A cyclic fluidization-based bio-macromolecule detection chip

Abstract

The application discloses a kind of biological macromolecule detection chips based on circulating fluidization, including functionalized microbead system, micro circulating fluidization system and electric impedance analysis system.Functionalized microbead system is composed of a variety of microbeads that can capture different types of macromolecular biomarkers, and the functionalized microbead system is dragged into a fluidized state under the action of the micro circulating fluidization system.When specific macromolecular biomarkers enter the chip, they will cause the functionalized microbead system in the fluidized state to undergo immune aggregation, thereby causing the electric impedance of the circulating fluidized bed to change.The electric impedance of the circulating fluidized bed is monitored by the electric impedance analysis system, enabling quantitative detection of biomarkers.The chip designed by the application can quickly and efficiently capture biological macromolecules and detect them without labeling.Compared with existing technologies, the chip designed by the application has the advantages of simple operation, fast detection speed and no need for labeling.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of macromolecular biomarker detection, and in particular to a circulating fluidized-based biomacromolecule detection chip. BACKGROUND

[0002] Macromolecular biomarkers include proteins, DNA, RNA, etc. Macromolecular biomarker detection technology mainly includes two parts of biomarker capture and biomarker concentration detection, and its application scenarios are very broad. For example, in blood routine examination, the concentration of specific types of macromolecular biomarkers (such as proteins, nucleic acids, etc.) in peripheral blood needs to be detected, so macromolecular biomarker detection technology is needed. For another example, nucleic acid detection needs to determine whether the sample contains specific nucleic acid sequences of a virus, so as to determine whether the detected sample contains the virus. In addition, macromolecular biomarker detection technology also has many applications in biology, environment, etc.

[0003] Most of the existing macromolecular biomarker quantitative analysis technologies are based on immune probe labeling, which needs multiple biochemical reactions to realize the fluorescence labeling of biomacromolecules, and realizes the concentration detection of macromolecular biomarkers through optical, acoustic, electrical, etc. This method is complicated, and the operation is complex and time-consuming, and the biochemical changes of the measured substance may be caused in the labeling process, thereby affecting the accuracy of the detection.

[0004] Most of the existing macromolecular biomarker capture systems are modified with specific antibodies or nucleic acid aptamers on the solid surface, and the required protein or nucleic acid is captured by the antibody or nucleic acid aptamer, so as to realize the capture of macromolecular biomarkers. In order to improve the capture efficiency, an external mass transfer system needs to be introduced, so as to realize the circulation of biomarkers on the modified solid surface. SUMMARY

[0005] The present application aims at the defects of the prior art, and provides a circulating fluidized-based biomacromolecule detection chip, which realizes efficient capture and quantitative analysis of specific types of macromolecular biomarkers without destroying the activity of macromolecular biomarkers.

[0006] To solve the above technical problems, the technical solutions of the present application are as follows:

[0007] The application discloses a circulating fluidized-based biomacromolecule detection chip, which is characterized by comprising a micro circulating fluidized system and a functionalized microbead system, wherein the functionalized microbead system comprises various types of microbead bodies and capture structures modified on the microbead bodies, the capture structures are specific antibodies or nucleic acid aptamers, the micro circulating fluidized system comprises a chip shell and a circulating fluidized driving device, an inner part of the chip shell forms a cavity, a lower end of the chip shell is provided with a circulating fluidized channel inlet, an upper end of the chip shell is provided with a circulating fluidized channel outlet, the circulating fluidized driving device is connected with the circulating fluidized channel inlet and the circulating fluidized channel outlet, buffer solution is circulated from bottom to top in the cavity through the circulating fluidized driving device, a side of the chip shell is provided with a microbead channel and a sample channel, and the functionalized microbead system and a sample to be detected are injected into the cavity through the microbead channel and the sample channel respectively.

