CN115266766A - Multi-target unmarked cancer detection radio frequency biosensor based on heterogeneous resonance unit - Google Patents

Abstract

The multi-target label-free cancer detection radio frequency biosensor based on the heterogeneous resonance unit is designed to solve the problem that the detection range of the existing biological detection of cancer is small and it is difficult to realize multi-target detection. The two ends of the microstrip line in the multi-target label-free cancer detection radio frequency biosensor of the present invention are connected to two feed lines, the first open-loop resonator and the second open-loop resonator are distributed on both sides of the connected microstrip line, and the open-loop resonance A silicon dioxide layer is deposited on the surface of the second open-loop resonator, an amino group is modified on the surface of the silicon dioxide layer, and a carboxyl-DNA aptamer is bonded through the amino group. , thiol-DNA aptamers are modified on the surface of the first ring-opening resonator. The invention adopts microwave detection technology, applies microwave devices as sensing units, realizes specific detection through surface orientation modification technology, and realizes label-free biosensing of multi-target aptamers with high sensitivity and high selectivity.

Description

Multi-target radiofrequency biosensor for label-free cancer detection based on heterogeneous resonant cell

technical field

The invention adopts advanced QFN encapsulation and biological surface directional modification technology, and proposes a radio frequency biosensor based on a heterogeneous resonance unit to realize multi-target and label-free cancer detection.

Background technique

With the continuous development of society and the expansion of population size, the prevalence of cancer has gradually increased, and it has become the primary factor affecting the development of human society and limiting the improvement of happiness index. Standardized cancer prevention physical examination can detect cancer early, and about one-third of cancer can reduce mortality and social medical burden through early detection and early treatment. Taking breast cancer and cervical cancer, which are the top four new cases among women in the world, as an example, the cure rate of breast cancer can be increased by about 40%, and the mortality rate of cervical cancer can be reduced by 20% to 60%. At present, the sensing detection of cancer cells is the main way to realize the early diagnosis of cancer, mainly including electrochemical method, enzyme-based colorimetric method, immunofluorescence method and so on. Compared with the existing biological detection technology, the microwave detection technology has a large detection range, simple operation, economy, and portability, and can be used in a wide range of applications. As well as label-free detection, it has significant advantages in the early screening of multiple types of cancer.

Contents of the invention

The purpose of the present invention is to solve the problem that the detection range of the existing biological detection cancer is small and it is difficult to realize multi-target detection, and to provide a multi-target label-free cancer detection radio frequency biosensor based on a heterogeneous resonance unit.

The multi-target radio frequency biosensor for cancer detection based on the heterogeneous resonance unit of the present invention includes a substrate, a first port feeder, a second port feeder, a connecting microstrip line, two open-loop resonators and two input and output microstrip line groups , the first port feeder, the second port feeder, the connecting microstrip line, two open-loop resonators and two input and output microstrip line groups are integrated on the substrate by thin film technology, the first port feeder, the second port feeder and the connection The microstrip line is located on the axis of the substrate, one end connected to the microstrip line communicates with the feeder line of the first port, and the other end connected to the microstrip line communicates with the feeder line of the second port, and the first open-loop resonator and the second open-loop resonator are distributed On both sides of the connecting microstrip line, the first input and output microstrip line group and the second input and output microstrip line group are distributed on both sides of the connecting microstrip line, and the first open-loop resonator and the first input and output microstrip line The openings of the line group are opposite and electromagnetically coupled, the openings of the second open-loop resonator and the second input-output microstrip line group are opposite and electromagnetically coupled, and the microstrip line is connected to the first open-loop resonator and the second open-loop resonator. for parallel line coupling;

Among them, the material of the two open-ring resonators and the two input and output microstrip line groups is gold, and a silicon dioxide layer is deposited on the surface of the second open-ring resonator, and amino groups are modified on the surface of the silicon dioxide layer, and the amino groups are connected through amino bonds. Carboxy-DNA aptamers, thiol-DNA aptamers are modified on the surface of the first ring-opening resonator.

