CN109459422A - Small molecule metabolites SERS detection device and method based on the high-elastic polymer of dielectric - Google Patents
Small molecule metabolites SERS detection device and method based on the high-elastic polymer of dielectric Download PDFInfo
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Abstract
The invention discloses a kind of small molecule metabolites SERS detection device and method based on the high-elastic polymer of dielectric.The present invention is mainly by being fixed on the high-elastic thin polymer film of dielectric of bottomless culture dish bottom, single metal nano particle is the SERS active layer of basic structural unit, isolation layer, culture medium and corresponding working electrode are composed, two sides applies voltage on the high-elastic thin polymer film of dielectric, film can be driven to generate deformation, the folding condition of SERS " hot spot " between the metal nanoparticle of regulation load on it, realize the absorption and enrichment of the selectivity of the small molecule metabolites in complex sample, finally the laser of film surface visible/near infrared is irradiated, signal is acquired by Raman spectrometer, the real-time detection of metabolin in culture medium can be achieved.Present invention omits the process that metabolite is separated and be enriched with from culture medium, it is not only able to avoid complicated operation, moreover it is possible to realize the real-time detection of small molecule metabolites.
Description
Technical field
The invention belongs to spectroscopy and field of bioanalysis, and in particular to a kind of small point based on the high-elastic polymer of dielectric
Sub- metabolin SERS detection device and method.
Background technique
Metabolism is one significant process of life entity, provides matter and energy necessary to physiological activity for life entity.Newly
The chemical small molecule metabolin (metabolite) generated in old metabolic process, can reflect organism real-time status and function, with
And the response of response environment variation, it is extremely important to the subtle mechanism etc. for understanding and decoding vital movement.To generation
The research for thanking to object is extracted from histocyte firstly the need of by it, and is identified, then according to existing biological Xue Zhi
Know, infers the metabolic pathway in surveyed histocyte.Metabolin extracts and identification method has nuclear magnetic resonance (nuclear
Magnetic resonance, NMR), chromatography (chromatography), mass spectrography, electrochemical assay, Capillary Electrophoresis
Deng.The method of mainstream is gas chromatograph-mass spectrometer (GC-MS) (GC-MS) and liquid chromatograph-mass spectrometer (LC-MS) at present, is deposited
In complex pretreatment, detection cycle length, testing result time lag, it is unable to real-time detection and the high problem of analysis cost.
Surface enhanced Raman scattering (SERS) spectrum has high detection sensitivity, it can be achieved that Single Molecule Detection, and
With very high selectivity, because each molecule has unique corresponding Raman spectrum fingerprint, it can be seen that it is in metabolism analyte detection
Aspect has broad application prospects.SERS effect is mainly derived from the metal Nano structure tip with surface phasmon characteristic
Very strong local electromagnetic field (SERS " hot spot ") is generated with gap location.The molecule being among SERS hot spot, Raman signal are strong
The approximate biquadratic for being increased to electric field strength gain times of degree (I ∝ | E |4/|E0|4).SERS humidification is only attached in SERS " hot spot "
It is nearby more significant, and SERS " hot spot " is one very narrow (nanometer/sub-nanometer scale), almost closed three-dimensional space.
Therefore, how to overcome its huge steric hindrance effectiveness, determinand molecule is made to enter this region, be to obtain high quality SERS letter
Number key.Another problem is that metabolite content is low, and matrix is complicated, how to be chemically modified to SERS hot spot surface, is increased
Add its energy-absorbing power to determinand molecule, exclusive PCR is also very crucial.
The high-elastic polymer of dielectric (DE) is used as a kind of electric field type electroactive polymer, and alive irritate down can produce outside
Very big deformation (maximum up to 380%).And after applied voltage revocation, and it can be promptly restored to reset condition, there is electromechanics
The advantages such as high conversion efficiency, elastic energy density be big, fast response time, light weight, price are low, be widely used in various drivers and
Energy collecting device.Further, since the high-elastic polymer environment of dielectric is adaptable, be readily formed with damage not easy to fatigue, be one
Kind can be used for the ideal material of sensor design.