[0008] Further, the application further comprises an electrical impedance analysis system, wherein the electrical impedance analysis system comprises a serpentine excitation electrode and a plurality of detection electrodes arranged in the cavity, the detection electrodes are sequentially arranged to form an array structure, and the electrical impedance analysis system can monitor the fluidized state, spatial position distribution and size of the microbead groups after immune condensation of the functionalized microbead system in the cavity in real time.

[0009] Further, the serpentine excitation electrode has a multilayer serpentine structure, can construct an excitation electric field with multiple layers of space, and the detection electrodes at different spatial positions can be sequentially and periodically connected and pass through current from surrounding serpentine excitation electrodes.

[0010] Further, the electrical impedance analysis system can apply a direct current detection signal and an alternating current detection signal to the cavity, the peak value of the alternating current detection signal is about 1V, and the frequency is 1KHz-1MHz.

[0011] Further, the functionalized microbead system is micrometer or nanometer scale polymer microbeads or magnetic beads, and the equivalent volume of the functionalized microbead system in the cavity is one fifth to one third of the volume of the cavity.

[0012] Further, the chip shell is manufactured by using a photoetching technology, a metal adhesive layer and an electrode layer are embedded on the chip shell by a magnetron sputtering method, the metal adhesive layer is made of titanium or cadmium, and the electrode layer is made of gold or platinum.

[0013] Compared with the prior art, the beneficial effects of the present application are that: using the detection chip designed in the present application, the efficient capture and quantitative analysis of specific types of macromolecular biomarkers can be realized without destroying the activity of the macromolecular biomarkers and without labeling the macromolecular biomarkers. Compared with the existing quantitative detection technology of macromolecular biomarkers based on fluorescent labeling, the present technology has the advantages of simple operation and shorter time consumption; compared with the existing macromolecular biomarker capture technology, the present technology introduces a micro circulating fluidization device to fluidize the microbeads and mix them with the sample to be tested, thereby greatly improving the contact efficiency of biological macromolecules and the surface of the modifier. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a structural schematic diagram of the embodiment of the present application when the microbeads are in a fluidized state.

[0015] Figure 2 It is a schematic diagram of the biological macromolecule recognition mechanism of the microbead immune aggregation of the embodiment of the present application.

[0016] Figure 3 It is a structural schematic diagram of the embodiment of the present application when the microbeads occur immune aggregation phenomenon.

[0017] Wherein: 1- serpentine excitation electrode, 2- detection electrode, 3- sample to be tested channel, 4- microbead channel, 5- circulating fluidization channel outlet, 6- circulating fluidization channel inlet, 7- chip shell, 8- cavity, 9- functionalized microbead system, 10- non-target macromolecular biomarker, 11- microbead body, 12- target macromolecular biomarker, 13- capture structure, 14- microbead group. DETAILED DESCRIPTION

[0018] In order to deepen the understanding of the present application, the present application will be further described below in combination with the drawings, and the embodiment is only used to explain the present application and does not constitute a limitation on the protection scope of the present application.

[0019] Figures 1-3 A specific embodiment of a biological macromolecule detection chip based on circulating fluidization is shown, which is used to detect whether a certain macromolecular biomarker is contained in the sample to be tested. As shown in Figure 1 The chip is composed of a functionalized microbead system 9, a micro circulating fluidization system, and a resistance impedance analysis system. The micro circulating fluidization system is composed of a chip shell 7, a circulating fluidization channel inlet 6, a circulating fluidization channel outlet 5, and a circulating fluidization driving device, and a cavity 8 is formed inside the chip shell 7. The resistance impedance analysis system includes a serpentine excitation electrode 1 and a detection electrode 2 embedded inside the cavity 8. The equivalent volume of the functionalized microbead system 9 is about one fifth to one third of the volume of the cavity 8, and the functionalized microbead system 9 enters and exits the cavity 8 of the detection chip through the microbead channel 4.

