CN102501495A - Substrate with PEI (polyetherimide) film on surface and preparation method and application thereof - Google Patents

Abstract

The invention discloses a substrate with a PEI (polyetherimide) film on surface of the substrate, wherein a hyperbranched polymer ethylenimine film is cross-linked via glutaraldehyde on the substrate surface; the substrate is a substrate with a surface containing hydroxyl, or a substrate with a surface which can form hydroxyl after treatment, such as a glass slide; and the film is not less than one layer, preferably 2-5 layers. The invention completes amino amplification via branch shape coupling on the substrate surface, and the obtained substrate has stable properties and a good broad-spectrum sterilizing effect. The preparation method comprises immersing the substrate in PBS (phosphate buffer solution) buffer solution, culturing at 37 DEG C for 30d, and using FT-IR (Fourier transform infrared spectroscopy) to analyze degradation rate (less than 10%). When the substrate contains one film layer on the surface, E. coli adsorption capacity is 5.10*10<4>CFU/cm<2>; and when the substrate contains five film layers, the E. coli adsorption rate increases to 1.10*10<7>CFU/cm<2>. A contact antibacterial ability test shows that the substrate can rapidly kill E. coli representing Gram-negative bacteria adsorbed on the substrate within 8min, and at the same time has good antibacterial effect on Gram-positive bacteria such as Bacillus subtilis. The invention has good practicability, and can produce good economic and social effects.

Description

There is substrate of PEI rete and its production and application on a kind of surface

Technical field

The present invention relates to a kind of substrate surface method of modifying and modify after product and application, being specifically related to a kind of surface has the ultra cladodification polymer of PEI(dimethyleneimine) substrate of rete and its production and application.

Background technology

According to existing report; The material that can effectively suppress bacterial infection has polycation, antibacterial peptide, antibiotic, enzyme and nano material (like silver, titanium dioxide, silica, magnesia, cupric oxide, zinc oxide, cadmium selenide/cadmium telluride nano particle or SWCN) etc.; Wherein, Can be used as the polymine that contains the polycation group and the derivative thereof of antiseptic paint; Because it has good stability, have no drug resistance, cytotoxicity is little and advantage such as broad spectrum antibacterial, is extensively modified on the dissimilar substrate surfaces.

Different according to the method that suppresses bacterial infection; Antiseptic paint mainly is divided into these two kinds of spacetabs type and contact-types; Traditional antimicrobial coating that utilizes the preparation of physical absorption method; Though can play a role,, lose antibacterial ability easily because its absorption affinity is not strong through the slowly-releasing approach; By contrast; The antimicrobial coating of covalent coupling is because its good stable property can still keep its anti-microbial property after repeatedly using.Therefore, work out antimicrobial coating steady in a long-term, occur in food industry for solution, the infected by microbes of hospital or public place is significant.

Summary of the invention

Goal of the invention: the deficiency to existing in the prior art, the purpose of this invention is to provide the substrate that there is the PEI rete on a kind of surface, so that it has stronger antibacterial ability, stable anti-microbial property.Another object of the present invention provides the preparation method of above-mentioned substrate.The present invention also has a purpose to provide a kind of purposes of above-mentioned substrate.

Technical scheme: in order to realize the foregoing invention purpose, the technical scheme that the present invention adopts is following:

There is the substrate of PEI rete on a kind of surface, through glutaraldehyde cross-linking ultra cladodification polymer dimethyleneimine rete is arranged at substrate surface; Said substrate is the substrate that contains hydroxyl on the surface, or is the surperficial substrate that can form hydroxyl after treatment, for example slide; This rete is no less than one deck, is preferably 2 ~5 layers.

Substrate can be slide, silicon rubber, silica, metal.When being slide, processing method commonly used is: slide is put into 98% concentrated sulfuric acid H < > 2 <> SO < > 4 <> With 30% oxydol H < > 2 <> O < > 2 <> (3:1, V/V) in the mixed solution, room temperature 15min, rinse well with deionized water the back.

