CN109562377A

CN109562377A - The high resolution system, kit, device and method using Combined culture base strategy for high-flux microorganism application

Info

Publication number: CN109562377A
Application number: CN201780040231.7A
Authority: CN
Inventors: A·哈洛克; K·曾格勒; P·克里斯蒂
Original assignee: General Automation Experiment Technology Co
Current assignee: General Automation Experiment Technology Co
Priority date: 2016-06-30
Filing date: 2017-06-27
Publication date: 2019-04-02
Also published as: JP2019520826A; CA3027158A1; EP3478415A1; AU2017289053A1; SG11201811782VA; WO2018005389A1; IL263875A

Abstract

Provide a kind of method of Selective agar medium.This method includes obtaining the micromachining device comprising multiple micropores；A variety of different culture mediums are loaded into multiple micropores, so that each micropore of multiple micropores includes a kind of culture medium, and multiple micropores include a variety of different culture mediums；At least one cell from sample is loaded into each micropore of multiple micropores；Micromachining device is incubated for the scheduled duration in predefined conditions；The content of multiple micropores on more multiple micropores；With based on comparing, at least one culture medium is determined from a variety of different culture mediums.

Description

The high-resolution using Combined culture base strategy for high-flux microorganism application System, kit, device and method

Cross reference to related applications

The application is that U.S. Non-provisional Patent application the 15/135,377th part submitted on April 21st, 2016 is prolonged It is continuous, and U.S. Provisional Patent Application the 62/357th, 135 priority submitted on June 30th, 2016 is required, they are every One disclosure is incorporated herein by reference in their entirety.

Technical field

The disclosure relates generally to the innovations of microbiology, micro Process, chemistry, optics, robot and information technology.More Body, this disclosure relates to be for high-throughput culture, screening, separation, sampling and/or identification biological entities and/or nutrients System, equipment, kit and method.

Background technique

Traditional technology and tool for cultivating the biological entities from environment He other samples is usually slow, laborious and high It is expensive.Even if cell and other biological entity still cannot usually cultivate success using these technologies and tool, cause information and/ Or the missing of product chance.Equally, screening is used for specific metabolite, enzyme, protein, nucleic acid, phenotype, mutation, metabolic pathway, base Cause, adaptability, the biological entities group of ability and/or curative effect are challenging, need complicated and expensive method.For example, micro- life Object is lived in the extremely environment of high risk.In order to survive, microorganism has been developed that a set of wonderful biochemical tool, packet Include new enzyme, unique metabolite, innovation genetic approach, and manipulate the strategy of its environment and microorganism neighbours, these Strong solution can bring new opinion and product (from the antibiotic of rescue life to improvement food production and safety Fertilizer).

Invention summary

According to some embodiments, the disclosure is provided for simplifying culture process, supporting high flux screening and/or exploitation new Opinion and product microbiology system, equipment, kit and the method according to some embodiments.For example, equipment can be with Including for receiving the micromachining device for containing the sample of one or more cells.Micromachining device is for cultivating one or more The high density microwell array of a cell.

In some embodiments, the method for Selective agar medium is provided.This method includes that acquisition includes the micro- of multiple micropores Processing unit (plant)；A variety of different culture mediums are loaded into multiple micropores, so that each micropore of the multiple micropore includes one Kind culture medium, and the multiple micropore includes a variety of different culture mediums；At least one cell from sample is loaded into described In each micropore of multiple micropores；Micromachining device is incubated for the scheduled duration in predefined conditions；It is more multiple micro- The content of multiple micropores on hole；With based on comparing, at least one culture medium is determined from a variety of different culture mediums.

In some embodiments, the method using the micromachining device Selective agar medium comprising multiple micropores, institute are provided Stating micromachining device includes a variety of different culture mediums being loaded on multiple micropores.This method include will from sample at least one A cell is loaded into each of the multiple micropore；When micromachining device being incubated for scheduled continue in predefined conditions Between；The content of more multiple micropores；With based on comparing, at least one of a variety of different culture mediums culture medium is determined.

