Biosensor and detection device

Abstract

The present invention discloses a biosensor and a detection device, belonging to the field of sensor technology. A peptide nucleic acid probe is arranged on the QCM biosensor chip of the biosensor, and biotin is arranged on the peptide nucleic acid probe. The detection device includes a network analyzer, a control terminal, a controller, a temperature control mechanism, an array solution pool mechanism and a liquid supply mechanism. The array solution pool mechanism and the liquid supply mechanism are both arranged in the temperature control mechanism, and a plurality of biosensors are arranged in the array solution pool mechanism. A biosensor and a detection device using the above structure improve the accuracy of microRNA detection, and perform multiple detections simultaneously through the array solution pool mechanism, keep the detection process in a constant temperature state through the temperature control mechanism, and accurately control the liquid inlet and flushing through the liquid supply mechanism.

Description

Technical Field

[0001] The present invention relates to the field of sensor technology, and in particular to a biosensor and a detection device. Background Art

[0002] The quartz crystal microbalance (QCM) is a precision mass measuring instrument that can measure mass to the nanogram level. It is widely used in the analysis of various material components in the gas and liquid phases in the fields of biology, chemistry, environment, aerospace, etc. In the prior art, the detection target of QCM can be biological molecules, such as nucleic acid molecules, proteins, and polysaccharides. The concentration of biotin can be obtained by measuring the resonant frequency offset of QCM without the need for fluorescent labeling or enzyme labeling. In the prior art, DNA probes are used to detect microRNA, which has poor stability and low detection accuracy. At the same time, there are many problems with existing detection devices. For example, the existing patent number CN201720399925.7 discloses a portable QCM test device that is easy to disassemble and wash, which achieves the purpose of easy cleaning through a structure that is easy to disassemble, but the prior art still has the following problems:

[0003] (1) The prior art device can only detect a sample once at a time. If multiple tests are required, the experiment needs to be repeated, which is time-consuming.

[0004] (2) The liquid method of adding the sample is not accurate enough. At the same time, the detection process requires constant temperature conditions. Temperature changes affect the stability of biological molecules and affect the accuracy of detection.

[0005] (3) It needs to be disassembled and cleaned, and in the actual process it needs to be blown dry with nitrogen, which is complicated to operate and easily contaminated. Summary of the invention

[0006] The purpose of the present invention is to provide a biomolecule detection device based on a QCM biosensor to solve the above problems.

[0007] To achieve the above object, the present invention provides a biosensor, including a QCM biosensor chip, on which a peptide nucleic acid probe is disposed, and on which a biotin for signal amplification is disposed.

[0008] The detection device based on the above-mentioned biosensor includes a network analyzer, a control terminal and a controller. The controller and the network analyzer are electrically connected to the control terminal. The device includes a temperature control mechanism, an array solution pool mechanism and a liquid supply mechanism. The array solution pool mechanism and the liquid supply mechanism are both arranged in the temperature control mechanism. A plurality of biosensors are arranged in the array solution pool mechanism. The plurality of biosensors are electrically connected to the network analyzer. The temperature control mechanism and the liquid supply mechanism are both electrically connected to the controller.

[0009] Preferably, the temperature control mechanism includes a thermostatic seat and a plurality of temperature sensors inserted into the array solution pool mechanism, the bottom and side walls of the thermostatic seat are embedded with fixed thermostatic plates arranged in parallel, and the temperature sensors and the fixed thermostatic plates are electrically connected to the controller.

[0010] Preferably, the thermostatic seat is in the shape of a dustpan, and a slide groove is provided at the bottom of the thermostatic seat.

[0011] Preferably, the array solution pool mechanism comprises a bottom plate and a top plate;

[0012] The top plate is provided with a plurality of test slots arranged in an array, a chip slot is provided on one side of the test slot, a sealing rubber ring is provided in the chip slot, a sensor jack, a liquid inlet and a liquid outlet are provided in the test slot, a thermostat slot is provided in the top plate on both sides of the test slot, a plug-in thermostat is provided in the thermostat slot, a biosensor is provided in the chip slot, and a temperature sensor is plugged in the sensor jack;

[0013] A sliding block is arranged at the bottom of the bottom plate, the sliding block is arranged in the sliding groove, threaded connection holes are opened at both ends of the top plate and the bottom plate, and the top plate and the bottom plate are fixedly connected by bolts.