A kind ofly prepare the method that there is the substrate of PEI rete on the surface, may further comprise the steps:

(1) substrate is immersed in 98% concentrated sulfuric acid and 30% hydrogen peroxide mixed solution that volume ratio is 3:1, room temperature reaction 15min takes out, and cleans with deionized water; Then substrate is immersed in volume ratio and is 1h in 1% the APTES aqueous solution, use the deionized water rinse substrate, use N again < > 2 <> Dry up, 110 ℃ are descended dry 30min, make the APTES-glass of APTES modification;

(2) APTES-glass being immersed volume ratio is in 5% the glutaraldehyde water solution, and room temperature reaction 2h cleans with deionized water; Immerse volume ratio again and be in 1% the PEI aqueous solution, room temperature reaction 2h cleans with deionized water; Be immersed in 1M NaBH again < > 4 <> 2h in the aqueous solution cleans with deionized water, makes the substrate G1.0-PEI-glass that there is individual layer PEI rete on the surface;

(3) G1.0-PEI-glass is repeated the operation of n step (2), make the substrate Gn-PEI-glass that there is multilayer PEI rete on the surface, wherein n is not less than 1.

There is the application of substrate in broad-spectrum sterilization of PEI rete on the surface.

Beneficial effect: compare with existing antiseptic paint; The outstanding advantage that surface of the present invention has the substrate of PEI rete to have comprises: through accomplishing amino the amplification at substrate surface through dendritic coupling; The substrate stable performance that makes has good broad-spectrum sterilization effect.Substrate is immersed in the PBS cushioning liquid, cultivates 30d for 37 ℃, analyze its degradation rate less than 10% with T-IR.When substrate surface contains one deck rete, < > E. coli <> Adsorbance is 5.10 * 10 < > 4 <> Individual/cm < > 2 <> When being 5 layers, < > E. coli <> Adsorbance is increased to 1.10 * 10 < > 7 <> Individual/cm < > 2 <> Detect through the contact antibacterial ability, the result shows within 8min, to kill fast and is adsorbed on on-chip representative Gram-negative bacteria < > E. coli <> , simultaneously to good antibacterial effect also being arranged as gram-positive bacterias such as bacillus subtilises.Have good practicability, can produce favorable economic benefit and social benefit.

Description of drawings

Fig. 1 is 2700 ~2885cm that modify different PEI number of plies substrates < >-1 <> With 2885 ~3100cm < >-1 <> The infrared absorbency figure at place; This figure is used for explanation-CH < > 2 <>-intensity and the PEI number of plies of infrared absorption peak between concern, among the figure-■-be asymmetric stretching vibration ,-zero-be symmetrical stretching vibration;

Fig. 2 is that LCSM characterizes Escherichia coli and on Gn-PEI-glass, adheres to figure;

Fig. 3 observes the colibacillary adsorbance of substrate surface figure as a result under the laser confocal microscope.

The specific embodiment

Below in conjunction with specific embodiment the present invention is done further explanation.

Employed medicine of following examples and instrument are:

Common slide, 3-aminopropyl triethoxysilane (APTES)(Sigma, SA); Glutaraldehyde (Aldrich, USA), fluorescein isothiocynate (FITC)(Sigma) Beijing Bo Aosen bio tech ltd, China), LIVE/DEAD BacLight bacterial viability Kit (L13152 probes); 1-pyrene methylamine hydrochloride (1-PAM)(Sigma, USA), sodium borohydride (NaBH < > 4 <> )(Aldrich, USA), gather dimethyleneimine (PEI)(M.W.60000, USA), the LB culture medium (1% tryptone yeast extract NaCl, pH=7.2), beef-protein medium (0.5% beef extract; 1% tryptone NaCl, pH=7.2), (deionized water is solvent (resistance Da Yu > to PBS cushioning liquid; 18M Ω /cm), 8.0g/L NaCl, 0.2g/L KCl, 1.2g/L Na < > 2 <> HPO < > 4 <> , 0.2g/L KH < > 2 <> PO < > 4 <> , pH=7.2)), the preparation method of L – agar solid medium is with agar (1% (w/w)) join in the LB culture medium, cooling forms solid medium behind the high-temperature sterilization.

Instrument: the mensuration utilization resolution ratio of transmitted infrared light spectrum is 0.25 cm < >-1 <> Bruker Vertex 70 spectrometers.

Embodiment 1

(1) slide is put into 98% concentrated sulfuric acid H < > 2 <> SO < > 4 <> With 30% oxydol H < > 2 <> O < > 2 <> (3:1, V/V) in the mixed solution, room temperature 15min, rinse well with deionized water the back.Then clean slide be immersed in the APTES aqueous solution (1%, v/v, pH=5.5) in 1h, with deionized water rinse substrate 3 times,, use high-purity N again to remove physical absorption < > 2 <> Dry up, put into 110 ℃ of baking oven 30min, make the sample APTES-glass of APTES modification.