In some embodiments, the method using the micromachining device Selective agar medium comprising multiple micropores, institute are provided Stating micromachining device includes at least one cell of each for being loaded onto multiple micropores.This method comprises: by a variety of different trainings Feeding base is loaded on multiple micropores；Micromachining device is incubated for the scheduled duration in predefined conditions；More multiple micropores On multiple cells；With based on comparing, at least one of a variety of different culture mediums culture medium is determined.

In some embodiments, a kind of method is provided, comprising: obtain the micromachining device comprising multiple micropores；It will be more Kind different culture medium is loaded into multiple micropores, so that each micropore of the multiple micropore includes a kind of culture medium, and institute Stating multiple micropores includes a variety of different culture mediums；Target biological entities (for example, at least one cell) are loaded into the multiple In each micropore of micropore；The content of multiple micropores on more multiple micropores；With based on comparing, cultivated from a variety of differences Base determines at least one culture medium.

In the method for above-mentioned various embodiments, drop printer (droplet printer), which can be used, to be cultivated Base is loaded into each micropore.Equally, drop printer also can be used at least one cell is loaded into each micropore.Or Person can be used pipette and at least one cell be loaded into each micropore.The content of more multiple micropores may include Assess the property of at least one observable of the content of multiple micropores.In some embodiments, more multiple micropores is interior Tolerant includes carrying out digital image analysis to the image of the content of multiple micropores.Can before loading at least one cell or Culture medium is loaded later.Loading at least one cell may include that multiple cells are loaded into each micropore.

It includes that a variety of different culture mediums are loaded into micromachining device in multiple micropores that a variety of different culture mediums, which are loaded into, On individual region micropore in, wherein each individual region include more than one micropore so that in each individual region It including identical culture medium, and include different culture mediums in the micropore of different zones.In some such embodiments, The content of more multiple micropores includes the statistic of the property of the content in the micropore for compare each individual region.

Multiple micropores can be sealed before being incubated for micromachining device.It, can not be close in some such embodiments Multiple micropores are sealed to allow liquid evaporation before the content of more multiple micropores.

In some embodiments, the content of more multiple micropores may include assessing content at multiple time points At least one property.

In some embodiments, the culture medium being loaded at least one of multiple micropores may include nutrients.? In some embodiments, the culture medium being loaded at least one of multiple micropores includes antibiotic.

In some embodiments, a kind of kit is provided comprising: the micromachining device comprising multiple micropores；With add The a variety of different culture mediums being downloaded in multiple micropores, each of multiple micropores include the training of one of a variety of different culture mediums Support base.In this kit, in the micropore for the individual region that a variety of different culture mediums can be loaded into micromachining device, In each individual region include multiple micropores so that include identical culture medium in micropore in each individual region, and It and include different culture mediums in the micropore of different zones.

In method described herein or any embodiment of kit, the surface of multiple micropores of micromachining device is close Degree can be at least 150 micropore/cm², at least 250 micropore/cm², at least 400 micropore/cm², at least 500 micropores/ cm², at least 750 micropore/cm², at least 1000 micropore/cm², at least 2500 micropore/cm², at least 5000 micropore/cm²、 At least 7500 micropore/cm², at least 10000 micropore/cm², at least 50000 micropore/cm², at least 100000 micropores/ cm²Or at least 160000 micropore/cm².In method described herein or any embodiment of kit, micromachining device The diameters of each micropore of multiple micropores can be about 5 μm to about 500 μm, about 10 μm to about 300 μm or about 20 μm to about 200μm。

By checking that the following drawings and detailed description, other systems, method and feature to those skilled in the art will It is obvious.All these others systems, method and feature are intended to be included in this specification, in the scope of the present invention It is interior, and protected by the attached claims.

Detailed description of the invention

It will be understood by those skilled in the art that attached drawing being mainly described property purpose, and it is not intended to limit invention described herein The range of theme.

The drawings are not necessarily drawn to scale；Under some cases, the various aspects of invention disclosed herein theme may be attached It is exaggerated in figure or amplifies display to promote the understanding to different characteristic.In the accompanying drawings, similar reference character generally refers to similar Feature (for example, function similar and/or the similar element of structure).

