CN104458694A - Method for enhancing Raman signals with nano super-crystal technology to identify microorganisms - Google Patents
Method for enhancing Raman signals with nano super-crystal technology to identify microorganisms Download PDFInfo
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Abstract
The invention discloses a method for enhancing Raman signals with a nano super-crystal technology to identify microorganisms. The method is characterized by comprising steps as follows: airing optically anisotropic precious metal nano-materials on silicon wafer substrates in a natural evaporation manner under humidity-controlled and temperature-controlled conditions so as to form direction-controllable and regularly-arranged three-dimensional super-crystals; enabling the to-be-tested microorganisms to be adsorbed on the three-dimensional super-crystals, amplifying the intrinsic Raman signals of the microorganisms by using a plasmon field of the three-dimensional super-crystals, and acquiring the intrinsic Raman signals of the to-be-tested microorganisms through detection with an enhanced Raman technology so as to identify the species of the to-be-tested microorganisms. According to the method, the nano super-crystals are used as the substrates, the microorganisms are adsorbed directly, the weak intrinsic Raman signals of the microorganisms are efficiently amplified, label-free quick identification of the microorganisms can be realized, and the long-cycle process for propagation and culture of the microorganisms is avoided.
Description
Technical field
The invention belongs to the detection field of microorganism, be specifically related to optically anisotropic noble metal nano and surpass crystal as high sensing surface enhancing Raman substrate, the faint intrinsic Raman signal of microorganism is efficiently amplified, comes the existence and the kind that effectively identify microorganism fast.
Background technology
At present, the microorganism recognition technology mainly colony counting method generally applied, its sense cycle is longer, usually needs three days to one week, and because it can only cause detection not accurate enough according to the form of microorganism to the category identification of microorganism, microorganism identification requirement fast and accurately cannot be met.
Summary of the invention
The present invention is the weak point for avoiding existing for above-mentioned prior art, there is provided a kind of and surpass crystal technique enhancing Raman signal to identify method of microorganism by nanometer, it is a kind of microorganism recognition technology of versatility, the efficient enhancing Raman ability of crystals antenna effect generation is surpassed by nanometer, amplify the intrinsic Raman signal that microorganism is faint, realize quick and precisely identifying the mark of exempting from of microorganism.
The present invention is that technical solution problem adopts following technical scheme:
The present invention surpasses crystal technique by nanometer and strengthens Raman signal to identify method of microorganism, is characterized in:
In containing the solution of optically anisotropic noble metal nanometer material, add orientation adjustment agent solution, obtain mixed solution; Dropped at the bottom of silicon wafer-based by described mixed solution, in light protected environment, control environment temperature and humidity, makes the solvent spontaneous evaporation between 15 ~ 30 hours in mixed solution, forms that direction is controlled, the three-dimensional of arranging rule surpasses crystal on namely at the bottom of silicon wafer-based;
Tested microorganism being adsorbed on described three-dimensional surpasses on crystal, utilize three-dimensional super crystal phasmon field to the amplification of the intrinsic Raman signal of microorganism, detect by strengthening Raman technology the intrinsic Raman signal obtaining tested microorganism, thus identify the bacterial classification class of tested microorganism.
The present invention surpasses crystal technique by nanometer and strengthens Raman signal to identify method of microorganism, and its feature is also:
Described optically anisotropic noble metal nanometer material is gold nanorods, Golden Triangle nanometer sheet, gold and silver core-shell nanometer rod or gold and silver nucleocapsid triangular nano sheet.
It is perpendicular to the three-dimensional super crystal of nanometer rods of arrangement at the bottom of silicon wafer-based, perpendicular to the three-dimensional super crystal of the nanometer sheet of arranging at the bottom of silicon wafer-based, the three-dimensional super crystal of nanometer rods being parallel to arrangement at the bottom of silicon wafer-based that described three-dimensional surpasses crystal, or is parallel to the super crystal of nanometer sheet three-dimensional of arrangement at the bottom of silicon wafer-based.
Described orientation adjustment agent solution is the aqueous solution of NaCl or the aqueous solution of sulfydryl-polyglycol;
When described orientation adjustment agent solution is the aqueous solution of NaCl, described three-dimensional surpasses crystal and arranges perpendicular at the bottom of silicon wafer-based;
When the aqueous solution that described orientation adjustment agent solution is sulfydryl-polyglycol, described three-dimensional surpasses crystal and is parallel to arrangement at the bottom of silicon wafer-based.