(2) APTES-glass is immersed glutaraldehyde solution (5%, v/v, pH=9.5) in, room temperature reaction 2h; Wash repeatedly with deionized water, immerse again the PEI aqueous solution (1%, v/v, pH=9.5) in, room temperature reaction 2h; Wash repeatedly with deionized water, glass substrate is immersed in NaBH < > 4 <> 2h in the aqueous solution, reaction back glass substrate with ethanol or deionized water rinsing repeatedly, thereby guarantee the physical absorption full scale clearance on the slide, make G1.0-PEI-glass, G1.0-PEI-glass has the substrate of 1 layer of PEI rete for the surface.

During preparation Gn-PEI-glass, only need G1.0-PEI-glass repeating step (2), repeating 1 time can increase by 1 layer of PEI film; Repeatedly repeat, can make multilayer PEI film substrate, i.e. Gn-PEI-glass, wherein n represents the rete number.

According to the method described above, preparing the rete number is 1,2,3,4 and 5 Gn-PEI-glass sample.Use Fourier transform infrared spectroscopy (FT-IR) characterize and detect Gn-PEI-glass, at 3393 cm < >-1 <> , 3300 cm < >-1 <> (N – H key range) and 2937 cm < >-1 <> , 2850 cm < >-1 <> (– CH < > 2 <> The – zone) uphill process is gradually arranged, so Gn-PEI-glass forms for the multilayer grafting.

Gn-PEI-glass is carried out the sign of AFM; The process that descends gradually from 63.2 ° to 56.1 ° appears in the contact angle of substrate water, through the sign of AFM to each circulation substrate, can find out; The clean smooth substrate of contrast; The protuberance nanometer polymer of dispersion of the same type is arranged on G1.0-PEI-glass and G5.0-PEI-glass surface, get identical size area, substrate RMS roughness is respectively 1.6nm, and 12.2nm can find out whole roughness increasing and raise with circulation.At the G1.0-PEI-glass substrate surface, nanometer polymer protuberance diameter 130 ± 40nm, height 32nm.But G5.0-PEI-glass, enlarged-diameter to 190 ± 60nm highly is increased to 64nm.Can find out that from magnitude range and intensity the formation of these protuberance nanometer polymers is the reasons owing to ultra dendritic PEI.Level of coverage for quantitative substrate surface PEI molecule has detected 2700 ~2885cm < >-1 <> With 2885 ~3100cm < >-1 <> The infrared absorbency (– CH at place < > 2 <> The – zone), as shown in Figure 1.Can see that in Fig. 1 along with the increase of PEI circulation, absorbance is straight line rising thereupon also, this is because PEI plays crucial effect in each circulation of crosslinked substrate surface.In addition,, it was put into PBS cushioning liquid continuous dipping 30 days, Zhui Zong – CH in order to detect the stability of G5.0-PEI-glass substrate polymeric membrane < > 2 <> The variation of – group infrared absorbency.Can find out, at the 1st day about 5% PEI molecule owing to coming off of physical absorption run off, after 30 days in, PEI molecule total amount presents coming off of gradual slow, but the quantity not sufficient 5% that comes off.The stable higher of Gn-PEI-glass substrate is described, hydrophobic ultra branch structure has better protect property to the hydrolysis of siloxanes.

Embodiment 2

At room temperature, be 5 * 10 with 100 μ l concentration < > 8 <> The bacterial suspension of cfu/mL is added drop-wise on the Gn-PEI-glass, leaves standstill 1h, and the Gn-PEI-glass substrate with flushing of PBS cushioning liquid and immersion, is used laser confocal microscope (LSCM) the following bacterium of observing its surface adsorption.

Escherichia coli (E.coli) activity dyes to bacterium with the LIVE/DEAD kit, and this kit comprises nuclei dyeing toner YTO9 and propidium iodide (PI).Under room temperature lucifuge environment,, 1:1) join 15min in the bacterial suspension of 100 μ l, at laser confocal microscope (LSCM with the YTO9 of 100 μ l and the mixed solution (v/v of PI) observe down.

