WO2017011940A1 - 太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法 - Google Patents

太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法 Download PDF

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WO2017011940A1
WO2017011940A1 PCT/CN2015/084344 CN2015084344W WO2017011940A1 WO 2017011940 A1 WO2017011940 A1 WO 2017011940A1 CN 2015084344 W CN2015084344 W CN 2015084344W WO 2017011940 A1 WO2017011940 A1 WO 2017011940A1
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biological sample
terahertz
metamaterial
avidin
sample
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PCT/CN2015/084344
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English (en)
French (fr)
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应义斌
徐文道
***
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浙江大学
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Priority to US15/106,710 priority Critical patent/US10031133B2/en
Priority to PCT/CN2015/084344 priority patent/WO2017011940A1/zh
Publication of WO2017011940A1 publication Critical patent/WO2017011940A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3581—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation
    • G01N21/3586—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation by Terahertz time domain spectroscopy [THz-TDS]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • the invention relates to a method for amplifying a terahertz signal of a biological sample, in particular to a method for amplifying a biological sample in combination with a terahertz metamaterial and a nano gold particle.
  • terahertz spectroscopy has gradually attracted people's attention. Since the vibration and rotational energy levels of many macromolecules fall in the terahertz band, terahertz waves are considered to be a very potential band for biological sample detection. For applications where terahertz spectroscopy has great application prospects, such as safety, biology, medicine, agriculture and material characterization, there are trace or even ultra-trace non-destructive testing requirements.
  • the technique is difficult to be used for rapid detection of trace samples.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the above background art, and provide a method for amplifying a biological sample signal combined with a terahertz metamaterial and a nano gold particle, and the method should have the characteristics of high sensitivity and quick and convenient detection.
  • the biological sample solution is added dropwise to the surface of the cleaned metamaterial, and each concentration is added at least three times, the amount of each drop is the same, and three reference sample points are arbitrarily set, as shown in FIG. 2, the reference sample points are both The position of the sample to be tested is different, and it is dried at room temperature after dropping;
  • the frequency shift of the transmission peak or the reflection peak is obtained from the terahertz time domain signal, and the transmittance or reflectance of all the sample points to be tested and the reference sample point are calculated, and calculated according to the frequency value corresponding to the lowest point of the transmittance or the reflectance.
  • Frequency shift of transmission peak or reflection peak The fast Fourier transform is used to convert the terahertz spectrum time domain signal of the biological sample to the frequency domain signal, and the transmittance or reflectance of the sample point to be tested is calculated from the frequency domain signal, and will be treated The absolute value obtained by subtracting the transmittance of the sample point from the reference sample point or the frequency value corresponding to the lowest point of the reflectance is used as the frequency shift of the transmission peak or the reflection peak to achieve amplification of the avidin signal.
  • the supermaterials in the steps 2) and 3) are cleaned by taking a complete terahertz metamaterial, washing it with deionized water, phosphate buffer, washing with deionized water, and drying with nitrogen.
  • the gold-labeled avidin solution in the step 1) is configured in the following manner:
  • the common avidin and the nano gold particles are mixed, and the ratio of the amount of the common avidin and the nano gold particles is 10:1 to 2500:1, as shown in Fig. 1, and shaken on a shaker under normal temperature conditions. And stored at a temperature of 0 to 4 ° C; the nano gold solution formed is a wine red;
  • the gold-labeled avidin is taken out, placed in a centrifuge tube, centrifuged by a centrifuge, and the excess common avidin in the supernatant of the centrifuge tube is removed, and the precipitate is repeatedly washed with deionized water, and finally deionized water is added and fully oscillated. A gold standard avidin solution was obtained.
  • the centrifugation speed of the centrifuge in the step 1.2) is 5000 to 10000 rpm, and the centrifugation time is 10 to 20 minutes.
  • the detection area of the sample point to be tested is greater than 1 mm 2 .
  • the biological sample uses common avidin, DNA or E. coli.
  • the concentration range of the gold-labeled avidin solution and the biological sample solution obtained in the step 1) is between 2 ⁇ 10 -10 and 10 ⁇ 10 -10 mol/L.
  • the amount of the gold sample avidin solution in the biological sample solution in the step 3) or the step 4) is 5 to 100 ul per drop.
  • the biological sample adopts DNA or Escherichia coli, and a plurality of gold-labeled avidin solutions of different concentrations are a gold-labeled avidin and a biotin-labeled biological sample complex solution.
  • the combination method and process are: the combination of gold-labeled avidin and biotin in the biotin-labeled biological sample, forming a gold-labeled avidin and biotin-labeled biological sample complex solution, gold-labeled avidin and organism
  • concentration ratio between the prime or biological sample is 1:1 or more, and the preferred ratio is 1:1 to 4:1.
  • the pH of the common avidin in the step 1.1) is 5 to 9, and the pH of the nano gold particles is 8 to 12.
  • the humidity of the measurement environment is ⁇ 0.2%.
  • the preferred avidin of the present invention may be selected from the avidin of A9275 manufactured by Sigma, but is not limited thereto.
  • the nano gold particles have a particle diameter of 8 to 90 nm.
  • the gold-labeled avidin of the present invention can be used for the binding reaction with biotin, and thus the signal amplification method can be widely applied in DNA hybridization and antibody-specific binding.
  • the nano gold particles of the present invention can directly amplify the sample signal without exciting the surface plasma, and the effect is remarkable.
