CN101576523A - Method for detecting tumour cells by adopting microelectrode array impedance biosensor chip - Google Patents
Method for detecting tumour cells by adopting microelectrode array impedance biosensor chip Download PDFInfo
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Abstract
本发明涉及一种利用微电极阵列阻抗生物传感器芯片检测肿瘤细胞的方法，利用纳米免疫磁微球富集分离技术俘获痰液或血液样品中的肿瘤细胞，形成磁微球－肿瘤细胞生物磁性组合体，借助外加磁铁产生磁力，将磁微球－肿瘤细胞生物磁性组合体在瞬间吸附于微电极阵列阻抗生物传感器芯片表面上，引起微传感电极的阻抗特性发生变化的原理，通过阻抗分析仪对微传感电极的阻抗特性进行并行扫描式测试，确定是否存在肿瘤细胞。本发明提供了一种无损、快速、简便的肿瘤细胞检测方法，适用于肿瘤早期诊断和治疗，也可对临床肿瘤患者的癌细胞转移复发进行诊断与监测，对重大疾病进行预测普查，同时可用于食品检测、药物筛选等领域。
The invention relates to a method for detecting tumor cells using a microelectrode array impedance biosensor chip, using nano-immune magnetic microsphere enrichment and separation technology to capture tumor cells in sputum or blood samples, and forming a magnetic microsphere-tumor cell biomagnetic combination body, with the help of an external magnet to generate magnetic force, the magnetic microsphere-tumor cell biomagnetic assembly is instantly adsorbed on the surface of the micro-electrode array impedance biosensor chip, which causes the impedance characteristics of the micro-sensing electrodes to change. Through the impedance analyzer The impedance characteristics of the micro-sensing electrodes are tested in parallel scanning to determine the presence of tumor cells. The invention provides a non-destructive, rapid and simple tumor cell detection method, which is suitable for early diagnosis and treatment of tumors, and can also be used for diagnosis and monitoring of cancer cell metastasis and recurrence in clinical tumor patients, and for predicting and generalizing major diseases. In food testing, drug screening and other fields.
Description
Technical field
The present invention relates to a kind of method of utilizing microelectrode array impedance biosensor chip detection tumour cell.Can be used for fields such as food inspection, drug screening, medical diagnosis on disease and treatment.Belong to biomedical engineering and electronic sensor technology interdisciplinary field.
Background technology
The method of pulmonary cancer diagnosis mainly contains pathology, iconography, immunology, molecular biology in general.The sample that is detected has sputum, lump, hydrothorax and blood etc.Just at present China's hospital image is learned detection level, in case generally believe and find the lung cancer lump, in most cases lump 〉=1cm, patient's the course of disease is not in early days.In above-mentioned every cytolgical examination, the most general with the application of blood, sputum cytoscopy especially, the sputum cancer cell detects the recall rate that helps to improve early-stage cases; The blood cancer cell detects and mainly solves clinical patients with lung cancer transfer and relapse prediction, prognosis judgement, specificity anti-recurrence treatment problem.And at present, the inspection of sputum cancer cell finally is to depend on optical technology, by the microscopic discriminatory analysis; The blood testing sample finally is that (Polymerase Chain Reaction PCR) carries out the gene quantification analysis by the PCR.Also have a kind of detection method of the electronic sensor technology based on piezoelectric quartz crystal and PZT piezoelectric, the big molecule of detection of biological need carry out immune response at sensor surface, thereby the quality of biomacromolecule is changed into corresponding electronic information.Owing to need carry out immune response on the surface of sensor, be difficult to blood and several cancer cells in the sputum complex system are separated and accumulate to the surface of sensor from thousands of other cells, sensitivity and verification and measurement ratio are very low.
