CN102937649A - Miniaturized magnetic fluxgate biosensor for serum tumor marker detection - Google Patents
Miniaturized magnetic fluxgate biosensor for serum tumor marker detection Download PDFInfo
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Abstract
The present invention provides a miniaturized magnetic fluxgate biosensor for serum tumor marker detection. The biosensor comprises a miniaturized magnetic fluxgate sensor positioned on a glass substrate, a biochip and a signal acquisition system, wherein the biochip comprises a Au film on the glass substrate, a bio-sensitive film and a magnetic tag, the Au film is a sputtered Cr/Au film, the bio-sensitive film is a self-assembled film having a nano-scale thickness, and can be linked with various monoclonal antibodies, and the magnetic tag comprises streptavidin-modified magnetic nanoparticles or magnetic beads formed by magnetic nanoparticles, and can be combined with biotin modified monoclonal antibodies or polyclonal antibodies. According to the biosensor, the detection principle adopts a double antibody sandwich method, and the detection sensitivity is that serum tumor marker antigen having a concentration up to 1 ng/ml can be detected.
Description
Technical field
The present invention relates to biology sensor, particularly, relate to the miniaturized fluxgate biology sensor that a kind of blood serum tumor markers detects, belong to medical detecting method and magnetic transducing technical field.
Background technology
Malignant tumour is one of principal disease that threatens now human health and life, and the incidence of disease has the trend that rises year by year.Clinical main lesion detection means comprise at present:
[1] laboratory examination: mainly comprise routine inspection, biochemistry and immunologic test, pathological examination of blood, urine, stool etc.
[2] radiological examination: comprise that mainly x-ray fluoroscopy, X-ray take the photograph sheet, mammography, CT scan, Magnetic resonance imaging etc.
[3] radio nuclide examination: i.e. isotopic examination comprises functional examination inspection, scanner uni gamma examination, radiommunoassay etc.
[4] ultrasonic inspection: comprise A type, B-type ultrasonography.
[5] endoscopy: comprise various rigid or optical fiber mirrors.
In the research and clinical practice of tumour, early detection, early diagnosis, early treatment are crucial.Conventional image method such as x-ray, CT, nuclear magnetic resonance, B ultrasonic etc. only can be found the lump more than the 0.5cm at present, and at this moment Partial tumors has been in middle and advanced stage, and transfer has occured the tumour that has, and most patients have lost best treatment opportunity.Blood serum tumor markers is at cancer screening, diagnosis, judging prognosis and lapse to, estimate aspects such as treating curative effect all has larger practical value.Blood serum tumor markers detects has become one of method of early detection tumour, developed at present multiple blood serum tumor markers detection technique and constantly be applied to clinical in immunoradiometric assay, enzyme labeled immunoassay analytical technology, chemiluminescence immunoassay, fluoroimmunoassay, liquid chip detection technique etc., these methods respectively have its advantage, once can check out Diagnostic Value of Several Serum Tumor Markers such as liquid chip, compare with repeated detection and reduced cost.But the weak point of these methods is to lack dirigibility, and single index susceptibility is low, specificity is relatively poor.All there is defective aspect detection speed, accuracy rate and the testing cost, therefore utilizing the biosensor technology of the advantage development of new tumor markers detection of multidisciplinary intersection to seem particularly important.