  • the nano gold of the present invention can be replaced by other metal nanoparticles, including nano silver particles, nano gold bars, nano silver coated gold particles, nano gold coated silver particles and the like.
  • the preferred terahertz time domain spectroscopy system of the present invention is recommended to use a z3 terahertz time domain spectroscopy system manufactured by z-omega.
  • the invention adopts Terahertz time-domain spectroscopy (THz-TDS), which is a new research and detection technology developed in recent years in the world.
  • THz-TDS Terahertz time-domain spectroscopy
  • a terahertz wave is an electromagnetic wave having a wavelength between microwave and infrared radiation and has a frequency of 0.1-10 THz.
  • the energy of terahertz radiation is very low, a large number of molecules, especially many organic macromolecules (DNA, protein, etc.) exhibit strong absorption and dispersion in this frequency band.
  • the metamaterial of the present invention is an artificially fabricated periodic structural material having properties that many natural materials cannot exhibit.
  • the research on metamaterials in the terahertz band has gradually attracted the attention of scholars. At present, it has been applied in communication and absorbers.
  • metamaterials have gradually played a dominant role in terahertz band detection applications.
  • the present invention utilizes terahertz metamaterial technology, which has the beneficial effects of:
  • the invention combines the terahertz metamaterial technology and the nano gold particle modification technology to amplify the sample signal by using the electric field local enhancement effect of the super material.
  • the invention simultaneously utilizes nano gold to change the effect of surface electric field distribution, and further enlarges the sample signal by the method of nano gold modification, so the detection sensitivity of the method is high.
  • the method of the invention can greatly improve the detection sensitivity; and the method is simple and rapid in operation, and can meet the increasing demand for rapid detection.
  • Figure 1 is a schematic diagram of the gold-labeled avidin of the metamaterial detection of the present invention.
  • FIG. 2 is a distribution diagram of a sample point and a reference sample point of a surface of a metamaterial according to Embodiment 1 of the present invention.
  • Example 3 is a graph showing the frequency shift of a resonance peak of a terahertz metamaterial caused by a sample of avidin and a gold avidin sample according to Example 1 of the present invention.
  • Example 4 is a graph showing the frequency shift of a resonance peak of a terahertz metamaterial caused by a nano gold sample in Example 3 of the present invention.
  • A is a common avidin
  • B is a nano gold
  • C is a metamaterial
  • D is a terahertz wave
  • E is a sample point to be tested
  • F is a reference sample point.
  • Nano-gold solution (pH about 10) 0.5mL, the two are mixed (the ratio of the amount of common avidin to nano-gold material is about 2500:1), shaken on a shaker at room temperature for 15 minutes, and in the refrigerator In the middle of storage, the storage temperature is 4 ° C, the storage time is greater than or equal to 0.5 hours;
  • the centrifuge tube containing the gold-labeled avidin was taken out from the refrigerator, and another centrifuge tube of the same type was taken to inject the same amount of deionized water, and then centrifuged, centrifuged at 10,000 rpm, and centrifuged for 15 minutes. After centrifugation, the excess common avidin is suspended in the upper layer, the gold-labeled avidin is in the lower layer of the centrifuge tube, the supernatant in the centrifuge tube is removed, and the precipitate is repeatedly washed with deionized water, and the excess common avidin is washed and removed;
  • concentrations of the gold-labeled avidin solution were separately disposed (in this example, 2 ⁇ 10 -10 mol / L, 4 ⁇ 10 -10 mol / L, 6 ⁇ 10 -10 mol / L, 8 ⁇ 10 - 10 mol/L and 10 ⁇ 10 -10 mol/L), take 10 ⁇ L of the solution, add dropwise to the surface of the cleaned metamaterial, add three times for each concentration, and set three reference sample points (without any sample), room temperature The air is dried (the amount of the corresponding gold-labeled avidin is 2fmol, 4fmol, 6fmol, 8fmol, 10fmol), and the detection area of the sample to be tested is about 4mm 2 ;
  • the terahertz time domain spectrum system starts to be filled with nitrogen gas, the humidity drops, and the laser can be measured after half an hour of heating; the lid for measuring the terahertz time domain spectrum system is turned on.
  • the metamaterial is placed in the detection optical path and fixed by the clamp; in the case of nitrogen filling, the sampling point and reference of the same metamaterial are collected on the spectrum of the terahertz time domain spectrum system with a spectral bandwidth of 0.1-3.5 THz.
  • the terahertz time-domain spectrum of the sample point is performed by the terahertz time domain spectrum system starts to be filled with nitrogen gas, the humidity drops, and the laser can be measured after half an hour of heating; the lid for measuring the terahertz time domain spectrum system is turned on.
  • the metamaterial is placed in the detection optical path and fixed by the clamp; in the case of nitrogen filling, the sampling point and reference of the same metamaterial are collected on the spectrum of the terahertz time domain spectrum system with a spectral bandwidth of 0.1
  • the measurement environment humidity requirement is ⁇ 0.2%, and the temperature is normal temperature; the terahertz time domain spectrum of the sample is measured one by one by the above method and saved, and the terahertz time domain spectrum data set of all the sample points to be tested and the reference sample point are obtained.