Fig. 1 is the perspective view for illustrating micromachining device or chip according to some embodiments.

Fig. 2A -2C is the top view of dimension for illustrating the micromachining device or chip according to some embodiments, side respectively View and end-view.

Fig. 3 A and 3B are illustrated according to the micromachining device of some embodiments or the decomposition view of chip and top view respectively Figure.

Fig. 4 A and 4B are the schematic diagrames for illustrating the film according to some embodiments.Fig. 4 C is the film according to some embodiments The image on surface, the film surface have the mark formed and contacting with hole array.

Fig. 5 A is the flow chart illustrated according to some embodiments from the cellifugal method of sample point.Fig. 5 B is to illustrate basis Schematic diagram of some embodiments from the cellifugal method of pedotheque point.

Fig. 6 is the flow chart for illustrating to separate and cultivate from sample according to some embodiments the method for cell.

Fig. 7 is the schematic diagram for illustrating to separate and cultivate from complex sample according to some embodiments the method for cell.

Fig. 8 A-8C is the schematic diagram for illustrating to be chosen according to some embodiments by a needle or multiple needles.

Fig. 9 A-9D is to show the image that hole is chosen according to some embodiments.

Figure 10 A-10D is the schematic diagram for illustrating to be used to choose according to some embodiments the tool of chip.

Figure 11 is the image in the hole chosen by thin layer agar, illustrates to pass through film or sealing according to some embodiments The selection of layer.

Figure 12 is the schematic diagram for illustrating the cross section of the chip 1200 according to some embodiments.

Figure 13 is the flow chart illustrated according to some embodiments for the method for screening.

Figure 14 is the schematic diagram for illustrating the screening technique according to some embodiments.

Figure 15 is a series of image of explanations according to the screening example of some embodiments.

Figure 16 A-16C is the image illustrated according to some embodiments from screening recycling.

Figure 17 A is the exploded view for illustrating the chip for screening according to some embodiments.Figure 17 B is according to some realities The fluorescent image of chip after applying option screening.Figure 17 C is to show according to some embodiments, chooses sample from chip after screening The image of the process of product.

Figure 18 is the flow chart for illustrating the method for counting according to some embodiments.

Figure 19 is the schematic diagram for illustrating the method for counting according to some embodiments.

Figure 20 is the schematic diagram for illustrating the directory system according to some embodiments.

Figure 21 A-21E is the schematic diagram for illustrating the chip for having hole specific chemical object according to some embodiments.

Figure 22 A and 22B are the different culture mediums printed in the different micropores of micro Process chip according to some embodiments Image.

Figure 23 A depicts the micropore for being divided into trizonal micromachining device, each region includes that 3x9 micropore is used in combination Different culture medium processing；Figure 23 B-23D is the micro-image of the representative micropore in each region shown in Figure 23 A.

Figure 24 is the digital image analysis knot for showing the average gray value according to some embodiments based on micropore content The figure of fruit.

Detailed description of the invention

The disclosure relates generally to for separating, cultivating, adaptability, sampling and/or screening biological entities and/or nutrients System, kit, device and method.It discloses for receiving comprising at least one biological entities (for example, at least one is thin Born of the same parents) sample micromachining device (or " chip ").Term " biological entities " can include but is not limited to organism, cell, thin Born of the same parents' ingredient, cellular products and virus, term " type " can be used for describing a taxon, including but not limited to activity classification Unit (OTU), genotype, phylotype, phenotype, the ecotype, history, behavior or interaction, product, variant and evolution are upper significant Unit.

Cell can be Archimycetes, bacterium and eucaryote (such as fungi).For example, cell can be microorganism, such as Aerobic, anaerobism or facultative aerobic microbiological.Virus can be bacteriophage.Other cell component/products can include but is not limited to It is protein, amino acid, enzyme, polysaccharide, atriphos (ATP), lipid, nucleic acid (such as DNA or RNA), nucleosides, nucleotide, thin After birth/cell wall, flagellum, pili, organelle, metabolin, vitamin, hormone, neurotransmitter and antibody.

