阅读说明：本技术 基于光力组装周期细胞结构的方法 (Based on the cyto-architectural method of luminous power assembling cycle ) 是由 辛洪宝 李宝军 李宇超 于 2019-08-30 设计创作，主要内容包括：本发明公开了一种基于光力组装周期细胞结构的方法,包括以下步骤：制定锥型光纤、配备混合悬浮液、安装锥形光纤、周期细胞结构组装与光传输性能探测；所述步骤S1制备的光纤锥形区直径在1~10μm,长度在2~50μm,锥形光纤末端锥角在70~120o；所采用的激光是近红外的激光和单色性好的可见光；根据组装的细胞链长度来决定所用激光的功率,当长度小于100μm时,功率要小于60mW。本发明所提供的方法实现了完全基于光力的周期细胞结构组装,组装过程不需要依赖于复杂的微结构衬底,组装的细胞结构具有可控的周期性,且具有单细胞精度的操控性,组装的细胞结构还可以实现灵活的移动,传输的信号可以被实时监测。(The invention discloses one kind to be based on the cyto-architectural method of luminous power assembling cycle, comprising the following steps: formulates tapered optical fiber, outfit mixing suspension, installation conical fiber, cycling cells structure and assembles and detect with optical transmission performance；The optical fiber tapered zone diameter of the step S1 preparation is at 1 ~ 10 μm, and length is at 2 ~ 50 μm, and conical fiber terminal bevel is in 70 ~ 120o；Used laser is the laser and the good visible light of monochromaticjty of near-infrared；The power of laser used is determined according to the cell chain length of assembling, when length is less than 100 μm, power is less than 60mW.The cycling cells structure assembling that method provided by the present invention realizes based entirely on luminous power, assembling process is not need to rely on complicated micro-structure substrate, the eucaryotic cell structure of assembling has controllable periodicity, and it is handling with unicellular precision, the eucaryotic cell structure of assembling can also realize flexible movement, and the signal of transmission can be by real-time monitoring.)

1. one kind is based on the cyto-architectural method of luminous power assembling cycle, which comprises the following steps:

Step S1: production conical fiber

The buffer layer and plastic wrapper that commercial single-mode fiber jumper is removed using optical fiber wire stripper, remove partial-length；It will removal The optical fiber of housing protrudes into glass capillary, end is pulled out after alcolhol burner flame envelope heats 155 ~ 160s, optical fiber starts to melt； Optical fiber is broken in flame upper end, optical fiber connector forms a tapered distal end, as conical fiber；Optical fiber tapered zone diameter At 1 ~ 10 μm, length is at 2 ~ 50 μm, and conical fiber terminal bevel is in 70 ~ 120o；

Step S2: it is equipped with mixing suspension

Escherichia coli are placed in LB culture medium in 37 DEG C of culture 12h, is then cleaned and is diluted with phosphoric acid hydrochloride buffer, Until concentration is 3.0 × 10⁶A/mL；Chlorella cells are diluted to 1.0 × 15 by deionized water⁶A/mL；Then by large intestine bar Bacterium cell and chlorella cells mixing, the volume ratio of the two suspension are 1:2；Mixing suspension is then added drop-wise to sheet glass On, and sheet glass is put in spare on microscopical two-dimensional shift platform；

Step S3: installation conical fiber

Two conical fibers are respectively placed in the glass capillary of experimental provision, the end of two conical fibers is submerged in In cell suspending liquid；

The experimental provision, including photo-coupler, two glass capillaries, two adjusting brackets, translation stage, light power meter, CCD are aobvious Micro mirror, computer；Described two glass capillaries are separately fixed on two adjusting brackets, the centerline collineation of two glass tubes, and two The degree of regulation of a adjusting bracket is 50nm；It is translation stage between two glass capillaries, can be placed on translation stage equipped with suspension The sheet glass of liquid, glass capillary is interior to place optical fiber, and the optical fiber in the glass capillary of left side is connected to photo-coupler, right side capillary Optical fiber in glass tube is connected on light power meter, is a CCD microscope above translation stage, and CCD microscope can be connected to electricity Brain；The good visible light of the laser and monochromaticjty of near-infrared is connected to optical fiber by 2 × 1 photo-couplers；

Step S4: the assembling of cycling cells structure is detected with optical transmission performance