Described tested microorganism is gram-positive bacteria, Gram-negative bacteria, fungi or mould.
When orientation adjustment agent is sulfydryl-polyglycol, by nanocrystalline surface chemistry orientation anchorage method, the direction that three-dimensional surpasses crystal is controlled, in this technology of preparing, used the reactivity as its end face of optically anisotropic noble metal nanometer material (for nanometer rods) or three arms of angle (for triangular nano sheet) to be better than the reactivity of side (for nanometer rods) or triangular facet (for triangular nano sheet), control the consumption of sulfydryl-polyglycol, use is pretended by golden mercapto key, replace noble metal nanometer material surfactant, shutoff is carried out to end face or three arms of angle, the surfactant only leaving side or triangular facet forms three-dimensional super crystal for the self assembly of nanometer rods or nanometer triangular plate.
When orientation adjustment agent is NaCl, by adjustment electrolyte ion activity physical adsorption process, the direction that three-dimensional surpasses crystal is controlled, in this technology of preparing, use electrolytical intervention, change the ion activity in solution, have adjusted the balance between the electrostatic repulsion forces of the surfactant of the coated noble metal nanometer material of surfactant and in-fighting attractive force, the self assembly that result in noble metal nanometer material forms three-dimensional phasmon and surpasses crystal.
Compared with the prior art, beneficial effect of the present invention is embodied in:
1, present invention utilizes nanometer and surpass crystal as substrate, directly adsorb microorganism, efficient amplify the faint intrinsic Raman signal of microorganism, can realize to microorganism exempt from mark quick identification, avoid the macrocyclic process spent by microbial reproduction cultivation;
2, the inventive method has versatility, stays out of any optical probe, and avoid chemistry of micro-organisms or the loaded down with trivial details process of biological functional, recognition methods is simple, has universality widely;
3, the present invention is by the selection of orientation adjustment agent, and the three-dimensional that can obtain direction controlled surpasses crystal.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of the three-dimensional super crystal of nanometer rods of the vertical silicon chip substrate arrangement of optical anisotropy gold and silver core-shell nanometer rod;
Fig. 2 is the scanning electron microscope (SEM) photograph that Escherichia coli are adsorbed on the three-dimensional super crystal of gold and silver core-shell nanometer rod of vertical silicon chip substrate arrangement;
Fig. 3 uses the three-dimensional super crystal a and non-super crystal b of the gold and silver core-shell nanometer rod of vertical silicon chip substrate arrangement to the identification of colibacillary enhancing Raman;
Fig. 4 is the scanning electron microscope (SEM) photograph of the three-dimensional super crystal of nanometer sheet of the parallel silicon chip substrate arrangement of optical anisotropy Golden Triangle nanometer sheet;
Fig. 5 is that the three-dimensional super crystal of nanometer sheet of the parallel silicon chip substrate arrangement of using Golden Triangle nanometer sheet is to the identification of saccharomycetic enhancing Raman.
Embodiment
Embodiment 1: strengthen colibacillary intrinsic Raman signal to identify Escherichia coli by the three-dimensional super crystal of gold and silver core-shell nanometer rod
The present embodiment, for strengthening colibacillary intrinsic Raman signal to identify Escherichia coli by the three-dimensional super crystal of gold and silver core-shell nanometer rod, carries out according to the following procedure:
1st step: by the gold chloride (HAuCl of 20mL, 0.5mM
4) cetyl trimethyl ammonium bromide (CTAB) of solution and 20mL, 0.2M is mixed to get mixed liquor under condition of ice bath; By the sodium borohydride (NaBH of 1.2mL, 0.02M
4) join in mixed liquor, under 1000rpm speed conditions, vigorous stirring 2 minutes, and 1 hour is left standstill at 25 DEG C, obtain the solution containing nm of gold seed;
By the silver nitrate (AgNO of CTAB and the 10mL of 200mL, 0.2M, 4mM
3) solution is mixed to form yellow growth liquid at ambient temperature; By the HAuCl of 200mL, 1mM
4solution adds in growth liquid, and along with the adding of ascorbic acid of 2.8mL, 0.08M, yellow growth liquid becomes colourless mixed liquor, shows that the gold of three valence states is reduced into the gold of a valence state.Join in above-mentioned colourless mixed liquor by the solution containing nm of gold seed of 480 μ L, after 5-10 minute, the color of solution becomes cinnabar redness from colourless, shows that gold nanorods is formed, and obtains gold nanorods solution.