Nutrients can be determining (such as culture medium chemically determine or synthesis) or uncertain (such as basis Or complex medium).Nutrients may include culture medium that either laboratory is prepared and/or that business manufactures (for example, two kinds Or the mixture of a variety of chemicals) ingredient.Nutrients may include either liquid nutrient media (i.e. nutrient broth), example The ingredient of the raw bacterial context soup in such as sea, bacteriolyze meat soup (such as Luria meat soup).Nutrients may include either fluid nutrient medium Ingredient is mixed to form solid medium and/or commercially available manufacture agar plate, such as blood agar with agar.

Nutrients may include or the ingredient of selective medium.For example, Selective agar medium can be used for only growing it is certain Biological entities or the biological entities only with certain properties (such as antibiotic-resistant or the certain metabolins of synthesis).Nutrients can wrap It includes or the ingredient of differential medium, is deposited by using specific indicator (such as dimethyl diaminophenazine chloride, phenol red, eosin Y or methylenum careuleum) Biochemical character under distinguishes a kind of biological entities and another biological entities or other kind of biological entities.

Nutrients may include or the ingredient of the extract from natural surroundings or culture medium.For example, nutrients can be with From the environment natural to certain types of biological entities, varying environment or multiple environment.Environment can include but is not limited to one Kind or a variety of biological tissues (such as connective tissue, muscle, nerve, epithelium, plant epidermis, blood vessel, elementary organization etc.), biology stream Body or other biological product (such as amniotic fluid, bile, blood, cerebrospinal fluid, earwax, exudate, fecal matter, gastric juice, interstitial fluid, Intracellular fluid, lymph, milk, mucus, rumen content, saliva, sebum, sperm, sweat, urine, vaginal fluid, vomitus Deng), microbial suspension, air (including such as gas with various content), supercritical carbon dioxide, soil (including such as mineral Matter, organic matter, gas, liquid, organism etc.), deposit (such as agricultural, ocean etc.), living organism (such as plant Object, insect, other small organisms and microorganism), dead organic matter, feed (such as herbage, beans, ensilage, crop residues Deng), mineral, oil or oil product (such as animal, plant, petrochemical industry), water (such as natural fresh water, drinking water, seawater etc.) And/or sewage (such as health, business, industry and/or agricultural effluent and rainwash).

Micromachining device can define the high density microwell array for cultivating at least one biological entities.Term is " highly dense Degree " can refer to the ability that system or method are distributed many experiments in constant region domains.For example, including " high density " experimental considerations unit Micromachining device may include about 150 micropore/cm²To about 160,000 micropores or more/cm², discussed further below. Other embodiments are shown in table 1.

Table 1

Micromachining device may include a series of substrate with functional layers.The functional layer of the series may include first Functional layer defines the experimental considerations unit (for example, hole) and at least one subsequent functional layer of the first array, defines aforementioned function The experimental considerations unit (such as micropore) of the subsequent array of each experimental considerations unit of layer.Each experimental considerations unit can be set to receive and train Support and/or screen biological entities and/or nutrients.Specifically, system as described herein, kit, device and method can be used for Automation and/or high flux screening are directed to the different condition of high-density cells matrix.For example, system as described herein, kit, Device and method can be used for the effect tested and more one or more different nutrients grow microorganism, and/or screening generation Thank object, enzymatic activity, mutation or other cell characteristics.

Fig. 1 is the perspective view for illustrating micromachining device or chip according to some embodiments.Chip 100 includes with micro- The substrate of mirror format slide forming, upper surface 102 have injection moulding feature.Feature includes four separated microwell arrays (microarray) 104 and injection label 106.Micropore in each microarray is arranged with grid pattern, in the edge of chip 100 week Enclose has seamless edge between microarray 104.

