CN102298965A - Micro-array containing retinaldehyde membrane protein, preparation method thereof, and application thereof - Google Patents
Micro-array containing retinaldehyde membrane protein, preparation method thereof, and application thereof Download PDFInfo
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- CN102298965A CN102298965A CN2011102001522A CN201110200152A CN102298965A CN 102298965 A CN102298965 A CN 102298965A CN 2011102001522 A CN2011102001522 A CN 2011102001522A CN 201110200152 A CN201110200152 A CN 201110200152A CN 102298965 A CN102298965 A CN 102298965A
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Abstract
The invention belongs to the technical fields of biomaterial and information material, and specifically relates to a micro-array containing retinaldehyde membrane protein, a preparation method thereof, and an application thereof. According to the invention, an electrodeposition method is adopted. Retinaldehyde membrane protein is orientedly deposited on a surface of a gold micro-array substrate. With hydrosulfide groups carried by retinaldehyde membrane protein mutants and a cross-linking technology, active protein molecules are connected with the micro-array through chemical bonds, and the active protein molecules are connected with each other through chemical bonds, such that a network formed by orderly and chemically cross-linked layers of active protein is obtained. The network is washed by using a surfactant, processed through ultrasonic treatment, and dried, such that a fixedly distributed micro-array containing active retinaldehyde membrane protein is obtained, wherein the micro-array is stable, easy to preserve, and has a specific orientation. The protein micro-array can be used in the storing and processing of photoelectrical information, and has good data-storing stability and safety. The protein micro-array contains cell-adhesion-resisting and non-cell-adhesion-resisting zones. With the micro-array provided by the invention, a novel material is provided for basic cell biology researches and tissue engineering applications.
Description
Technical field
The invention belongs to biomaterial, information material technical field, be specifically related to a kind of microarray that contains the retinene memebrane protein and its production and application.
Background technology
In optical information storage and process field, retinene protein such as bacteria rhodopsin (bacteriorhodopsin with photolytic activity response, be called for short BR) and mutant be functional protein with good application prospect, the composite membrane that they and polymer mixed make can be applied in the optical information storage and (comprise two dimensional storage, three-dimensional storage and holographic memory etc.) and optical information processing aspects such as (comprising the light conversion, light filtration and Signal Regulation etc.).（Hampp,?N.?Chem.?Rev.?2000,?100,?1755-1776）。
The microarray system that contains active retinene protein has in the past designed microarray and retinene is proteins deposited in microarray surface.For physics contacts, reactive protein is deposited as a monoblock membrane structure in whole microarray surface, is not real microarray between active retinene memebrane protein and the microarray.Though perhaps be microarray, but retinene protein is by there not being chemical bond to connect between the retinene memebrane protein molecule, can not tolerant T riton etc. the processing of scaling agent, be easy to be subjected to ectocine and lose original form even destroyed, be unfavorable for long-term preservation.
Summary of the invention
The object of the present invention is to provide a kind of protein microarray that keeps the retinene protein active and its production and application.
The microarray that contains the retinene memebrane protein provided by the invention is to be deposited on the multilayer film arrays of immobilized protein that orientation is arranged that forms on the substrate gold microarray by the retinene membrane protein molecule.Wherein, between retinene memebrane protein molecule and the substrate gold microarray, all be connected into an integral body by chemical bond-linking between the retinene memebrane protein molecule, the retinene memebrane protein on each microarray forms the chemical crosslinking network of orientation.
Among the present invention, described retinene protein is by the bacteria rhodopsin of genetic engineering acquisition or the mutant of archaerhodopsin 4.Introducing in the mutein has halfcystine, is between two the spirals connections in seven transbilayer helix born of the same parents outsides (aminoterminal) or born of the same parents inboard (c-terminus), thereby obtains to have active mercapto groups.Be connected by covalent bond between mercapto groups in the retinene protein mutant and the golden microarray.
Among the present invention, crosslinked between retinene membrane protein molecule layer and the layer by the effect of coupling agent.
Among the present invention, described substrate gold microarray can pass through design and adjust the microarray size, and scope is 1 micron to 900 microns, can obtain the retinene memebrane protein array of the pixel composition of different sizes thus.
