CN102115025B - Method for preparing polystyrene micro-sphere micro-array by ultrasonic focusing micro-jet process - Google Patents
Method for preparing polystyrene micro-sphere micro-array by ultrasonic focusing micro-jet process Download PDFInfo
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- CN102115025B CN102115025B CN 201110002410 CN201110002410A CN102115025B CN 102115025 B CN102115025 B CN 102115025B CN 201110002410 CN201110002410 CN 201110002410 CN 201110002410 A CN201110002410 A CN 201110002410A CN 102115025 B CN102115025 B CN 102115025B
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Abstract
The invention relates to the field of preparing micro-array biochips, in particular to a method for preparing a polystyrene micro-sphere micro-array by an ultrasonic focusing micro-jet process. The method comprises the following steps: preparing a suspension of polystyrene micro-spheres; cleaning a reagent reservoir, pouring the suspension of polystyrene micro-spheres into the reagent reservoir and ensuring that an ultrasonic focusing micro-jet nozzle is in a jet-ready state; taking a micro-array substrate, clamping the micro-array substrate on a micro-displacement bench, and arranging the micro-displacement bench below an ultrasonic focusing micro-jet device; sending an operating command; sending a jet command to make individual jet points of the suspension of polystyrene micro-spheres; setting a row distance, a column distance and a jet point number of the micro-array; making a polystyrene micro-sphere micro-array; and stamping and fixing the polystyrene micro-sphere micro-array on a flat plate after the polymerization of poly(dimethylsiloxane) (PDMS). With the technical scheme, the method can achieve the purpose of nozzle-free, non-blocking, non-contact and low-consumption preparation of polystyrene micro-sphere micro-arrays.
Description
Technical field
The present invention relates to the micro-array biochip preparation field, relate in particular to a kind of method for preparing the polystyrene microsphere microarray.
Background technology
Along with reaching its maturity of the controlled polymer microsphere technology of preparing of monodispersity and particle diameter, the application of polymer microsphere more and more widely, and is wherein the highest in the using value of the monodisperse polystyrene microsphere of 2～30 μ m with particle diameter especially.With the polystyrene microsphere is carrier; Can carry various characteristics functional group or functional materials such as activated protein, nucleic acid, magnetic metal on it; The polystyrene microsphere that carries characteristic functional group or functional material is carried out array or patterning, can be applicable to fields such as medicine and pharmacology, bioengineering, food chemical industry, information engineering and microelectric technique.Particularly, significant to the research of clinical diagnosis, check, immunological technique, cytology etc. at biomedical sector.
At present, under the prior art condition, the method that p-poly-phenyl ethene microballoon carries out array and prepares the polystyrene microsphere microarray mainly contains contact point sample, contact spray appearance, and is the semiconductor approach etc. of representative with photoetching and seal.Above-mentioned polystyrene microsphere microarray exists cost of manufacture height, complex process, biologically active to be difficult for problems such as maintenance, spotting needle or shower nozzle are prone to stop up, reagent dosage is big, thereby is unfavorable for promoting the use of.
Summary of the invention
The object of the present invention is to provide the method that adopts the little spray of focus ultrasonic to be equipped with the polystyrene microsphere microarray, to solve the problems of the technologies described above.
The technical problem that the present invention solved can adopt following technical scheme to realize:
Adopt the little spray of focus ultrasonic to be equipped with the method for polystyrene microsphere microarray, it is characterized in that, may further comprise the steps:
1) produces little spray reagent: preparation polystyrene (PS) microballoon aaerosol solution; The particle size range of the used polystyrene microsphere of little spray range of choice as required is 5～20 μ m, because the density of polystyrene microsphere is bigger than water, and general 1.05g/cm
3,, need preparation polystyrene microsphere aaerosol solution earlier for satisfying little spray needs.