Biology sensor is to utilize bioactive materials (such as enzyme, protein, DNA, antibody, antigen, biological membrane etc.) and physical chemistry transducer (such as galvanochemistry, optics, machinery, electricity, magnetic etc.) to organically combine a kind of biological information that consists of to detect analysis tool.Because biology sensor has widely purposes in health care, medical diagnosis on disease, food safety detection etc., countries in the world scientists' further investigation and energetically research and development have been subject to; Yet large-scale application and the popularization of biology sensor that still had some effects limit is too many such as the analysis operation step of conventional biosensor, analytical cycle long, expensive, optical detection apparatus that volume is large, expensive and need well-trained professional just can finish etc.In recent years, because the continuous progress of immunomagnetic bead technique and the development of sensor technology, scientists have proposed to be used for the biology sensor of new generation that the biological informations such as biology, medical science, science of heredity, toxicology are surveyed with Magnetic Sensor and in conjunction with the magnetic labels development.Utilize Magnetic Sensor and be [Baselt D.R. such as Baselt by US Naval Research Laboratory the earliest in conjunction with the theory that magnetic labels is used for detectable biomolecule, Lee G.U., Natesan M., Metzger S.W., Sheehan P.E., Colton R., A biosensor based onmagnetoresistancetechnology, Biosens.Bioelectr.17 (1998) 731.] proposed in 1998, the upsurge of magnetic biosensor research that Open from This Side.The Magnetic Sensor that is used for magnetic nano-particle (magnetic bead) detection of having reported at present mainly contains electromagnetic sensor, superconducting quantum interference device, fluxgate sensor, magnetoresistive transducer, Hall element, giant magnetoresistance sensor and giant magnetic impedance sensor.Fluxgate sensor is as a kind of low-intensity magnetic field detection means, be widely used as attitude sensor especially at satellite launch, carrier rocket, spacecraft etc. in recent years, reach greatly the shortcomings such as long-time stability are poor yet traditional fluxgate sensor has large, the high weight of volume, power consumption.Since the nineties, the develop rapidly of MEMS (micro electro mechanical system) (MEMS) technology is for the development of miniaturized fluxgate sensor provides an effectively reliably approach.Adopt MEMS technology development microminiaturization, integrated fluxgate sensor to become the focus of domestic and international research and development.Compare with traditional fluxgate sensor, MEMS fluxgate sensor compact conformation, volume is little, quality is light, Installation and Debugging are simple, be not afraid of shock impacts, it is little affected by variation of ambient temperature, shows wide application prospect in magnetic field detection and biomedical context of detection.
Find through the literature search to prior art, (the Diechhoff J. such as Dieckhoff, Schilling M., Ludwig F.) at " Appl.Phys.Lett. " (U.S.'s applied physics wall bulletin) " Vol.99, pp.112501,2011 " propose to survey magnetic nano-particle with fluxgate sensor, proved the feasibility of fluxgate sensor at biomedical detection field, but this research work has just obtained the response of fluxgate sensor to the Fe3O4 magnetic nano-particle, does not have realization to the detection of biomarker.
Along with the development of MEMS (micro electro mechanical system) (MEMS) technology, utilize the micro-fluidic chip of MEMS technology preparation to be applied to biological and medical science detection field, and the MEMS technology can be used to make microminiaturization, integrated fluxgate sensor equally.Fixing the combining with miniaturized fluxgate sensor of biological information made up novel blood serum tumor markers detection system, utilize the magnetic nano particle subtab that blood serum tumor markers is detected, have important Research Significance and clinical value.By document and patent retrieval, do not have to find about miniaturized fluxgate sensor is used for the correlative study achievement that blood serum tumor markers detects.
Summary of the invention
For defective of the prior art, the purpose of this invention is to provide the miniaturized fluxgate biology sensor that a kind of blood serum tumor markers detects.This biology sensor adopts the MEMS processing technology to make, and can be produced on supporting testing circuit, realizes miniaturization, the low cost of whole system, and makes detection system have high sensitivity and response speed, and be easy to produce in enormous quantities.The detection sensitivity of this biology sensor can detect blood serum tumor markers up to the antigen of 1ng/ml.
To achieve these goals, the miniaturized fluxgate biology sensor that blood serum tumor markers of the present invention detects comprises miniaturized fluxgate sensor, biochip, applies the solenoid coil of axial magnetic field and the signal acquiring system that is connected with miniaturized fluxgate sensor, wherein: described biochip is positioned on the sensitive axes of miniaturized fluxgate sensor, described biochip by be positioned at Au film on the glass substrate, be positioned at the bio-sensitive film on the Au film, the magnetic labels that is positioned on the bio-sensitive film forms.
Preferably, the Cr/Au film that described Au film is sputter, its thickness are 100-500nm, are used for modifying self-assembled film.
Preferably, described bio-sensitive film is the self-assembled film of one deck nanometer grade thickness, and self-assembled film is 11-sulfydryl undecanoic acid, then forms through the EDC+NHS activation, bio-sensitive film can connect various monoclonal antibodies, monoclonal antibody can with detected antigen combination.
Preferably, described magnetic labels is the magnetic nano-particle of streptavidin modification or the magnetic bead that is made of magnetic nano-particle.Can combine with monoclonal antibody or the polyclonal antibody through biotin modification.The monoclonal antibody of biotin modification or polyclonal antibody can with detected antigen combination.
Preferably, the described solenoid coil that applies axial magnetic field, this solenoid coil are one to be wound on the solenoid coil on the PVC, produce axial uniform magnetic field by external direct supply; The sensitive axes of miniaturized fluxgate sensor is placed on the axial location of solenoid coil, and biochip is positioned at same plane with the sensitive axes of miniaturized fluxgate sensor and closely links to each other.