The present invention can pass through biology, physics, three kinds of method associatings of chemistry, prepares to have optically active retinene protein microarray.Concrete steps are:
1, utilize the photoetching method making to have the substrate gold microarray of pattern.Microarray can be adjusted the microarray size by design, from 1 micron to 900 microns, can obtain the golden microarray that different big or small pixels are formed thus.
2, by electro-deposition method, on substrate gold microarray, deposit the retinene protein molecule, be formed with the multilayer film retinene arrays of immobilized protein of orientation.Soon the retinene protein molecule is fixed on the golden microarray and forms protein microarray.The used retinene protein of protein microarray is the bacteria rhodopsin of genetic engineering acquisition or the mutant of archaerhodopsin 4.Introduce halfcystine in the mutein, thereby obtain to have active mercapto groups.Be connected by covalent bond between mercapto groups in the retinene protein mutant and the golden microarray.
3, by coupling agent with crosslinked between retinene rete and the rete, specifically be to make amino and activated carboxylic formation amido link connect into an integral body by adding coupling agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl) and N-hydroxy-succinamide (NHS), the retinene memebrane protein on each microarray forms the chemical crosslinking network of orientation.
4, the protein microarray that obtains is soaked in surfactant Triton-X 100 solution, removes not chemical crosslinking protein molecule.Comprise removal not with the crosslinked retinene protein of golden patterned surfaces, molecule uncrosslinked between the retinene protein also is removed.The retinene memebrane protein layer that connects through chemical bond can keep under the situation that scaling agents such as Triton are handled and being connected of substrate.
The preparation method of the retinene memebrane protein microarray that the present invention relates to, the detail operations step is as follows:
In the step 1, utilize photoetching technique to make substrate gold microarray.Utilize template and photoresist with on pattern is from the template transfer to matrix.Initial matrix is glass sheet, with the glass sheet surface wiped clean, utilizes piranha (with the volume ratio 3:1 mixing concentrated sulphuric acid and hydrogen peroxide) solution-treated subsequently.Glass sheet is soaked wherein 30 min, and N is used behind supersound washing 15 min three times in water flushing back in pure water
2Dry up, in acetone, soak ultrasonic 10 min, N
2Dry up and be placed on 60 ℃ of baking ovens, can obtain the slide that surfactivity can be stronger.(SBC-12 KYKY Technology Development Ltd.) spreads the cadmium layer on glass sheet with sputter, and thickness is about 2 nm.With the slide that is covered with the cadmium layer of drying at sol evenning machine (Kw-4A Microelectronics Center, Chinese Academy of Sciences) upward rotates with rotating speed 3000 rpm, drip and go up 6 of positive photoetching rubbers (RZJ-304), stop behind 20 s, so just spread one deck positive photoetching rubber in the one side of glass, thickness probably is 1.2 μ m.Glass dries by the fire 20 min in 100 ℃ of baking ovens, photoresist and glass matrix are adhered to firmly.Put into litho machine then and under the covering of template, use 7 mw/cm
2Light intensity shine 30 s, the glass sheet behind the irradiation soaks 45 s in positive photoetching rubber developer solution RZX-308 developer, soak 1 min with clear water again, in 115 ℃ baking oven the heating 30 min, remove developer solution and the water and the correction pattern of sticking together.Use sputter (SBC-12 KYKY Technology Development Ltd.) on glass sheet, to spread the gold layer afterwards, spread the Au that thickness is about 13 nm on the photoresist of pattern earlier having, the existence of Cr can increase the adhesion of Au and glass effectively, in the sequence of operations of aftertreatment, the Au layer can not drop and be wiped easily.At last, glass sheet is immersed in supersound washing 15 min in the acetone, the metal level on photoresist and the glue is removed together, so only has been left the metal level that photoresist that development step dissolves away is partly being spread, just our pattern of just designing on template.Sample is used N after cleaning with pure water
2Dry up, be positioned in the sealing double dish.Provided the photo of the golden pattern of template and preparation among Fig. 1.