Antibiotic property detects and adopts U.S. ASTM standard :E2149-01 Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents under Dynamic Contact Conditions.With Gn-PEI-glass substrate (1.5 * 2.5cm < > 2 <> ) be immersed in that (cell quantity is about 7 * 10 in the cell suspending liquid of 5mL < > 5 <> ), alternative blank glass substrate is put into the cell suspending liquid 1h of another 5mL of same concentration equally as contrast.Suitably change in the L-agar solid medium after the dilution and cultivate, each visible bacterial plaque all is that single bacterial clone duplicates formation, can infer antibacterial effect through the counting bacterial plaque.The bactericidal effect of substrate surface modification can obtain through the detection to L-agar solid medium bacterial plaque number among Ncontrol and the Nsample.

The adhere to utilization LCSM of Escherichia coli on Gn-PEI-glass characterizes, as shown in Figure 2, and contrast APTES-glass (5.10 * 10 < > 4 <> Units/cm < > 2 <> ), the Gn-PEI substrate adsorbs colibacillary quantity to be increased (from 1.53 * 10 gradually < > 6 <> To 1.10 * 10 < > 7 <> Units/cm < > 2 <> ), the G3.0-PEI polymeric membrane is near saturated.Bacillus coli cells adhering on Gn-PEI-glass is because electrostatic adsorption forms, and bacteria cell wall has negative electrical charge outward, and the Gn-PEI-glass substrate surface has a large amount of positive charges.Along with the increase of Gn-PEI-glass circulation, the positively charged increase of substrate surface, thus adsorb more Escherichia coli.In addition, through regulating the pH value, can effectively regulate colibacillary adsorbance.Get identical G5.0-PEI substrate, when pH5.5, colibacillary adsorbance rises to 1.13 * 10 < > 8 <> Units/cm < > 2 <> , and when pH9.5, adsorbance drops to 1.51 * 10 < > 4 <> Units/cm < > 2 <> The adsorbance of pH5.5 substrate is about 10 times of common physiological status, and this is because amino provides proton to produce positive charge (NH < > 3 <> < > + <> ).The result has proved that better a large amount of absorption are because the mutual Electrostatic Absorption of negative electrical charge that the positive charge on Gn-PEI-glass surface and bacteria cell wall have forms.

Escherichia coli (E. coli) activity dyes to bacterium with the LIVE/DEAD kit; This kit comprises can be under fluorescence microscope to be dyed nucleus green coloring agent SYTO9 (maximum excitation/emission wavelength) and dead cell is dyed red propidium iodide PI(maximum excitation/emission wavelength, 490/635nm).YTO9 can dye green with the bacterium with intact cell film, and PI can dye redness with the dead cell of imperfect cell membrane.

After the Escherichia coli absorption, use normal/dead two kinds of fluorescent staining mark bacterial cell permeability of the membranes, can see that the cell that is adsorbed is all dead.So can use with quadrat method and follow the trail of the dynamic process that bacterium contacts with the Gn-PEI-glass surface, blank glass sample, APTES-glass and Gn-PEI-glass surface dripped Escherichia coli suspension (5 * 10 of same amount < > 8 <> Cell/mL); The back adds YTO9 and the mixed dyeing liquor of PI; At room temperature put altogether and cultivate 15min; Do not wash afterwards and directly carry out observing under the laser confocal microscope the colibacillary adsorbance of substrate surface; As shown in Figure 3; Simultaneously, also use the representative of having handled gram-positive bacteria with quadrat method--bacillus subtilis.Escherichia coli on the blank glass sample all are being green under fluorescence microscope behind the 30min, it is strong active to illustrate that test has with Escherichia coli, has complete cell membrane.Bacterium is because cell membrane begins to break and becomes redness on the Gn-PEI-glass substrate behind the 10min, and contrast APTES-glass has only seldom a part of bacterium to become redness, illustrates that the PEI polymeric membrane has very strong contact sterilization ability to bacterium.Simultaneously, for antibacterial ability analysis, use the standard method of ASTM to detect again to Gn-PEI.Among Fig. 8, dead colibacillary amount reaches 1.65 * 10 in G1.0-PEI-glass < > 5 <> Units/cm < > 2 <> , and dead colibacillary amount is 8.93 * 10 among the APTES-glass < > 4 <> Units/cm < > 2 <> Therefore, PEI has conclusive effect in antibacterial tests.Yet the antibacterial effect of Gn-PEI polymeric membrane not only is decided by the cycle-index of PEI, and (slight rising is by 1.65 * 10 < > 5 <> To 1.74 * 10 < > 5 <> Units/cm < > 2 <> ).