  • the transmittance or reflectance can be obtained by the following formula:
  • T represents the transmittance
  • E (sample-T) represents the electric field strength of the sample point to be tested in the transmission mode
  • E (reference-T) represents the electric field strength of the reference sample point in the transmission mode
  • R represents the reflectance
  • E (sample-R) represents the electric field strength of the sample point to be tested in the reflection mode
  • E (reference-R) represents the electric field strength of the reference sample point in the reflection mode.
  • the gold-labeled avidin was taken out from the refrigerator, and another centrifuge tube of the same type was taken to inject the same amount of deionized water, and then centrifuged, centrifuged at a speed of 15000 rpm, and the centrifugation time was 10 minutes. After centrifugation, the excess common avidin is suspended in the upper layer, the gold-labeled avidin is in the lower layer of the centrifuge tube, the supernatant in the centrifuge tube is removed, and the precipitate is repeatedly washed with deionized water, and the excess common avidin is washed and removed;
  • a centrifuge tube containing DNA was added, and PBS buffer was added to prepare a DNA solution of 6 ⁇ mol/L.
  • the biotin-labeled target DNA sequence in this example is: 5'-TATCCTGAGACCGCGTTTTTTTTTT-C6-Biotin-3'.
  • Pipette 500 ⁇ L of DNA solution add to the gold-labeled avidin, fully react for 3 hours, take another centrifuge tube of the same type and inject the same amount of deionized water. After leveling, centrifuge the centrifuge to rotate at 5000 rpm and centrifuge for 20 times. minute.
  • the excess biotin-labeled target DNA is suspended in the upper layer, and the biotin-labeled target DNA-gold-labeled avidin complex is placed in the lower layer of the centrifuge tube, the supernatant in the centrifuge tube is removed, and the precipitate is repeatedly washed with deionized water. Wash and remove excess biotin-labeled target DNA, add 0.5 mL of deionized water, and oscillate to obtain a biotin-labeled target DNA-gold-based avidin complex;
  • biotin-labeled target DNA sequence in this example is: 5'-TATCCTGAGACCGCGTTTTTTTTTTTT-C6-Biotin-3', and the biotin-labeled target DNA sequence is not limited thereto in practice.
  • the target DNA of this example is: 5'-TATCCTGAGACCGCGTTTTTTTTTTTTTTTTTT-C6-3'.
  • a target DNA solution with a certain concentration gradient is separately disposed (in this embodiment, 2 ⁇ 10 -10 mol/L, 4 ⁇ 10 -10 mol/L, 6 ⁇ 10 -10 mol/L, 8 ⁇ 10 -10 mol/L). And 10 ⁇ 10 -10 mol/L), take 5 ⁇ L of the solution, add dropwise to the surface of the cleaned metamaterial, add three times for each concentration, and set three reference sample points (without any sample), and dry at room temperature.
  • the detection area of the sample point to be tested is about 1 mm 2 ;
  • the terahertz time domain spectrum system starts to be filled with nitrogen gas, the humidity drops, and the laser can be measured after half an hour of heating; the lid for measuring the terahertz time domain spectrum system is turned on.
  • the metamaterial is placed in the detection optical path and fixed by the clamp; in the case of nitrogen filling, the sampling point and reference of the same metamaterial are collected on the spectrum of the terahertz time domain spectrum system with a spectral bandwidth of 0.1-3.5 THz.
  • the terahertz time-domain spectrum of the sample point is performed by the terahertz time domain spectrum system starts to be filled with nitrogen gas, the humidity drops, and the laser can be measured after half an hour of heating; the lid for measuring the terahertz time domain spectrum system is turned on.
  • the metamaterial is placed in the detection optical path and fixed by the clamp; in the case of nitrogen filling, the sampling point and reference of the same metamaterial are collected on the spectrum of the terahertz time domain spectrum system with a spectral bandwidth of 0.1
  • the measurement environment humidity requirement is ⁇ 0.2%, and the temperature is normal temperature; the terahertz time domain spectrum of the sample is measured one by one by the above method and saved, and the terahertz time domain spectrum data set of all the sample points to be tested and the reference sample point are obtained.
  • the fast Fourier transform is used to convert the terahertz spectrum time domain signal of the sample to the frequency domain signal, and the frequency domain signal is used to obtain the transmittance or reflectance of the sample point to be tested. Find a frequency value corresponding to the lowest point of transmittance or reflectance, and subtract the frequency value of the sample point to be tested from the frequency value of the reference sample point to obtain a frequency shift of the transmission peak or the reflection peak.
  • E. coli antibody (pH about 9) at a concentration of 1 mg/mL into a clean centrifuge tube, and then pipette the nanoparticle at a concentration of 20 nmol/L.