Described signal acquiring system can be implemented in and apply one in the drive coil and make magnetic core be in the alternating current of state of saturation and the second harmonic signal that the output signal of receiving coil obtains after filtering is detected.The second harmonic signal size is directly proportional with the external magnetic field, therefore can measure the magnetic field size of magnetic labels generation and the relation between the detectable antigens.
Biology sensor provided by the invention, the principle of its detectable antigens are to adopt the double antibodies sandwich method.Adopt self-assembled film technology immobilized monoclonal antibody, monoclonal antibody is combined with antigen, is combined with biotinylated polyclonal antibody with the magnetic labels of streptavidin, because the immune response of Ag-Ab, magnetic labels is tagged on the surface of Au film.In case the detected biomolecule (antigen) of denier is arranged when existing, under the outside magnetic field effect, the disperse magnetic field that magnetic labels produces will cause the voltage signal of sensor to change, thereby realize the high-sensitivity detection to corresponding biomolecule.
Compared with prior art, the present invention has following beneficial effect:
(1) technology of preparing of Magnetic Sensor can with semi-conductive CMOS technical compatibility, thereby can realize the integrated magnetic sensor array, low, the low price of cost, be easy to the batch production;
(2) signal can obtain by integrated cmos circuit processing, and directly biological information is converted to electric signal, can realize instant analysis, and have very high detection sensitivity;
(3) biological sample carrying magnetic not itself can provide a very low noise magnetic measurement environment, and with respect to labels such as fluorescence molecule, radioisotope, enzymes, magnetic labels is highly stable;
(4) in single chemical examination, when can realizing the multiobjective analysis thing, sensor array detects, have advantage quick, highly sensitive detection.
(5) the blood serum tumor markers detection system among the present invention have detection speed fast, reusable, without particular surroundings with deposit the advantages such as requirement, volume is little, sensitivity is high; Do not need to depend on operating personnel's biology medical science experience and numerous and jumbled, expensive fluorescence detection device, just can realize specific biochemical analysis or medical diagnosis on disease, this will be conducive to realize biomedical disease detecting system portable, with low cost, quick diagnosis, will produce huge economic benefit and social value.
Description of drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is structural representation of the present invention.
Fig. 2 is along A-A sectional view shown in Figure 1.
Fig. 3 is along B-B sectional view shown in Figure 1.
Fig. 4 is principle of biochip figure of the present invention.
Among the figure: 1 is glass substrate, 2 is drive coil, and 3 is receiving coil, and 4 is magnetic core, 5 is pin, 6 is polyimide foam insulation, and 7 is the bottom coil of drive coil, and 8 is the bonding conductor of drive coil, 9 is the top layer coil of drive coil, 10 is the bottom coil of receiving coil, and 11 is the bonding conductor of receiving coil, and 12 is the top layer coil of receiving coil.13 is glass substrate, and 14 is the Au film, and 15 is self-assembled film, and 16 is the EDC+NHS active layer, and 17 is monoclonal antibody, and 18 is antigen, and 19 is monoclonal antibody or polyclonal antibody, and 20 is magnetic bead, and 21 is biotin, and 22 is streptavidin.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.Following examples will help those skilled in the art further to understand the present invention, but not limit in any form the present invention.Should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.
The miniaturized fluxgate biology sensor that present embodiment provides a kind of blood serum tumor markers to detect.This biology sensor comprises miniaturized fluxgate sensor, biochip, applies the solenoid coil of axial magnetic field and the signal acquiring system that is connected with miniaturized fluxgate sensor.
Described miniaturized fluxgate sensor is positioned on the glass substrate, can adopt existing techniques in realizing, be the sensor of putting down in writing in 200910046099.8 the Chinese patent such as number of patent application, can certainly adopt other can realize the miniaturized fluxgate sensor of function of the present invention.Following application number is that the sensor construction of 200910046099.8 records illustrates embodiments of the invention.