The invention adopts microwave detection technology, uses microwave devices as sensing units, and accurately distinguishes the measured objects mainly through surface directional modification of biological aptamers, and judges the sample concentration through the strong resonance behavior exhibited by the microwave sensor and the tested sample. The microwave sensor has the following unique advantages: first, specific detection is achieved through surface directional modification technology, which solves the problem that microwave sensing cannot distinguish biological macromolecules well; The microwave signal as the detection reference overcomes the problem that traditional electrochemical biosensors are difficult to perform multi-target recognition; thirdly, the detection performance is further improved through the selection of the detection sensitive frequency of the microwave device and the optimized design of the device, which provides a higher level for the improvement of the sensitivity of the microwave sensor. Feasibility; Fourth, the introduction of an active active feedback circuit with signal amplification effect and a window-type leadless integrated surface mount packaging process reduces the interference of environmental changes on sensor performance and keeps the sensor chip in good condition. sensitivity and reliability.

The invention takes the research on the surface directional modification technology of biological aptamers as the entry point, and aims to solve the problem of non-specific recognition in the microwave sensing technology. The research results of the present invention will provide a theoretical research basis for the biosensing of microwave measurement and biomodification cooperative detection, and provide a reference scheme for the processing, modification, packaging and detection process of such sensors, and will promote the development of microwave detection in the field of biological macromolecules. development is important.

The present invention researches multi-objective and high-sensitivity microwave bio-detection technology comprehensively and multi-levelly from the three aspects of heterogeneous resonant unit processing and surface directional modification, microwave device design, and microwave sensor micro-nano processing technology, and realizes the detection of mammary glands. For the detection of cancer and cervical cancer tumor DNA, the sensitivity is as high as 0.1MHz/nM, and the detection limit is lower than 0.3nM. The invention constructs a model of microwave dual-frequency resonance unit with signal amplification effect, reveals the cooperative detection mechanism of microwave detection and biological modification in the field of biosensing, and provides theoretical guidance for the specific synthesis and modification of materials based on microwave detection characteristics . In addition, this work has established a preliminary device real-time feedback evaluation system, laying a solid material foundation and device foundation for the future realization of reliable miniaturized and integrated microwave biosensors.

Aiming at the dual-frequency resonance sensitive unit and multi-target aptamer, the present invention proposes an optimization strategy for modifying the opposite side of the resonant unit, combined with microwave multi-dimensional signal technology, to realize the high-sensitivity and high-selectivity label-free biosensing of the multi-target aptamer . The sensor also uses an active feedback loop, using the surface mount and window-type leadless integrated packaging process (QFN package), which can not only compensate the power loss of the resonator, significantly improve the quality factor of the sensor, but also facilitate the realization of microwave The low cost, miniaturization and integration of biosensors is a new and promising method for early screening of cancer.

Description of drawings

Fig. 1 is a schematic diagram of the overall structure of the multi-target label-free cancer detection radio frequency biosensor based on the heterogeneous resonance unit of the present invention;

Fig. 2 is a processing flow chart of the multi-target label-free cancer detection radio frequency biosensor based on the heterogeneous resonance unit of the present invention;

Fig. 3 is a schematic diagram of the directional modification of the biological aptamer surface in the multi-target label-free cancer detection radio frequency biosensor based on the heterogeneous resonance unit of the present invention;

Fig. 4 is the S-parameter amplitude change test diagram of the multi-target label-free cancer detection radio frequency biosensor in the embodiment, wherein the ellipse on the left represents breast cancer, the ellipse on the right represents cervical cancer, ● represents 3nM, * represents 2nM, and the hexagon represents 1nM;

Fig. 5 is the S-parameter phase change test diagram of multi-target label-free cancer detection RF biosensor in the embodiment, wherein the left ellipse represents breast cancer, the right ellipse represents cervical cancer, ● represents 3nM, * represents 2nM, and hexagon represents 1nM;

Fig. 6 is a diagram of the packaged microwave biosensor testing system in the embodiment.