The nano immune magnetic bead is even, spherical, as to have superparamagnetism and containment vessel nano particle, and magnetic bead can binding active proteins matter (antibody) form Ag-Ab-immunomagnetic beads biomagnetism assembly, can be attracted by magnet again.By extraneous magneticaction, this biomagnetism assembly can with other separating substances, reach the purpose of effective separation specific cell composition.The volume of nanometer magnetic bead is very little, neither can or not influence cell function and biodegradable again by damaging cells, can be used to immediately analyze and experiment subsequently.For the complex system (blood or sputum) that has problems such as tumour cell rareness, nano immune magnetic bead technology is a kind of effective concentration and separation technology, for the early diagnosis of tumour provides a kind of new technical thought.
Chinese invention patent ZL2005100262113 has proposed a kind of " utilizing the method for probe of piezoelectric sensing chip controlled from biologic magnetic detecting cancer cell ", wherein said probe of piezoelectric sensing chip controlled from biologic magnetic is the beam type sensor chip of lead zirconate titanate piezoelectric membraneous material, biomagnetism assembly by magnetic ball-cancer cell is adsorbed onto on the piezoelectric sensing chip surface, convert the quality and the quantity of cancer cell to be measured to corresponding electronic information, whether exist in order to detecting cancer cell.Sensing chip in this invention adopts the beam type structure, and complex manufacturing technology, crudy be difficult to guarantee, and detects informational needs and convert electronic information to and could detect, and need carry out numerous and diverse data acquisition and processing (DAP) work.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of method of utilizing microelectrode array impedance biosensor chip detection tumour cell is proposed, can be used for the tumour cell in blood or the sputum sample is detected fast and accurately, effectively improve the positive rate of cancer early stage patient, and simple to operation, cost is lower.
For achieving the above object, the present invention utilizes nano immune magnetic microsphere concentration and separation technology to capture tumour cell in sputum or the blood sample, form magnetic microsphere-tumour cell biomagnetism assembly, produce magnetic force by adding magnet, magnetic microsphere-tumour cell biomagnetism assembly was adsorbed on the microelectrode array impedance biosensor chip surface in moment, cause the principle that the impedance operator of micro sensing electrode changes, to the scan-type test that walks abreast of the impedance operator of micro sensing electrode, determine whether to exist tumour cell by electric impedance analyzer.
The present invention utilizes the method for microelectrode array impedance biosensor chip detection tumour cell, comprises following concrete steps:
1, adopt the microelectrode array impedance biosensor chip that is made of 4 microelectrode array detecting units, each microelectrode array detecting unit comprises supporting layer, basalis, micro sensing electrode layer, insulation course and liquid storage tank five-layer structure; Described micro sensing electrode layer has the micro sensing electrode of 16～100 passages, and the range of size of each micro sensing electrode is 10 μ m～50 μ m, and the micro sensing electrode separation is 10 μ m～30 μ m.
2, extract the sputum or the blood sample of 1-5 milliliter, join in the microelectrode array impedance biosensor chip liquid storage tank, after liquefaction is handled, extracting 1-5ulA type specificity proteantigen again joins in the microelectrode array impedance biosensor chip liquid storage tank, tumour cell in sputum in the liquid storage tank or the blood sample is repaired by A type specificity proteantigen, formed A type specificity proteantigen-tumour cell life assemblage body.
3, a kind of Type B specific protein antibody is repaired Fe
3O
4On the magnetic microsphere, through immune response in 3-10 minute, form antibody-magnetic microsphere, again this antibody magnetic microballoon 1-5ul is joined in the microelectrode array impedance biosensor chip liquid storage tank, make antibody-magnetic microsphere capture A type specificity proteantigen-tumour cell life assemblage body in the liquid storage tank, and carrying out immune response at 25-30 ℃, reaction time 2-6 minute, the reaction back formed magnetic microsphere-tumour cell biomagnetism assembly.
4, below microelectrode array impedance biosensor chip supporting layer, produce magnetic force, magnetic microsphere-tumour cell biomagnetism assembly all is adsorbed onto on the microelectrode array impedance biosensor chip surface by applying magnet; By miniature three-electrode system, with the micro sensing electrode is working electrode, is to electrode with miniature platinum electrode, is reference electrode with miniature Ag/AgCl electrode,, survey and determine whether to exist tumour cell the scan-type test that walks abreast of the impedance operator of micro sensing electrode by electric impedance analyzer.