Summary of the invention
In order to solve the problems, such as background technique, the present invention provides a kind of small points based on the high-elastic polymer of dielectric
Sub- metabolin SERS detection device and method, the deformation generated using the high-elastic polymer of applied voltage driving dielectric, regulation load
The folding condition in local electromagnetic field " hot spot " region in noble metal nano structure on it, to realize small molecule metabolites
Selective enrichment and detection.
Technical scheme is as follows:
One, a kind of small molecule metabolites SERS detection device based on the high-elastic polymer of dielectric, comprising:
Culture dish and the high-elastic polymer film of dielectric, the high-elastic polymer film of dielectric are sealingly fastened in culture after being pre-stretched
Ware bottom and as culture dish bottom surface；
SERS active layer, the SERS active layer are formed in the high-elastic polymer film upper surface of dielectric；
Microculture layer, the microculture layer are mainly made of culture medium, and culture medium is formed in culture dish；
Isolation layer is formed on SERS active layer, is completely cut off between SERS active layer and culture medium；
Working electrode, the working electrode include round loop electrode in the culture dish and coated on the high-elastic polymer of dielectric
The flexible electrode of film bottom.
The SERS active layer is that basic structural unit array of closely arranging is constituted by single metal nano particle, can
See/near infrared light excitation under, metal nanoparticle array generate surface plasma body resonant vibration, metal nanoparticle gap formation office
Domain electromagnetic field " hot spot ", the Raman scattering of molecule enhanced strength among " hot spot ", i.e. surface enhanced Raman scattering effect, from
And it is detected by Raman spectrometer.
Under voltage effect tensile deformation can occur for the high-elastic polymer film of dielectric, cause on the high-elastic polymer film of dielectric
The metal nanoparticle gap on surface becomes larger, and testing molecule is facilitated to enter, after removing voltage, the deformation of the high-elastic polymer film of dielectric
It disappears, metal nanoparticle gap restPoses；The material of the preparation high-elastic polymer film of dielectric is polyacrylic acid, silicon
One of rubber, polyurethane and polyacrylic acid pyrrolidones ethyl ester.
The isolation layer is made of mercaptan or the phospholipid bilayer self-assembled film containing sulfydryl, for completely cutting off culture medium and big
Moieties, including organic micromolecule compound and large biological molecule, because large biological molecule cannot be closed between nano particle
In gap, therefore it is able to achieve and selective enrichment and detection is carried out to complex fluid sample small molecular compound.
The culture dish is the container for cultivating microorganism, no bottom.
The culture medium, which is able to achieve, to carry out microorganism to increase numerous and culture.
Gather respectively as the positive and negative anodes of power supply for high-elastic to dielectric at the both ends that the round loop electrode and flexible electrode are drawn
Compound film applies driving voltage；The material for preparing of the flexible electrode is graphite or conductive hydrogel.
The partial size of the metal nanoparticle is 10nm-200nm, and material is one or more of gold, silver, platinum, copper
Mixed metal, pattern are one of cube, rodlike, spherical, ellipsoid or polyhedron regular shape.
Two, using a kind of preparation method of above-mentioned SERS detection device
It will be fixed on bottomless culture dish bottom after the high-elastic polymer film stretching of dielectric first, then in the high-elastic polymerization of dielectric
Preparation forms the SERS active layer constituted using single metal nano particle as basic structural unit on object film；Then in SERS activity
Isolation layer is formed on layer, in the high-elastic polymer film lower surface coating flexible electrode of dielectric；Finally according to the difference of microorganism, training
It supports and culture medium is added in ware, form microculture layer.
The method for preparing the SERS active layer has following three kinds:
Method one: hydrophilic treated will be carried out after the high-elastic polymer film cleaning of dielectric, be placed on horizontal plane, then in upper table
Face adds water to form the moisture film for covering the entire high-elastic polymer film surface of dielectric, the metal nanoparticle that will be scattered in organic solvent
It injects in moisture film, forms one layer of close-packed arrays metal nanoparticle array in the air liquid interface of moisture film, after water film evaporation
To the metal nanoparticle array closely arranged.
Method two: the single-layer metal of one layer of close ordered arrangement is covered on hard substrate by the way of the self assembly of interface
Nano-grain array, then flexible material is overlying on single-layer metal nano-grain array, single-layer metal is received after final removing
Rice grain array is transferred on the high-elastic polymer film of dielectric.