Specific implementation method

Specific Embodiment 1: In this embodiment, the multi-target label-free cancer detection RF biosensor based on the heterogeneous resonance unit includes a substrate, a first port feeder 1, a second port feeder 2, a connecting microstrip line 3, and two open-loop resonators And two input and output microstrip line groups, the first port feeder 1, the second port feeder 2, the connecting microstrip line 3, two open-loop resonators and two input and output microstrip line groups are integrated on the substrate by thin film technology , the first port feeder 1, the second port feeder 2 and the connecting microstrip line 3 are located on the axis of the substrate, one end of the connecting microstrip line 3 communicates with the first port feeder 1, and the other end of the connecting microstrip line 3 communicates with the second The port feeder 2 is connected, the first open-loop resonator 4-1 and the second open-loop resonator 4-2 are distributed on both sides of the connecting microstrip line 3, the first input and output microstrip line group 5-1 and the second input The output microstrip line group 5-2 is distributed on both sides of the microstrip line 3, and the openings of the first open-loop resonator 4-1 and the first input and output microstrip line group 5-1 are opposite and electromagnetically coupled, and the second The open-loop resonator 4-2 and the opening of the second input-output microstrip line group 5-2 are opposite and electromagnetically coupled, connecting the microstrip line 3 with the first open-loop resonator 4-1 and the second open-loop resonator 4- 2 are parallel line coupling;

The material of the two open-ring resonators and the two input-output microstrip line groups is gold, and a silicon dioxide layer is deposited on the surface of the second open-ring resonator 4-2, and amino groups are modified on the surface of the silicon dioxide layer. The amino group (chemically) bonds the carboxyl-DNA aptamer, and modifies the thiol-DNA aptamer on the surface of the first ring-opening resonator 4-1.

This embodiment utilizes the characteristic that the biological aptamer can specifically recognize, so that the modified microwave detection region can produce a specific reaction to the target detection substance. Establish the aptamer permittivity tensor analytical relationship under the action of incident waves, detect the change of aptamer permittivity according to microwave perturbation theory and Debye equation, and predict the detection range and sensitivity under different input biological parameters.

In this embodiment, the multi-target label-free cancer detection radio frequency biosensor is a radio frequency biosensor composed of a basic microwave resonant circuit, and the biological aptamer surface directional modified open-loop resonator is used. Compared with the existing terahertz cancer detector, it can Detection of specific types of cancer macromolecules is more conducive to assisting in the diagnosis of early cancers, saving detection time and costs. The biosensor does not need to be combined with genetic engineering to cultivate specific strains, nor does it need a supporting fluorescence detection system to analyze signals, avoids ultraviolet radiation, makes the production process and operation easier, has low environmental requirements, and simplifies the cumbersome steps of microbial sensor detection of cancer.

The microwave detection method used in this embodiment adopts active feedback amplification and QFN packaging, which improves the detection sensitivity and anti-interference ability. Compared with the existing photoelectrochemical gene sensor, it has good environmental adaptability and does not need sealing. The test results are more reliable.

Specific embodiment two: the difference between this embodiment and specific embodiment one is that the first port feeder 1, the second port feeder 2, the connecting microstrip line 3, two open-loop resonators and two input and output microstrip line groups adopt The photolithography process is integrated on the substrate.

Embodiment 3: This embodiment is different from Embodiment 1 or Embodiment 2 in that the substrate is a silicon substrate.

Embodiment 4: This embodiment differs from Embodiments 1 to 3 in that the thickness of the gold layer of the two open-loop resonators and the two input-output microstrip line groups is 100-300 nm.

Embodiment 5: This embodiment differs from Embodiments 1 to 4 in that the openings of the first split-loop resonator 4-1 and the second split-loop resonator 4-2 are sharp-angled.

Embodiment 6: This embodiment differs from Embodiments 1 to 5 in that a silicon dioxide layer with a thickness of 50-100 nm is deposited on the surface of the second open-loop resonator 4-2 by magnetron sputtering.

Embodiment 7: The difference between this embodiment and one of Embodiments 1 to 6 is that the method for modifying the surface of the silicon dioxide layer with amino groups is to soak the second ring-opening resonator 4-2 with a silicon dioxide layer in silane distillate. in the joint solution.

The silane coupling agent used in this embodiment is 3-amino-18 alkyl-trimethoxysilane, and its volume content in the aqueous solution is 5%.