Fig. 2A -2C is top view, side view and the end view for illustrating the dimension of the chip 100 according to some embodiments respectively Figure.In Fig. 2A, the top surface of chip 100 is about 25.5mm multiplied by 75.5mm.In Fig. 2 B, the end of chip 100 is about that 25.5mm multiplies With 0.8mm.In Fig. 2 C, the side of chip 100 is about 75.5mm multiplied by 0.8mm.

It, can be at least part of micromachining device application film after micromachining device is added in sample.Fig. 3 A is from root According to the decomposition view of micromachining device 300 shown by top view in Fig. 3 B of some embodiments.Device 300 includes accommodating example Such as the chip of the array with hole 302 of edaphon.Film 304 is placed in above 302 array of hole.Gasket 306 is placed on film 304 Side.Polycarbonate cap 308 with filling hole 310 is placed in 306 top of gasket.Finally, applying band 312 to lid 308.

Film can cover at least part of micromachining device, including one or more experimental considerations units, hole or micropore.Example Such as, after micromachining device being added in sample, at least a film can be applied at least one micropore of high density microwell array.It can To apply multiple films to the multiple portions of micromachining device.For example, can be applied to the individual branch of high density microwell array Individual film.

Film can be connected, attachment, partially attachment, fixed, sealing and/or part are sealed to micromachining device, near A kind of few biological entities are retained at least one micropore of high density microwell array.It is it is, for example, possible to use being laminated that film is reversible Ground is fixed to micromachining device.Film can be punctured, be removed, separated, is partially separated, removed and/or part is removed to obtain height At least one of at least one micropore of density microwell array biological entities.

A part of cell mass at least one experimental considerations unit, hole or micropore can be attached on film (via such as inhaling It is attached).If so, the cell mass at least one experimental considerations unit, hole or micropore can be sampled by stripping film, so that at least one The part cell mass in a experimental considerations unit, hole or micropore is still attached on film.

Fig. 4 A and 4B are the schematic diagrames for illustrating the film according to some embodiments.Fig. 4 A shows the side view of chip 400, Define filled with content hole array and in hole array encapsulating chip 400 film 402 so that when from chip 400 remove When, the surface of the film 402 contacted with chip 400 with the trace in each hole and has in the hole of attachment (such as sticking) thereon Tolerant sample, as shown in Figure 4 B.Fig. 4 C is that have the film table that the trace to be formed is contacted with hole array according to some embodiments The image in face.

Film can be it is the infiltration of impermeable, semipermeable, permselective, otherness and/or partial penetration, In at least one micropore to allow at least one nutrients diffusion high density microwell array.For example, film may include natural Material and/or synthetic material.In some embodiments, the sufficiently small film in selection aperture will be will at least protect some or all cells It stays in micropore.For mammalian cell, aperture can be several microns, and still retain cell.However, in some embodiment party In case, aperture can be less than or equal to about 0.2 μm, such as 0.1 μm.Film diameter and aperture depend on material.It is, for example, possible to use Hydrophily polycarbonate membrane, diameter can be about 10mm to about 3000mm, and aperture can be about 0.01 μm to about 30.0 μm.No The aperture of permeable membrane is close to 0.In the embodiment with impermeable membrane, any nutrients must provide before being sealed with film In micropore.Gas-permeable but the impermeable film of liquid may allow that oxygen enters micropore and carbon dioxide goes out from micropore It goes.Film can have labyrinth, and aperture can determine or not know.However, hole can be nanoscale.Selective membrane its His factor may include cost, sealability and/or sterilization capability.

Substrate can define the micro channel array that the second surface opposite with the first surface is extended to from first surface. Microchannel can have the first opening in first surface and the second opening in second surface.It can be at least part of The first film is applied on one surface, so that at least some of at least one microchannel cell mass is attached to the first film.It can be at least The second surface of a part applies the second detachable film, so that at least some of at least one microchannel cell mass is attached to the Two films.The cell mass at least one microchannel is sampled by removing the first film, so that at least one at least one microchannel A little cell masses are still adhered to the first film, and/or the second film of removing, so that at least some of at least one microchannel cell mass It is still adhered to the second film.