Method three: being mixed to form mutual exclusive interface with the metal nanoparticle being scattered in water phase for organic solvent,
Ethyl alcohol is injected into water phase, inducing metal nano particle forms one layer of close-packed arrays metal at organic solvent/aqueous phase interface and receives
Hard substrate insertion organic solvent/aqueous phase interface after hydrophilic treated is proposed behind lower section, is closely arranged by rice grain array
Metal nanoparticle array, then single-layer metal nano-grain array is transferred on the high-elastic polymer film of dielectric.
The method of the coating flexible electrode is uniformly to coat graphite or conductivity water in the high-elastic polymer film lower surface of dielectric
Gel forms conductive region.
Three, a kind of application method of the small molecule metabolites SERS detection device based on the high-elastic polymer of dielectric
The application method the following steps are included:
1) sample needed for being added in culture dish is cultivated, and generates metabolin, metabolin passes through isolation after a period of time
Layer enters SERS active layer；
2) tensile deformation occurs for the high-elastic polymer film of dielectric after the high-elastic polymer film both ends on-load voltage of dielectric, leads to it
The metal nanoparticle gap of upper surface becomes larger, and promotes SERS " hot spot " to open, testing molecule is facilitated to enter；After removing voltage,
The deformation of the high-elastic polymer film of dielectric disappears, and metal nanoparticle gap restPoses, and SERS " hot spot " is promoted to be closed, will
Testing molecule is enclosed in " hot spot " region；
3) after above steps may be repeated multiple times 2), the laser of dielectric high-elastic polymer film surface visible/near infrared is carried out
Irradiation, metal nanoparticle gap form local electromagnetic field " hot spot ", are enclosed in the drawing of the testing molecule between metal nanoparticle
Graceful signal is amplified, and Raman spectrometer acquires signal, obtains the testing result of testing molecule in sample.
The beneficial effects of the present invention are:
The present invention provides a kind of collection microcultures and metabolin to be detected on integrated SERS detection device, to omit
The process that metabolite is separated and be enriched with from culture medium is not only able to avoid complicated operation, moreover it is possible to realize that small molecule is metabolized
The real-time detection of object.
Detailed description of the invention
Fig. 1 is structural schematic diagram of the invention.
Fig. 2 is a kind of embodiment flow diagram produced by the present invention.
Fig. 3 is typical metal nano-array scanning electron microscope (SEM) photograph of the invention.
Fig. 4 is the detection schematic diagram that the present invention is used for medium molecule metabolites.
Fig. 5 is present invention detection Pseudomonas aeruginosa metabolite response Raman spectrogram (I: before culture；II: after culture；III:
After culture after the making alive driving high-elastic thin polymer film deformation of dielectric).
Attached drawing 6 is that the present invention is based on the flow diagrams that gas/liquid self assembly mode prepares apparatus of the present invention.
Attached drawing 7 is that the present invention is based on the flow diagrams that liquid liquid self assembly mode prepares apparatus of the present invention.
In figure: 1, culture dish, 2, the high-elastic thin polymer film of dielectric, 3, SERS active layer, 4, isolation layer, 5, flexible electrode,
6, round loop electrode, 7, conducting wire, 8, culture medium, 9, moisture film, 10, syringe, 11, microculture layer, 12, small molecule to be measured metabolism
Object, 13, chaff interferent in culture medium, 14, rigid substrate, 15, container, 16, toluene.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.
As shown in Figure 1, the present invention includes culture dish 1 and the high-elastic polymer film 2 of dielectric, the high-elastic polymer film 2 of dielectric is through pre-
1 bottom of culture dish is sealingly fastened in after stretching, SERS active layer 3 is formed at the high-elastic 2 upper surface center of polymer film of dielectric, training
It supports base 8 to be formed in inside culture dish 1, for isolation layer 4 between SERS active layer 3 and culture medium 8, round loop electrode 6 is set to culture
In ware 1, a flexible electrode 5 is arranged at high-elastic 2 bottom of polymer film of dielectric.
Embodiment 1
Preparation flow is as shown in Figure 2:
(1) preparation of the high-elastic thin polymer film of dielectric: the high-elastic polymer of dielectric is formed after the high-elastic polymer pre-stretching of dielectric
Film is fixed on 1 bottom of culture dish by film 2, cleans film using water, Plasma.
