CN105588820B - The method that micro bacterium living is detected based on Terahertz Meta Materials - Google Patents
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- 239000000463 material Substances 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 28
- 241001467578 Microbacterium Species 0.000 title claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 52
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 239000002184 metal Substances 0.000 claims abstract description 13
- 229910052751 metal Inorganic materials 0.000 claims abstract description 13
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 12
- 239000010703 silicon Substances 0.000 claims abstract description 12
- 238000001228 spectrum Methods 0.000 claims abstract description 12
- 238000001328 terahertz time-domain spectroscopy Methods 0.000 claims abstract description 7
- 230000005540 biological transmission Effects 0.000 claims abstract description 4
- 230000000737 periodic effect Effects 0.000 claims abstract description 4
- 238000001035 drying Methods 0.000 claims abstract description 3
- 239000000758 substrate Substances 0.000 claims description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 2
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims 1
- 239000010931 gold Substances 0.000 claims 1
- 229910052737 gold Inorganic materials 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 description 12
- 230000008859 change Effects 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- 238000006073 displacement reaction Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 244000144987 brood Species 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003990 capacitor Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 239000006781 columbia blood agar Substances 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007373 indentation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 230000004044 response Effects 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
Classifications
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- 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
- 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
- G01N21/59—Transmissivity
-
- 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
- G01N21/59—Transmissivity
- G01N2021/5903—Transmissivity using surface plasmon resonance [SPR], e.g. extraordinary optical transmission [EOT]
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- Spectroscopy & Molecular Physics (AREA)
- General Health & Medical Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The invention discloses the methods that micro bacterium living is detected based on Terahertz Meta Materials, specific method measures transmitted spectrum under the conditions of 18~25 DEG C, detection cell relative humidity are less than 2% using terahertz time-domain spectroscopy instrument after drying for bacterium solution is taken to add to Terahertz Meta Materials center using transmission mode；Wherein Terahertz Meta Materials are made of periodic arrangement metal openings resonant ring in silicon base and silicon base, method of the invention can quickly, the micro bacterium living of markless detection.
Description
Technical field
The invention belongs to detection fields, and in particular to the method that micro bacterium living is detected based on Terahertz Meta Materials.
Background technology
Conventional bacteria detection method is according to the form and metabolic characteristics after Bacteria Culture, carries out the identification of kind.Usually
The time of several days is needed to obtain the species of pathogenic bacteria, and the time required for some more slow bacteriums of growth is longer.It is based on
The reaction of nucleic acid amplifying technique, such as PCR can more quickly identify pathogen type, but complicated nucleic acid is needed to carry
It takes process and prepares matched primer in advance.Matrix-assisted laser desorption ionization (MALDI-TOF-MS)
The pure bacterium colony after culture can be directly detected to avoid nucleic acid extraction, but sensitivity is relatively low, and sensitivity is only 105-106CFU.More
Importantly, nucleic acid amplifying technique and MALDI-TOF-MS are the specific nucleic acid and albumen of detection bacterium respectively, it is impossible to be distinguished
The survival condition of bacterium to be checked, because specific component may be from viable bacteria and also may be from dead bacterium.Detect dead bacterium to infectious disease
Sick diagnostic significance is little, and may cause the generation of false positive results.Therefore, it is thin to establish a kind of easy, quick micro work
The detection method of bacterium has very important significance for the diagnosis of clinical infectious disease.
Terahertz (Terahertz, THz) radiation refers to frequency in 0.1~10THz, and wavelength is between 30~3000 μm
Electromagnetic wave, due to its wave band be located at microwave and it is infrared between, in those early years also referred to as far ir ray (Far-infrared
rays).Terahertz time-domain spectroscopy instrument (THz-TDS, Terahertz time-domain spectroscopy) is higher due to it
Signal-to-noise ratio, can directly acquire the advantages that refractive index and absorption coefficient and be widely used in biomedical research, including cancer into
Picture, residual antibiotic composition detection and protein dynamics research.Its specific structure having due to different bacterium is in THz frequency ranges
There is down different dielectric response features, and the viable bacteria of bacterium of the same race and dead bacterium have apparent otherness spectrum, are terahertz
Hereby ripple provides correlation theory support and opportunity for Bacteria Detection.But most of brood body bacteriums are strong due to intracellular water
Absorption cover effect, can not show specificity absworption peak, absorption coefficient spectrum be relative smooth curve, Zhi Nengtong
Numerical values recited is crossed to be differentiated.Further, since Terahertz wavelength (being 300 μm at 1THz) and bacterium size (usual 1-2 μm) are deposited
The length dimension mismatch the problem of, according to Rayleigh scattering principle, low scattering section can be caused.It is, thus, sought for a kind of method,
Solve the problems, such as that Terahertz wavelength is not matched that with bacterium size.
The content of the invention
In view of this, one of the objects of the present invention is to provide the sides that micro bacterium living is detected based on Terahertz Meta Materials
Method need not mark, and can quickly detect, and method is simple.