[0014] Preferably, both the fixed thermostatic plate and the pluggable thermostatic plate include a plurality of semiconductor heating plates and semiconductor cooling plates that are alternately arranged in parallel.

[0015] Preferably, the liquid supply mechanism comprises a peristaltic pump and a sample placement plate;

[0016] A slider is arranged at the bottom of the sample placement plate, and a plurality of placement holes are opened on the sample placement plate, and a temperature conducting plate is fixed in the placement holes;

[0017] The sample container placed on the temperature conducting plate is connected to the peristaltic pump through a liquid taking conduit, the liquid taking conduit is provided with a liquid taking valve, the peristaltic pump is connected to the liquid inlet hole through a liquid inlet conduit, the liquid inlet conduit is provided with a liquid inlet valve, the liquid outlet is connected to a liquid discharge pipe, the liquid discharge pipe is provided with a liquid discharge valve;

[0018] The peristaltic pump is electrically connected to the controller via a motor driver, and the liquid extraction valve, the liquid inlet valve and the liquid discharge valve are all electrically connected to the controller.

[0019] Preferably, the QCM biosensor chip uses an 8 MHz AT-cut QCM chip.

[0020] Preferably, a drying mechanism is also included, the drying mechanism includes a drying fan, the drying fan is connected to a nitrogen tank, the air outlet of the drying fan is connected to the test slot through an air pipe, and the drying fan is electrically connected to the controller.

[0021] Therefore, the present invention adopts a biosensor and a detection device of the above structure, which has the following features:

[0022] Beneficial effects:

[0023] (1) A peptide nucleic acid probe is used, and biotin is set on the peptide nucleic acid probe for signal amplification to improve the detection accuracy of microRNA.

[0024] (2) The array solution pool mechanism is used to achieve multiple tests on the same sample or simultaneous testing of multiple samples, greatly reducing the time spent on repeated experiments.

[0025] (3) The temperature control mechanism is used to keep the detection process in a constant temperature state to ensure detection accuracy.

[0026] (4) The liquid supply mechanism is used to accurately control the liquid intake and flushing, and the drying mechanism is used to perform drying operations to avoid long natural drying time. The operation is simple and not easily contaminated.

[0027] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic structural diagram of a detection device with a biosensor without a drying mechanism according to the present invention;

[0029] Figure 2 It is a side view of the thermostatic seat of the present invention;

[0030] Figure 3 The layout diagram of semiconductor cooling sheet and semiconductor heating sheet;

[0031] Figure 4 A schematic diagram showing the solution pool structure of a test tank;

[0032] Figure 5 It is a schematic diagram of the structure of a detection device with a drying mechanism and a biosensor;

[0033] Figure 6 The resonance frequency data graph is for different concentrations of microRNA.

[0034] Reference numerals

[0035] 1. Network analyzer; 2. Control terminal; 3. Controller; 4. Temperature control mechanism; 41. Constant temperature seat; 411. Slide; 42. Temperature sensor; 43. Fixed constant temperature plate; 44. Plug-in constant temperature plate; 45. Semiconductor heating plate; 46. Semiconductor cooling plate; 5. Array solution pool mechanism; 51. Bottom plate; 52. Top plate; 521. Test slot; 522. Chip slot; 523. Sealing rubber ring; 524. Liquid inlet hole; 525. Liquid outlet hole; 526. Temperature regulating slot; 6. Liquid supply mechanism; 61. Peristaltic pump; 62. Sample placement plate; 63. Temperature conduction plate; 64. Liquid extraction valve; 65. Liquid inlet valve; 66. Liquid discharge valve; 7. Biosensor; 8. Blow-drying mechanism; 81. Blow-drying fan; 82. Nitrogen filling. DETAILED DESCRIPTION

[0036] Example

[0037] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0038] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0040] In the description of the present invention, it should be noted that the terms "upper", "lower", "inside", "outside", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings, or are directions or positional relationships in which the product of the invention is usually placed when in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as a limitation on the present invention.