The gold nanorods solution of fresh preparation, leaves standstill 24 hours, under 10000rpm speed conditions centrifugal 20 minutes, removes supernatant, is again dissolved in 40mL ultrapure water for subsequent use.Under 15-25 DEG C of condition, by the gold nanorods solution dilution of 1mL in the CTAB solution of 5mL, 0.1M, then add 0.75mL, 4mM AgNO successively
3solution and 0.1mL, 0.1M ascorbic acid, add the NaOH solution of 0.2mL, 0.1M wherein after slight oscillatory, contributes to silver ion at the surface of nanometer gold bar formation silver layer to form alkaline environment; The color of last solution becomes green from cinnabar redness, shows the formation of gold and silver core-shell nanometer rod.
2nd step: gold and silver core-shell nanometer rod solution prepared by above-mentioned steps, under 10000rpm rotating speed, centrifugal 10 minutes, remove supernatant, again be dissolved in 50 μ L 0.01M NaCl solution, get 10 μ L drop in the process of lower 1 hour of the concentrated sulphuric acid/hydrogen peroxide (3:1) 80 DEG C of conditions after clean silicon chip on.Then, under being placed in the environment of room temperature 25 DEG C and 80% humidity lucifuge, treat that it slowly dries for 24 hours naturally.After the process of slow evaporation, understand at the bottom of silicon wafer-based and form a coffee ring, the gold and silver core-shell nanometer rod be distributed on coffee ring is formed with regularly arranged three-dimensional and surpasses crystal.
Fig. 1 gives the scanning electron microscope (SEM) photograph of the super crystal of three-dimensional manometer rod of the vertical silicon chip substrate arrangement of the optical anisotropy gold and silver core-shell nanometer rod within the scope of large scale.From Fig. 1 clearly can observe that gold and silver core-shell nanometer rod is vertically at the bottom of silicon wafer-based, the three-dimensional defining tight regular array surpasses crystal.
3rd step: Escherichia coli are cultivated 12 hours at 37 DEG C in bacteriolyze meat soup (LB) nutrient culture media, be dispersed in ultrapure water, under 5000rpm speed conditions after centrifugal treating, remove supernatant, again be dispersed in ultrapure water, form 1mL, 0.005OD Escherichia coli liquid, then the Escherichia coli liquid of 10 μ L is got, drop on the three-dimensional super crystal of the gold and silver core-shell nanometer rod prepared, after within 1 hour, naturally drying, carry out Raman test, its testing conditions is detection power 0.85mW, excitation wavelength 785nm, acquisition time 10S.
Fig. 2 gives the scanning electron microscope (SEM) photograph of the three-dimensional super crystal of gold and silver core-shell nanometer rod that Escherichia coli attachment is combined in vertical silicon chip substrate arrangement.Clearly can observe that from figure the Escherichia coli that length scale is about 1 μm are adsorbed on super plane of crystal.
As a comparison, prepare the three-dimensional gold and silver core-shell nanometer rod of non-super crystal as follows, and by Escherichia coli absorption thereon:
Gold and silver core-shell nanometer rod solution above-mentioned steps prepared, under 10000rpm rotating speed, centrifugal 10 minutes, removes supernatant, is again dissolved in 50 μ L ultrapure water solution, gets 10 μ L and drop on silicon chip.Then put into air blast hair dryer, under temperature is arranged on the environment of 40 DEG C, treat its 20 minutes rapid airings.After the process of rapid evaporation, can not at the bottom of silicon wafer-based on formed a coffee ring, thus define the non-super crystalline substrate of disorder distribution.Then by the Escherichia coli liquid of above-mentioned preparation, get the Escherichia coli liquid of 10 μ L, drop in the non-super crystalline substrate prepared, after within 1 hour, naturally drying, carry out Raman test, its testing conditions is detection power 0.85mW, excitation wavelength 785nm, acquisition time 10S.
Fig. 3 gives the three-dimensional super crystal of gold and silver core-shell nanometer rod that non-super crystal (curve b) and vertical silicon chip substrate arrange, and (curve a) is composed colibacillary Raman identification.From figure, a spectral line can observe colibacillary intrinsic Raman shuttle belt, and its position is at 752cm
-1, 1001cm
-1, 1449cm
-1faint intrinsic Raman signal, by the Raman excitation of three-dimensional super crystal phasmon field, relative to the Raman spectrogram that the gold and silver core-shell nanometer rod substrate of disorder distribution obtains, strengthened significantly.