[0020] Preferably, the above-mentioned chip is manufactured by photolithography technology to manufacture the chip shell 7 and engrave the sample channel 3, the microsphere channel 4, the circulating fluid channel inlet 6 and the circulating fluid channel outlet 5 on the surface of the chip shell 7 respectively. The metal adhesive layer and the electrode layer of the electrical impedance analysis system are embedded on the chip shell 7 by the magnetron sputtering method, the material of the metal adhesive layer is titanium or cadmium, the material of the electrode layer is gold or platinum, and the serpentine excitation electrode 1 and the detection electrode 2 are installed, so as to complete the manufacture of the chip main body. Then the circulating fluid driving device is introduced, and the chip can work.

[0021] Preferably, the functionalized microsphere system 9 includes a microsphere body 11 and a capture structure 13 modified on the microsphere body 11. According to different target macromolecular biomarkers, the capture structure 13 can be selected from specific antibodies or nucleic acid aptamers. By adding a hydroxylating agent or a carboxylating agent to the reagent of the magnetic beads or the polymer microspheres, a chemical group can be modified on the surface of the magnetic beads or the polymer microspheres, and then a corresponding antibody or nucleic acid aptamer surface modification reagent is added to the reagent, so as to connect the capture structure 13 on the surface of the microsphere body 11. Through the specific binding of the capture structure 13 and the target macromolecular biomarker, the target biomarker can be captured, and the immunological aggregation phenomenon occurs.

[0022] Before the chip works, the minimum fluidization speed of the functionalized microsphere system 9 needs to be determined. First, the functionalized microsphere system 9 with surface modified specific antibodies or nucleic acid aptamers is introduced into the cavity 8 from the microsphere channel 4 and is packaged. The equivalent volume of the introduced functionalized microsphere system 9 is about one fifth to one third of the volume of the chip cavity 8. Due to the action of gravity, the introduced functionalized microsphere system 9 is deposited at the bottom of the chip cavity 8.

[0023] Then, the buffer solution is introduced into the chip cavity 8 from the bottom to the top through the circulating fluid channel inlet 6, and the flow rate of the buffer solution is slowly changed from small to large. When the flow rate of the solution is small, the upward fluid drag force on the functionalized microsphere system 9 is smaller than the downward gravity, so the functionalized microsphere system 9 is still deposited at the bottom of the chip cavity 8. As the flow rate of the solution gradually increases, the upward fluid drag force on the microspheres gradually increases, and when the flow rate reaches the threshold value, i.e. the minimum fluidization speed, the upward fluid drag force on the functionalized microsphere system 9 is equal to its own gravity, at this time the functionalized microsphere system 9 starts to suspend in the cavity 8 and enters the fluidized state. In the experimental process, the fluidization state of the functionalized microsphere system 9 in the cavity 8 is detected in real time by the electrical impedance analysis system as the flow rate gradually increases, so as to determine the minimum fluidization speed and provide a basis for subsequent experiments.

[0024] When the chip is working, first, the functionalized microbead system 9 with surface modified specific antibodies or nucleic acid aptamers is introduced through the microbead channel 4, and the buffer solution is introduced through the circulation fluidization channel inlet 6. The flow rate of the buffer solution is gradually increased until the minimum fluidization velocity, so that the functionalized microbead system 9 in the cavity 8 enters the fluidization state. Figure 1

[0025] Subsequently, the sample solution to be tested containing non-target macromolecular biomarkers 10 and target macromolecular biomarkers 12 is introduced from the sample channel 3 to be tested. The sample solution to be tested is mixed with the buffer solution under the action of the micro circulation fluidization system, fully contacts the surface of the functionalized microbead system 9, and realizes circulation flow. In this embodiment, the target macromolecular biomarkers 12 can specifically bind to the capture structure 13, i.e. antibodies or nucleic acid aptamers, thereby causing an immune aggregation phenomenon, as shown in Figure 2