(2) preparation of metal nanoparticle organosol: the metal nanoparticle being prepared using wet chemistry method is molten
Glue is transferred in organic solvent, and the specific method is as follows: 20mL metal nanoparticle colloidal sol and the sulfydryl-dissolved with 0.1mg/mL is poly-
The chloroformic solution of ethylene glycol (average molecular weight 5000) mixes；After solution layering, 1mL methanol is rapidly joined in water phase, it can
Metal nanoparticle is transferred in chloroformic solution；It is washed repeatedly and removes excessive sulfydryl-polyethylene glycol in solution, by metal
Nanoparticle concentration is stand-by into 200 μ L chloroforms.
(3) preparation of SERS active layer 3: by the above-mentioned high-elastic thin polymer film of cleaned dielectric together with culture dish 1 as
On levelling bench, a certain amount of water is added, forms the moisture film 9 for covering the entire high-elastic polymeric film surface of dielectric；Utilize injection
Device 10, the injection step 2 on moisture film gas liquid interface) obtained metal nanoparticle organosol, it forms orderly metal and receives
Rice grain array can form the SERS active layer 3 using single metal nano particle as basic structural unit, such as after removing moisture film
Fig. 3 show metal nano array scanning electron microscope.
(4) preparation of isolation layer: the solution of a certain amount of mercaptan or the phospholipid bilayer containing sulfydryl is added dropwise in SERS
On active layer 3, fine and close self-assembled film is generated in metal nanoparticle upper surface, forms isolation layer 4 for stopping interfering substance.
(5) preparation of working electrode: by flexible conducting material, (it is high-elastic poly- that carbon rouge, graphite, conductive hydrogel are coated on dielectric
Object film lower surface is closed, is formed flexible electrode 5 (with a thickness of 100-200 micron), and connecting wire 7, formation working electrode will be with
The conducting wire of circular ring shape, which is placed in sample, forms another working electrode；
(6) preparation of microculture layer 11: according to the culture medium of the different strain to be cultivated preparation heterogeneity.With LB
For agar medium, by after 3mL high pressure sterilization, the LB agar of molten condition be added dropwise on SERS isolation layer 4, at room temperature into
Row solidify after up to required microculture layer 11, can direct inoculated bacteria cultivated.The proportion of LB agar are as follows: 1L is pure
10g peptone, 5g yeast extract, 10g NaCl, 1.5wt% agar are added in water.
(7) cultural method microculture: is determined according to the specific type of the microorganism to be cultivated.By taking Pseudomonas aeruginosa as an example,
After the bacterium solution that OD value is 1.0 is diluted 1000 times, 2 μ L are added dropwise on microculture layer 11,30 DEG C of constant temperature are cultivated.
(8) small molecule metabolites are detected: as shown in figure 4, two working electrodes of step 5) preparation are before applying voltage
State applies voltage (500-10000V) on two working electrodes as shown in Fig. 4 II as shown in Fig. 4 I, and driving dielectric is high-elastic poly-
It closes object film 2 to expand, causes metal nanoparticle gap thereon to be opened, small molecule metabolites 12 to be measured is made to enter gap；Such as
Shown in Fig. 4 III, after removing above-mentioned voltage, the high-elastic thin polymer film of dielectric restPoses, and leads to metal nano thereon
Grain gap-closing, small molecule metabolites 12 to be measured are enclosed in the gap of nano particle.To reach better molecule trapping effect
Fruit, the above process can be repeated several times.
(9) using the Raman spectrum of Raman spectrometer acquisition film surface, testing molecule in sample can Raman detection: be measured
Type and content, as shown in Figure 5, after dielectric high-elastic 2 making alive of thin polymer film driving, Pseudomonas aeruginosa metabolite (green pus
Toxin) Raman signal be significantly enlarged (Fig. 5 III), the sensitivity of detection is improved, and there is selectivity, just for small
Molecular compound.It (is as shown in fig. 5i the Raman signal before Pseudomonas aeruginosa metabolite culture；As Fig. 5 II show green pus bar
Raman signal after bacterium metabolite culture).