Term " high throughput " can refer to that system or method can be realized the quick execution of parallel or sequence many experiments Ability.The example of " high throughput " system may include there is the automation equipment of cytobiology technology with prepare, be incubated for and/ Or a large amount of chemistry, heredity, pharmacology, optics and/or image analysis are carried out, to screen one or more biological entities extremely A kind of few metabolin, enzyme, protein, nucleic acid, phenotype, mutation, metabolic pathway, gene, adaptability and ability, as begged for herein By.According to some embodiments, " high throughput " can refer to that scale is at least about 96 experiments at least about 10,000,000 While experiment or almost experiment simultaneously.

System disclosed herein, kit, device and method can be used for for biological entities (such as cell) matrix not With the high flux screening of condition." Kong Zhongkong " concept can pass through manufacture (for example, micro Process) any need or desired level Substrate with multiple function layers or chip realize (that is, hole in hole in hole in hole).First functional layer can be with Define experimental considerations unit (such as hole) array.Each experimental considerations unit is indicated by defining subsequent experimental considerations unit (such as micropore) array Second functional layer.This allows to carry out multiple experiments or test simultaneously on a single chip, to allow high-throughput operation.

For example, in figures 3 a and 3b, gasket 306 is placed in 304 top of film, the film 304 is applied to according to some embodiment party The array in the hole 302 on the micromachining device 300 of case.Gasket 306 only has an opening.However, in further embodiment party In case, it can be placed above device (with or without film) multiple compared with minipad or with more than one with smaller opening The single gasket of a smaller opening, to form the functional layer being located therein or the larger experimental considerations unit battle array with subsequent functional layer Column or subsequent experimental considerations unit array (such as hole 302).

Using multiple function layers, such as can simultaneously or almost simultaneously test more than a kind of nutrients or nutritional preparation.Example Such as, same format can be used to screen the special ability of metabolin or cell or avoid making microbial compared with other nutrients Rely on nutrients derived from environment.

Experimental considerations unit is the predetermined site on micromachining device surface.For example, can be with the surface of design chips to consolidate cell It is scheduled in the predetermined site of the first array.These predetermined sites can be hole, micropore, microchannel and/or specified fixation site. For example, surface can be manufactured to define microwell array.It can be by determining wall or addition wall by array part in substrate.Example Such as, surface can be manufactured to define the hole of the first array first, wherein manufacturing the inner surface in each hole again to define second array Micropore, microchannel or fixed site.In another example, surface can be manufactured to define microwell array, answered to the surface With another substrate (such as agar, plastics or another material) to be separated by the surface and by the micropore that it is defined.Each hole, Micropore, microchannel and/or fixed site can be set to receive and grow at least one cell；However, in use, it is any to give Fixed hole, micropore, microchannel or fixed site can actually receive or not receive and/or grow one or more cells.It is real The type of verification certificate member is commutative.For example, the embodiment of micropore explicitly described herein is also intended to open wherein micropore at least partly The embodiment for replacing with microchannel, fixed site and/or other kinds of experimental considerations unit.

One or more parts of micromachining device can be selected with surface chemical modification agent, processing and/or coating are to have There is specific surface chemistry.For example, the substrate surface of at least one portion can be set to first surface feature or second Surface characteristics, the first surface feature repel cell and/or reduce the tendency of cell adherence on the surface, the second surface Feature attracts cell and/or increases the tendency of cell adherence on the surface.According to the type of target cell, material and/or coating can To be hydrophobic and/or hydrophilic.At least part top surface of substrate be can handle to repel target with first surface feature The tendency of cell and/or the adherency of reduction target cell on the surface.Meanwhile it can handle each experimental considerations unit, hole or micropore at least A part of inner surface occupies experimental considerations unit, hole or micro- to attract target cell and/or increase target cell with second surface feature The tendency in hole.Substrate surface can have multiple portions, with different surfaces feature.

