CN106918543A - A kind of device and method for detecting single gold nano grain surface biomolecules - Google Patents
A kind of device and method for detecting single gold nano grain surface biomolecules Download PDFInfo
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- 239000010931 gold Substances 0.000 title claims abstract description 79
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 79
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 71
- 230000008859 change Effects 0.000 claims abstract description 39
- 239000006059 cover glass Substances 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 11
- 238000010438 heat treatment Methods 0.000 claims description 37
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 29
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 29
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 29
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 29
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 29
- 230000003287 optical effect Effects 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 17
- 239000012530 fluid Substances 0.000 claims description 10
- 238000010521 absorption reaction Methods 0.000 claims description 8
- 239000010453 quartz Substances 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 230000005670 electromagnetic radiation Effects 0.000 claims description 7
- 238000012544 monitoring process Methods 0.000 claims description 5
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- 238000005266 casting Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 238000011065 in-situ storage Methods 0.000 abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 3
- 238000011897 real-time detection Methods 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000004140 cleaning Methods 0.000 abstract description 2
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- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 235000013339 cereals Nutrition 0.000 description 52
- 108020004414 DNA Proteins 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
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- 239000012498 ultrapure water Substances 0.000 description 5
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- 230000000694 effects Effects 0.000 description 4
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- 238000001228 spectrum Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 3
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- 239000003574 free electron Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000011946 reduction process Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
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- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical class CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 1
- 230000008836 DNA modification Effects 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 238000002983 circular dichroism Methods 0.000 description 1
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- 239000002082 metal nanoparticle Substances 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 239000002052 molecular layer Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
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- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1023—Microstructural devices for non-optical measurement
-
- 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
- G01N21/171—Systems in which incident light is modified in accordance with the properties of the material investigated with calorimetric detection, e.g. with thermal lens detection
-
- 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
- G01N21/19—Dichroism
-
- 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
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
- G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
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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
- G01N21/171—Systems in which incident light is modified in accordance with the properties of the material investigated with calorimetric detection, e.g. with thermal lens detection
- G01N2021/1712—Thermal lens, mirage effect
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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
- G01N21/21—Polarisation-affecting properties
- G01N2021/216—Polarisation-affecting properties using circular polarised light
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- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Dispersion Chemistry (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to a kind of apparatus and method for carrying out biomolecule and its conformation detection based on single gold nano grain local surface plasma resonance property, gold nano grain is covered first be loaded on the cover glass of cleaning and assembled with the micro-fluidic chip of customization, then the liquid in micro-fluidic chip reservoir cavity is switched by peristaltic pump, the light thermal detection built using laboratory is attached to the biomolecule and its conformation change on gold nano grain surface, and the biomolecule can be DNA or polypeptide.Compared with prior art, the present invention can realize in situ, real-time detection, and with high sensitivity, high s/n ratio, few amount of reagent is semi-automatic, the features such as low concentration.
Description
Technical field
The present invention relates to biomolecule and the detection technique field of conformation, and in particular to one kind is for detecting single gold nano
The device and method of particle surface biomolecule.
Background technology
In related to disease biomolecule detection development with application process, people for detection method sensitivity,
Detectable limit, portable degree proposes higher and higher requirement.For example in the detection of nucleic acid, traditional PCR method and equipment is difficult to
Realize short cycle, the detection of miniaturization；In the detection of the tumor markers related to diagnosing tumor, mark content is very low, passes
Radio isotope is used in system method such as radiommunoassay etc., has infringement to human body and environment, and cumbersome.It is near
The development of year nanometer technique, to solve the problems, such as the above-mentioned biomolecule detection related to disease in terms of provide opportunity.