  • Gold solution (pH about 8) 0.5mL, the two are mixed (the ratio of the amount of E. coli antibody to nano gold material is about 10:1), and shaken on a shaker for 15 minutes under normal temperature conditions;
  • the E. coli antibody was coupled to the nano gold from the refrigerator, and another centrifuge tube of the same type was taken to inject the same amount of deionized water, and then centrifuged by a centrifuge, the rotation speed was 5000 rpm, and the centrifugation time was 20 minutes. The supernatant in the centrifuge tube is removed, and the precipitate is repeatedly washed with deionized water;
  • E. coli antibody coupled with nano gold to capture E. coli
  • E. coli solution with a concentration of 10 8 CFU/mL was added to the above-mentioned E. coli antibody-conjugated nano gold solution, and the reaction was allowed to stand for 2 hours to obtain a complex of E. coli antibody-conjugated nano gold and Escherichia coli;
  • concentration gradients of the target E. coli solution (2*10 6 4*CFU/mL, 6*10 6 CFU/mL, 8*10 6 CFU/mL, and 10 7 CFU/mL in this example) were respectively configured. Take 100 ⁇ L of the solution, add it to the surface of the cleaned supermaterial, add three times for each concentration, and set three reference sample points (without any sample), and dry at room temperature. The detection area of the sample to be tested is greater than 10 mm 2 ;
  • E. coli antibody Five concentration gradients of E. coli antibody were coupled to the complex of nano gold and E. coli, respectively (in this example, 2*10 6 4*CFU/mL, 6*10 6 CFU/mL, 8*10 6 CFU/ mL and 10 7 CFU/mL), take 100 ⁇ L of solution, add dropwise to the surface of the cleaned metamaterial, add three times for each concentration, and set three reference sample points (without any sample), dry at room temperature, and test The detection area of the sample point is greater than 10 mm 2 ;
  • the concentration of nanogold in the complex of E. coli antibody-coupled nanogold and E. coli is at least 10-10 mol/L.
  • the terahertz time domain spectrum system starts to be filled with nitrogen gas, the humidity drops, and the laser can be measured after half an hour of heating; the lid for measuring the terahertz time domain spectrum system is turned on.
  • the metamaterial is placed in the detection optical path and fixed by the clamp; in the case of nitrogen filling, the sampling point and reference of the same metamaterial are collected on the spectrum of the terahertz time domain spectrum system with a spectral bandwidth of 0.1-3.5 THz.
  • the terahertz time-domain spectrum of the sample point is performed by the terahertz time domain spectrum system starts to be filled with nitrogen gas, the humidity drops, and the laser can be measured after half an hour of heating; the lid for measuring the terahertz time domain spectrum system is turned on.