Among the present invention, described microelectrode array impedance biosensor chip selects for use biocompatibility better polymerization thing material (as Parylene or polyimide) as basalis and insulating layer material, select for use inertia noble metal (gold, silver, platinum, iridium etc.) as the micro sensing electrode material, select for use the SU-8 photoresist as the liquid storage tank material, select for use monocrystalline silicon as support layer material.
Basalis thickness in the described microelectrode array impedance biosensor chip is 5～20 μ m.
Micro sensing electrode layers thickness in the described microelectrode array impedance biosensor chip is 0.4～1 μ m.
Thickness of insulating layer in the described microelectrode array impedance biosensor chip is 2～4 μ m.
Liquid storage tank height in the described microelectrode array impedance biosensor chip is 50～500 μ m, and the liquid storage tank bottom size is 250 μ m * 250 μ m～850 * 250 μ m, and thickness is 50～200 μ m.
The present invention can carry out quick scan-type testing impedance to hyperchannel micro sensing electrode, and a plurality of microelectrode array detecting units can detect simultaneously to different sputums or blood sample, the tumour cell of quantity rareness can be detected, realize whether there is tumour cell in accurate fast detecting sputum or the blood sample, greatly reduced the possibility of omission.There is higher sensitivity in the inventive method to tumour cell, when tumour cell is carried out fast detecting, need not tumour cell is fixed on sensor surface, for detecting, tumour cell provides a kind of harmless, quick, easy detection method, be applicable to early diagnosis of tumor and treatment, also can diagnose and monitor, major disease is predicted generaI investigation, can be used for fields such as food inspection, drug screening simultaneously clinical tumor patient's cancer metastasis recurrence.
Description of drawings
Fig. 1 utilizes the fundamental diagram of microelectrode array impedance biosensor chip detection tumour cell for the present invention.
Among Fig. 1,1 is supporting layer; 2 is basalis; 3 is the micro sensing electrode layer; 4 is insulation course; 5 is liquid storage tank; 6 are lead-in wire; 7 is lead solder-joint; 8 is miniature platinum electrode; 9 is miniature Ag/AgCl contrast electrode; 10 is electric impedance analyzer; 11 is the biomagnetism assembly of magnetic bead-tumour cell.
Fig. 2 is the structural representation of microelectrode array impedance biosensor chip described in the present invention.
Among Fig. 2,1 is supporting layer; 2 is basalis; 3 is the micro sensing electrode layer; 4 is insulation course; 5 is liquid storage tank; 7 is lead solder-joint.
Embodiment
Below in conjunction with drawings and Examples technical scheme of the present invention is described in detail.
A kind of method of utilizing microelectrode array impedance biosensor chip detection tumour cell that the present invention proposes, its basic principle of work as shown in Figure 1.For a sensor based on impedance operator, the combination of its electric capacity, inductance and resistance characteristic can produce a specific impedance signal under normal operation.If the variation of sensor surrounding environment causes any variation of above-mentioned characteristic, all can cause the change of impedance.By measuring the characteristic of this impedance transducer, will obtain a series of new impedance operators with frequency change.The present invention has adopted a kind of microelectrode array impedance biosensor chip, and its structure as shown in Figure 2.This sensor chip is made of 4 microelectrode array detecting units, and each microelectrode array detecting unit comprises supporting layer 1, basalis 2, micro sensing electrode layer 3, insulation course 4 and liquid storage tank 5 five-layer structures.Described micro sensing electrode layer is by the micro sensing electrode of 16～100 passages, and the range of size of each micro sensing electrode is 10 μ m～50 μ m, and electrode separation is 10 μ m～30 μ m.