The laser of near-infrared is passed through into conical fiber, laser is focused in conical fiber end, further to neighbouring large intestine bar Bacterium cell generates optical gradient forces and realizes luminous power capture, and after capturing cell, laser can be transmitted further in cell, captures large intestine Bacillus；By mobile conical fiber, the Bacillus coli cells of capture are moved near chlorella cells, laser is in Escherichia coli The luminous power generated after the outgoing of end captures chlorella, so that chlorella and Escherichia coli be linked together；It is similar, it can be with More cells are captured, thus the cycling cells structure that package assembly is controllable；The laser of the near-infrared of transmission passes through right side taper Fiber coupling can carry out real-time optical transport detection into light power meter；The laser of near-infrared can make the period of assembling thin Born of the same parents' structure is stabilized in a liquid, the good visible light of monochromaticjty is coupled into conical fiber, it can be seen that laser is in light wave Lead middle transmission.

2. according to claim 1 a kind of based on the cyto-architectural method of luminous power assembling cycle, which is characterized in that the step In rapid S1, the range of partial-length is 25 ~ 45cm.

3. according to claim 1 a kind of based on the cyto-architectural method of luminous power assembling cycle, which is characterized in that the step In rapid S1, the optical fiber of flame upper end is broken using the speed of 11 ~ 20mm/s.

4. according to claim 1 a kind of based on the cyto-architectural method of luminous power assembling cycle, which is characterized in that the step In rapid S1, optical patchcord core diameter is 9 μm, and covering is 125 μm；The length of glass capillary is 120mm, internal diameter 0.9mm, tube wall Thick 0.1mm.

5. according to claim 1 a kind of based on the cyto-architectural method of luminous power assembling cycle, which is characterized in that the step The laser of near-infrared needs to meet cell and absorbs weak wavelength to it in rapid S4, so that the damage to cell is reduced, it is further excellent Select the laser, 808nm laser, 1064nm laser of 980nm；The monochromaticjty good visible light can further preferably 644nm The green light of feux rouges, the blue light of 473nm or 532nm.

6. according to claim 1 a kind of based on the cyto-architectural method of luminous power assembling cycle, which is characterized in that the step In rapid 4, Bacillus coli cells can connect single chlorella cells, also can connect two chlorella cells；According to assembling Cell chain length determines that the power of laser used, required power increase with needing the cell chain length assembled to increase, when When length is less than 100 μm, power is less than 60mW.

Technical field

The invention belongs to particles and biological cell assembled formation technical field, are related to a kind of based on luminous power assembling cycle cell The method of structure.

Background technique

Different cells are assembled into specified shape according to particular space arrangement to lead in organizational project, test cell line, cell It is all significant in many biomedical applications such as letter.The method for being conventionally used to cell assembling is mainly that the cells are fixed in thing On the substrate first formulated, such as micro- art of printing, photoetching, dielectrophoresis, electrochemical deposition etc..However, these methods generally require use To with fine microstructure substrate or electrode determine the position of cell, furthermore it is smart to lack unicellular control for these methods Degree.Although there are also the eucaryotic cell structure assembling that method can be realized unicellular precision, such as pen nano-photoetching art, holography Optical tweezer light capture technique, DNA encoding technology etc..But these methods can not achieve controllable groups while to variety classes cell Dress, to cannot achieve the signal communication research to variety classes cell.In addition, the eucaryotic cell structure of these methods assembling is fixed It in some position, cannot further manipulate, limit further applying for Cell microstructure.Although luminous power, especially light is terraced Power is spent, is applied to the capture, movement and manipulation of particle and cell, but there is no realize to particle and biological cell Controllable assembly.In order to solve these problems, the present invention provides one kind to be based on the cyto-architectural method of luminous power assembling cycle.

Summary of the invention

In order to achieve the above object, the present invention provides a kind of based on the cyto-architectural method of luminous power assembling cycle, solves Existing micro-structure substrate is complicated when carrying out cell assembling in the prior art, lacks unicellular control precision, can not achieve to not Eucaryotic cell structure with Controllable assembly while cell types, assembling is fixed on some position and not can be carried out asking of further manipulating Topic.