  • the metamaterial is placed in the detection optical path and fixed by the clamp; in the case of nitrogen filling, the sampling point and reference of the same metamaterial are collected on the spectrum of the terahertz time domain spectrum system with a spectral bandwidth of 0.1
  • the measurement environment humidity requirement is ⁇ 0.2%, and the temperature is normal temperature; the terahertz time domain spectrum of the sample is measured one by one by the above method and saved, and the terahertz time domain spectrum data set of all the sample points to be tested and the reference sample point are obtained.
  • the frequency domain signal obtains the transmittance or reflectivity of the sample point to be tested. Find a frequency value corresponding to the lowest point of transmittance or reflectance, and subtract the frequency value of the sample point to be tested from the frequency value of the reference sample point to obtain a frequency shift of the transmission peak or the reflection peak.
  • nanogold particles of the order of the amount of material on the order of fmol can cause significant peak shifts in the terahertz metamaterial. Therefore, the nanogold is linked to avidin, DNA or E. coli, and as long as the amount of the nanogold particles is on the order of fmol, avidin, DNA or Escherichia coli can be detected.

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Abstract

公开了一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法。配置不同浓度的多个生物样品溶液和金标亲和素溶液，在超材料表面滴加生物样品溶液后常温下晾干，在超材料表面滴加金标亲和素溶液后常温下晾干，采集超材料表面所有待测样品点与参考样品点的太赫兹时域信号，由太赫兹时域信号计算所有待测样品点与参考样品点的透射率或反射率，并根据透射率或反射率最低点对应的频率值计算得到透射峰或反射峰的频移。联用太赫兹超材料与纳米金颗粒修饰，利用超材料电场局域增强效应放大样品信号；并利用纳米金改变电场分布效应，通过纳米金修饰进一步放大样品信号，检测灵敏度高，操作简便快速，能满足日益增长的快速检测需求。

Description

太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法 技术领域
本发明涉及一种生物样品的太赫兹信号放大方法，尤其涉及一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法。
背景技术
随着检测技术的发展，波谱检测技术由于其检测快速简便而逐渐引起了国内外学者的广泛关注。太赫兹波谱技术作为一种新兴的波谱技术已经逐渐引起人们的注意。由于许多大分子的振动、转动能级都落在太赫兹波段，太赫兹波被认为是一种对生物样品检测非常具有潜力的波段。对于太赫兹波谱技术具有较大应用前景的领域，如安全、生物、医药、农业和材料表征等应用方面，存在微量甚至是超微量的无损检测需求。然而，由于太赫兹波源能量低和直接检测灵敏度有限的劣势，导致该技术很难用于微量样品的快速检测。
发明内容
本发明所要解决的技术问题是克服上述背景技术的不足，提供一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，该方法应具有灵敏度性高、检测快速方便的特点。
本发明采用的技术方案包括如下步骤：
1)配置不同浓度的多个生物样品溶液和不同浓度的多个金标亲和素溶液；
2)超材料表面滴加生物样品溶液；
将生物样品溶液滴加在一清洗过的超材料表面，每个浓度滴加至少三次，每次滴加量相同，并任意设置三个参考样品点，如图2所示，参考样品点均与待测样品点位置不同，滴加后常温下晾干；
3)超材料表面滴加金标亲和素溶液；
将金标亲和素溶液滴加在另一清洗过的超材料表面，每个浓度滴加至少三次，每次滴加量相同，并任意设置三个参考样品点，如图2所示，参考样品点均与待测样品点位置不同，滴加后常温下晾干；
4)采集超材料表面所有待测样品点与参考样品点的太赫兹时域信号；在充氮气氛围下，将生物样品放在待测样品点上，在太赫兹时域波谱***的波谱频宽为0.1-3.5THz区间分别采集同一超材料上待测样品点与参考样品点的太赫兹 时域信号；
5)由太赫兹时域信号得到透射峰或反射峰的频移，计算所有待测样品点与参考样品点的透射率或反射率，并根据透射率或反射率最低点对应的频率值计算得到透射峰或反射峰的频移：利用快速傅里叶变换将生物样品的太赫兹波谱时域信号转换到频域信号，由频域信号计算得到待测样品点的透射率或者反射率，将待测样品点与参考样品点的透射率或者反射率最低点对应的频率值相减得到的绝对值作为透射峰或反射峰的频移，实现对亲和素信号的放大。
所述步骤2)和3)中超材料采用以下方式清洗：取一块完整的太赫兹超材料，先后用去离子水、磷酸盐缓冲液清洗后，再用去离子水清洗，并用氮气吹干。