The method of the invention is being used for the tumour cell testing process, also comprises the biomagnetism assembly 11 of the lead-in wire 6 of micro sensing electrode, the lead solder-joint 7 that is used to be electrically connected, miniature platinum electrode 8, miniature Ag/AgCl contrast electrode 9, electric impedance analyzer 10 and magnetic bead-tumour cell.
Below three embodiment provided the specific operation process of the method for utilizing microelectrode array impedance biosensor chip detection tumour cell in detail, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
1) adopt the microelectrode array impedance biosensor chip that is made of 4 microelectrode array detecting units, each microelectrode array detecting unit comprises supporting layer, basalis, micro sensing electrode layer, insulation course and liquid storage tank five-layer structure; Described micro sensing electrode layer has the micro sensing electrode of 16 passages, and the range of size of each micro sensing electrode is 10 μ m, and the micro sensing electrode separation is 10 μ m;
2) extract 1 milliliter sputum or blood sample, join in the microelectrode array impedance biosensor chip liquid storage tank, after liquefaction is handled, extracting 1ul A type specificity proteantigen again joins in the microelectrode array impedance biosensor chip liquid storage tank, tumour cell in sputum in the liquid storage tank or the blood sample is repaired by A type specificity proteantigen, formed A type specificity proteantigen-tumour cell life assemblage body;
3) a kind of Type B specific protein antibody is repaired Fe
3O
4On the magnetic microsphere, through immune response in 8 minutes, form antibody-magnetic microsphere, again this antibody magnetic microballoon 1ul is joined in the microelectrode array impedance biosensor chip liquid storage tank, make antibody-magnetic microsphere capture A type specificity proteantigen-tumour cell life assemblage body in the liquid storage tank, and carry out immune response at 28 ℃, in 4 minutes reaction time, the reaction back forms magnetic microsphere-tumour cell biomagnetism assembly;
4) below microelectrode array impedance biosensor chip supporting layer, produce magnetic force, magnetic microsphere-tumour cell biomagnetism assembly all is adsorbed onto on the microelectrode array impedance biosensor chip surface by applying magnet; By miniature three-electrode system, with the micro sensing electrode is working electrode, is to electrode with miniature platinum electrode, is reference electrode with miniature Ag/AgCl electrode,, survey and determine whether to exist tumour cell the scan-type test that walks abreast of the impedance operator of micro sensing electrode by electric impedance analyzer.Impedance as the micro sensing electrode increases before the detection to some extent, illustrates in sputum or the blood sample to have tumour cell.
The impedance biosensor of microelectrode array described in the present invention chip can be made by following concrete technology:
1, choosing monocrystalline silicon is supporting layer, above it the deposition or spin coating one deck have biocompatibility thin polymer film as basalis, THICKNESS CONTROL is at 5 μ m～20 μ m;
2, above basalis, adopt photoetching, sputter or evaporation and peel off (Lift-off) technology and make patterned micro sensing electrode layer, make contact conductor layer and lead solder-joint layer simultaneously; The metal of making the micro sensing electrode layer comprises inertia noble metals such as gold, silver, platinum, iridium; The micro sensing electrode layers thickness is controlled at 0.4 μ m～1 μ m;
3, above the micro sensing electrode layer, again the deposition or spin coating have biocompatibility thin polymer film as insulation course, THICKNESS CONTROL is at 2 μ m～4 μ m;
4,, the insulation course of micro sensing electrode layer and lead solder-joint layer top is removed, thereby exposed " conductive window " by reactive ion etching (RIE), laser ablation or photoetching process;
5, select the SU-8 photoresist for use, above insulation course, by photoetching, exposure, developing process, above each microelectrode array detecting unit, make a liquid storage tank, the liquid storage tank height is controlled at 50～500 μ m, the liquid storage tank bottom size is 250 μ m * 250 μ m～850 * 250 μ m, and THICKNESS CONTROL is at 50～200 μ m, thereby finishes the manufacturing process of chip.
