was completed within 5 minutes. This rapid analysis was possible because of the minimum distance and volume from the injection valve to the cell. The delay at the beginning of the signal is very small. This delay occurs when the front part of the injected sample is diluted with the buffer solution. In this design, the connector has no dead volume, minimizes the distance and volume from the injection valve to the cell, and minimizes the volume inside the flow cell. The delay could be reduced.

11 is a diagram illustrating a change in frequency according to the concentration of a sample. When the samples containing different concentrations of heptoglobin are injected, the frequency change is graphed. It can be seen that the frequency change is very accurate and sensitive according to the heptoglobin concentration. In addition, the three-sided open chamber structure facilitated the operation required for the experiment, and the connection structure of the syringe in the pump was able to obtain the effect of simplifying the replenishment of the buffer solution.

As described above, although the present invention has been described by way of limited embodiments and drawings, the present invention is not limited thereto and is intended by those skilled in the art to which the present invention pertains. Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

According to the present invention, a biosensor chip using a QCM is connected to a circuit, and a batch type cell and a flow type cell can be interchanged with each other. There is an effect that enables the measurement of all of the test specimens.

In addition, according to the present invention, it is effective to increase the signal stability by adopting a structure in which the cells containing the oscillator circuit and the biosensor chip sensitive to the ambient environment, such as temperature, are isolated in separate chambers.

Claims (10)

As a quartz crystal microbalance (QCM) biosensor device, Self-assembly formed on any one metal thin film layer of a disk-shaped quartz crystal microbalance (QCM), the metal thin film layer provided on each side of the disk-shaped crystal crystal, the metal thin film layer provided on both sides of the quartz crystal microbalance a biosensor chip having a monolayer and an acceptor immobilized on the self-assembled monolayer; A cell having a space for accommodating the biosensor chip therein and allowing a gaseous or liquid sample to contact the receptor of the biosensor chip; A connection line electrically connected to the metal thin film layers provided on each side of the biosensor chip and extending out of the cell; An oscillator connected to a connection line of the biosensor chip and converting vibration information of the biosensor chip into an electrical signal and outputting the electrical signal; And A frequency meter for measuring the frequency of the biosensor chip from the electrical signal output from the oscillator; Including; The cell has a cell lower end for receiving and fixing the biosensor chip, A silicon pedestal for fixing the biosensor chip is attached to the lower end of the cell, At the lower end of the cell, a cell upper end (hereinafter referred to as a flow-type cell upper end) for allowing a flowing sample to contact the receptor of the biosensor chip or a cell upper end for allowing a stationary sample to contact the receptor of the biosensor chip. (Hereinafter referred to as the top of a well cell) is selectively combined, The upper end of the flow type cell and the upper end of the well type cell may be detachable from the lower end of the cell while the biosensor chip accommodated in the lower end of the cell is fixed. Crystalline microbalance biosensor device. The method according to claim 1, The upper part of the flow cell, A hole for inserting a sample inlet tube and a sample outlet tube; One end of the sample inlet tube and one end of the sample outlet tube are inserted into the holes, respectively. Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 2, The upper part of the flow cell, And a space (hereinafter referred to as a flow type sample contact space) for contacting the biosensor chip with the sample introduced through the sample introduction tube and the sample introduction hole. Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 3, The upper part of the flow cell, A circular groove is provided around the flow sample contact space, A circular ring is attached to the circular groove to prevent the sample from leaking when the lower end of the cell and the upper end of the flow cell are coupled to each other. Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 1, The lower end of the cell, When receiving the biosensor chip, the bottom electrode of the received biosensor chip is provided with a circular groove that does not directly contact the bottom surface of the cell, The circular groove is attached to the circular ring for supporting the biosensor chip Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 1, The well cell upper end, With a space (hereinafter referred to as a well type sample contact space) in which the injected sample comes into contact with the biosensor chip Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 6, The well cell upper end, A circular groove is provided around the well-type sample contact space, A circular ring is attached to the circular groove to prevent the sample from leaking when the lower end of the cell and the upper end of the flow cell are coupled to each other. Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 1, The oscillator is connected to the connection line of the biosensor chip, the oscillator is located in a chamber separate from the chamber (chamber) in which the cell containing the biosensor chip is located Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 2, Opposite end of one end of the sample inlet tube is connected to a syringe pump for injecting a solvent to be delivered to the cell Crystalline crystal microbalance biosensor device characterized in that. The method according to claim 9, One point of the sample inlet tube is provided with an injection valve for controlling the injection of the sample Crystalline crystal microbalance biosensor device characterized in that.

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