Embodiment 8: The difference between this embodiment and one of Embodiments 1 to 7 is that the surface modification of the first ring-opening resonator 4-1 is to immerse the first ring-opening resonator 4-1 in mercapto-DNA. in the DNA solution.

Embodiment 9: This embodiment differs from Embodiments 1 to 8 in that the input and output microstrip line groups are connected to the active feedback circuit.

Embodiment 10: The difference between this embodiment and Embodiments 1 to 9 is that the heterogeneous resonance unit-based radio frequency biosensor for multi-target label-free cancer detection is packaged in a QFN (quad flat no-lead package) process.

Embodiment: In this embodiment, the multi-target label-free cancer detection radio frequency biosensor based on the heterogeneous resonance unit includes a substrate, a first port feeder 1, a second port feeder 2, a connecting microstrip line 3, two open-loop resonators and two One input and output microstrip line group, first port feeder 1, second port feeder 2, connecting microstrip line 3, two open-loop resonators and two input and output microstrip line groups are integrated on high-impedance silicon by photolithography On the substrate, the first port feeder 1, the second port feeder 2 and the connecting microstrip line 3 are located on the axis of the substrate, one end of the connecting microstrip line 3 communicates with the first port feeder 1, and the other end of the connecting microstrip line 3 communicates with the The second port feeder 2 is connected, the first open-loop resonator 4-1 and the second open-loop resonator 4-2 are distributed on both sides of the connecting microstrip line 3, the first input and output microstrip line group 5-1 and the second The two input-output microstrip line groups 5-2 are distributed on both sides of the microstrip line 3, and the first open-loop resonator 4-1 is opposite to the opening of the first input-output microstrip line group 5-1, and the second open-loop resonator 4-1 is opposite to the opening of the first input-output microstrip line group 5-1. The ring resonator 4-2 is opposite to the opening of the second input-output microstrip line group 5-2, and the connecting microstrip line 3 is parallel to the first open-loop resonator 4-1 and the second open-loop resonator 4-2 line coupling;

The material of the two open-loop resonators and the two input-output microstrip line groups is gold, and a silicon dioxide layer is deposited on the surface of the second open-loop resonator 4-2 by using a magnetron sputtering process. Amino groups are modified on the surface of the layer, carboxyl-DNA aptamers are bonded through the amino groups, and mercapto-DNA aptamers are modified on the surface of the first ring-opening resonator 4-1.

In this embodiment, the amino groups are modified on the surface of the silica layer by immersing the second ring-opening resonator 4-2 sputter-deposited with the silica layer in the silane coupling agent solution, and then soaking in the carboxyl-DNA aptamer solution middle. To modify the surface of the first ring-opening resonator 4-1 with the mercapto-DNA aptamer is to soak the first ring-opening resonator 4-1 in the mercapto-DNA aptamer solution.

Both the carboxyl-DNA aptamer and the thiol-DNA aptamer in this example were purchased from Beijing Saibaisheng Gene Technology Co., Ltd.

The modification process of the sensitive material in this embodiment:

The sensitive material in this embodiment is the DNA aptamer of breast cancer and cervical cancer, and the specific detection of the microwave sensor is realized according to the principle of complementary base pairing of DNA. The surface-directed modification process of the aptamer is as follows: a layer of amino groups is modified on the surface of silica, combined with the modifiability of the end group of the suitable ligand, the sulfhydryl-DNA aptamer is immobilized on the gold surface through the action of the gold sulfhydryl bond and the amide bond, respectively. , the carboxy-DNA aptamer was immobilized on the amino-silica surface to improve the overall detection sensitivity. In solution, intermolecular van der Waals forces cause the sulfhydryl group to have a special affinity for gold. Therefore, sulfhydryl-modified DNA can be directional modified on the surface of Au, while carboxyl-modified DNA can be directional modified on the surface of aminated silica due to the condensation reaction. The surface properties of the resonant unit can be adjusted by adjusting the selection of the modification material, the concentration of the modification material, and the modification time. The concentration of the DNA aptamer solution can be calculated according to the cross-sectional area of the DNA aptamer, the expected device surface coverage and the device surface area to calculate the number of moles of the DNA aptamer. The specific calculation formula is as follows:

DNA amount (mol) = (device surface area × expected coverage) / cross-sectional area of DNA aptamer.