In order to solve the above technical problems, the technical scheme adopted by the invention is that, one kind being based on luminous power assembling cycle cell The method of structure, comprising the following steps:

One kind being based on the cyto-architectural method of luminous power assembling cycle, comprising the following steps:

Step S1: production conical fiber

The buffer layer and plastic wrapper that commercial single-mode fiber jumper is removed using optical fiber wire stripper, remove partial-length；It will removal The optical fiber of housing protrudes into glass capillary, end is pulled out after alcolhol burner flame envelope heats 155 ~ 160s, optical fiber starts to melt； Optical fiber is broken in flame upper end, optical fiber connector forms a tapered distal end, as conical fiber；Optical fiber tapered zone diameter At 1 ~ 10 μm, length is at 2 ~ 50 μm, and for conical fiber terminal bevel in 70 ~ 120o, this makes capture rate and manipulation sensitivity more It is high；

Step S2: it is equipped with mixing suspension

Escherichia coli are placed in LB culture medium in 37 DEG C of culture 12h, is then cleaned and is diluted with phosphoric acid hydrochloride buffer, Until concentration is 3.0 × 10⁶A/mL；Chlorella cells are diluted to 1.0 × 15 by deionized water⁶A/mL；Then by large intestine bar Bacterium cell and chlorella cells mixing, the volume ratio of the two suspension are 1:2；Mixing suspension is then added drop-wise to sheet glass On, and sheet glass is put in spare on microscopical two-dimensional shift platform；

Step S3: installation conical fiber

Two conical fibers are respectively placed in the glass capillary of experimental provision, the end of two conical fibers is submerged in In cell suspending liquid；

The experimental provision, including photo-coupler, two glass capillaries, two adjusting brackets, translation stage, light power meter, CCD are aobvious Micro mirror, computer；Described two glass capillaries are separately fixed on two adjusting brackets, the centerline collineation of two glass tubes, and two The degree of regulation of a adjusting bracket is 50nm；It is translation stage between two glass capillaries, can be placed on translation stage equipped with suspension The sheet glass of liquid, glass capillary is interior to place optical fiber, and the optical fiber in the glass capillary of left side is connected to photo-coupler, right side capillary Optical fiber in glass tube is connected on light power meter, is a CCD microscope above translation stage, and CCD microscope can be connected to electricity Brain；The good visible light of the laser and monochromaticjty of near-infrared is connected to optical fiber by 2 × 1 photo-couplers；

Step S4: the assembling of cycling cells structure is detected with optical transmission performance

The laser of near-infrared is passed through into conical fiber, laser is focused in conical fiber end, further to neighbouring large intestine bar Bacterium cell generates optical gradient forces and realizes luminous power capture, and after capturing cell, laser can be transmitted further in cell, captures large intestine Bacillus；(it is mobile that it is not limited to optical fiber herein, it is motionless to be also possible to optical fiber, and translation stage is dynamic or two by mobile conical fiber Person moves, but only this method of moving fiber has more controllability, and optical fiber can be moved to specified any position, realizes To specified cell capture and assembling, and easy to operate), the Bacillus coli cells of capture are moved near chlorella cells, The luminous power that laser generates after Escherichia coli end is emitted captures chlorella, so that chlorella and Escherichia coli are connected to one It rises；Similar, more cells can be captured, thus the cycling cells structure that package assembly is controllable；The near-infrared of transmission swashs Light is coupled in light power meter by right side conical fiber, can carry out real-time optical transport detection；The laser of near-infrared can be with It is stabilized the cycling cells structure of assembling in a liquid, the good visible light of monochromaticjty is coupled into conical fiber, it can be with See that laser transmits in optical waveguide.

Further, in the step S1, the range of partial-length is 25 ~ 45cm.

Further, in the step S1, the optical fiber of flame upper end is broken using the speed of 11 ~ 20mm/s.

Further, in the step S1, optical patchcord core diameter is 9 μm, and covering is 125 μm；The length of glass capillary For 120mm, internal diameter 0.9mm, thickness of pipe wall 0.1mm.

Further, the laser of near-infrared needs to meet cell and absorbs weak wavelength to it in the step S4, to subtract Few damage to cell, laser, 808nm laser, the 1064nm laser of further preferred 980nm；Good visible of the monochromaticjty Light can further preferably the feux rouges of 644nm, the blue light of 473nm or 532nm green light, these three laser be current laboratory most Common, monochromaticjty is good and production cost is minimum.

Further, in the step 4, Bacillus coli cells can connect single chlorella cells, also can connect two A chlorella cells；Determine that the power of laser used, required power are assembled with needs according to the cell chain length of assembling Cell chain length increases and increases, and when length is less than 100 μm, power is less than 60mW.