Gold nano grain is one of most commonly used nano material of research in recent years, and it has good biocompatibility, changes
The optical property of stability and uniqueness is learned, there is very big development potentiality in terms of biomolecule detection, diagnosis and treatment.Especially
It is that gold nano grain shows special local surface plasma resonance characteristic (Localized surface plasmon
Resonance, LSPR).Under extraneous electromagnetic field effect, the free electron on gold nano grain surface produces polarization, when extraneous electricity
Magnetic field occurs when alternately, and the polarised direction of gold nano grain surface electronic also occurs alternately change, this cause free electron also with
Generation reciprocating motion, when the change frequency of electromagnetic field and the proper motion frequency of metal nanoparticle surface free electron are equal
When can produce resonance cause surface plasma collective concussion be greatly enhanced, cause gold nano grain surface generation strong electromagnetic
, and finally enhance the radiation characteristic for such as absorbing and scattering.
The surface plasma resonance frequency of gold nano grain can be with the size of particle, intergranular distance, surrounding environment
The factor such as dielectric constant change.Therefore, can be used for for gold nano grain to sense some biomolecule such as albumen by we
Matter, the presence of DNA and its conformation change.
Local surface plasma resonance has application in many fields, but most of is all that the dark field microscope applied enters
Row detection.The such as most basic absorption peak shift by detecting gold nano grain, it is possible to judge the change of its surrounding medium
Change.First pass through the detection single stranded DNA that golden sulfide linkage modifies upper band sulfydryl on gold nano grain surface, it is complementary therewith when being added in system
Target dna when, DNA hybridization causes the dielectric constant on gold nano grain surface to change, so that gold nano grain
There is red shift Deng from primitive absworption peak.Scattering light is collected by CCD spectrometers, the degree according to scattering light red shift can be chased after in real time
Track DNA hybridization process.But noise is exactly very big in itself for the detection of individual particle, and collected during DNA conformation changes complete
Spectrum carries out data fitting again, the method for finding peak shift, and uncertainty is than larger.Because gold nano grain in change procedure
The degree that local plasmon body absorbs red shift is also smaller, and the absworption peak red shift amount of document report reaches maximum also to be only had
2.0nm。
The content of the invention
The purpose of the present invention is exactly to provide a kind of high sensitivity, Gao Xin for the defect for overcoming above-mentioned prior art to exist
Make an uproar than, few amount of reagent, semi-automatic for gold nano grain surface biomolecules and the detection means and method of conformation.
The purpose of the present invention can be achieved through the following technical solutions：One kind is for detecting single gold nano grain surface
The device of biomolecule, described device include opto-thermal system, the dark field microscope for finding and selecting single gold nano grain,
The micro-fluidic chip sample being placed on dark field microscope sample stage and the avalanche diode for detecting optical signal, the light
Hot systems transmitting is heated light and detection light and is focused on micro-fluidic chip sample, is reflected from micro-fluidic chip sample
In detection light backtracking to avalanche diode, the heating light and detection light reach confocal point when at micro-fluidic chip sample.
Device of the invention uses dark field microscope, can find out proper single gold nano grain in micro-fluidic chip sample,
Then the single gold nano grain is individually analyzed.By the way that light and the confocal point of detection light will be heated, when micro-fluidic chip sample
Product can form similar thermal lens after absorbing heating light, so as to cause the change of the dispersion angle and polarization direction of detection light, institute
The microenvironment situation residing for Gold Samples nano particle is understood with the change by analyzing detection light.
Described opto-thermal system includes detection Optical Transmit Unit and heating Optical Transmit Unit, the detection Optical Transmit Unit
The heating light that the detection light and heating light emitting units emitting launched go out overlaps at dichroscope, focuses on micro-fluidic chip
On sample.
Green laser that described heating Optical Transmit Unit includes setting gradually, chopper, polarizer, No. one 1/
2 wave plates and a beam expander, the heating light that beam expander is obtained are penetrated on dichroscope, and the reflection for passing through dichroscope
Focus on micro-fluidic chip sample.The wavelength of the laser that green laser sends is 520~550nm.
Described heating light is circularly polarized light, and the frequency for heating light is 400~1000HZ, heat the power of light for 0~
50mW。
Described detection Optical Transmit Unit includes the red laser, No. two polarizers, No. two 1/2 ripples that are successively set on
Piece, No. two beam expanders and Amici prism, project detection light and pass through dichroscope in the Amici prism, and are overlapped with heating light,
It is then focused on micro-fluidic chip sample, detection light focuses on backtracking after micro-fluidic chip sample, and passes through dichroic
Mirror reaches Amici prism, then injects in avalanche diode, obtains Photothermal Signals.The wavelength of the laser that red laser sends is
620~650nm.