For achieving the above object, the present invention provides following technical solution：
The method that micro bacterium living is detected based on Terahertz Meta Materials, takes bacterium solution to add to Terahertz Meta Materials center, dries
Terahertz time-domain spectroscopy instrument is measured under the conditions of 18~25 DEG C, detection cell relative humidity are less than 2% using transmission mode afterwards
Transmitted spectrum；The Terahertz Meta Materials are made of periodic arrangement metal openings resonant ring in silicon base and silicon base.
Preferably, the drying dries 10min under the conditions of being 42 DEG C.
Preferably, the bacterium solution additive amount is at least 10 for thalline quantity3CFU.It is furthermore preferred that taking the 10 μ L bacterial concentrations to be
105The bacterium solution of CFU/ml is added to Terahertz Meta Materials center.
Preferably, each it is made of five metal openings resonant rings, central indentation size is 2~3 μm.
It is furthermore preferred that the thickness of the silicon base is 470 μm, the metal thickness of the metal openings resonant ring is 200nm.
The beneficial effects of the present invention are：The invention discloses the sides that micro bacterium living is detected based on Terahertz Meta Materials
Method, this method is simple, need not mark, and can quickly detect, and Terahertz Meta Materials have multiple absworption peaks, therefore can realize
Various bacteria differentiates, and the sample size needed is few, greatly improves detection sensitivity, detection sensitivity reaches 103CFU, it is real
Effective discriminating of existing trace of bacteria.
Description of the drawings
In order to make the purpose of the present invention, technical solution and advantageous effect clearer, the present invention provides drawings described below：
Fig. 1 is the optical microscope picture of Terahertz Meta Materials.
Fig. 2 is to detect micro Escherichia coli transmitted light spectrogram using Terahertz Meta Materials.
Fig. 3 is the transmitted light spectrogram that micro Escherichia coli viable bacteria and dead bacterium are detected using Terahertz Meta Materials.
Fig. 4 is the transmitted light for covering Escherichia coli and staphylococcus aureus simulated using finite element method
Spectrogram.
Specific embodiment
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.It is not specified in embodiment specific
The experimental method of condition, usually according to normal condition or according to the condition proposed by manufacturer.
Terahertz Meta Materials are that the second wavelength metallic structure being produced on Semiconductor substrate or dielectric forms, and are had
The artificial composite material for the special electromagnetic property that nature material does not possess.It is micro- that pervious functionality Meta Materials are concentrated mainly on manipulation
Ripple, far infrared and visible light frequency band, rarely found Meta Materials are used for the relevant report of Terahertz frequency range, and the present invention is applicable in by preparing
In the Meta Materials of Terahertz frequency range, opportunity of the Meta Materials for Terahertz frequency range vacancy is made up.
The Terahertz Meta Materials of the present invention are that the metal openings resonant ring of periodic arrangement on a silicon substrate is formed, silicon chip
For 470 μ m-thicks, the golden film on silicon chip is thick for 200nm, and each cycle is made of five metal openings resonant rings, intermediate notch
Size is 2-3 μm, is matched (Fig. 1) with bacterium size dimension.Wherein Meta Materials are prepared by existing photoetching technique.
If the split ring resonator (SRR) in Meta Materials is seen as a LC circuit element, Meta Materials are total to
Vibration frequency is represented byWherein L and C is inductance and capacitance respectively.Inductance is mainly prepared
The geometric parameter of Meta Materials is determined, and the effective dielectric constant of capacitance and capacitor is closely related.When bacterium is covered in super material
When expecting surface, the change of effective dielectric constant results in the change of capacitance, has ultimately resulted in the displacement of resonant frequency.Due to difference
Bacterium terahertz wave band have different dielectric individual features, dielectric constant change on difference cause resonant frequency displacement number
Difference in value, so as to the discriminating being used between bacterium kind.The Local field enhancement distribution of Meta Materials and high q-factor resonance make it to material
Expect that the substance on surface is very sensitive so that Terahertz Meta Materials can be used for the detection of trace bacterium.In addition, because material internal clearance
Size and bacterium size dimension match, be micron order, Terahertz Meta Materials are suitable for Bacteria Detection.
The method that micro bacterium living is detected using Terahertz Meta Materials, is as follows：
(1) Bacteria Culture：The reference culture Escherichia coli frozen are inoculated in Columbia Blood Agar tablet, are in temperature
37℃、CO2Overnight incubation in the constant temperature incubator that volume fraction is 5%, selects pollution-free, the typical single bacterium colony of form after culture,
Using the abundant rinse removal medium component of sterile saline, subsequent centrifugal enrichment is resuspended in sterile saline, according to
Absorbance (OD600) and bacterium solution in every milliliter colony forming single-digit value (cfu/mL) standard curve, measure absorbance, match somebody with somebody
It is set to 105The concentration of CFU/ml；
(2) sample measures：Reference signal of the transmitted spectrum of High Resistivity Si as sample is measured, is brought in itself with eliminating silicon chip
Then the influence of F-P standing waves takes 10 μ L bacterium solutions to add to Terahertz Meta Materials center, is positioned on hot plate under the conditions of 42 DEG C respectively
10min is dried, then with terahertz time-domain spectroscopy instrument (THz-TDS, Terahertz time-domain spectroscopy)
It is measured under room temperature (18~25 DEG C) using transmission mode, in order to avoid the strong inhalation effects testing result of vapor,
Obturator is configured in the optical path and pours nitrogen-rich gas, and detection cell relative humidity is made to be down to less than 2%；And to measure not
This white Meta Materials of Terahertz of sample-adding compare, and the results are shown in Figure 2.