Chemical vapor deposition, electroporation, corona treatment and/or electrochemical deposition Applied Surface Chemistry can be used to change Property agent.Surface chemical modification agent can control surface potential, Lund current potential, zeta potential, configuration of surface, hydrophobicity and/or hydrophilic Property.Surface chemical modification agent may include salt water, polyelectrolyte, metal, polymer, antibody and/or plasma.For example, table Face chemical modifier may include octadecyl trichlorosilane alkane.Surface chemical modification agent may include dynamic copolymers, such as poly- Ethylene oxide (20) sorbitan mono-laurate and/or polyethylene glycol P (1,13,3- tetramethyl butyl)-phenyl ether.It comes to the surface Learning modifying agent may include static copolymer, such as poloxamer188, poly (L-lysine), and/or the poly- (L- of poly(ethylene glycol)- Lysine) block copolymer.

Equipment for screening the different condition for being directed to cellular matrices may include with the surface for defining microwell array Substrate.The part of microwell array is segmented into time array (such as by large hole or wall).Substrate can be with micro Process.Each micropore It can receive and grow at least one biological entities (such as cell).The matrix of gained biological entities (such as cell) can be life The high-density matrix of object entity.First array and/or second array can be plane, substantially planar and/or more planes (such as on idler wheel).

Term " high-resolution " can refer to that system or method distinguish the ability that can must largely test.For example, " high-resolution The system or method of rate " can select an experimental considerations unit from the micromachining device comprising high density experimental considerations unit, wherein described The diameter of experimental considerations unit is about 1nm to about 800 μm.The substrate of micromachining device or chip may include about or more than 10,000, 000 micropore.For example, microwell array may include at least 96 positions, at least 1,000 position, at least 5,000 position, At least 10,000 positions, at least 50,000 positions, at least 100,000 positions, at least 500,000 positions, at least 1, 000,000 position, at least 5,000,000 positions or at least 10,000,000 positions.

The superficial density of micropore is about 150 micropore/cm²To about 160,000 micropore/cm²Or it is higher.Micromachining device Or the micropore surface density of the substrate of chip is at least 150 micropore/cm², at least 250 micropore/cm², at least 400 micropores/ cm², at least 500 micropore/cm², at least 750 micropore/cm², at least 1,000 micropore/cm², at least 2,500 micropores/ cm², at least 5,000 micropore/cm², at least 7,500 micropore/cm², at least 10,000 micropore/cm², at least 50,000 Micropore/cm², at least 100,000 micropore/cm²Or at least 160,000 micropore/cm²。

The size of micropore can be from nanoscale (such as diameter is about 1 to about 100 nanometer) to micron order or bigger.For example, The diameter of each micropore can be 1 μm to about 800 μm, diameter be about 25 μm to about 500 μm or diameter be about 30 μm to about 100 μm. The diameter of micropore can be about or less than 1 μm, about or less than 5 μm, about or less than 10 μm, about or less than 25 μm, about or less than 50 μ M, about or less than 100 μm, about or less than 200 μm, about or less than 300 μm, about or less than 400 μm, about or less than 500 μm, about or Less than 600 μm, about or less than 700 μm or about or less than 800 μm.

The depth of micropore can be about 500 μm to about 5000 μm, depth be about 1 μm to about 500 μm or depth be about 25 μm To about 100 μm.The depth of micropore can be about 1 μm, about 5 μm, about 10 μm, about 25 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 1000 μm, about 1,500 μm, about 2,000 μm, about 3,000 μm or about 5,000 μm.

The opening of each micropore or cross section can be any shape, such as round, hexagon or square.Each micropore can be with Including side wall.It is not circular micropore for opening or cross section, the diameter of micropore as described herein refers to same homalographic Annular effective diameter.For example, the square micropore for being 10x10 microns for side length, having same homalographic, (100 squares micro- Rice) ring diameter be 11.3 microns.The cross-sectional view of side wall can be straight, oblique, and/or curved.It can be in high density The unique location specific label of at least one described further below is put at least one micropore of microwell array to promote Into contacting for identifying species and type and the specific micropore of high density microwell array.At least one unique label can be put into And/or it is placed in at least side of micropore bottom and/or micropore.At least one unique label may include nucleic acid molecules, have For the sum with target specificity nucleotide sequence with the annealing of the target nucleic acids segment of at least one biological entities for reflecting Determine the location specific nucleotide sequence of at least one micropore of high density microwell array.