1) adopt the microelectrode array impedance biosensor chip that is made of 4 microelectrode array detecting units, each microelectrode array detecting unit comprises supporting layer, basalis, micro sensing electrode layer, insulation course and liquid storage tank five-layer structure; Described micro sensing electrode layer has the micro sensing electrode of 36 passages, and the range of size of each micro sensing electrode is 30 μ m, and the micro sensing electrode separation is 20 μ m;
2) extract 3 milliliters sputum or blood sample, join in the microelectrode array impedance biosensor chip liquid storage tank, after liquefaction is handled, extracting 3ul A type specificity proteantigen again joins in the microelectrode array impedance biosensor chip liquid storage tank, tumour cell in sputum in the liquid storage tank or the blood sample is repaired by A type specificity proteantigen, formed A type specificity proteantigen-tumour cell life assemblage body;
3) a kind of Type B specific protein antibody is repaired Fe
3O
4On the magnetic microsphere, through immune response in 10 minutes, form antibody-magnetic microsphere, again this antibody magnetic microballoon 3ul is joined in the microelectrode array impedance biosensor chip liquid storage tank, make antibody-magnetic microsphere capture A type specificity proteantigen-tumour cell life assemblage body in the liquid storage tank, and carry out immune response at 25 ℃, in 6 minutes reaction time, the reaction back forms magnetic microsphere-tumour cell biomagnetism assembly;
4) below microelectrode array impedance biosensor chip supporting layer, produce magnetic force, magnetic microsphere-tumour cell biomagnetism assembly all is adsorbed onto on the microelectrode array impedance biosensor chip surface by applying magnet; By miniature three-electrode system, be working electrode with the micro sensing electrode, be to electrode with miniature platinum electrode, be reference electrode with miniature Ag/gCl electrode, by electric impedance analyzer to the scan-type test that walks abreast of the impedance operator of micro sensing electrode.Impedance as the micro sensing electrode increases before the detection to some extent, illustrates in sputum or the blood sample to have tumour cell.
1) adopt the microelectrode array impedance biosensor chip that is made of 4 microelectrode array detecting units, each microelectrode array detecting unit comprises supporting layer, basalis, micro sensing electrode layer, insulation course and liquid storage tank five-layer structure; Described micro sensing electrode layer has the micro sensing electrode of 100 passages, and the range of size of each micro sensing electrode is 50 μ m, and the micro sensing electrode separation is 30 μ m;
2) extract 5 milliliters sputum or blood sample, join in the microelectrode array impedance biosensor chip liquid storage tank, after liquefaction is handled, extracting 5ul A type specificity proteantigen again joins in the microelectrode array impedance biosensor chip liquid storage tank, tumour cell in sputum in the liquid storage tank or the blood sample is repaired by A type specificity proteantigen, formed A type specificity proteantigen-tumour cell life assemblage body;
3) a kind of Type B specific protein antibody is repaired Fe
3O
4On the magnetic microsphere, through immune response in 3 minutes, form antibody-magnetic microsphere, again this antibody magnetic microballoon 5ul is joined in the microelectrode array impedance biosensor chip liquid storage tank, make antibody-magnetic microsphere capture A type specificity proteantigen-tumour cell life assemblage body in the liquid storage tank, and carry out immune response at 30 ℃, in 2 minutes reaction time, the reaction back forms magnetic microsphere-tumour cell biomagnetism assembly;
4) below microelectrode array impedance biosensor chip supporting layer, produce magnetic force, magnetic microsphere-tumour cell biomagnetism assembly all is adsorbed onto on the microelectrode array impedance biosensor chip surface by applying magnet; By miniature three-electrode system, be working electrode with the micro sensing electrode, be to electrode with miniature platinum electrode, be reference electrode with miniature Ag/AgCl electrode, by electric impedance analyzer to the scan-type test that walks abreast of the impedance operator of micro sensing electrode.Impedance as the micro sensing electrode increases before the detection to some extent, illustrates in sputum or the blood sample to have tumour cell.
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