The results show that Escherichia coli transmitted spectrum is different from the projection spectrum of blank sample, resonant frequency generates partially
It moves, the reason is that when Escherichia coli are covered in Meta Materials surface, the change of effective dielectric constant results in the change of capacitance, finally
Result in the displacement of resonant frequency.Wherein offset is 12.5GHz, shows the method for the present invention and can detect 103The sample of CFU
This, sensitivity reaches 103CFU。
Then according to above-mentioned identical method detection Escherichia coli viable bacteria and dead bacterium, the dead bacterium preparation method of Escherichia coli be
1h is boiled at 100 DEG C, the results are shown in Figure 3.The results show that the resonant frequency production of the transmitted spectrum of Escherichia coli viable bacteria and dead bacterium
Micro offset is given birth to.It the reason is that since viable bacteria is different from the water content of dead bacterium, is covered each by when Meta Materials surface, is effectively situated between
Caused by the knots modification difference of electric constant results in capacitance change difference.It is therefore possible to use the method detection of the present invention is micro-
Dead bacterium and viable bacteria are measured, need not be marked.
In order to which the method for proving the present invention can differentiate various bacteria, transmitted light is simulated using finite element method
Spectrum, the results are shown in Figure 4.
The results show that the resonant frequency of the transmitted spectrum of Escherichia coli and staphylococcus aureus generates offset.It is former
Because being since the change of the effective dielectric constant of material surface microenvironment causes the change of capacitance, the resonant frequency generation of material is red
Move, and different bacterium has tera-hertz spectra a different dielectric individual features, the difference of dielectric constant can epitope be red shift amount
Difference.
Then the resonant frequency shift amount of Escherichia coli and staphylococcus aureus is compared, the results are shown in Table 1.
The comparison of table 1, Escherichia coli and the resonant frequency shift of staphylococcus aureus amount
As shown in Table 1, △ f1, △ f2 and △ f3 are the numerical value of first and second and three formant frequency displacements respectively.Due to
There are multiple SRR, material has multiple formants, detecting in itself in each cycle of the taken Terahertz Meta Materials of experiment
It is more suitable for when specimen types are more, because only needing the red shift amount at single peak, difference can distinguish bacterium, this
It is particularly important when detection specimen types are more.Therefore, different bacteriums can be distinguished using the method for the present invention, be not required to
It marks.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (4)
1. the method for micro bacterium living is detected based on Terahertz Meta Materials, it is characterised in that：Bacterium solution is taken to add to Terahertz Meta Materials
Center uses Transmission Modes using terahertz time-domain spectroscopy instrument after drying under the conditions of 18 ~ 25 DEG C, detection cell relative humidity are less than 2%
Formula measures transmitted spectrum；The Terahertz Meta Materials are by periodic arrangement metal openings resonant ring institute group in silicon base and silicon base
Into；Each cycle is made of five metal openings resonant rings, and five metal openings resonance ring-shaped are for square box and in concentric rows
Row, the opening are symmetrically disposed on square box, and the openings of sizes is 2 ~ 3 μm, and the metal is gold.
2. the method for micro bacterium living is detected based on Terahertz Meta Materials according to claim 1, it is characterised in that：The baking
It does to dry 10 min under the conditions of 42 DEG C.
3. the method for micro bacterium living is detected based on Terahertz Meta Materials according to claim 1, it is characterised in that：The bacterium
Liquid additive amount is at least 10 for thalline quantity3CFU。
4. the method for micro bacterium living is detected based on Terahertz Meta Materials according to claim 1, it is characterised in that：The silicon
The thickness of substrate is 470 μm, and the metal thickness of the metal openings resonant ring is 200nm.
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| CN109324011B (en) * | 2018-09-19 | 2021-11-05 | 杭州快格科技有限公司 | Method for detecting live bacteria based on nano material |
| CN109239007B (en) * | 2018-10-29 | 2019-07-09 | 中国人民解放军陆军军医大学第一附属医院 | Functionalization Terahertz slit nano-antenna for markless detection cell excretion body |
| CN110455743B (en) * | 2019-08-19 | 2021-08-06 | 中央民族大学 | Method for detecting aflatoxins B1 and B2 by utilizing terahertz waveband metamaterial sensor |
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