In the step 2, retinene protein orientation is arranged is deposited on microarray surface by electro-deposition method.Described retinene protein is made up of 248 amino acid, c-terminus (C-end) both sides in outer aminoterminal (N-end) of born of the same parents and the born of the same parents are arranged, neutrallty condition is whole down electronegative, and this both sides distribution is uneven, C-end institute is electronegative more more, so the dipole moment of whole protein molecule is to point to the C-end by the N-end.Utilize this physical characteristics and dc electrodeposition method can make retinene protein be formed with the layer of orientation.Because retinene protein belt negative electricity, under electric field action, just be enriched in anode, owing to both sides CHARGE DISTRIBUTION inequality, electronegative more C-end will be towards anode (also can change this orientation by the pH value that changes electrolytic solution, make the N-end towards anode) under neutrallty condition again.The retinene memebrane protein that is adopted in the above-mentioned retinene protein microarray is the mutant (does not contain sulfydryl the wild-type bacterium rhodopsin) of bacteria rhodopsin itself, has sulfydryl because of containing halfcystine (Cys) in the mutant.Retinene protein carries out chemical crosslinking with sulfydryl that can pass through after matrix contacts to be had and the golden microarray on the stromal surface.Crosslinked between protein and the substrate molecule can be that all protein and matrix participate in, and also can be that partially protein or substrate molecule participate in.The preparation method of obvious microarray of the present invention also is applicable to other material that itself just has sulfydryl.
In the step 3, by coupling agent with the retinene layer and the layer between crosslinked.The retinene protein microarray that has deposited is carried out dried, and the sulfydryl that makes mutein and gold are with firm covalent bonds.The mixed solution that adopts 1-ethyl-(3-dimethylaminopropyl) the phosphinylidyne diimmonium salt hydrochlorate of 8-10 mg/mL and 4-6 mg/mL N-hydroxy-succinamide then is with the protein microarray submergence, and the reaction time is 50-70 minute.Guarantee the retinene protein layer with the layer between combine with firm amido link.
In the step 4, utilize surfactant Triton-X 100 solution to soak and remove uncrosslinked retinene protein molecule.It is that the surfactant Triton-X 100 of 1.5-2.5% handled 40-50 hour that above-mentioned retinene microarray is placed mass concentration.Can remove not and the crosslinked retinene protein of golden patterned surfaces, molecule uncrosslinked between the retinene protein also is removed.The microarray that is obtained can stand that scaling agent such as Triton is handled and protein does not come off.
Among the above-mentioned preparation method, described electro-deposition method is deposition retinene protein molecule on substrate gold microarray, specifically be to use the retinene suspension liquid of protein, this suspending liquid is suspended in the aqueous solution by the retinene protein dry powder and obtains, and the concentration of retinene protein is 2-5 mg/mL in the general suspending liquid.
Among the above-mentioned preparation method, described retinene suspension liquid of protein disperses by ice-water bath is ultrasonic, and according to system different choice different parameters, ultrasonic power is 100-200 W generally speaking.On the ultrasonic time 3-5 s, interval times 30 s-50 s, ultrasonic number of times 10-100 time can evenly be disperseed the mixed liquor of clear after ultrasonic.
Among the above-mentioned preparation method, it is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate that the coupling agent that adopts is generally the water crosslinking chemical, and N-hydroxy-succinamide has good aqueous solubility, to not influence of activity of proteins, can not cause albuminous degeneration.The mild condition of cross-linking reaction, condition and ranges such as pH, temperature and illumination can not cause the sex change of albumen.
Among the above-mentioned preparation method, the dried after the polymerization does not have specific (special) requirements, just place under room temperature and common humidity can, obtains smooth uniform retinene protein multilayer film after the drying.