[0026] The principle and process of the immune aggregation phenomenon are as follows: as shown in Figure 2 , one end of the target macromolecular biomarker 12 can be combined with the capture structure 13 modified on the surface of one microbead body 11, and the other end can be combined with the capture structure modified on the surface of another microbead body. By combining the two ends of the target macromolecular biomarker 12 with the capture structures 13 on the surfaces of two microbead bodies 11, respectively, the two microbead bodies 11 can be connected together. Since the surface of one microbead body 11 is modified with multiple capture structures 13, and the sample to be tested contains target macromolecular biomarkers 12, a large number of microbead bodies 11 are connected together under the action of the target macromolecular biomarkers 12, forming a microbead group 14, i.e. the immune aggregation phenomenon occurs. The target macromolecular biomarkers 12 can cause the immune aggregation phenomenon of the microbeads, and the non-target macromolecular biomarkers 10 cannot be combined with the capture structure 13, so they will not cause the immune aggregation phenomenon of the functionalized microbead system 9.

[0027] After the immune aggregation phenomenon occurs, since the drag force of the circulation fluidization system on the microbead group 14 is not enough to support it to continue to maintain the fluidization state, the microbead group 14 will be deposited at the bottom of the detection cavity 8, as shown in Figure 3 The microbead group 14 that has occurred the immune aggregation will cause the change of the electrical impedance of the solution system in the cavity 8. By monitoring the change of the electrical impedance of the solution system in the cavity 8 through the electrical impedance analysis system, whether the target macromolecular biomarkers 12 are contained in the solution to be tested and the quantity or concentration of the target macromolecular biomarkers 12 can be determined.

[0028] ​​The electrical impedance analysis system can monitor the microbead fluidization state, the spatial position distribution of the microbeads and the size of the immune-aggregated microbead groups in the chip in real time. The serpentine excitation electrode 1 has a multi-layer serpentine structure, which is used to construct an excitation electric field with spatial multi-layers and to enhance the electric field intensity in the specified detection area inside the cavity 8, thereby improving the detection sensitivity and spatial resolution. The detection electrode array comprises a plurality of detection electrodes 2 at different spatial positions, the detection electrodes 2 are connected with a conditioning circuit through a multi-control switch, the conditioning circuit is connected with a data acquisition card through a multi-control switch, and the two multi-control switches are connected through a clock, so that when the multi-control switch of the detection electrode array points to a certain detection electrode 2, the multi-control switch of the data acquisition card also points to the corresponding data acquisition card input port, and finally a multiplexer is formed among the detection electrode 2, the conditioning circuit and the data acquisition card. When the chip enters the working state, the detection electrodes 2 at different positions and the corresponding data acquisition card input ports are connected in sequence and periodically under the action of the multiplexer. When the corresponding detection electrode 2 at a certain spatial position is connected, the serpentine excitation electrode 1 structure near the detection electrode 2 has a current flowing to the detection electrode 2, and other detection electrodes do not have a current due to being not connected, so that the detection current of the to-be-detected area is enhanced, and the crosstalk caused by the surrounding detection electrodes is avoided, thereby enhancing the amplitude of the detection response signal and the signal-to-noise ratio. By processing the electric signal flowing through the detection electrode 2, the fluidization state of the microbeads at the corresponding spatial position can be obtained, and it can be judged whether the microbead groups 14 and the size of the microbead groups 14 occur immune-aggregation at the position.

[0029] Preferably, the serpentine excitation electrode 1 of the electrical impedance analysis system applies a direct current detection signal and an alternating current detection signal to the cavity 8 system inside the chip. The peak-to-peak value of the alternating current detection signal is about 1V, and the frequency is 1KHz-1Mhz.