As Figure 1-3, in the present embodiment, miniaturized fluxgate sensor comprises glass substrate 1, drive coil 2, receiving coil 3, magnetic core 4, pin 5, polyimide foam insulation 6, two groups of three-dimensional spiral line pipe drive coils 2 that link to each other of symmetrical coiling on the closed rectangle magnetic core 4 are with one group of three-dimensional spiral line pipe of drive coil 2 vertical coilings receiving coil 3; Drive coil 2 is positioned on the glass substrate 1, is connected to form by bonding conductor 8 by bottom coil 7, top layer coil 9, and the two ends of drive coil 2 connect pin; Receiving coil 3 is positioned on the glass substrate 1, is connected to form by bonding conductor 11 by bottom coil 10, top layer coil 12, and the two ends of receiving coil 3 connect pin 5, and drive coil 2 and receiving coil 3 are all by polyimide foam insulation 6 and magnetic core 4 insulation isolation.Magnetic core 4, drive coil 2 and receiving coil 3 also are coated and fixed as a whole fully by polyimide insulative, support, with air insulated, sensor surface only exposes pin 5.Magnetic core 4 can be NiFe film, amorphous and nano-crystal soft-magnetic band material, and adopts MEMS technique to process.
During work, a logical alternating current makes magnetic core 4 be in state of saturation in drive coil 2, and when not having the external magnetic field, receiving coil 3 is exported without any signal; When having the external magnetic field to exist, receiving coil 3 has output signal, and signal is even-order harmonic, can obtain second harmonic signal after filtering.The second harmonic signal size is directly proportional with the external magnetic field, therefore can measure the external magnetic field size and Orientation.
As shown in Figure 4, in the present embodiment, biochip by be positioned at Au film 14 on the glass substrate 13, be positioned at the bio-sensitive film on the Au film 14, the magnetic labels that is positioned on the bio-sensitive film forms.
In the present embodiment, Au film 14 is the Cr/Au film of sputter, and its thickness is 100-500nm, is used for modifying self-assembled film.
In the present embodiment, bio-sensitive film adopts self-assembled film 15, and self-assembled film 15 is 11-sulfydryl undecanoic acid, then forms active layer 16 through the EDC+NHS activation, active layer 16 can connect various monoclonal antibodies 17, monoclonal antibody 17 can with detected antigen combination.
In the present embodiment, magnetic labels is the magnetic nano-particle of streptavidin 22 modifications or the magnetic bead 20 that is made of magnetic nano-particle.Can combine with monoclonal antibody or the polyclonal antibody 19 modified through biotin 21.The monoclonal antibody that biotin 21 is modified or polyclonal antibody 19 can with detected antigen 18 combinations.
In the present embodiment, the solenoid coil that applies axial magnetic field is one to be wound on the solenoid coil on the PVC, and its diameter and length are 10cm, can produce the axial uniform magnetic field of 0-100Oe by external direct supply; The sensitive axes of miniaturized fluxgate sensor is placed on the axial location of solenoid coil, and biochip is positioned at same plane with the sensitive axes of miniaturized fluxgate sensor and closely links to each other.
In the present embodiment, signal acquiring system comprises signal generator, power amplifier, wave filter, lock-in amplifier, oscillograph etc., all is some general instrument and equipment or circuit, can adopt existing techniques in realizing, also can buy as required or designed, designed, no longer describe in detail.
The above-mentioned biosensor production method of present embodiment is as follows:
(1) make miniaturized fluxgate sensor: method detailed can adopt existing techniques in realizing, is 200910046101.1 patent of invention such as the existing patent No..
(2) method for making of biochip is as follows:
● sputter Cr/Au film on glass substrate, get rid of photoresist, oven dry, exposure, development, etching Cr/Au film, remove photoresist, make the Au film be exposed to the outside.
● the preparation of bio-sensitive film on the Au film.Bio-sensitive film is the self-assembled film of one deck nanometer grade thickness, and self-assembled film is 11 sulfydryl undecanoic acids, then forms through the EDC+NHS activation.
● fixing of tumor markers monoclonal antibody.The tumor markers monoclonal antibody solution of debita spissitudo is splashed into above the Au film, 37 ℃ of lower cultivations 40 minutes of steam bath; Then with the cleaning of PBS solution, the sealing of BSA solution, at last with the cleaning of PBS solution, drying at room temperature.
● the antigen point sample.The antigenic solution of debita spissitudo is splashed into Au film surface, 37 ℃ of lower cultivations 40 minutes of steam bath, then clean with PBS solution, dry under the room temperature.
● fixing of biotinylated antibody.The biotinylated antibody solution of debita spissitudo is splashed into Au film surface, and then 22 ℃ of cultivations of room temperature clean with PBS solution, dry under the room temperature.
● magnetic labels is fixed.Magnetic labels is the magnetic nano-particle of streptavidin modification or the magnetic bead that is made of magnetic nano-particle.The magnetic labels of debita spissitudo is splashed into Au film surface, cultivate under the room temperature, then clean drying at room temperature with PBS solution.