[0041] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "setting", "installation" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0042] Some embodiments of the present invention are described in detail below in conjunction with the accompanying drawings.

[0043] Example 1

[0044] like Figure 1 As shown, a detection device with a biosensor includes a network analyzer 1, a control terminal 2, a controller 3, a temperature control mechanism 4, an array solution pool mechanism 5 and a liquid supply mechanism 6.

[0045] The model of the network analyzer 1 in this embodiment is KEYSIGHT E5061B, the sweep frequency range of the network analyzer 1 is set to between 7.92M and 7.99MHz, the number of sweep frequency points is set to the maximum number 1601, the control terminal 2 is a personal computer, and the controller 3 is a single-chip microcomputer controller. The controller 3 and the network analyzer 1 are both electrically connected to the personal computer to realize data collection, processing and display.

[0046] The biosensor 7 includes a QCM biosensor chip, on which a peptide nucleic acid (PNA) probe is arranged. PNA (peptide nucleic acid) has the ability to specifically bind to RNA of a specific sequence, and follows the principle of complementary base pairing. PNA is uncharged and is more likely to bind to negatively charged target RNA than negatively charged DNA and RNA probes. The backbone of PNA is a peptide chain, which is much more stable than RNA and DNA. In order to reduce the detection limit and improve the sensitivity of detection, biotin (nanogold is used in this embodiment) for amplifying the signal is arranged on the peptide nucleic acid probe.

[0047] In order to ensure that the detected biological molecules are in a constant temperature environment, a temperature control mechanism 4 is used to place an array solution pool mechanism 5 and a liquid supply mechanism 6, such as Figure 2 As shown, the temperature control mechanism includes a thermostatic seat 41 and a plurality of temperature sensors 42 inserted on the array solution pool mechanism 5. In this embodiment, the thermostatic seat 41 is in the shape of a dustpan, and a slide groove 411 is provided at the bottom of the thermostatic seat 41. The bottom and side walls of the thermostatic seat 41 are both embedded with fixed thermostatic sheets 43 arranged in parallel, and the temperature sensors 42 and the fixed thermostatic sheets 43 are both electrically connected to the controller 3.

[0048] In order to perform multiple tests on the same sample or to test multiple samples at the same time, this embodiment uses an array solution pool mechanism 5 for testing. The array solution pool mechanism 5 includes a bottom plate 51 and a top plate 52. In this embodiment, the top plate 52 is provided with three parallel test slots 521. Figure 4 As shown, a chip slot 522 is provided on one side of the test slot 521. The biosensor 7 uses an 8MHz AT-cut QCM chip, and the resonance frequency will be between 7.92M and 7.99MHz, and is placed in the chip slot 522. A sealing rubber ring 523 is provided in the chip slot 522 to prevent chip damage. The test slot 521 is provided with a sensor jack, a liquid inlet 524, and a liquid outlet 525. A thermostatic slot 526 is provided in the top plate 52 on both sides of the test slot 521. A plug-in thermostatic sheet 44 is provided in the thermostatic slot 526. The biosensor 7 is provided in the chip slot 522, and the temperature sensor 42 is plugged in the sensor jack. A slider is provided at the bottom of the bottom plate 51, and the slider is provided in the slide slot 41, so that the array solution pool mechanism 5 can be adjusted to the position of the thermostatic seat 41, which is convenient for maintenance or installation. Both ends of the top plate 52 and the bottom plate 51 are provided with threaded connection holes, and the top plate 52 and the bottom plate 51 are fixedly connected by bolts, adopting a sandwich structure, which is convenient for replacing the biosensor 7 and cleaning the top plate 52 and the bottom plate 51. Figure 3 As shown, the fixed thermostatic sheet 43 and the plug-in thermostatic sheet 44 both include a plurality of semiconductor heating sheets 45 and semiconductor cooling sheets 46 alternately arranged in parallel, and the semiconductor heating sheets 45 and semiconductor cooling sheets 46 are both electrically connected to the controller 3, and the controller 3 uses the PID control principle to adjust the temperature. The controller 3 controls the start and stop and working power of the semiconductor heating sheets 45 and semiconductor cooling sheets 46 at the corresponding positions according to the data of the temperature sensor 42. A plurality of biosensors 7 are electrically connected to the network analyzer 1 for collecting frequency data.