For step 1, " the removal fiber lengths selection of optical fiber wire stripper 25 ~ 45cm, optical patchcord are made in conical fiber Core diameter is 9 μm, and covering is 125 μm, the length of glass capillary is 120mm, internal diameter 0.9mm, thickness of pipe wall 0.1 mm, 11 ~ 20mm/s ", wherein " 25 ~ 45cm " is convenient for protruding into this section of optical fiber in glass capillary, to facilitate manipulation and fixed light It is fine；" optical patchcord core diameter is 9 μm, and covering is 125 μm, the length of glass capillary is 120mm, internal diameter 0.9mm, thickness of pipe wall 0.1mm " this be the intrinsic parameter of the intrinsic and used glass capillary of optical fiber；The draw rate of " 11 ~ 20mm/s " can pull out The fiber taper shape used in experiment, speed is too fast, and the shape of pull-out is difficult tapered, and speed is too slow, and the optical fiber of pull-out is too Carefully.

For step 4, the laser of 980nm can be used by capturing different cells, it is not limited to cell of the invention, The laser of 980nm is to be used to capture for generating luminous power, can also select the laser of other near-infrareds, such as 808nm, 1064nm Deng；And the laser of 644nm is transmitted in the cell that these are assembled for observing light, it, can also because 644nm is red laser To select other visible lights, such as the blue light of 473nm, the green light of 532nm.

The principle of the present invention is that the capture and assembling of cell are realized using optical scattering power, does not use light scattering force and spoke Penetrate power.Cell is captured under the action of optical gradient forces, and laser transmits in the cell of capture, further poly- in cell end Coke generates new optical gradient forces, to capture next cell, realizes the assembling of more many cells.Luminous power assembling cycle cell knot Structure is directly controlled by luminous power, does not need complicated micro Process structure, and the eucaryotic cell structure assembled is directly by moving fiber come real It is existing, there is unicellular precision controllability；Glass capillary can ensure that conical fiber protrudes into cell suspending liquid as the crow flies, moreover it is possible to make Conical fiber is conditioned the flexible adjusting position of frame；Right side conical fiber is connected on light power meter, is used to monitoring transmission at any time Optical signal；Microscope is used for real-time observation experiment process, and for CCD for capturing experiment picture, it is laggard that experimental data reaches computer The further analysis of row；Left side conical fiber is used for the detection of output optical signal for assembling eucaryotic cell structure, right side conical fiber； Wherein the laser of 980nm assembles cell for luminous power, and the laser of 644nm is for observing light in the cycling cells structure of assembling Transmit situation.

The beneficial effects of the present invention are:

The cycling cells structure assembling based entirely on luminous power is realized, assembling process is not need to rely on complicated micro-structure lining Bottom, the eucaryotic cell structure of assembling have controllable periodicity, and handling with unicellular precision, and the eucaryotic cell structure of assembling may be used also Flexible mobile to realize, the signal of transmission can be by real-time monitoring.Biological cell assembling is ground with field shaping technique To study carefully and provides new approaches, cell assembling process does not need complicated micro-nano manufacture craft, and assembling process is simple controllable, easy to operate, The eucaryotic cell structure of assembling can be manipulated flexibly.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with It obtains other drawings based on these drawings.

Fig. 1 is the cyto-architectural building flow chart (being followed successively by a, b, c, d) of luminous power assembling cycle.

Fig. 2 is the Experimental equipment for assembling eucaryotic cell structure and signal transduction and detection.

Fig. 3 is conical fiber scanning electron microscope (SEM) photograph.

Fig. 4 is the cycling cells structure and optical transmission performance phenogram (I and II in Fig. 4 of the different length of assembling Illustration gives the cyto-architectural enlarged drawing of assembling).

Fig. 5 is that the Escherichia coli of assembling connect the eucaryotic cell structure and optical transmission performance phenogram of two chlorellas.

Fig. 6 is the iuntercellular of assembling away from structure and optical transmission performance phenogram with arithmetic progression length.

Fig. 7 be cycling cells structure optical propagation loss curve graph (illustration be measured cyto-architectural signal Figure).

Fig. 8 is the cyto-architectural experimentation picture that mobile Escherichia coli connect single chlorella.

Fig. 9 is the cyto-architectural experimentation picture that mobile Escherichia coli connect two chlorellas.

Specific embodiment

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other Embodiment shall fall within the protection scope of the present invention.