Fabrication, packaging and testing process of microwave biosensor:

Firstly, the thin-film integrated passive device technology such as photolithography and thermal evaporation is used to prepare the resonant unit with the gold ring as the metal layer, and then the silicon dioxide is deposited on the gold surface by the magnetron sputtering process. The physical size, electromagnetic parameters, material properties and surface topography and structure information of the finished product are fed back by using ellipsometer, proslometer, AFM, SEM and other semiconductor measuring instruments. By adjusting the evaporation rate, rotation speed, oxygen supply and other technological processes, a uniform and dense heterogeneous resonance unit is prepared. An active feedback loop is connected in series with the sensor's SMA port via a coaxial cable to compensate for dissipated electromagnetic power. Then, stick an adhesive tape on the substrate, place the chip to be packaged on the adhesive tape with the front facing down; plastic seal the packaged chip around it, and smooth the back of the chip so that the back of the chip is flush with the top surface of the plastic package; The substrate and the adhesive tape form a package with the front and back of the chip exposed and the surroundings provided with a first plastic seal. Using the improved window-opening QFN packaging technology, the sensor prepared by the integrated passive device process is bonded to the PCB board in a safe and low-cost way. The way of exposing the top and bottom of the chip increases the heat dissipation area of the chip and improves the components and Thermal performance between components.

The multi-target radio frequency biosensor for label-free cancer detection in this embodiment mainly includes a heterogeneous dual-frequency microwave resonator, a biological aptamer surface modification layer and an active feedback amplifier circuit. The dual frequency is controlled by the size of two different open-loop resonators. And it adopts the window-type leadless integrated packaging process.

The processing method of the heterogeneous dual-frequency microwave resonator designed by the present invention is shown in Fig. 2 . Firstly, the resonant unit with the gold ring as the metal layer is prepared by thin-film integrated passive device technology such as photolithography and thermal evaporation, and then silicon dioxide is deposited on the gold surface by magnetron sputtering and dry etching. Subsequently, combined with the advantages of heterogeneous materials and aptamers' modifiability, the surface of the resonant unit and the end groups of the aptamers were respectively modified, and the simple, accurate and stable surface directional modification was realized through gold sulfhydryl bonds and amide bonds, ensuring High-precision identification and detection of various aptamers, as shown in Figure 3. Finally, the physical dimensions, electromagnetic parameters, material properties, and surface topography information of the finished product are fed back by semiconductor measuring instruments such as ellipsometers, profilometers, AFMs, and SEMs, and a preliminary real-time feedback evaluation system for devices is constructed. By adjusting the evaporation rate, rotation speed, oxygen supply and other technological processes, a uniform and dense heterogeneous resonance unit is prepared.

According to Fig. 4 and Fig. 5, it can be seen that when the test liquid contains the corresponding DNA to be tested, the frequency, amplitude and phase of the resonance point of the radio frequency sensor change with the DNA concentration. Moreover, the change ranges of the two resonance points do not overlap, and the changes of different targets can be clearly distinguished. When the content of the aptamer adsorbed by the resonant unit changes, the equivalent circuit parameters of the sensor will also change accordingly, resulting in changes in the resonant frequency of the sensor, the amplitude of the scattering parameters, and the phase. In addition, this embodiment will accurately construct the equivalent circuit model of the microwave-coupled feed type open-loop resonant unit, and based on the Debye equation, the existing analytical model of the dielectric constant tensor of the aptamer is corrected, and the dielectric constant tensor of the aptamer is clarified. Quantitative relationship with incident wave intensity, frequency, and aptamer concentration, and then construct aptamer biological parameters and dielectric constant tensors, device material properties, structural parameters, and multidimensional electromagnetic parameters of microwave resonator devices such as resonance frequency and scattering parameter amplitude The standard function mapping model of value, phase, etc., so as to judge the content and type of the detection target.

Fig. 6 is a device test diagram after adding an active feedback amplification loop. The active feedback loop is connected in series with the SMA port of the sensor via a coaxial cable, and the chip is ribbon bonded or wire bonded to the metal leads of the QFN package. The metal lead pitch is 0.5mm, and the size of the QFN box is 4mm×4mm. While protecting the device, the design facilitates subsequent experimental testing, and finally realizes a miniaturized and integrated radio frequency biosensor device with high quality factor.