Described detection light is linearly polarized light.
The quartz slide of described micro-fluidic chip sample including bottom, middle PDMS layer and it is covered in PDMS layer
The cover glass of top, gold nano grain to be detected covers load on the cover slip, and the quartz slide and PDMS layer pass through plasma
Bonding, the cover glass is combined with PDMS layer by absorption affinity.
Then described PDMS layer is poured by casting mould and formed, and the center of the PDMS layer sets a diameter of 8mm's
Reservoir cavity, the inlet opening and fluid hole of a diameter of 1mm are respectively provided with the both sides of reservoir cavity, and the inlet opening passes through width
For 0.4mm, depth for the feed liquor flow path groove of 0.1mm is connected with reservoir cavity, the fluid hole is 0.2mm by degree, depth is
The liquid flow path groove that goes out of 0.1mm is connected with reservoir cavity, using different in width feed liquor flow path groove and go out liquid flow path groove, build certain
Current difference in order to the liquid of middle reservoir cavity can be full of, the cover glass covers reservoir cavity and exposes inlet opening
And fluid hole.
Small wavelength is moved through the change that detection light is converted into polarization direction and light intensity by the present apparatus, is used in combination lock
Phase amplifier carries out noise reduction process to signal, can improve sensitivity and signal to noise ratio.
A kind of method of the single gold nano grain surface biomolecules of detection carried out using detection means as described above, bag
Include following steps：
(1) exposed gold nano grain is covered the bottom lower surface for being loaded in cover glass, cover glass is then covered in PDMS
On layer and exposed gold nano grain is located in reservoir compartment body, micro-fluidic chip sample is obtained, and micro-fluidic chip sample is put
Put on the sample stage of dark field microscope；
(2) suitable single exposed gold nano grain is selected, then opto-thermal system adds to micro-fluidic chip electromagnetic radiation
Hot light and detection light, and the Photothermal Signals of the single exposed gold nano grain are detected by avalanche diode；
(3) in the inlet opening by peristaltic pump to PDMS layer add with biomolecule solution, then opto-thermal system to
Micro-fluidic chip electromagnetic radiation detection light and heating light, upper gold nano is modified by avalanche diode real-time monitoring biomolecule
The change of its Photothermal Signals in grain surface process；
(4) by peristaltic pump to buffer solution is added in PDMS layer inlet opening, by many of unmodified upper gold nano grain surface
The solution of remaining biomolecule is rinsed out, then adds the solution that biomolecule conformation can be induced to change by peristaltic pump,
Then opto-thermal system is to micro-fluidic chip electromagnetic radiation detection light and heats light, by avalanche diode real-time monitoring biomolecule
The change of its Photothermal Signals in gold nano grain surface conformation change procedure；
(5) in analytical procedure (2), (3) and (4) each Photothermal Signals change, obtain biomolecule type and conformation become
The information of change.
Present analysis chemistry and biological detectable limit are exactly unimolecule.There is the molecule of assemblage level complicated conformation to move
Mechanics, molecule interaction and spatial dispersion phenomenon, many this characteristics are obtained by statistical average result, and this has just lacked and has divided
Information under son and nanoscale.And individual particle level research it can be found that assemblage level institute it is not detectable, such as molecular layer
Secondary and nanoscale mechanism and information.Know that the details of molecular dynamics understands that us biological function is on a molecular scale
Necessary.However, seldom laboratory apparatus can provide the activity that platform removes detection molecules in solution to us at present, generally
All it is to use fluorescent marker, the mark modification of analyte may influence biological processes, and this promotes unmarked new detection
Method is born.This method detects unlabelled biomolecule using single gold nano grain, with resolution ratio and letter very high
Make an uproar ratio.