2) clean the interior reagent pond of focus ultrasonic micro-spray device, said polystyrene microsphere aaerosol solution is added in the said reagent pond, but adopt examination spray method to confirm that the focus ultrasonic mini sprinkler of said focus ultrasonic micro-spray device is in spray regime;
3) get the microarray substrate, and with its clamping on micropositioner, the microarray substrate on the said micropositioner is arranged on the below of said focus ultrasonic micro-spray device;
4) the injection frequency control with said focus ultrasonic mini sprinkler switches to industrial computer by frequency controller, sends movement instruction, makes the step setting in motion of micropositioner by acceleration-maintenance-deceleration;
Send jeting instruction, make said focus ultrasonic mini sprinkler send high-frequency ultrasonic and focus on polystyrene microsphere aaerosol solution liquid level, form single drop and spray and be sprayed on the said microarray substrate, make the single specking of polystyrene microsphere solution;
5) line space, column pitch and the specking number of setting microarray;
Movement instruction, the jeting instruction in the step 4) sent in circulation, makes the polystyrene microsphere microarray;
The flat board that 6) will scribble dimethyl silicone polymer (PDMS) prepolymer impresses the polystyrene microsphere microarray that has just made; Thereby the polystyrene microsphere two dimensional structure after the array is quoted to said flat board, treated just the polystyrene microsphere microarray to be fixed on this flat board after the PDMS polymerization.Be beneficial to follow-up application need.
Among the present invention, the microarray substrate is arranged on the below, adopts downward spray regime to spray, and compares with the spray regime that makes progress, and has institute's liquid droplets and arrives the characteristics that substrate forms specking to quicken " falling " mode, and its positioning accuracy will effectively improve; Because the reduction of the required kinetic energy of liquid droplets; The required pulse width also will reduce; And pulsewidth reduce to make also corresponding the reducing of drop (specking) diameter, consequently except that the polystyrene microsphere aaerosol solution reduce consume, the density of specking further improves on the microarray; And the also relative minimizing of the quantity of polystyrene microsphere in the single specking, this helps preparing high-throughout biochip.In addition; The present invention compares with existing polystyrene microsphere micro-array biochip preparation method; Following advantage is arranged: the focus ultrasonic mini sprinkler with as the polystyrene microsphere aaerosol solution of little spray reagent does not take place directly to contact, thereby effectively avoids in the actual use the bioactive infringement to the characteristic functional group of lift-launch on it; Because the space between liquid level and lens must occupy with liquid, replace this space of little spray reagent filling and do coupling liquid with pure water, must effectively reduce little spray reagent dosage, further reduce the cost of manufacture of micro-array biochip; Noseless spray regime has avoided shower nozzle by the generation of little spray reagent clogging.
In the step 1), the mass fraction of said polystyrene microsphere aaerosol solution is preferably 10%.According to the difference of polystyrene microsphere aggregation number in the single specking of polystyrene, the mass fraction of the polystyrene microsphere aaerosol solution that little spray is used can be prepared arbitrarily as required.
In the step 1), be to improve suspension stability, add concentration and be 2% polyvinyl alcohol (PVA) aqueous solution as dispersant;
The compound method of 2% polyvinyl alcohol water solution is: the selective polymerization degree is 1700 polyvinyl alcohol, is continuous stirring under 75 ℃～80 ℃ situation in temperature, is dissolved in water behind 15min～20min and makes.
The addition of said dispersant is about 15% of a said polystyrene microsphere aaerosol solution.
Step 2) in, the method for said examination spray method is following: the ping that said focus ultrasonic mini sprinkler produces frequency 20MHz, continues 20 μ s is set; Utilize micro syringe to adjust little spray reagent solution liquid level, when it is positioned at focus ultrasonic energy transducer focus zone, just form the continuous injection of 50Hz.
Microarray substrate in the step 3) adopts slide.
In the step 4), move into place and reach stable state, after said movement instruction sends, send jeting instruction again after waiting for 2s for guaranteeing micropositioner.
In the step 4), during accelerated motion, acceleration maximum impulse frequency values is 10000c/s
2
In the step 5), set microarray line space and column pitch and be 200 μ m.