Embodiment 2: indirect branch method
As shown in fig. 6, metal nanoparticle array is prepared on rigid substrate 14 in the way of gas/liquid self assembly, then will
Metal nanoparticle array is transferred on the high-elastic thin polymer film of dielectric.
Concrete operations are as follows:
(1) substrate cleans: cleaning rigid substrate 14 using water, Plasma；
(2) preparation of metal nanoparticle organosol: identical as embodiment 1 step (2)；
(3) it the preparation of SERS active layer 3: by cleaned rigid substrate 14 as on levelling bench, is added a certain amount of
Water forms the moisture film 9 for covering entire 14 surface of rigid substrate.Using syringe 10, the injection step on moisture film gas liquid interface
2) the metal nanoparticle organosol being prepared forms orderly metal nanoparticle array, after removing moisture film, can be formed
It is the SERS active layer 3 of basic structural unit by single metal nano particle；
(4) nano particle shifts: by metal nanoparticle array that step 3) is prepared and being fixed on 1 bottom of culture dish
The high-elastic thin polymer film 2 of dielectric contact, i.e., metal nanoparticle array is transferred on the high-elastic thin polymer film of dielectric.
(5) remaining operation step is same as Example 1.
Embodiment 3: indirect branch method
As shown in fig. 7, metal nanoparticle battle array is prepared on rigid substrate 14 in the way of liquid/liquid interface self assembly
Column, then metal nanoparticle array is transferred on the high-elastic thin polymer film 1 of dielectric.
Concrete operations are as follows:
(1) substrate cleans: identical as embodiment 2 step (1)；
(2) preparation of metal nanoparticle: identical as embodiment 2 step (2)；
(3) cleaned rigid substrate 14 preparation of SERS active layer 3: is sunk into 15 bottom of container；In container 15, add
Enter 10mL metal nanoparticle colloidal sol and 10mL toluene 16；After solution layering after, using syringe 10, in metal-sol with
Alcohol, inducible metal nanoparticle form compact arranged gold in toluene/water phase interface to the rate injection 12mL of 300 μ L/min
Metal nano-particle array can be fished for the rigid substrate 14 through over cleaning, after being completely dried, can be formed by single metal nanometer
Particle is the SERS active layer 3 of basic structural unit.
(4) remaining operation step is same as Example 1.
Although the present invention is disclosed with specific embodiment, it is not intended to limit the invention, the technology of any this field
Personnel, the displacement of made equivalent assemblies under the premise of not departing from the spirit and scope of the present invention, or according to the invention patent
Equivalent variations made by protection scope and modification, should still belong to the scope that this patent is covered.
Claims (10)
1. a kind of small molecule metabolites SERS detection device based on the high-elastic polymer of dielectric, it is characterised in that:
Including culture dish (1) and the high-elastic polymer film of dielectric (2), the high-elastic polymer film of dielectric (2) seals after being pre-stretched
It is fixed on culture dish (1) bottom and as culture dish (1) bottom surface；
Including SERS active layer (3), the SERS active layer (3) is formed in the high-elastic polymer film upper surface of dielectric；
Including microculture layer (11), the microculture layer (11) is mainly made of culture medium (8), culture medium (8) shape
At in culture dish (1)；
Including isolation layer (4), the isolation layer (4) is mainly made of mercaptan or the phospholipid bilayer self-assembled film containing sulfydryl,
Be formed on SERS active layer (3), completely cut off between SERS active layer (3) and culture medium (8), for completely cut off culture medium (8) and
Interfering substance in culture medium (8) in SERS active layer (3) outside；
Including working electrode, the working electrode includes round loop electrode (6) in culture dish (1) and high-elastic coated on dielectric
The flexible electrode (5) of polymer film (2) bottom.
2. the small molecule metabolites SERS detection device according to claim 1 based on the high-elastic polymer of dielectric, feature
It is, the SERS active layer (3) is that basic structural unit array of closely arranging is constituted by single metal nano particle, can
See/near infrared light excitation under, metal nanoparticle array generate surface plasma body resonant vibration, metal nanoparticle gap formation office
Domain electromagnetic field " hot spot ", the Raman scattering of molecule enhanced strength among " hot spot ", to be detected by Raman spectrometer.