Photo-thermal method is the laser for using two beam different wave lengths, and the laser that can be absorbed by the sample is heating light, and it is inspection that no
Light-metering, when sample energy absorption temperature rising after can form similar thermal lens, so as to cause detection light dispersion angle and partially
Shake the change in direction, so by analyzing the microenvironment situation residing for Gold Samples nano particle knowable to the change i.e. of detection light.
Can by the change of the material of the method real-time monitoring detection zone, and because this method need not touch sample, be
A kind of detection method of non-intrusion type, is provided simultaneously with high sensitivity and signal to noise ratio.On the books, the photo-thermal in unimolecule PTI articles
The signal to noise ratio of method can reach 15, and sensitivity can strengthen 700 times of relative to traditional dark field microscope method, and this is also us
The reason for biomolecule being detected with photo-thermal method.
Contain chiral biomolecule or the biomolecule with conformation change in the solution of the biomolecule.
Polypeptide has chirality, but monomolecular photo-thermal effect is small and in ultraviolet region, gold nano grain photo-thermal effect
It is high not have chirality still, by peptide modified on gold nano grain surface so that being organized in visible region has chirality, will add
Thermo-optic modulation is circularly polarized light and realizes being switched fast for left-handed and right-circularly polarized light, and biomolecule is justified to left-handed and dextrorotation
The degree of absorption difference of polarised light can cause molecule Photothermal Signals to change, and reach the purpose of detection absorbance difference.Due to Jenner
The Photothermal Signals of rice grain are relatively very strong so can cause overall big to the change of left-handed and right-handed rotation degree of absorption difference.This is than passing
The circular dichroism detector of system has sensitivity higher, lower concentration is can apply to, with signal to noise ratio higher.
Single stranded DNA rich in bases G, by sulfydryl-gold chemical bond in-situ modification on gold nano grain surface, further leads to
It is the buffer solution containing potassium ion to cross peristaltic pump switching liquid, and induction ssDNA forms G4 structures.The modification and the formation of G4 of DNA, all
The refractive index of the environment on gold nano grain periphery is increased, the absworption peak that result in gold nano grain occurs red shift, so as to can change
Become Photothermal Signals.Therefore we change by the Photothermal Signals for being easier to measurement, and indirect detection DNA molecular is in gold nano grain table
The modification in face, and the follow-up DNA conformation changes induced by potassium ion.Traditional being observed with dark field microscope combines CCD
Collect full spectrum carries out data fitting again, the method for finding peak shift, and uncertainty is than larger.The method signal to noise ratio and sensitivity
Have and significantly lifted.
Compared with prior art, beneficial effects of the present invention are embodied in following several respects：
(1) change of biomolecule and its conformation on single nanogold particle is detected by the present apparatus, detection sensitivity is high；
(2) lock-in amplifier is connected behind avalanche diode carries out the noise reduction process of signal, can improve sensitivity and
Signal to noise ratio；
(3) special micro-fluidic chip sample is used, less dosage is used, it is possible to biomolecule and its conformation is realized
In situ, real-time detection.
Brief description of the drawings
Fig. 1 is the connection diagram of apparatus of the present invention；
Fig. 2 is the structural representation of micro-fluidic chip sample of the present invention；
Fig. 3 is the testing result of step (2) in embodiment 1；
Fig. 4 is the detection figure of Photothermal Signals after addition DNA in embodiment 1 and cushioning liquid.
Wherein, 1 is green laser, and 2 is chopper, and 3 is a polarizer, and 4 is 1/2 wave plate, and 5 expand for No. one
Device, 6 is red laser, and 7 is No. two polarizers, and 8 is No. two 1/2 wave plates, and 9 is No. two beam expanders, and 10 is Amici prism, and 11 are
Dichroscope, 12 is dark field microscope, and 13 is avalanche diode, and 14 is lock-in amplifier, and 15 is speculum, and 16 is cover glass,
17 is quartz slide, and 18 is PDMS layer, and 19 is reservoir cavity, and 20 is inlet opening, and 21 is feed liquor flow path groove, and 22 is fluid hole,
23 is liquid flow path groove.
Specific embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed implementation method and specific operating process, but protection scope of the present invention is not limited to following implementations
Example.