Said focus ultrasonic micro-spray device; Comprise a frame; Said frame is provided with a focus ultrasonic mini sprinkler, a micropositioner, and said micropositioner is provided with a microarray substrate, comprises that also one contains the coupling liquid pool of water; The showerhead branch of said focus ultrasonic mini sprinkler is arranged in the said coupling liquid pool; The below of said coupling liquid pool is provided with an agent plate, the agent plate under the said focus ultrasonic mini sprinkler be provided with one be used to place the taper of little spray reagent the reagent pond, the upper surface and the lower surface of said agent plate run through in said reagent pond;
The bottom surface of said coupling liquid pool is a transparent plastic film, and said transparent plastic film separates said coupling liquid pool and said reagent pond;
Said microarray substrate is arranged on the vertex of a cone below in said reagent pond.
Above-mentioned design is provided with the through reagent of taper pond under the focus ultrasonic mini sprinkler, and injects little spray reagent through runner, makes it form free surface and unlikely by the surface tension of liquid and leaks down; Between little spray reagent and coupling liquid pool, isolate with transparent plastic film; After profit uses water as coupling liquid transmission sound wave; Little spray reagent in the reagent pond has overcome the surface tension of liquid; Thereby form small drop and eject, and this liquid-drop diameter is fully by the acoustical behavior decision of ultrasonic frequency and liquid.
The preferred 0.05ml of volume in said reagent pond, the preferred 2mm of vertex of a cone diameter.So that satisfy the injection needs.
Beneficial effect: owing to adopt technique scheme, adopt that method of the present invention can realize not having nozzle, the preparation of no obstruction, noncontact, low consumed polystyrene microsphere microarray, the approach that makes new advances for the manufacturing exploration of micro-array biochip.
Description of drawings
Fig. 1 is an overall flow block diagram of the present invention;
Fig. 2 is the principle schematic of focus ultrasonic micro-spray device of the present invention;
Fig. 3 is the structural representation of polystyrene microsphere microarray of the present invention;
Fig. 4 for the present invention impress before structural representation between polystyrene microsphere microarray and the flat board;
Fig. 5 impresses the structural representation between back polystyrene microsphere microarray and the flat board for the present invention.
The specific embodiment
For technological means, creation characteristic that the present invention is realized, reach purpose and effect and be easy to understand and understand, further set forth the present invention below in conjunction with concrete diagram.
With reference to Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, adopt the little spray of focus ultrasonic to be equipped with the method for polystyrene microsphere microarray, may further comprise the steps: the first step, produce little spray reagent: preparation polystyrene (PS) microballoon aaerosol solution.According to the difference of polystyrene microsphere aggregation number in the single specking 5, the mass fraction of the polystyrene microsphere aaerosol solution that little spray is used can be prepared arbitrarily as required.The present invention preferably adopts the polystyrene microsphere aaerosol solution of 10% mass fraction.The particle size range of the used polystyrene microsphere of little spray range of choice as required is 5～20 μ m, and preferable particle size is the monodisperse polystyrene microsphere (monodisperse system number＜5%) of 15 μ m.Be to improve suspension stability, add concentration and be 2% polyvinyl alcohol (PVA) aqueous solution as dispersant, the addition of dispersant is about 15% of a whole aaerosol solution.The compound method of 2% polyvinyl alcohol water solution is: the selective polymerization degree is 1700 polyvinyl alcohol, and preferred PVA1788 (degree of polymerization is 1700) is a continuous stirring under 75 ℃～80 ℃ situation in temperature, is dissolved in water behind 15min～20min and makes.
Second step, clean the reagent pond 4 in the focus ultrasonic micro-spray device, the polystyrene microsphere aaerosol solution is added in the reagent pond 4, but adopt examination spray method to confirm that the focus ultrasonic mini sprinkler 1 of focus ultrasonic micro-spray device is in spray regime.The method of examination spray method is following: it is that 50Hz, amplitude are that the continuous signal of 2VRMS is input in the pulse signal source that the frequency controller that focus ultrasonic mini sprinkler 1 is set sends frequency; Producing width is 20 μ s; Amplitude is the pulse of 5V, and the 20MHz signal that frequency synthesizer is exported carries out impulse modulation; Pulse-modulated signal inputs to the focus ultrasonic transducer after power amplification, produce the ping of frequency 20MHz, lasting 20 μ s thus; Utilize micro syringe to adjust little spray reagent solution liquid level, when it is positioned at focus ultrasonic energy transducer focus zone, just form the continuous injection of 50Hz.