Amino on the Gn-PEI polymeric membrane is induced at the cation of bacterium surface and positive charge, upsets the anion on the bacteria cell wall, thereby causes the breakage of cell membrane, causes K < > + <> With leaking of other cellular contents, final result is the death of bacterium.In the SEM Electronic Speculum picture, dead its cellular morphology shrinkage of cell, membranolysis, content leaks.And the normal cell figure is full, and cell is complete.Along with the increase of PEI circulation, the amount of Gn-PEI polymeric membrane absorption bacterium increases, and sterilization speed is accelerated, and antibacterial effect is better.

Embodiment 3

The preparation method of Gn-PEI-glass is with embodiment 1, and substrate is a silicon rubber, and the processing method of silicon rubber is: silicone rubber plate is put into 98% concentrated sulfuric acid H < > 2 <> SO < > 4 <> With 30% oxydol H < > 2 <> O < > 2 <> (3:1, V/V) in the mixed solution, room temperature 15min, rinse well with deionized water the back.

The antibacterial experiment of Gn-PEI-glass is with embodiment 2, and the result shows that can obtain with the slide is suitable effect of Gn-PEI-glass and the similarity rules that substrate makes.

Embodiment 4

The preparation method of Gn-PEI-glass is with embodiment 1, and substrate is a silica, and the processing method of silica is: oxidized silicon chip is put into 98% concentrated sulfuric acid H < > 2 <> SO < > 4 <> With 30% oxydol H < > 2 <> O < > 2 <> (3:1, V/V) in the mixed solution, room temperature 15min, rinse well with deionized water the back.

The antibacterial experiment of Gn-PEI-glass is with embodiment 2, and the result shows that can obtain with the slide is suitable effect of Gn-PEI-glass and the similarity rules that substrate makes.

Embodiment 5

The preparation method of Gn-PEI-glass is with embodiment 1, and substrate is a metal, and the processing method of metal is air plasma processing method commonly used.Sheet metal commonly used comprises iron plate, aluminium flake, aluminium alloy, ferroalloy etc.

The antibacterial experiment of Gn-PEI-glass is with embodiment 2, and the result shows that can obtain with the slide is suitable effect of Gn-PEI-glass and the similarity rules that substrate makes.

Claims (7)

1. there is the substrate of PEI rete on a surface, it is characterized in that: through glutaraldehyde cross-linking ultra cladodification polymer dimethyleneimine rete is arranged at substrate surface; Said substrate is the substrate that contains hydroxyl on the surface, or can form the substrate of hydroxyl after treatment for the surface.

2. there is the substrate of PEI rete on surface according to claim 1, it is characterized in that: this rete is 2 ~5 layers.

3. there is the substrate of PEI rete on surface according to claim 1, it is characterized in that: said substrate is slide, silicon rubber, silica, metal.

4. one kind prepares the method that there is the substrate of PEI rete on the described surface of claim 1, it is characterized in that, may further comprise the steps:

(1) substrate is immersed in 98% concentrated sulfuric acid and 30% hydrogen peroxide mixed solution that volume ratio is 3:1, room temperature reaction 15min takes out, and cleans with deionized water; Then substrate is immersed in volume ratio and is 1h in 1% the APTES aqueous solution, use the deionized water rinse substrate, use N again < > 2 <> Dry up, 110 ℃ are descended dry 30min, make the APTES-glass of APTES modification;

(2) APTES-glass being immersed volume ratio is in 5% the glutaraldehyde water solution, and room temperature reaction 2h cleans with deionized water; Immerse volume ratio again and be in 1% the PEI aqueous solution, room temperature reaction 2h cleans with deionized water; Be immersed in 1M NaBH again < > 4 <> 2h in the aqueous solution cleans with deionized water, makes the substrate G1.0-PEI-glass that there is individual layer PEI rete on the surface;

(3) G1.0-PEI-glass is repeated the operation of n step (2), make the substrate Gn-PEI-glass that there is multilayer PEI rete on the surface, wherein n is not less than 1.

5. there is the application of substrate in broad-spectrum sterilization of PEI rete on the described surface of claim 1.

6. there is the application of substrate in killing Gram-negative bacteria of PEI rete on the described surface of claim 1.

7. there is the application of substrate in killing gram-positive bacteria of PEI rete on the described surface of claim 1.

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