Embodiment 1
A kind of device for detecting single gold nano grain surface biomolecules, its structure is as shown in figure 1, including photo-thermal
System, the dark field microscope 12 for finding single gold nano grain, be placed on it is micro-fluidic on the sample stage of dark field microscope 12
Chip sample and the avalanche diode 13 for detecting optical signal, opto-thermal system include detection Optical Transmit Unit and heating light
Transmitter unit, the heating light that the detection light and heating light emitting units emitting that detection Optical Transmit Unit is launched go out is by dichroic
After mirror 11 is integrated, it is radiated on micro-fluidic chip sample.
Heating Optical Transmit Unit includes the green laser 1,1/2 ripple of polarizer 3, of chopper 2, that set gradually
Piece 4 and a beam expander 5, the heating light that beam expander 5 is obtained are penetrated on dichroscope 11, and by dichroscope 11
Penetrate on micro-fluidic chip sample.Heating light is circularly polarized light, and the frequency for heating light is 400~1000HZ, heating light
Power is 0~50mW.
Detection Optical Transmit Unit includes the wave plate 8, two of the polarizer 7,2 of red laser 6, two 1/2 being successively set on
Number beam expander 9 and Amici prism 10, project detection light and pass through dichroscope 11 in Amici prism 10, and are overlapped with heating light, so
After penetrate on micro-fluidic chip sample, detection light is irradiated to backtracking after micro-fluidic chip sample, and passes through dichroscope 11
Amici prism 10 is reached, is then injected in avalanche diode 13, obtain optical signal.Detection light is linearly polarized light.The present apparatus in order to
Reduce floor space, the direction of transfer of laser is changed using some speculums 15, the angle that the present embodiment laser changes is
90°。
Small wavelength is moved through the change that detection light is converted into polarization direction and light intensity by the present apparatus, is used in combination lock
Phase amplifier 14 carries out noise reduction process to signal, can improve sensitivity and signal to noise ratio.
Wherein, the structure of micro-fluidic chip sample is as shown in Fig. 2 the quartz slide 17 including bottom, middle PDMS
Layer 18 and the cover glass 16 being covered in above PDMS layer 18, gold nano grain to be detected are covered and are loaded on cover glass 16, and quartz is carried
Slide 17 and PDMS layer 18 are bonded by plasma, and cover glass 16 is combined with PDMS layer 18 by absorption affinity.PDMS layer 18 passes through
Then casting mould pours and forms, and the center of PDMS layer 18 sets the reservoir cavity 19 of a diameter of 8mm, the two of reservoir cavity 19
Side is respectively provided with the inlet opening 20 and fluid hole of a diameter of 1mm, and inlet opening 20 is 0.4mm by width, depth is for 0.1mm's enters
Liquid flow path groove 21 is connected with reservoir cavity 19, fluid hole by width be 0.2mm, depth for 0.1mm go out liquid flow path groove 23 with
Reservoir cavity 19 is connected, using different in width feed liquor flow path groove 21 and go out liquid flow path groove 23, build certain current difference in order to
The liquid of middle reservoir cavity 19 can be full of, cover glass 16 covers reservoir cavity 19 and exposes inlet opening 20 and fluid hole
22。
The detection of single gold nano grain surface biomolecules, including following steps are carried out using said apparatus：
(1) blank coverglass is chosen, and cover glass is cleaned, cleaning process is：It is ultrasonic in following liquid successively
30min, 2%Hellmanex washing lotion；Ultra-pure water；Acetone；Ultra-pure water；Methyl alcohol；Ultra-pure water, to remove the organic of cover glass surface
Thing, dust etc..Ultrasound 30min in ultra-pure water again is soaked after 12h after high pure nitrogen drying in the concentrated sulfuric acid, high pure nitrogen dries up standby
With；Then cover glass is surface-treated：By 6 hours in methyl alcohol (order of spectrum) solution of cover glass immersion silane coupler.