The 3rd step, get microarray substrate 2, microarray substrate 2 adopts slide, and on micropositioner, the microarray substrate 2 on the micropositioner is arranged on the below of focus ultrasonic micro-spray device with the slide clamping.
The 4th goes on foot, and the injection frequency of focus ultrasonic mini sprinkler 1 is controlled switched to industrial computer by frequency controller, sends movement instruction, makes the step setting in motion of micropositioner by acceleration-maintenance-deceleration, and during accelerated motion, acceleration maximum impulse frequency values is 10000c/s
2Send jeting instruction, make focus ultrasonic mini sprinkler 1 send high-frequency ultrasonic and focus on polystyrene microsphere aaerosol solution liquid level, form single drop and spray and be sprayed on the microarray substrate 2, make the single specking 5 of polystyrene microsphere solution.For guaranteeing that micropositioner moves into place and reaches stable state, after movement instruction sends, send jeting instruction again after waiting for 2s.
The 5th step, line space, column pitch and the specking number of setting microarray; Movement instruction, the jeting instruction in the 4th step sent in circulation, makes the polystyrene microsphere microarray.Set microarray line space and column pitch and be 200 μ m.With reference to Fig. 3, the diameter range of each specking 5 is in 60～100 μ m, controlled amount.Polystyrene microsphere aggregation number in each specking 5 is by the concentration control of polystyrene microsphere aaerosol solution, and the polystyrene microsphere aggregation number is four among the figure.
The 6th step; With reference to Fig. 4 and Fig. 5; To scribble the polystyrene microsphere microarray that 6 pairs of the flat boards of dimethyl silicone polymer (PDMS) prepolymer 61 have just made impresses; Thereby the polystyrene microsphere two dimensional structure after the array is quoted on dull and stereotyped 6, treated just the polystyrene microsphere microarray to be fixed on this flat board 6 after the PDMS polymerization.Be beneficial to follow-up application need.
With reference to Fig. 2, the focus ultrasonic micro-spray device comprises frame; Frame is provided with focus ultrasonic mini sprinkler 1, micropositioner, and micropositioner is provided with microarray substrate 2, also comprises the coupling liquid pool 3 that contains water; The showerhead branch of focus ultrasonic mini sprinkler 1 is arranged in the coupling liquid pool 3; The below of coupling liquid pool 3 is provided with agent plate, and the agent plate under the focus ultrasonic mini sprinkler 1 is provided with the reagent pond 4 of the taper that is used to place little spray reagent, and the upper surface and the lower surface of agent plate run through in reagent pond 4.The bottom surface of coupling liquid pool 3 is a transparent plastic film 31, and will be coupled liquid pool 3 and reagent pond 4 of transparent plastic film 31 separates.Microarray substrate 2 is arranged on the vertex of a cone below in reagent pond 4.Above-mentioned design is provided with the through reagent of taper pond 4 under focus ultrasonic mini sprinkler 1, the preferred 0.05ml of the volume in reagent pond 4, the preferred 2mm of vertex of a cone diameter.Be provided with little spray reagent in the reagent pond 4, make it form free surface and unlikely by the surface tension of liquid and leak down; Isolate with transparent plastic film 31 at little spray reagent and 3 of liquid pools of coupling; After profit uses water as coupling liquid transmission sound wave; Little spray reagent in the reagent pond 4 has overcome the surface tension of liquid; Thereby form small drop and eject, and this liquid-drop diameter is fully by the acoustical behavior decision of ultrasonic frequency and liquid.