[0030] In addition to being used for detecting whether a certain type of macromolecular biomarker is contained in the to-be-detected sample, the detection chip can also be used for multiple detection of biological macromolecules, such as simultaneously detecting whether a certain protein and a certain RNA single strand are contained in the to-be-detected sample. At this time, the functionalized microbead system 9 should be composed of multiple types of microbeads, the surfaces of which are respectively modified with antibodies or nucleic acid aptamers that can specifically bind to target biological macromolecules, and the different types of microbeads should have a certain size difference. Since the immune aggregation of microbeads of different sizes will cause different degrees of electrical impedance change, whether different types of macromolecular biomarkers are contained in the to-be-detected sample and the quantity thereof can be judged and quantitatively characterized by analyzing the electrical impedance change of the solution system in the cavity 8 of the detection chip.

[0031] After detection, the functionalized microbead system 9 and the buffer solution can be led out through the microbead channel 4, the internal cavity 8 of the detection chip is cleaned, and after new microbeads are injected, the next detection work can be carried out.

[0032] The above detailed description is only for illustrating the technical concept and structural features of the present application, and is intended to enable the skilled in the art to implement it, but the above content does not limit the protection scope of the present application, and any equivalent changes or modifications made according to the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. A cyclic fluidization-based bio-macromolecule detection chip, characterized by: The micro circulating fluidization system comprises a chip shell (7) and a circulating fluidization driving device, the inside of the chip shell (7) forms a cavity (8), the lower end of the chip shell (7) is provided with a circulating fluidization channel inlet (6), the upper end is provided with a circulating fluidization channel outlet (5), the circulating fluidization driving device is connected with the circulating fluidization channel inlet (6) and the circulating fluidization channel outlet (5), a buffer solution is circulated in the cavity (8) from bottom to top by the circulating fluidization driving device, the side of the chip shell (7) is provided with a microbead channel (4) and a sample channel (3), the functionalized microbead system (9) and the sample to be detected are injected into the cavity (8) through the microbead channel (4) and the sample channel (3) respectively. The electric impedance analysis system comprises a serpentine excitation electrode (1) and a plurality of detection electrodes (2) arranged in the cavity (8), the detection electrodes (2) are arranged in an array structure, and the electric impedance analysis system can monitor the fluidization state, spatial position distribution and size of the microbead group (14) after immune condensation of the functionalized microbead system (9) in the cavity (8) in real time. The serpentine excitation electrode (1) has a multi-layer serpentine structure and can form an excitation electric field with multiple layers of space, and the detection electrodes (2) at different spatial positions can be connected in sequence and periodically and through the current from the surrounding serpentine excitation electrode (1). One end of the target macromolecular biomarker (12) can be combined with the capture structure (13) on the surface of one microbead body (11), and the other end can be combined with the capture structure on the surface of another microbead body, and the surface of one microbead body (11) is modified with a plurality of capture structures (13); when the corresponding detection electrode (2) at a certain spatial position is connected, the structure of the serpentine excitation electrode (1) near the detection electrode (2) appears current flowing to the detection electrode (2), and other detection electrodes do not have current because they are not connected, so that the detection current of the detection region is enhanced, and crosstalk caused by surrounding detection electrodes is avoided.

2. The cyclic fluidized-based bio-macromolecule detection chip according to claim 1, wherein: The electric impedance analysis system can apply a direct current detection signal and an alternating current detection signal to the cavity (8), the peak value of the alternating current detection signal is 1V, and the frequency is 1KHz~1MHz.

3. The cyclic fluidized-based bio-macromolecule detection chip according to claim 1, wherein: The functionalized microbead system (9) is a micrometer or nanometer scale polymer microbead or magnetic bead, and the equivalent volume of the functionalized microbead system (9) in the cavity (8) is one fifth to one third of the volume of the cavity (8).

4. The cyclic fluidized-based bio-macromolecule detection chip according to claim 1, wherein: The chip shell (7) is manufactured by using a photoetching technology, a metal adhesive layer and an electrode layer are embedded on the chip shell (7) by a magnetron sputtering method, the material of the metal adhesive layer is titanium or cadmium, and the material of the electrode layer is gold or platinum.

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