Wherein, the methanol solution of silane coupler, using the 3- aminopropyl triethoxysilanes that concentration of volume percent is 5%, then
The ultrasound 30min in methyl alcohol (order of spectrum) and ultra-pure water, standby after high pure nitrogen drying successively.The silanization treatment is in order to allow lid
Surface of glass slide band amino group, is easy to subsequently cover load with gold nano grain；The cover glass that surface treated is crossed is in certain body
24h or so is soaked in the long-pending gold nano grain aqueous solution, the surface self-organization of cover glass is formed one layer of exposed gold nano
, then be covered in cover glass on PDMS layer and exposed gold nano grain be located in reservoir compartment body, and PDMS layer is covered by grain
On quartz slide, micro-fluidic chip sample is obtained, and micro-fluidic chip sample is placed on the sample stage of dark field microscope
On；
(2) by the selected suitable single exposed gold nano grain of dark field microscope, then opto-thermal system is to micro-fluidic
Chip sample launches detection light, heating light hot spot is overlapped with the single gold nano grain on micro-fluidic chip sample, and pass through
Avalanche diode detects the Photothermal Signals of the single exposed gold nano grain；
(3) in the inlet opening by peristaltic pump to PDMS layer add with biomolecule solution, then opto-thermal system to
Micro-fluidic chip electromagnetic radiation detection light and heating light, upper gold nano is modified by avalanche diode real-time detection biomolecule
The change of grain surface Photothermal Signals during this；By taking the single stranded DNA rich in bases G as an example, selection one can be with for the present embodiment
The DNA sequence dna of G4 is formed, the DNA with Tris-HCl (10mM pH=7.40) buffers into 2 μM stores liquid, is passed through
After peristaltic pump injection micro-fluidic chip reservoir cavity, DNA can be by sulfydryl-gold chemical bond in-situ modification in gold nano grain table
Face,
(4) will be carried to the buffer solution containing potassium ion that concentration is 500nM is added in PDMS layer inlet opening by peristaltic pump
The solution of biomolecule is all replaced, and then opto-thermal system is to micro-fluidic chip electromagnetic radiation detection light and heats light, by snow
Collapse the change that diode detection single stranded DNA occurs Photothermal Signals during cluster forms the serobilas of G- tetra- in the presence of potassium ion
Change；
(5) in analytical procedure (2), (3) and (4) reflected light signal change, obtain the type information of biomolecule.
Wherein, Fig. 3 is the testing result of step 2, it can be seen that on same sample at gold nano grain
(GNP) Photothermal Signals with heating luminous power increase and increase, and at blank background (Background) Photothermal Signals little with
Heating light changes and changes, it was demonstrated that signal is actually from the Photothermal Signals in gold nano grain.
As shown in figure 4, in this experiment, we have carried out the DNA modification of solution situ to single gold nano grain, hair
The Photothermal Signals of existing individual particle kept stable in Tris-HCl cushioning liquid, there is minor variations.When introduce DNA solution it
Afterwards, signal starts slowly constantly to reduce, and at about 90 minutes, signal was reduced near minimum, maintains stabilization.This be because
It is that DNA molecular is modified after gold nano grain surface, the effective refractive index around nano particle becomes big, causes the spectral peak of LSPR
Red shift, while absorption of the nano particle to exciting light is also reduced, so that signal reduction.
Claims (10)
1. a kind of device for detecting single gold nano grain surface biomolecules, it is characterised in that described device includes light
Hot systems, the dark field microscope for finding and selecting single gold nano grain, be placed on it is micro- on dark field microscope sample stage
Fluidic chip sample and the avalanche diode for detecting optical signal, the opto-thermal system transmitting heating light and detection light are simultaneously gathered
Jiao on micro-fluidic chip sample, the detection light backtracking reflected from micro-fluidic chip sample to avalanche diode
In, the heating light and detection light reach confocal point when at micro-fluidic chip sample.
2. the device for detecting single gold nano grain surface biomolecules according to claim 1, it is characterised in that
Described opto-thermal system includes detection Optical Transmit Unit and heating Optical Transmit Unit, what the detection Optical Transmit Unit was launched
The heating light that detection light and heating light emitting units emitting go out overlaps at dichroscope, focuses on micro-fluidic chip sample.