[0049] In order to provide each test slot 521 with a stable flow rate and a fixed volume of test liquid or flushing liquid, the liquid supply mechanism 6 of this embodiment includes a peristaltic pump 61 and a sample placement plate 62. A slider is provided at the bottom of the sample placement plate 62. The sample placement plate 62 is provided with a plurality of placement holes, and a temperature conducting plate 63 (made of copper) is fixed in the placement holes. The sample container placed on the temperature conducting plate 63 is connected to the peristaltic pump 61 through a liquid collection conduit, and a liquid collection valve 64 is provided on the liquid collection conduit. The peristaltic pump 61 is connected to the liquid inlet hole 524 through a liquid inlet conduit, and a liquid inlet valve 65 is provided on the liquid inlet conduit. The liquid outlet hole 525 is connected to a discharge pipe, and a discharge valve 66 is provided on the discharge pipe. The peristaltic pump 61 is electrically connected to the controller 3 through a motor driver, and the liquid collection valve 64, the liquid inlet valve 65 and the discharge valve 66 are all electrically connected to the controller 3.

[0050] like Figure 5As shown, a drying mechanism 8 is also provided, which includes a drying fan, which is connected to a nitrogen tank 82, an air outlet of the drying fan is connected to the test slot 521 through an air pipe, and the drying fan is electrically connected to the controller 3 to achieve nitrogen drying after flushing.

[0051] The specific experimental process is as follows:

[0052] Bovine serum albumin solid produced by BIOSHARP was prepared into a 0.1% BSA solution in water for testing.

[0053] Test process:

[0054] Prepare the medicines as follows:

[0055] 1. Sodium dihydrogen phosphate produced by Fuchen Chemical was prepared into 1M PBS buffer and the pH was adjusted to 7 with NaOH.

[0056] 2. The single-stranded RNA produced by Ruibo Bio is 5 nmol in total.

[0057] The sequence is: 5-auaagacgagcaaaaagcuugu-3.

[0058] Use 1 M PBS to prepare 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 50 μmol / L, and 100 μmol / L microRNA solutions.

[0059] 4. Ethanol and sterile water.

[0060] First, rinse and blow dry.

[0061] The liquid supply mechanism 6 sequentially provides ethanol and sterile water to flush the three test slots 521 for 10 minutes. After flushing, the drying mechanism 8 blows the test slots 521 dry.

[0062] Then, the temperature control mechanism 4, the controller 3 and the control terminal 2 are started, the sample container is placed on the plate to ensure that the temperature of the test sample is constant, and the sample container is connected to the peristaltic pump 61 through the liquid collection tube.

[0063] Finally, the peristaltic pump 61, the liquid extraction valve 64 and the liquid inlet valve 65 are started to provide 20 microliters of 0.05 μmol / L, 0.1 μmol / L and 0.2 μmol / L microRNA solutions to the three test slots 521 respectively. The network analyzer 1 is used to continuously measure for 20 minutes to obtain the data of the output resonance frequency of the three concentrations of RNA. After the test is completed, the drain valve is started to drain the liquid, and the test slot is rinsed with 1M PBS buffer through the liquid supply mechanism for 2 minutes. The next group of microRNA solutions with different concentrations are tested. The above steps are repeated until all concentrations of microRNA solutions are tested. Finally, the test slot is rinsed with ethanol and sterile water.

[0064] The experimental results are as follows Figure 6 shown.