The microarray of the retinene memebrane protein for preparing among the present invention has following photovoltaic applications as biological photoreceptor: one, retinene memebrane protein microarray has proton pump function, photochromic and photoelectric effect under the optical excitation condition, and these effects can be used as Conversion of energy motor, information-storing device and light reaction switch.Its two, retinene protein can the outside forms the proton transmembrane gradient from a side pump in the cell to cell with proton under the optical excitation.This proton transmembrane gradient can be used as energy and is beneficial to synthetic ATP enzyme by biosome, can be used as photocell element simultaneously and carries out the solar energy collecting utilization.They are three years old, retinene protein can be converted to metastable M intermediate state from ground state under the optical excitation, and the maximum absorption spectrum value of two states can clearly be separated, and can be used as the optical information storer and carries out information stores, this storage can be instantaneous storage, also can be long-time storage.Its four, retinene protein can produce the charge separation phenomenon under the optical excitation.This phenomenon time of origin is the microsecond yardstick, and charge separation phenomenon moment can return to original state.This character can be used as the photoelectric response switch and uses.Its five, retinene protein can produce the charge separation phenomenon under the optical excitation, produces little electric current thus.Various kinds of cell is seeded to the influence that can be subjected to little electric current on this retinene protein microarray, and inoculating cell is changed.
In addition, among the present invention the microarray of retinene memebrane protein also biomaterial carry out the application that anti-cell sticks the aspect.Natural retinene albuminous membranae can be resisted separately sticking of various kinds of cell, comprises l cell NIH3T3 cell, mouse osteocyte precursor MC3T3-E1 and rat mescenchymal stem cell MSC.Natural retinene albuminous membranae can be united sticking of use opposing various kinds of cell with high molecular polymer polyglycol (PEG) simultaneously, comprise l cell NIH3T3 cell, mouse osteocyte precursor MC3T3-E1 and rat mescenchymal stem cell MSC.
Characteristics of the present invention are to be applied on the basis of the protein microarray of optical storage and processing in preparation in the past, improved preparation technology, utilize active function groups on retinene protein and the matrix with various molecule crosslinked, form the cross-linked network of globality, fundamentally solved the protein molecule orientation deposition in the microarray preparation, orderly arrangement problems, the homogeneity and the stability of microarray have been improved, and can satisfy the needs of single-point control, make material obtain very big improvement at the application performance of optical information storage and processing.
Characteristics of the present invention are that between retinene protein layer and the layer be effect by crosslinking chemical, allow active function groups reacts to each other between the retinene protein molecule, make to connect by chemical bond between the protein molecule that whole rete forms a chemical crosslinking network.Protein molecule is as the unit of microarray, when forming oriented layer as layer with layer between the molecule that is connected be fixed in the microarray, can realize the stable and accurate long preservation of data, can not move and cause loss of data and mistake because of the protein molecule position.
Characteristics of the present invention are that bioactive protein microarray has the characteristic of aspects such as photochromic and photoelectricity under optical excitation, can be widely used in energy conversion, information stores, aspects such as optics regulating switch.Can form the proton transmembrane gradient in the outside in cell membrane as retinene protein under the optical excitation, synthetic ATP provides energy for biosome, can be used as photocell element simultaneously and carries out the solar energy collecting utilization.Retinene protein can be converted to metastable M intermediate state from ground state under the optical excitation, and the maximum absorption spectrum value of two states can clearly be separated, and can be used as the optical information storer and carries out information stores.Retinene protein can produce the charge separation phenomenon of microsecond yardstick under the optical excitation, and this character can be used as the photoelectric response switch and uses.The charge separation phenomenon can produce little electric current simultaneously, and various kinds of cell is seeded to the influence that can be subjected to little electric current on this retinene protein microarray, and the inoculating cell growth conditions is changed.
Characteristics of the present invention are that natural retinene albuminous membranae can be separately or unite with high molecular polymerization polyglycol etc. and to use sticking of opposing various kinds of cell, comprise l cell NIH3T3 cell, mouse osteocyte precursor MC3T3-E1 and rat mescenchymal stem cell MSC.
Description of drawings
Fig. 1 is the photo of the golden pattern of template and preparation.Wherein, (a) golden little pattern photo of obtaining by photoetching process and soft lithographic method of original mask photo (b).Pixel size is 120 x, 120 square microns, and each pixel independently exists, and is connected to the edge part by 10 microns leads.Black part is divided into light tight zone in the mask, and white portion is a transmission region.Golden pattern on the slide is made of positive photoetching rubber and is obtained.