3. the device for detecting single gold nano grain surface biomolecules according to claim 2, it is characterised in that
Described heating Optical Transmit Unit include set gradually green laser, chopper, a polarizer, 1/2 wave plate with
And a beam expander, the heating light that beam expander is obtained penetrated on dichroscope, and reflection by dichroscope is focused on
On micro-fluidic chip sample.
4. the device for detecting single gold nano grain surface biomolecules according to claim 3, it is characterised in that
Described heating light is circularly polarized light, and the frequency for heating light is 400~1000HZ, and the power for heating light is 0~50mW.
5. the device for detecting single gold nano grain surface biomolecules according to claim 3, it is characterised in that
Described detection Optical Transmit Unit includes the red laser, No. two polarizers, No. two 1/2 wave plates, No. two expansions that are successively set on
Beam device and Amici prism, project detection light and pass through dichroscope in the Amici prism, and are overlapped with heating light, are then focused into
On micro-fluidic chip sample, detection light focuses on backtracking after micro-fluidic chip sample, and reaches light splitting through dichroscope
Prism, then injects in avalanche diode, obtains Photothermal Signals.
6. the device for detecting single gold nano grain surface biomolecules according to claim 5, it is characterised in that
Described detection light is linearly polarized light.
7. the device for detecting single gold nano grain surface biomolecules according to claim 1, it is characterised in that
Quartz slide of the described micro-fluidic chip sample including bottom, middle PDMS layer and the lid being covered in above PDMS layer
Slide, gold nano grain to be detected covers load on the cover slip, and the quartz slide and PDMS layer are bonded by plasma, described
Cover glass is combined with PDMS layer by absorption affinity.
8. the device for detecting single gold nano grain surface biomolecules according to claim 7, it is characterised in that
Then described PDMS layer is poured by casting mould and formed, and the center of the PDMS layer sets the reservoir cavity of a diameter of 8mm,
Be respectively provided with the inlet opening and fluid hole of a diameter of 1mm in the both sides of reservoir cavity, the inlet opening by width be 0.4mm,
Depth is connected for the feed liquor flow path groove of 0.1mm with reservoir cavity, and the fluid hole is 0.2mm by width, depth is 0.1mm's
Go out liquid flow path groove to be connected with reservoir cavity, the cover glass covers reservoir cavity and exposes inlet opening and fluid hole.
9. the single gold nano grain surface biological of detection that a kind of use detection means as described in claim 1~8 is any is carried out
The method of molecule, it is characterised in that methods described includes following steps：
(1) exposed gold nano grain is covered the bottom lower surface for being loaded in cover glass, then cover glass is covered on PDMS layer
And exposed gold nano grain is located in reservoir compartment body, obtains micro-fluidic chip sample, and micro-fluidic chip sample is placed on
On the sample stage of dark field microscope；
(2) suitable single exposed gold nano grain is selected, then opto-thermal system heats light to micro-fluidic chip electromagnetic radiation
And detection light, and the Photothermal Signals of the single exposed gold nano grain are detected by avalanche diode；
(3) solution with biomolecule is added in the inlet opening by peristaltic pump to PDMS layer, then opto-thermal system is to miniflow
Control chip sample transmitting detection light and heating light, upper gold nano grain table is modified by avalanche diode real-time monitoring biomolecule
The change of its Photothermal Signals during face；
(4) by peristaltic pump to buffer solution is added in PDMS layer inlet opening, by the unnecessary of unmodified upper gold nano grain surface
The solution of biomolecule is rinsed out, then adds the solution that biomolecule conformation can be induced to change by peristaltic pump, then
Opto-thermal system is to micro-fluidic chip electromagnetic radiation detection light and heats light, by avalanche diode real-time monitoring biomolecule in gold
The change of its Photothermal Signals during nano grain surface conformation change；
(5) in analytical procedure (2), (3) and (4) each Photothermal Signals change, obtain the type and conformation change of biomolecule
Information.
10. the method for detecting single gold nano grain surface biomolecules according to claim 9, its feature exists
In containing chiral biomolecule or the biomolecule with conformation change in the solution of the biomolecule.
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