The Raman different in view of the optically anisotropic noble metal nano particles of different-shape strengthens characteristic, and the noble metal nanometer material therefore in this microorganism recognition technology can be selected from the optical anisotropy nano particle of gold nanorods, Golden Triangle nanometer sheet, gold and silver core-shell nanometer rod, gold and silver nucleocapsid triangular nano sheet.The Raman that the difference surpassing the nano-antenna effect of crystal due to the three-dimensional strengthening Raman identification for microorganism causes strengthens the difference of ability, therefore the super crystal of this microorganism recognition technology can be selected from vertical silicon sheet substrate arrangement nanometer rods surpasses crystal, nanometer sheet surpasses crystal or the nanometer rods of parallel silicon chip substrate arrangement, nanometer sheet surpass crystal.Because the Species differences of microorganism determines the difference of its intrinsic optic signal, so the microorganism of this microorganism recognition technology can be selected from gram-positive bacteria, Gram-negative bacteria, fungi, mould.
Embodiment 2: strengthen Raman signal to identify saccharomycete by the three-dimensional super crystal of Golden Triangle nanometer sheet
Raman signal is strengthened to identify saccharomycetic concrete steps by the three-dimensional super crystal of Golden Triangle nanometer sheet in the present embodiment:
1st step: 1mL, 0.01M chlorauric acid solution and 1mL, 0.01M citric acid three sodium solution are diluted with water to 40mL, mixed liquor is mixed to get under condition of ice bath, add 1mL, 0.1M sodium borohydride again, under 1000rpm speed conditions, vigorous stirring 2 minutes, at 25 DEG C, leave standstill 2-4 hour, obtain the solution containing nm of gold seed.
Then, 225mL, 0.05M CTAB, 1mL, 0.1M KI, 1.25mL, 0.1M ascorbic acid, 1.25mL, 0.1M NaOH, 6.25mL, 0.01M chlorauric acid solution is mixed to form mixing growth liquid mutually.Subsequently, from the conical flask C filling 225mL mixing growth liquid, get 22.5mL in tap web bottle B, in tap web bottle B, get 2.25mL in conical flask A.Finally, in tap web bottle A, add the solution containing nm of gold seed of the above-mentioned preparation of 225 μ L, pour solution in A bottle into B bottle rapidly, then B bottle solution is transferred to C bottle immediately.After 5-10 minute, the color of solution becomes aubergine from colourless, shows that Golden Triangle nanometer sheet is formed.The Golden Triangle nanometer sheet growth liquid of fresh preparation, leaves standstill 24 hours, collects the green solution bottom tap web bottle, then under 6000rpm speed conditions centrifugal treating 20 minutes, be again dissolved in 20mL ultrapure water for subsequent use.
2nd step: get 1mL Golden Triangle nanometer sheet solution, add 50uL, 1mg/mL sulfydryl-polyglycol solution (molecular weight 6000MW), under 25 DEG C of conditions, react 2 hours, 6000rpm rotating speed 10 minutes, centrifugal segregation supernatant, is dissolved in 200 μ L ultrapure waters again, gets 10uL and drops on the clean silicon chip of the concentrated sulphuric acid/hydrogen peroxide (3:1) 80 DEG C of conditions process in lower 1 hour.Then, under being placed in the environment of room temperature 25 DEG C and 80% humidity lucifuge, treat that it slowly dries for 24 hours naturally.After the process of slow evaporation, can be formed at the bottom of silicon wafer-based and form a coffee ring, the Golden Triangle nanometer sheet be distributed on coffee ring is formed with regularly arranged super crystal.
Fig. 4 gives the scanning electron microscope (SEM) photograph of the three-dimensional super crystal of nanometer sheet of the parallel silicon chip substrate arrangement of the optical anisotropy gold-nano-piece within the scope of large scale.From Fig. 4 can clearly observe Golden Triangle nanometer sheet parallel at the bottom of silicon wafer-based, the three-dimensional defining tight regular array surpasses crystal.