Based on the above description, the purpose of the present invention is to design a multi-target, label-free, radio frequency biosensor for cancer detection, through aptamer surface directional modification technology and active/passive double feedback mechanism, to achieve specific cancer markers Object recognition can be used for early screening of cancer.

Claims (10)

1. The radio frequency biosensor for detecting the multi-target unmarked cancer based on the heterogeneous resonance unit is characterized by comprising a substrate, a first port feeder line (1), a second port feeder line (2), a connecting microstrip line (3), two open-loop resonators and two input and output microstrip line groups, wherein the first port feeder line (1), the second port feeder line (2), the connecting microstrip line (3), the two open-loop resonators and the two input and output microstrip line groups are integrated on the substrate by adopting a thin film process, the first port feeder line (1), the second port feeder line (2) and the connecting microstrip line (3) are positioned on the axis of the substrate, one end of the connecting microstrip line (3) is communicated with the first port feeder line (1), the other end of the connecting microstrip line (3) is communicated with the second port feeder line (2), the first open-loop resonator (4-1) and the second open-loop resonator (4-2) are distributed on two sides of the connecting microstrip line (3), the first input and output microstrip line groups (5-1) and the second input and output microstrip lines (5-2) are distributed on two sides of the connecting microstrip line groups of the microstrip line (3), the first open-loop resonator (4-2) and the second open-2) are coupled oppositely, the connecting microstrip line (3) is coupled with the first open-loop resonator (4-1) and the second open-loop resonator (4-2) in parallel lines;

the two open-loop resonators and the two input and output microstrip line groups are made of gold, a silicon dioxide layer is deposited on the surface of the second open-loop resonator (4-2), amino groups are modified on the surface of the silicon dioxide layer, carboxyl-DNA aptamers are connected through amino bonds, and mercapto-DNA aptamers are modified on the surface of the first open-loop resonator (4-1).

2. The radio frequency biosensor for multi-target unmarked cancer detection based on heterogeneous resonance units as claimed in claim 1, wherein the first port feed line (1), the second port feed line (2), the connecting microstrip line (3), the two open-loop resonators and the two input and output microstrip line groups are integrated on the substrate by photolithography process.

3. The heterogeneous resonant cell-based multi-target label-free cancer detection radiofrequency biosensor of claim 1, wherein the substrate is a silicon substrate.

4. The radio frequency biosensor for detecting cancer without marking targets based on heterogeneous resonance units as claimed in claim 1, wherein the thickness of gold layer of two open-loop resonators and two input-output microstrip line groups is 100-300 nm.

5. The heterogeneous resonance unit based multiple target unmarked cancer detection radio frequency biosensor according to claim 1, wherein the openings of the first open loop resonator (4-1) and the second open loop resonator (4-2) are of pointed structure.

6. The heterogeneous resonance unit-based radio frequency biosensor for multi-target label-free cancer detection according to claim 1, wherein a silicon dioxide layer with a thickness of 50-100 nm is deposited on the surface of the second open-loop resonator (4-2) by magnetron sputtering process.

7. The heterogeneous resonance unit-based radio frequency biosensor for multi-target label-free cancer detection according to claim 1, wherein the surface of the silica layer is modified with amino groups by immersing the second open-loop resonator (4-2) with the silica layer in a silane coupling agent solution.

8. The heterogeneous resonance unit-based radio frequency biosensor for multi-target label-free cancer detection according to claim 1, wherein the surface modification of the thiol-DNA in the first open-loop resonator (4-1) is performed by soaking the first open-loop resonator 4-1 in a solution of thiol-DNA.

9. The heterogeneous resonance unit-based multi-target label-free cancer detection radio-frequency biosensor according to claim 1, wherein the input and output microstrip line set is connected to an active feedback circuit.

10. The heterogeneous resonance unit-based multi-target label-free cancer detection radio frequency biosensor of claim 1, wherein the heterogeneous resonance unit-based multi-target label-free cancer detection radio frequency biosensor is packaged using QFN process.

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