The side SEM photo of protein array on the little pattern of Fig. 2 gold electrode.The substrate slide is positioned at the left side, and protein array is in the right side.The position of arrow indication is the thickness of albuminous membranae.Wherein, (a) two protein spots of middle demonstration, (b) protein spots of middle demonstration.Illustration is a synoptic diagram separately.
Embodiment
Further describe the present invention below by embodiment, but be not limited to this embodiment.
The preparation of embodiment 1 gold medal microarray
Utilize photoetching technique to realize substrate gold microarray.Utilize template and photoresist with on pattern is from the template transfer to matrix.Initial matrix is glass sheet, with the glass sheet surface wiped clean, utilizes piranha (with the volume ratio 3:1 mixing concentrated sulphuric acid and hydrogen peroxide) solution-treated subsequently.Glass sheet is soaked wherein 30 min, and N is used after the supersound washing 15 minutes three times in water flushing back in pure water
2Dry up, in acetone, soak ultrasonic 10 min, N
2Dry up and be placed on 60 ℃ of baking ovens, can obtain the slide that surfactivity can be stronger.(SBC-12 KYKY Technology Development Ltd.) spreads the cadmium layer on glass sheet with sputter, and thickness is about 2 nm.With the slide that is covered with the cadmium layer of drying at sol evenning machine (Kw-4A Microelectronics Center, Chinese Academy of Sciences) upward rotates with rotating speed 3000 rpm, drip and go up 6 of positive photoetching rubbers (RZJ-304), stop behind 20 s, so just spread one deck positive photoetching rubber in the one side of glass, thickness probably is 1.2 microns.Glass dries by the fire 20 min in 100 ℃ of baking ovens, photoresist and glass matrix are adhered to firmly.Put into litho machine then and under the covering of template, use 7 mw/cm
2Light intensity shine 30 s, the glass sheet behind the irradiation soaks 45 s in positive photoetching rubber developer solution RZX-308 developer, soak 1 min with clear water again, in 115 ℃ baking oven the heating 30 min, remove developer solution and the water and the correction pattern of sticking together.Use sputter (SBC-12 KYKY Technology Development Ltd.) on glass sheet, to spread the gold layer afterwards, spread the Au that thickness is about 13 nm on the photoresist of pattern earlier having, the existence of Cr can increase the adhesion of Au and glass effectively, in the sequence of operations of aftertreatment, the Au layer can not drop and be wiped easily.
At last, glass sheet is immersed in supersound washing 15 min in the acetone, the metal level on photoresist and the glue is removed together, so only has been left the metal level that photoresist that development step dissolves away is partly being spread, just our pattern of just designing on template.Sample is used N after cleaning with pure water
2Dry up, be positioned in the sealing double dish.Provided the photo of the golden pattern of template and preparation among Fig. 1.
The preparation of embodiment 2 bacteria rhodopsin mutant BR-D36C microarraies
Utilize the method for genetic engineering point mutation, obtain bacteria rhodopsin mutant BR-D36C, promptly the 36th aspartic acid is replaced by halfcystine, halfcystine contains sulfydryl and is in two join domains between the spiral, therefore the mutant that obtains has sulfydryl at protein surface, can carry out chemical reaction with gold grain.
Be suspended in the pure water again after the BR-D36C process washing of extracting is centrifugal, concentration is 2 mg/mL, utilize ultrasonic help to disperse, form uniform suspending liquid, be injected into the intermediate space part of two plate electrodes then, the sealing back adds the DC voltage of 3V, powered-down behind 15 min on two electrodes, on anode, can obtain the purple protein layer of film forming, place 30 min and allow the sulfydryl and the reaction of gold compare fully.Be immersed in 1h in the mixed solution of 10 mg/mL EDC HCl and 6 mg/mL NHS after the reacted sample drip washing, be immersed in 2 %(w/w again after cleaning with pure water) Triton X-100 in, placing after 48 hours ultrasonic 10 min in ultrasonic pond so that remove the surface does not have the protein molecule that chemistry connects and the material of other adhesions.At room temperature dry at last.For the reaction of sulfydryl in confirming to prepare with gold, we have done same albuminous membranae with wild type BR, and the also soaking and washing by surfactant.