Focus ultrasonic mini sprinkler 1 of the present invention can adopt the parameter of modulation pulsewidth 20 μ s, injection frequency 1000Hz to spray, and the injection object is a water.Focus ultrasonic mini sprinkler 1 generally includes ultrasonic transducer and sphere acoustic lens, and the piezoelectric ceramic piece of ultrasonic transducer adopts the piezoelectric ceramic piece of resonant frequency 20MHz, diameter 5mm, the preferred 3mm of the spherical radius of sphere acoustic lens.Used key instrument of focus ultrasonic micro-spray device of the present invention and parameter are following:
(1) cleanliness factor is 100 grades, and temperature, humidity is adjustable;
(2) micropositioner: the German PI C-844 of company type direct current generator micropositioner, its smallest incremental displacement accuracy 0.1 μ m, omnidistance displacement accuracy 1 μ m, maximum speed 5cm/s, range 102mm;
(3) pulse signal generator: Nantong Nan Feng NF1511A, pulsewidth is adjustable continuously at 0～100 μ s;
(4) frequency controller: Liaoning wound difficult to understand RF-600, frequency 0～50Hz;
(5) frequency synthesizer: Germany breathes out and steps HM8134, and modulating mode is the GATE pattern.
More than show and described basic principle of the present invention and principal character and advantage of the present invention.The technical staff of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification just explains principle of the present invention; Under the prerequisite that does not break away from spirit and scope of the invention, the present invention also has various changes and modifications, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (9)
1. adopt the little spray of focus ultrasonic to be equipped with the method for polystyrene microsphere microarray, it is characterized in that, may further comprise the steps:
1) produces little spray reagent: preparation polystyrene (PS) microballoon aaerosol solution;
2) clean the interior reagent pond of focus ultrasonic micro-spray device, said polystyrene microsphere aaerosol solution is added in the said reagent pond, but adopt examination spray method to confirm that the focus ultrasonic mini sprinkler of said focus ultrasonic micro-spray device is in spray regime;
Said focus ultrasonic micro-spray device; Comprise a frame; Said frame is provided with a focus ultrasonic mini sprinkler, a micropositioner, and said micropositioner is provided with a microarray substrate, comprises that also one contains the coupling liquid pool of water; The showerhead branch of said focus ultrasonic mini sprinkler is arranged in the said coupling liquid pool; The below of said coupling liquid pool is provided with an agent plate, the agent plate under the said focus ultrasonic mini sprinkler be provided with one be used to place the taper of little spray reagent the reagent pond, the upper surface and the lower surface of said agent plate run through in said reagent pond;
The bottom surface of said coupling liquid pool is a transparent plastic film, and said transparent plastic film separates said coupling liquid pool and said reagent pond;
Said microarray substrate is arranged on the vertex of a cone below in said reagent pond;
3) get the microarray substrate, and with its clamping on micropositioner, the microarray substrate on the said micropositioner is arranged on the below in the said focus ultrasonic micro-spray device;
4) the injection frequency control with said focus ultrasonic mini sprinkler switches to industrial computer by frequency controller, sends movement instruction, makes the step setting in motion of micropositioner by acceleration-maintenance-deceleration;
Industrial computer sends jeting instruction, makes said focus ultrasonic mini sprinkler send high-frequency ultrasonic and focuses on polystyrene microsphere aaerosol solution liquid level, forms single drop and sprays, and on said microarray substrate, makes the single specking of polystyrene microsphere solution;
5) line space, column pitch and the specking number of setting microarray;
Movement instruction, the jeting instruction in the step 4) sent in circulation, makes the polystyrene microsphere microarray;
The flat board that 6) will scribble dimethyl silicone polymer (PDMS) prepolymer impresses the polystyrene microsphere microarray that has just made; Thereby the polystyrene microsphere two dimensional structure after the array is quoted to said flat board, treated just the polystyrene microsphere microarray to be fixed on this flat board after the PDMS polymerization.
2. the little spray of employing focus ultrasonic according to claim 1 is equipped with the method for polystyrene microsphere microarray, it is characterized in that, in the step 1), the mass fraction of said polystyrene microsphere aaerosol solution is 10%.
3. the little spray of employing focus ultrasonic according to claim 1 and 2 is equipped with the method for polystyrene microsphere microarray, it is characterized in that, in the step 1), be to improve suspension stability, adds concentration and be 2% polyvinyl alcohol (PVA) aqueous solution as dispersant;
The compound method of 2% polyvinyl alcohol water solution is: the selective polymerization degree is 1700 polyvinyl alcohol, is continuous stirring under 75 ℃～80 ℃ situation in temperature, is dissolved in water behind 15min～20min and makes.