所述步骤1)中金标亲和素溶液采用以下方式配置：
1.1)原料混合保存；
取普通亲和素和纳米金颗粒进行混合，普通亲和素和纳米金颗粒混合的物质的量之比为10:1～2500:1，如图1所示，常温条件下在摇床上进行振荡，并在0～4℃温度下中进行保存；其形成的纳米金溶液为酒红色；
1.2)金标亲和素提取；
将金标亲和素取出，放入离心管，经离心机离心后，去除离心管上清液中多余的普通亲和素，并用去离子水反复清洗沉淀，最后再加入去离子水并充分振荡得到金标亲和素溶液。
所述步骤1.2)中离心机的离心转速为5000～10000rpm，离心时间为10～20分钟。
所述步骤4)中采集太赫兹时域信号时，待测样品点的检测面积大于1mm2。
所述的生物样品采用普通亲和素、DNA或者大肠杆菌。
所述步骤1)配置得到的金标亲和素溶液和生物样品溶液的浓度范围均在2×10-10～10×10-10mol/L之间。
所述步骤3)中生物样品溶液或者步骤4)中金标亲和素溶液的每次滴加量为5～100ul。
所述的生物样品采用DNA或者大肠杆菌，不同浓度的多个金标亲和素溶液为金标亲和素与生物素标记后生物样品复合物溶液。
复合方式和过程是：金标亲和素与生物素标记后生物样品中的生物素特异性结合，形成金标亲和素与生物素标记后生物样品复合物溶液，金标亲和素与生物素或者生物样品之间的浓度比例关系为大于等于1:1，优选的比例为1:1-4:1。
所述步骤1.1)中普通亲和素的pH为5～9，纳米金颗粒的pH为8～12。
所述步骤4)中本发明的太赫兹时域光谱***采集太赫兹时域信号时测量环境的湿度为<0.2％。
优选的本发明普通亲和素具体实施中可选用Sigma公司生产的货号为A9275的亲和素，但不限于此。
所述的纳米金颗粒粒径为8-90nm。
本发明金标亲和素可以用于与生物素的结合反应，因此该信号放大方法能在DNA杂交、抗体特异性结合方面有广泛应用。
本发明的纳米金颗粒能够在不激发其表面等离子体的情况下，直接对样品信号进行放大，并且效果显著。
本发明的纳米金可以替换为其他金属纳米粒子，包括纳米银颗粒，纳米金棒，纳米银包金颗粒，纳米金包银颗粒等。
具体是实施中优选的本发明的太赫兹时域波谱***推荐采用z-omega公司生产的型号为z3的太赫兹时域波谱***。
本发明采用太赫兹时域波谱技术(Terahertz time-domain spectroscopy，THz-TDS)，其是国际上近年来发展起来的一项新的研究与检测技术。至今，太赫兹时域波谱技术已经在国防、医药、化学、食品、材料等方面有着许多的应用。太赫兹波是一种波长介于微波与红外辐射之间的电磁波，其频率为0.1-10THz。尽管太赫兹辐射的能量很低，但是大量的分子，尤其是许多有机大分子(DNA、蛋白质等)在这一频段内，表现出强烈的吸收和色散。
本发明的超材料是一种人工制作的周期性结构材料，具有许多自然界材料无法表现出的性质。近年来，太赫兹波段下超材料的研究逐渐引起广大学者的关注，目前已经在通讯、吸收器等方面有一定应用。近年来，超材料已经逐渐在太赫兹波段检测应用中发挥优势作用。
由此本发明利用太赫兹超材料技术，其具有的有益效果是：
本发明联用太赫兹超材料技术与纳米金颗粒修饰技术，利用超材料的电场局域增强效应放大样品信号。
本发明同时利用纳米金能改变表面电场分布的效应，通过纳米金修饰的方法进一步放大样品信号，因此该方法检测灵敏度高。
与传统的压片技术相比，本发明方法能大大提高检测灵敏度；并且本方法操作简便快速，能满足日益增长的快速检测需求。
附图说明
图1为本发明超材料检测金标亲和素原理图。
图2为本发明实施例1的超材料表面待测样品点和参考样品点分布图。
图3为本发明实施例1普通亲和素与金标亲和素样品引起的太赫兹超材料谐振峰频移图。
图4为本发明实施例3中纳米金样品引起的太赫兹超材料谐振峰频移图。
其中，A为普通亲和素，B为纳米金，C为超材料，D为太赫兹波，E为待测样品点，F为参考样品点。
具体实施方式
下面结合实施实例对本发明作进一步说明，但本发明并不限于以下实施例。
本发明的实施例如下：
实施例1
(1)超材料清洗；
用镊子夹取一块完整的超材料，先后用去离子水，磷酸盐缓冲液(Sigma公司)，去离子水清洗3次，并用氮气吹干；
(2)金标亲和素配置；
换上干净手套，用移液枪移取浓度为1mg/mL的普通亲和素(pH约为7)300μL于一支干净的离心管中，再用移液枪移取浓度为20nmol/L的纳米金溶液(pH约为10)0.5mL，将两者进行混合(普通亲和素与纳米金的物质的量之比约2500:1)，常温条件下在摇床上振荡15分钟，并在冰箱中进行保存，保存温度为4℃，保存时间大于等于0.5小时；
(3)金标亲和素提取；
将装有金标亲和素的离心管从冰箱中取出，另取一支同型号的离心管注入等量去离子水，配平后经离心机离心，转速为10000rpm，离心时间为15分钟。离心后多余的普通亲和素悬浮在上层，金标亲和素则在离心管下层，去除离心管中上清液，并用去离子水反复清洗沉淀，清洗并取走多余的普通亲和素；
(4)获得金标亲和素溶液；
清洗金标亲和素后，往离心管中加入500μL去离子水，通过振荡将金标亲和素溶于去离子水中；
(5)超材料表面滴加普通亲和素溶液；
分别配置五个浓度梯度的普通亲和素溶液(本实施实例中为2×10-10mol/L， 4×10-10mol/L，6×10-10mol/L，8×10-10mol/L和10×10-10mol/L)，取10μL溶液，滴加在清洗过的超材料表面，每个浓度滴加三次，并设置三个参考样品点(没有任何样品)，常温下晾干(相应的普通亲和素的物质的量为2fmol，4fmol，6fmol，8fmol，10fmol)，待测样品点的检测面积约为4mm2；
(6)超材料表面滴加金标亲和素溶液；
分别配置五个浓度梯度的金标亲和素溶液(本实施实例中为2×10-10mol/L，4×10-10mol/L，6×10-10mol/L，8×10-10mol/L和10×10-10mol/L)，取10μL溶液，滴加在清洗过的超材料表面，每个浓度滴加三次，并设置三个参考样品点(没有任何样品)，常温下晾干(相应的金标亲和素的物质的量为2fmol，4fmol，6fmol，8fmol，10fmol)，待测样品点的检测面积约为4mm2；
(7)采集超材料表面所有待测样品点与参考样品点的太赫兹时域波谱；
打开激光，电脑，控制器以及氮气阀门，此时太赫兹时域波谱***内开始充进氮气，湿度下降，激光预热半小时后方可进行测量；打开太赫兹时域波谱***测量用的盖子，并将超材料放进检测光路中，用夹具固定；在充氮气的情况下，在太赫兹时域波谱***的波谱频宽为0.1-3.5THz区间分别采集同一超材料上待测样品点与参考样品点的太赫兹时域波谱。其中测量环境湿度要求<0.2％，温度为常温；用以上方法逐个测量样本的太赫兹时域波谱并保存，获得所有待测样品点与参考样品点的太赫兹时域波谱数据组。