In the case study on implementation, the array of wild type BR preparation is after the process cleaning of surfactant, and protein is substantially all washed off, and only remaining golden pattern is on glass matrix, and the sample of BR-D36C has just well kept protein layer.We to the sample of D36C with scanning electron microscopic observation side view, do not have protein to connect between two points as can be seen, illustrate that the arrays of immobilized protein each point is independently, do not join together, and the thickness of definite albumin layer is about 5 μ m, see Fig. 2.Known BR bulk of molecule is 2.5 nm x, 3.5 nm x, 4.5 nm.The BR group of molecules is synthesized tripolymer, is arranged in the film fat with the form of two-dimentional hexagoinal lattice.Consider the thickness of the final protein array that forms, the BR array point of Xing Chenging comprises nearly thousand layers of BR film as can be known.Adjust the concentration of BR suspension and the voltage that electro-deposition is adopted, can obtain the BR film microarray of the different numbers of plies (which floor is to the hundreds of layer).
The photovoltaic applications of embodiment 3 bacteria rhodopsin mutant BR-D36C microarraies
In cuvette, put into 3mL electrolyte solution (100 mM NaCl, 20 mM KCl), pH is adjusted into 7.00, the gold electrode glass sheet that is connected with protein is as working electrode, a slice does not have the gold electrode glass sheet of protein as contrast electrode, insert in the electrolytic solution, at a distance of 6-8 mm, respectively be wired to amplifier, see through green color filter (seeing through light ≈ 570 nm) irradiation electrode elicitor protein layer with a flashlamp YINYAN BY-26AZ, voltage signal is input to digital storage oscilloscope RIGAL DS5102CA 100 MHz 1 GS/s after amplifying, at last by the computer processing signals.
Can see a negative peak and a slow slightly posivtive spike fast, these two peaks are typical B 1 and B2 components in the opsin photosignal, these two peaks only appear in the signal of the unified protein film that is orientated, B1 has represented the formation of the photic isomery of retinene and K intermediate and the quick distributed process of associated electric charge, and B2 then is a directed transmittance process of having represented the protein interior proton.The appearance of this signal shows that the protein in the sample has good activity and the higher degree of orientation is arranged.Show that simultaneously the protein microarray of having made connects and photosignal can be derived the formation photocurrent by micro wiring, this signal can be used as the basis of the application of biometric information sensor, biobattery and molecular switch.
The character that the anti-cell of embodiment 4 bacteria rhodopsins sticks
The bacteria rhodopsin memebrane protein can form anti-cell and stick the zone.The bacteria rhodopsin hanging drop adds to and forms the unordered BR diaphragm of multilayer on the golden slide.Choose mouse osteocyte precursor MC3T3-E1 in an embodiment respectively, l cell NIH3T3 cell and rat mescenchymal stem cell MSC are as experimental cell, and inoculating cell density is 10
5Individual/hole (6 orifice plate).The result shows that it is better than known anti-cell pasting material bovine serum albumin(BSA) (BSA) that short time interior (cultivating 8 h behind the inoculating cell) protein microarray superficial cell sticks performance, can compare favourably with high molecular polymerization anti-cell pasting material PEG.(cultivated 14 days behind the inoculating cell) under the long-time cultivation situation, it is stronger than PEG that the protein microarray superficial cell sticks performance.What usually PEG formed anti-ly sticks the longest anti-cell in zone to stick the time is 7 days.
The application of the anti-cell pasting material of embodiment 5 bacteria rhodopsin mutant BR-D36C microarraies
The retinene protein microarray has strong anti-cell and sticks characteristic.Choose mouse osteocyte precursor MC3T3-E1 among the embodiment respectively, l cell NIH3T3 cell and rat mescenchymal stem cell MSC are as experimental cell, and inoculating cell density is 10
5Individual/hole (6 orifice plate).The result shows that (8 h are cultivated in the inoculation back) and long-time (cultivated 14 days the inoculation back) cellular incubation all shows good anti-cell and sticks performance in the short time.This retinene protein can be combined with high molecular polymerization anti-cell pasting material PEG in addition, form compound anti-cell and stick the research that the surface can be used for basic cell biology aspect.