4. the little spray of employing focus ultrasonic according to claim 3 is equipped with the method for polystyrene microsphere microarray, it is characterized in that, the addition of said dispersant is 15% of a said polystyrene microsphere aaerosol solution.
5. the little spray of employing focus ultrasonic according to claim 1 is equipped with the method for polystyrene microsphere microarray; It is characterized in that; Step 2) in, the method for said examination spray method is following: the ping that said focus ultrasonic mini sprinkler produces frequency 20MHz, continues 20 μ s is set; Utilize micro syringe to adjust little spray reagent solution liquid level, when it is positioned at focus ultrasonic energy transducer focus zone, just form the continuous injection of 50Hz.
6. the little spray of employing focus ultrasonic according to claim 1 is equipped with the method for polystyrene microsphere microarray; It is characterized in that, in the step 4), move into place and reach stable state for guaranteeing micropositioner; After said movement instruction sends, send jeting instruction again behind the wait 2s.
7. be equipped with the method for polystyrene microsphere microarray according to claim 1 or the little spray of 6 described employing focus ultrasonics, it is characterized in that in the step 4), during accelerated motion, acceleration maximum impulse frequency values is 10000c/s
2
8. the little spray of employing focus ultrasonic according to claim 1 is equipped with the method for polystyrene microsphere microarray, it is characterized in that, in the step 5), sets microarray line space and column pitch and is 200 μ m.
9. the little spray of employing focus ultrasonic according to claim 1 is equipped with the method for polystyrene microsphere microarray, it is characterized in that, the volume in said reagent pond is 0.05ml, and the vertex of a cone diameter in said reagent pond is 2mm.
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| CN105858595B (en) * | 2016-04-06 | 2018-01-23 | 西南交通大学 | Flatness self-compensating large area micro Process Multi-tip array preparation method |
| CN108014895B (en) * | 2018-01-16 | 2023-05-30 | 北京工商大学 | Ultrasonic energy-gathering crushing method and device |
| CN113070106A (en) * | 2020-01-05 | 2021-07-06 | 天津大学 | Ultrasonic pipetting device and method for realizing ultrasonic pipetting based on phased array technology |
| CN115416282A (en) * | 2022-07-18 | 2022-12-02 | 广东工业大学 | Ultrasonic microstructure three-dimensional forming method |
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| US6162264A (en) * | 1996-06-17 | 2000-12-19 | Dai Nippon Printing Co., Ltd. | Process for producing porous coating layer electrode plate for secondary battery with nonaqueous electrolyte process for producing same and sheet for peeling active material layer |
| CN1701641A (en) * | 2002-09-24 | 2005-11-23 | 夏普株式会社 | Method and apparatus for manufacturing active-matrix organic EL display, active-matrix organic EL display, method for manufacturing liquid crystal array, liquid crystal array, method and apparatus for |
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| CN1980742A (en) * | 2004-07-06 | 2007-06-13 | 犹他大学研究基金会 | Spotting device and method for high concentration spot deposition on microarrays and other microscale devices |
| CN101301816A (en) * | 2007-05-11 | 2008-11-12 | 施乐公司 | Ink jet printhead having a movable redundant array of nozzles |
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| US20100059906A1 (en) * | 2008-09-05 | 2010-03-11 | E. I. Du Pont De Nemours And Company | High throughput electroblowing process |
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|---|---|---|---|---|
| US6162264A (en) * | 1996-06-17 | 2000-12-19 | Dai Nippon Printing Co., Ltd. | Process for producing porous coating layer electrode plate for secondary battery with nonaqueous electrolyte process for producing same and sheet for peeling active material layer |
| CN1701641A (en) * | 2002-09-24 | 2005-11-23 | 夏普株式会社 | Method and apparatus for manufacturing active-matrix organic EL display, active-matrix organic EL display, method for manufacturing liquid crystal array, liquid crystal array, method and apparatus for |
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| CN1836907A (en) * | 2005-03-23 | 2006-09-27 | 精工爱普生株式会社 | Droplet discharging head, droplet discharging device and manufacturing method of microarray |
| CN101301816A (en) * | 2007-05-11 | 2008-11-12 | 施乐公司 | Ink jet printhead having a movable redundant array of nozzles |
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