(8)计算所有待测样品点的透射率或反射率，并寻找透射率或反射率最低点对应的频率值；利用快速傅里叶变换将样品的太赫兹波谱时域信号转换到频域信号，利用频域信号得到待测样品点的透射率或者反射率。
其中，透过率或反射率可以由以下公式得到：
T＝(E(sample-T)/E(reference-T))2
R＝(E(sample-R)/E(reference-R))2
上述公式中，T表示透过率，E(sample-T)表示透射模式下待测样品点的电场强度，E(reference-T)表示透射模式下参考样品点的电场强度，R表示反射率，E(sample-R)表示反射模式下待测样品点的电场强度，E(reference-R)表示反射模式下参考样品点的电场强度。
寻找透射率或者反射率最低点对应的频率值，并将待测样品点的该频率值与参考样品点的该频率值相减，得到透射峰或反射峰的频移，如图3所示。
实施例2
(1)超材料清洗；
用镊子夹取一块完整的超材料，先后用去离子水，磷酸盐缓冲液(Sigma 公司)，去离子水清洗3次，并用氮气吹干；
(2)金标亲和素配置；
换上干净手套，用移液枪移取浓度为0.8mg/mL的普通亲和素(pH约为5)300μL于一支干净的离心管中，再用移液枪移取浓度为20nmol/L的纳米金溶液(pH约为9)0.5mL，将两者进行混合(普通亲和素与纳米金的物质的量之比约2000:1)，常温条件下在摇床上振荡15分钟，并在冰箱中进行保存，保存温度为0℃，保存时间大于等于0.5小时；
(3)金标亲和素提取；
将金标亲和素从冰箱中取出，另取一支同型号的离心管注入等量去离子水，配平后经离心机离心，转速为15000rpm，离心时间为10分钟。离心后多余的普通亲和素悬浮在上层，金标亲和素则在离心管下层，去除离心管中上清液，并用去离子水反复清洗沉淀，清洗并取走多余的普通亲和素；
(4)获得金标亲和素溶液；
清洗金标亲和素后，往离心管中加入500μL去离子水，通过振荡将金标亲和素溶于去离子水中；
(5)金标亲和素与生物素(biotin)标记的目标DNA结合；
取一支装有DNA的离心管，加入PBS缓冲液配置成6μmol/L的DNA溶液。本实施实例中生物素标记的目标DNA序列为：5’-TATCCTGAGACCGCGTTTTTTTTTT-C6-Biotin-3’。可以通过生工公司合成。移取500μLDNA溶液，加入到金标亲和素中，充分反应3小时，另取一支同型号的离心管注入等量去离子水，配平后经离心机离心，转速为5000rpm，离心时间为20分钟。离心后多余的生物素标记的目标DNA悬浮在上层，生物素标记的目标DNA-金标亲和素复合物则在离心管下层，去除离心管中上清液，并用去离子水反复清洗沉淀，清洗并取走多余的生物素标记的目标DNA，加入0.5mL去离子水，振荡溶解得到生物素标记的目标DNA-金标亲和素复合物；
本实施实例中生物素标记的目标DNA序列为：5’-TATCCTGAGACCGCGTTTTTTTTTT-C6-Biotin-3’，实际操作中生物素标记的目标DNA序列不限于此。
(6)超材料表面滴加目标DNA溶液；
本实施实例目标DNA为：5’-TATCCTGAGACCGCGTTTTTTTTTT-C6-3’。
分别配置一定浓度梯度的目标DNA溶液(本实施实例中为2×10-10mol/L，4×10-10mol/L，6×10-10mol/L，8×10-10mol/L和10×10-10mol/L)，取5μL溶液，滴加在清洗过的超材料表面，每个浓度滴加三次，并设置三个参考样品点(没有 任何样品)，常温下晾干，待测样品点的检测面积约为1mm2；
(7)超材料表面滴加金标亲和素与生物素标记的目标DNA复合物；
分别配置五个浓度梯度的金标亲和素与生物素标记的目标DNA复合物(本实施实例中为2×10-10mol/L，4×10-10mol/L，6×10-10mol/L，8×10-10mol/L和10×10-10mol/L)，取5μL溶液，滴加在清洗过的超材料表面，每个浓度滴加三次，并设置三个参考样品点(没有任何样品)，常温下晾干，待测样品点的检测面积约为1mm2；
(8)采集超材料表面所有待测样品点与参考样品点的太赫兹时域波谱；
打开激光，电脑，控制器以及氮气阀门，此时太赫兹时域波谱***内开始充进氮气，湿度下降，激光预热半小时后方可进行测量；打开太赫兹时域波谱***测量用的盖子，并将超材料放进检测光路中，用夹具固定；在充氮气的情况下，在太赫兹时域波谱***的波谱频宽为0.1-3.5THz区间分别采集同一超材料上待测样品点与参考样品点的太赫兹时域波谱。其中测量环境湿度要求<0.2％，温度为常温；用以上方法逐个测量样本的太赫兹时域波谱并保存，获得所有待测样品点与参考样品点的太赫兹时域波谱数据组。
(9)计算所有待测样品点的透射率或反射率，并寻找透射率或反射率最低点对应的频率值；
利用快速傅里叶变换将样品的太赫兹波谱时域信号转换到频域信号，利用频域信号得到待测样品点的透射率或者反射率。寻找透射率或者反射率最低点对应的频率值，并将待测样品点的该频率值与参考样品点的该频率值相减，得到透射峰或反射峰的频移。
实施例3
(1)超材料清洗；
用镊子夹取一块完整的超材料，先后用去离子水，磷酸盐缓冲液(Sigma公司)，去离子水清洗3次，并用氮气吹干；
(2)大肠杆菌抗体偶联纳米金；
换上干净手套，用移液枪移取浓度为1mg/mL的大肠杆菌抗体(pH约为9)2μL于一支干净的离心管中，再用移液枪移取浓度为20nmol/L的纳米金溶液(pH约为8)0.5mL，将两者进行混合(大肠杆菌抗体与纳米金的物质的量之比约10:1)，常温条件下在摇床上振荡15分钟；
(3)大肠杆菌抗体偶联纳米金的提取；
将大肠杆菌抗体偶联纳米金从冰箱中取出，另取一支同型号的离心管注入等量去离子水，配平后经离心机离心，转速为5000rpm，离心时间为20分钟。 去除离心管中上清液，并用去离子水反复清洗沉淀；
(4)获得大肠杆菌抗体偶联纳米金溶液；
清洗大肠杆菌抗体偶联纳米金后，往离心管中加入500μL去离子水，通过振荡将大肠杆菌抗体偶联纳米金溶于去离子水中；
(5)大肠杆菌抗体偶联纳米金捕获大肠杆菌；
取浓度为108CFU/mL的大肠杆菌溶液0.1mL，加入到上述的大肠杆菌抗体偶联纳米金溶液，静置反应2小时，得到大肠杆菌抗体偶联纳米金与大肠杆菌的复合物；
(6)超材料表面滴加目标大肠杆菌溶液；
分别配置五个浓度梯度的目标大肠杆菌溶液(本实施实例中为2*1064*CFU/mL，6*106CFU/mL，8*106CFU/mL和107CFU/mL)，取100μL溶液，滴加在清洗过的超材料表面，每个浓度滴加三次，并设置三个参考样品点(没有任何样品)，常温下晾干，待测样品点的检测面积大于10mm2；