[0065] When the RNA concentration is 0.05 to 0.2 μmol / L, the resonance frequency decreases with the increase of RNA concentration, which shows that the biosensor in this embodiment is more accurate when the RNA concentration is less than 0.2 μmol / L. The length of the line segment on each point value indicates the size of the variance. When the RNA concentration increases further, the resonance frequency no longer maintains a downward trend. When the RNA concentration exceeds 0.2 μmol / L, the frequency no longer shows a downward trend. The reasons are as follows:

[0066] 1. Because the binding between the tested RNA and the PNA probe has reached saturation, increasing the RNA concentration will not increase the mass of nucleic acid attached to the electrode surface.

[0067] 2. The measurement of RNA with different concentrations is not simultaneous. The RNA sample with higher concentration is measured later, and it is likely to have been degraded. The number of test slots can be increased to reduce the error caused by degradation.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A detection device based on a biosensor, characterized in that: The biosensor comprises a QCM biosensor chip, on which a peptide nucleic acid probe is arranged, and on which a biotin for signal amplification is arranged; The detection device includes a network analyzer, a control terminal and a controller, wherein the controller and the network analyzer are electrically connected to the control terminal, the detection device also includes a temperature control mechanism, an array solution pool mechanism and a liquid supply mechanism, wherein the array solution pool mechanism and the liquid supply mechanism are both arranged in the temperature control mechanism, wherein a plurality of biosensors are arranged in the array solution pool mechanism, wherein the plurality of biosensors are electrically connected to the network analyzer, and the temperature control mechanism and the liquid supply mechanism are both electrically connected to the controller; The temperature control mechanism includes a thermostatic seat and a plurality of temperature sensors inserted on the array solution pool mechanism, the bottom and side walls of the thermostatic seat are embedded with fixed thermostatic plates arranged in parallel, and the temperature sensors and the fixed thermostatic plates are electrically connected to the controller; The thermostatic seat is dustpan-shaped, and a slide groove is provided at the bottom of the thermostatic seat; The array solution pool mechanism includes a bottom plate and a top plate; The top plate is provided with a plurality of test slots arranged in an array, a chip slot is provided on one side of the test slot, a sealing rubber ring is provided in the chip slot, a sensor jack, a liquid inlet and a liquid outlet are provided in the test slot, a thermostat slot is provided in the top plate on both sides of the test slot, a plug-in thermostat is provided in the thermostat slot, a biosensor is provided in the chip slot, and a temperature sensor is plugged in the sensor jack; A slider is arranged at the bottom of the bottom plate, the slider is arranged in the slide groove, threaded connection holes are opened at both ends of the top plate and the bottom plate, and the top plate and the bottom plate are fixedly connected by bolts; Both the fixed thermostatic plate and the plug-in thermostatic plate include a plurality of semiconductor heating plates and semiconductor cooling plates arranged alternately in parallel; It also includes a drying mechanism, which includes a drying fan. The drying fan is connected to a nitrogen tank. The air outlet of the drying fan is connected to the test slot through an air pipe, and the drying fan is electrically connected to a controller.

2. A detection device according to claim 1, characterized in that: The liquid supply mechanism includes a peristaltic pump and a sample placement plate; A slider is arranged at the bottom of the sample placement plate, and a plurality of placement holes are opened on the sample placement plate, and a temperature conducting plate is fixed in the placement holes; The sample container placed on the temperature conducting plate is connected to the peristaltic pump through a liquid taking conduit, the liquid taking conduit is provided with a liquid taking valve, the peristaltic pump is connected to the liquid inlet hole through a liquid inlet conduit, the liquid inlet conduit is provided with a liquid inlet valve, the liquid outlet is connected to a liquid discharge pipe, the liquid discharge pipe is provided with a liquid discharge valve; The peristaltic pump is electrically connected to the controller via a motor driver, and the liquid extraction valve, the liquid inlet valve and the liquid discharge valve are all electrically connected to the controller.

3. A detection device according to claim 2, characterized in that: The QCM biosensor chip uses an 8MHz AT-cut QCM chip.

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