Biology sensor provided by the invention, the principle of its detectable antigens are to adopt the double antibodies sandwich method.Adopt self-assembled film technology immobilized monoclonal antibody, monoclonal antibody is combined with antigen, is combined with biotinylated polyclonal antibody with the magnetic labels of streptavidin, because the immune response of Ag-Ab, magnetic labels is tagged on the surface of Au film.In case the detected biomolecule (antigen) of denier is arranged when existing, under the outside magnetic field effect, the disperse magnetic field that magnetic labels produces will cause the voltage signal of sensor to change, thereby realize the high-sensitivity detection to corresponding biomolecule.
More than specific embodiments of the invention are described.It will be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (9)
1. the miniaturized fluxgate biology sensor that blood serum tumor markers detects is characterized in that comprising miniaturized fluxgate sensor, the biochip that is positioned on the glass substrate, the solenoid coil that applies axial magnetic field and the signal acquiring system that is connected with miniaturized fluxgate sensor; Wherein: described biochip is positioned on the sensitive axes of miniaturized fluxgate sensor, forms by being positioned at Au film on the glass substrate, the bio-sensitive film on the Au film, magnetic labels on the bio-sensitive film; Described bio-sensitive film is the self-assembled film of one deck nanometer grade thickness.
2. the miniaturized fluxgate biology sensor of blood serum tumor markers detection according to claim 1 is characterized in that, the Cr/Au film that described Au film is sputter is used for modifying self-assembled film.
3. the miniaturized fluxgate biology sensor of blood serum tumor markers detection according to claim 2 is characterized in that described Cr/Au film, its thickness are 100-500nm.
4. the miniaturized fluxgate biology sensor that detects of blood serum tumor markers according to claim 1, it is characterized in that, described bio-sensitive film is 11-sulfydryl undecanoic acid, then form through the EDC+NHS activation, bio-sensitive film can connect various monoclonal antibodies, monoclonal antibody can with detected antigen combination.
5. the miniaturized fluxgate biology sensor that detects of blood serum tumor markers according to claim 4, it is characterized in that, described magnetic labels is the magnetic nano-particle of Streptavidin modification or the magnetic bead that is made of magnetic nano-particle, can combine with monoclonal antibody or the polyclonal antibody through biotin modification, the monoclonal antibody of biotin modification or polyclonal antibody can with detected antigen combination.
6. the miniaturized fluxgate biology sensor that detects of blood serum tumor markers according to claim 5, it is characterized in that, described biochip adopts self-assembled film technology immobilized monoclonal antibody, monoclonal antibody is combined with antigen, magnetic labels with streptavidin is combined with biotinylated polyclonal antibody, because the immune response of Ag-Ab, magnetic labels is tagged on the surface of Au film, in case when having the detected biomolecule of denier to exist, under the outside magnetic field effect, the disperse magnetic field that magnetic labels on the biochip produces will cause the variation of miniaturized fluxgate sensor output voltage, thereby realize the high-sensitivity detection to corresponding biomolecule.
7. the miniaturized fluxgate biology sensor that detects of each described blood serum tumor markers according to claim 1-6, it is characterized in that, the described solenoid coil that applies axial magnetic field is one to be wound on the solenoid coil on the PVC, produces axial uniform magnetic field by external direct supply; The sensitive axes of miniaturized fluxgate sensor is placed on the axial location of solenoid coil, and biochip is positioned at same plane with the sensitive axes of miniaturized fluxgate sensor and closely links to each other.
8. the miniaturized fluxgate sensor that detects of each described blood serum tumor markers according to claim 1-6, it is characterized in that, described miniaturized fluxgate sensor, its core material are NiFe film, amorphous or nano-crystal soft-magnetic band material, and adopt MEMS technique to make.
9. the miniaturized fluxgate sensor that detects of each described blood serum tumor markers according to claim 1-6, it is characterized in that, described signal acquiring system can be implemented in and apply one in the drive coil and make magnetic core be in the alternating current of state of saturation and the second harmonic signal that the output signal of receiving coil obtains after filtering is detected, the second harmonic signal size is directly proportional with the external magnetic field, therefore can measure the magnetic field size of magnetic labels generation and the relation between the detectable antigens.
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| CN104007151A (en) * | 2014-05-05 | 2014-08-27 | 北京航空航天大学 | Gene circuit based on lung-cancer marker detection |
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| CN114509563A (en) * | 2022-04-18 | 2022-05-17 | 合肥工业大学 | Giant magnetoresistance sensor combined with microfluidic technology, and manufacturing method and application thereof |
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