(7)超材料表面滴加步骤(5)中得到的大肠杆菌抗体偶联纳米金与大肠杆菌的复合物；
分别配置五个浓度梯度的大肠杆菌抗体偶联纳米金与大肠杆菌的复合物(本实施实例中为2*1064*CFU/mL，6*106CFU/mL，8*106CFU/mL和107CFU/mL)，取100μL溶液，滴加在清洗过的超材料表面，每个浓度滴加三次，并设置三个参考样品点(没有任何样品)，常温下晾干，待测样品点的检测面积大于10mm2；
需要保证此处大肠杆菌抗体偶联纳米金与大肠杆菌的复合物中纳米金的浓度至少在10-10mol/L的量级。
(8)采集超材料表面所有待测样品点与参考样品点的太赫兹时域波谱；
打开激光，电脑，控制器以及氮气阀门，此时太赫兹时域波谱***内开始充进氮气，湿度下降，激光预热半小时后方可进行测量；打开太赫兹时域波谱***测量用的盖子，并将超材料放进检测光路中，用夹具固定；在充氮气的情况下，在太赫兹时域波谱***的波谱频宽为0.1-3.5THz区间分别采集同一超材料上待测样品点与参考样品点的太赫兹时域波谱。其中测量环境湿度要求<0.2％，温度为常温；用以上方法逐个测量样本的太赫兹时域波谱并保存，获得所有待测样品点与参考样品点的太赫兹时域波谱数据组。
(9)计算所有待测样品点的透射率或反射率，并寻找透射率或反射率最低点对应的频率值；
利用快速傅里叶变换将样品的太赫兹波谱时域信号转换到频域信号，利用 频域信号得到待测样品点的透射率或者反射率。寻找透射率或者反射率最低点对应的频率值，并将待测样品点的该频率值与参考样品点的该频率值相减，得到透射峰或反射峰的频移。
如附图4所示，物质的量在fmol量级的纳米金颗粒能使得太赫兹超材料出现明显的峰的偏移。因此，将纳米金与亲和素、DNA或者大肠杆菌相连，只要纳米金颗粒的物质的量在fmol量级，亲和素、DNA或者大肠杆菌就能被检测到。
上述具体实施方式用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明做出的任何修改和改变，都落入本发明的保护范围。

Claims (13)

  1. 一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于包括如下步骤：
    1)配置不同浓度的多个生物样品溶液和不同浓度的多个金标亲和素溶液；
    2)超材料表面滴加生物样品溶液：将生物样品溶液滴加在一清洗过的超材料表面，每个浓度滴加至少三次，每次滴加量相同，并任意设置三个参考样品点，参考样品点均与待测样品点位置不同，滴加后常温下晾干；
    3)超材料表面滴加金标亲和素溶液：将金标亲和素溶液滴加在另一清洗过的超材料表面，每个浓度滴加至少三次，每次滴加量相同，并任意设置三个参考样品点，参考样品点均与待测样品点位置不同，滴加后常温下晾干；
    4)采集超材料表面所有待测样品点与参考样品点的太赫兹时域信号：在充氮气氛围下，将生物样品放在待测样品点上，在波谱频宽为0.1-3.5THz区间分别采集同一超材料上待测样品点与参考样品点的太赫兹时域信号；
    5)由太赫兹时域信号得到透射峰或反射峰的频移：利用快速傅里叶变换将生物样品的太赫兹波谱时域信号转换到频域信号，由频域信号计算得到待测样品点的透射率或者反射率，将待测样品点与参考样品点的透射率或者反射率最低点对应的频率值相减得到的绝对值作为透射峰或反射峰的频移，实现对亲和素信号的放大。
  2. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤2)和3)中超材料采用以下方式清洗：取一块完整的太赫兹超材料，先后用去离子水、磷酸盐缓冲液清洗后，再用去离子水清洗，并用氮气吹干。
  3. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤1)中金标亲和素溶液采用以下方式配置：
    1.1)原料混合保存；
    取普通亲和素和纳米金颗粒进行混合，常温条件下在摇床上进行振荡，并在0～4℃温度下中进行保存；
    1.2)金标亲和素提取；
    将金标亲和素取出，放入离心管，经离心机离心后，去除离心管上清液中多余的普通亲和素，并用去离子水反复清洗沉淀，最后再加入去离子水并充分振荡得到金标亲和素溶液。
  4. 根据权利要求3所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述的普通亲和素和纳米金颗粒混合的物质的量之比为10:1～2500:1。
  5. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤1.2)中离心机的离心转速为5000～15000rpm，离心时间为10～20分钟。
  6. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤4)中采集太赫兹时域信号时，待测样品点的检测面积大于1mm2。
  7. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述的生物样品采用普通亲和素、DNA或者大肠杆菌。
  8. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤1)配置得到的金标亲和素溶液的浓度范围在2×10-10～10×10-10mol/L之间。
  9. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤1)配置得到的生物样品溶液的浓度范围在2×10-10～10×10-10mol/L之间。
  10. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤3)中生物样品溶液或者步骤4)中金标亲和素溶液的每次滴加量为5～100mL。
  11. 根据权利要求3所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤1.1)中普通亲和素的pH为5～9，纳米金颗粒的pH为8～10。
  12. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述步骤4)中采集太赫兹时域信号时测量环境的湿度为<0.2％。
  13. 根据权利要求1所述的一种太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法，其特征在于：所述的生物样品采用DNA或者大肠杆菌，不同浓度的多个金标亲和素溶液为金标亲和素与生物素标记后生物样品复合物溶液。
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