3rd step: saccharomycete is cultivated 20 hours at 30 DEG C in yeast extract tryptose D-glucose (YPD) nutrient culture media, be dispersed in ultrapure water, under 4000rpm speed conditions after centrifugal treating, remove supernatant, again be dispersed in ultrapure water, form 1mL, 0.005OD yeast liquid, then the yeast liquid of 10 μ L is got, drop on the three-dimensional super crystal of the Golden Triangle nanometer sheet prepared, after within 1 hour, naturally drying, carry out Raman test, its testing conditions is detection power 0.85mW, excitation wavelength 785nm, acquisition time 10S.
The three-dimensional super crystal of Golden Triangle nanometer sheet that Fig. 5 gives parallel silicon chip substrate arrangement is composed saccharomycetic Raman identification.Can observe saccharomycetic intrinsic Raman shuttle belt from figure, its position is at 1240cm
-1, 1375cm
-1faint intrinsic Raman signal, by the Raman excitation of super crystal phasmon field, be enhanced significantly, and intrinsic Raman signal is significantly different from colibacillary intrinsic Raman signal in Fig. 3.
Compared with general colony counting method micrometer biology, the invention has the advantages that, employ the optically anisotropic noble metal nanometer material with obvious Raman enhancement effect, constructed that direction is controlled, the three-dimensional of arrangement height rule surpasses crystal, high reappearance be provided, the faint Raman signal of intrinsic that regular optics focus efficiently amplifies microorganism.Relative to general colony counting method, this recognition technology at present, make use of the intrinsic intrinsic signal of different microorganisms, accurately can identify the kind of microorganism.
Claims (5)
1. surpass crystal technique by nanometer and strengthen Raman signal to identify a method of microorganism, it is characterized in that:
In containing the solution of optically anisotropic noble metal nanometer material, add orientation adjustment agent solution, obtain mixed solution; Dropped at the bottom of silicon wafer-based by described mixed solution, in light protected environment, control environment temperature and humidity, makes the solvent spontaneous evaporation between 15 ~ 30 hours in mixed solution, forms that direction is controlled, the three-dimensional of arranging rule surpasses crystal on namely at the bottom of silicon wafer-based;
Tested microorganism being adsorbed on described three-dimensional surpasses on crystal, utilize three-dimensional super crystal phasmon field to the amplification of the intrinsic Raman signal of microorganism, detect by strengthening Raman technology the intrinsic Raman signal obtaining tested microorganism, thus identify the bacterial classification class of tested microorganism.
2. method according to claim 1, is characterized in that: described optically anisotropic noble metal nanometer material is gold nanorods, Golden Triangle nanometer sheet, gold and silver core-shell nanometer rod or gold and silver nucleocapsid triangular nano sheet.
3. method according to claim 1, it is characterized in that: it is perpendicular to the three-dimensional super crystal of nanometer rods of arrangement at the bottom of silicon wafer-based, perpendicular to the three-dimensional super crystal of the nanometer sheet of arranging at the bottom of silicon wafer-based, the three-dimensional super crystal of nanometer rods being parallel to arrangement at the bottom of silicon wafer-based that described three-dimensional surpasses crystal, or is parallel to the super crystal of nanometer sheet three-dimensional of arrangement at the bottom of silicon wafer-based.
4. method according to claim 1, is characterized in that: described orientation adjustment agent solution is the aqueous solution of NaCl or the aqueous solution of sulfydryl-polyglycol;
When described orientation adjustment agent solution is the aqueous solution of NaCl, described three-dimensional surpasses crystal and arranges perpendicular at the bottom of silicon wafer-based;
When the aqueous solution that described orientation adjustment agent solution is sulfydryl-polyglycol, described three-dimensional surpasses crystal and is parallel to arrangement at the bottom of silicon wafer-based.
5. method according to claim 1, is characterized in that: described tested microorganism is gram-positive bacteria, Gram-negative bacteria, fungi or mould.
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| WO2021046741A1 (en) * | 2019-09-11 | 2021-03-18 | 亳州市新健康科技有限公司 | Substrate material for high-efficiency surface-enhanced raman scattering and preparation method |
| CN114858777A (en) * | 2022-04-25 | 2022-08-05 | 哈尔滨医科大学 | Method for label-free detection of bacteria based on surface enhanced Raman spectroscopy and application thereof |
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| CN115452799B (en) * | 2022-04-25 | 2023-08-18 | 哈尔滨医科大学 | Method for monitoring medicine without label by utilizing surface enhanced Raman spectrum technology and application thereof |
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