Claims (10)
1. a microarray that contains the retinene memebrane protein is characterized in that it being to be deposited on the multilayer film arrays of immobilized protein that orientation is arranged that forms on the substrate gold microarray by the retinene membrane protein molecule; Wherein, between retinene memebrane protein molecule and the substrate gold microarray, all be connected into an integral body by chemical bond-linking between the retinene memebrane protein molecule, the retinene memebrane protein on each microarray forms the chemical crosslinking network of orientation.
2. according to the described microarray that contains the retinene memebrane protein of claim 1, it is characterized in that described retinene protein for by the bacteria rhodopsin of genetic engineering acquisition or the mutant of archaerhodopsin 4, has active mercapto groups in this mutein; Be connected by covalent bond between mercapto groups in the retinene protein mutant and the golden microarray.
3. according to the described microarray that contains the retinene memebrane protein of claim 1, it is characterized in that between described retinene membrane protein molecule layer and the layer crosslinked by the effect of coupling agent.
4. according to the described microarray that contains the retinene memebrane protein of claim 1, it is characterized in that described substrate gold microarray range of size is 1 micron to 900 microns.
5. as the described preparation method who contains the microarray of retinene memebrane protein of claim 1, it is characterized in that concrete steps are:
(1) utilize the photoetching method making to have the substrate gold microarray of pattern;
(2) by electro-deposition method, on substrate gold microarray, deposit the retinene protein molecule, be formed with the multilayer film retinene arrays of immobilized protein of orientation; The used retinene protein of protein microarray is the bacteria rhodopsin of genetic engineering acquisition or the mutant of archaerhodopsin 4; Has active mercapto groups in this mutein; Be connected by covalent bond between mercapto groups in the retinene protein mutant and the golden microarray;
(3) by coupling agent with retinene protein molecule layer and the layer between crosslinked, described coupling agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy-succinamide; This coupling agent makes amino and activated carboxylic formation amido link connects into an integral body, and the retinene memebrane protein on each microarray forms the chemical crosslinking network of orientation;
(4) the retinene protein microarray that obtains is soaked in surfactant Triton-X 100 solution, remove not chemical crosslinking protein molecule, comprise not and retinene protein molecule that golden microarray gold patterned surfaces is crosslinked and retinene protein between uncrosslinked retinene protein molecule.
6. as the described preparation method who contains the microarray of retinene memebrane protein of claim 1, it is characterized in that described electro-deposition method deposition retinene protein molecule on substrate gold microarray, use the retinene suspension liquid of protein, this suspending liquid is suspended in the aqueous solution by the retinene protein dry powder and obtains, and the concentration of retinene protein is 2-5 mg/mL in the suspending liquid.
7. the preparation method who contains the microarray of retinene memebrane protein as claimed in claim 6, it is characterized in that described retinene suspension liquid of protein disperses by ice-water bath is ultrasonic, ultrasonic power is 100-200 W, ultrasonic time 3-5 s, on the interval times 30 s-50 s, ultrasonic number of times 10-100 time.
8. the preparation method who contains the microarray of retinene memebrane protein as claimed in claim 5, the mass concentration that it is characterized in that described surfactant Triton-X 100 solution is 1.5-2.5%, the processing time is 40-50 hour.
9. the preparation method who contains the microarray of retinene memebrane protein as claimed in claim 5, it is characterized in that described by coupling agent with retinene protein molecule layer and the layer between crosslinked, be to adopt 1-ethyl-(3-dimethylaminopropyl) the phosphinylidyne diimmonium salt hydrochlorate of 8-10 mg/mL and the mixed solution of 4-6 mg/mL N-hydroxy-succinamide, protein microarray is immersed in this mixed solution, and the reaction time is 50-70 minute.
10. application that contains the microarray of retinene memebrane protein as biological photoreceptor as claimed in claim 1, or as the application of anti-cell pasting material.
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Application publication date: 20111228 |