3. the small molecule metabolites SERS detection device according to claim 1 based on the high-elastic polymer of dielectric, feature
It is, under voltage effect tensile deformation can occur for the high-elastic polymer film of dielectric (1), lead to the high-elastic polymer film of dielectric
(1) the metal nanoparticle gap of upper surface becomes larger, and testing molecule is facilitated to enter, after removing voltage, the high-elastic polymer film of dielectric
(1) deformation disappears, and metal nanoparticle gap restPoses；The material of the preparation high-elastic polymer film of dielectric (2)
For one of polyacrylic acid, silicon rubber, polyurethane and polyacrylic acid pyrrolidones ethyl ester.
4. the small molecule metabolites SERS detection device according to claim 1 based on the high-elastic polymer of dielectric, feature
It is, the both ends of the round loop electrode (6) and flexible electrode (5) extraction are used for respectively as the positive and negative anodes of power supply to dielectric height
It plays polymer film (2) and applies driving voltage；The material for preparing of the flexible electrode (5) is graphite or conductive hydrogel.
5. the small molecule metabolites SERS detection device according to claim 1 based on the high-elastic polymer of dielectric, feature
It is, the partial size of the metal nanoparticle is 10nm-200nm, and material is the mixing of one or more of gold, silver, platinum, copper
Metal, pattern is one of cube, rodlike, spherical, ellipsoid and polyhedron regular shape.
6. a kind of preparation method of any SERS detection device of application claim 1-5, which is characterized in that will be situated between first
The high-elastic polymer film (2) of electricity is fixed on bottomless culture dish (1) bottom after stretching, then on the high-elastic polymer film of dielectric (2)
Preparation forms the SERS active layer (3) constituted using single metal nano particle as basic structural unit；Then in SERS active layer
(3) isolation layer (4) are formed on, in the high-elastic polymer film of dielectric (2) lower surface coating flexible electrode (5)；Finally according to microorganism
Difference, culture medium (8) are added in the culture dish (1), are formed microculture layer (11).
7. a kind of preparation method according to claim 6, which is characterized in that the method for preparing the SERS active layer (3)
For hydrophilic treated will be carried out after the high-elastic polymer film of dielectric (2) cleaning, it is placed on horizontal plane, then adds water to be formed in upper surface
The metal nanoparticle being scattered in organic solvent is injected water by the moisture film for covering the high-elastic polymer film of entire dielectric (2) surface
In film, one layer of close-packed arrays metal nanoparticle array is formed in the air liquid interface of moisture film, is obtained after water film evaporation close
The metal nanoparticle array of arrangement.
8. a kind of preparation method according to claim 6, which is characterized in that the side for preparing SERS active layer (3)
Method is to be covered with the single-layer metal nano particle battle array of one layer of close ordered arrangement on hard substrate by the way of the self assembly of interface
Column, then flexible material is overlying on single-layer metal nano-grain array, by single-layer metal nano-grain array after final removing
It is transferred on the high-elastic polymer film of dielectric (2).
9. a kind of preparation method according to claim 6, which is characterized in that the side for preparing SERS active layer (3)
Method is that organic solvent is mixed to form mutual exclusive interface with the metal nanoparticle being scattered in water phase, is injected into water phase
Ethyl alcohol, inducing metal nano particle form one layer of close-packed arrays metal nanoparticle array at organic solvent/aqueous phase interface, will
It is proposed after below hard substrate insertion organic solvent/aqueous phase interface after hydrophilic treated, the metal nano closely arranged
Single-layer metal nano-grain array, is then transferred on the high-elastic polymer film of dielectric (2) by grain array.
10. a kind of preparation method according to claim 7, which is characterized in that the method for the coating flexible electrode (5) is
Graphite or conductive hydrogel are uniformly coated in the high-elastic polymer film of dielectric (2) lower surface, forms conductive region.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109916879A (en) * | 2019-04-11 | 2019-06-21 | 重庆大学 | Material composition Raman spectra detection process based on the reunion enhancing of automatically controlled nano wire |
| CN110261367A (en) * | 2019-07-10 | 2019-09-20 | 中国科学技术大学 | A kind of SERS substrate, preparation method, regulate and control SERS substrate hotspot architecture method and SERS substrate application |
| CN114888276A (en) * | 2022-05-12 | 2022-08-12 | 集美大学 | Preparation method and application of chain-like high polymer modified nano particles |
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