For example, the substrate of micromachining device or chip can have having a size of from about 4 inches multiplied by 4 inches of surface.Surface The array of about 100,000,000 micropores can be defined.Microwell array can be divided into about 100 subdivisions by wall and/or substrate can be with boundary The array in 100 holes is concludeed a contract or treaty, wherein a total of about 100,000,000 micropores of about 1,000,000 defined in each subdivision or hole micropore.It is testing Under the service condition of different nutrients, the microorganism from environmental sample can be loaded on chip so that single microbial or Microorganism cluster is divided into the micropore of chip, and each micropore is located at the bottom of large hole.Each large hole may include experimental considerations unit, make Can parallel on the same chip or about 100 kinds of different nutrients of sequential testing, wherein each hole provides up to 1,000,000 surveys Try example.

Target cell can be Archimycetes, bacterium and eucaryote (such as fungi, plant or animal).For example, target cell can To be microorganism, such as aerobic, anaerobism and/or facultative aerobic microbiological.It can parallel on the composition of target cell or sequence Test different nutrients are to analyze and more such as growth to cell mass, cell component and/or cellular products or other influences. It can screen cell component, product and/or the ability of target cell composition, such as one or more viral (such as bacteriophages), Cell surface (such as cell membrane or cell wall), metabolin, vitamin, hormone, neurotransmitter, antibody, amino acid, enzyme, albumen Matter, polysaccharide, ATP, lipid, nucleosides, nucleotide, nucleic acid (such as DNA or RNA), phenotype, mutation, metabolic pathway, gene and adaptation Property.

Cell composition may include environmental sample extract and/or diluent.Environmental sample extract and/or diluent Can include but is not limited to one or more biological tissues (such as connective tissue, muscle, nerve, epithelium, plant epidermis, blood vessel, Elementary organization etc.), biofluid or other biological product (such as amniotic fluid, bile, blood, cerebrospinal fluid, earwax, exudate, excrement Object, gastric juice, interstitial fluid, intracellular fluid, lymph, milk, mucus, rumen content, saliva, sebum, sperm, sweat, urine, Vaginal fluid, vomitus etc.), microbial suspension, air (including such as gas with various content), supercritical carbon dioxide, Soil (including such as minerals, organic matter, gas, liquid, organism etc.), is lived at deposit (such as agricultural, ocean etc.) Organic matter (such as plant, insect, other small organisms and microorganism), dead organic matter, feed (such as herbage, beans, blueness Store feed, crop residues etc.), mineral, oil or oil product (such as animal, plant, petrochemical industry), water (such as natural fresh water, Drinking water, seawater etc.) and/or sewage (such as health, business, industry and/or agricultural effluent and rainwash).

To before the celliferous composition of micromachining device application (such as load) packet, method may include thin by combining Born of the same parents and environmental sample extract and/or diluent prepare composition.Method may further include liquefaction environmental sample extract And/or diluent.The concentration of cell in composition can be adjusted to the target of one each experimental considerations unit, hole or micropore cell Distribution.

If sample includes cell and/or virus, can be after applying to micromachining device by the cell cracking in sample To discharge nucleic acid molecules.It can be split with chemical treatment such as alkali exposure, detergent, ultrasonic wave, protease enzyme K or lysozyme exposure Solve cell.It can also be by heating lytic cell.

Fig. 5 A is the flow chart illustrated according to some embodiments from the cellifugal method of sample point.In step 500, it obtains Obtain sample.In step 502, at least one physical technique (such as mixing and/or ultrasonic treatment) and chemical technology (such as chela Mixture, detergent and/or enzyme) sample is homogenized and/or dispersed.In step 504, using for exampleIt is non- Particulate media (can obtain from Progen Biotechnik GmbH, Heidelberg, Germany) is separated by density centrifugation Cell in homogeneous and/or dispersion sample.

Fig. 5 B is the schematic diagram illustrated according to some embodiments from the cellifugal method of pedotheque point.Small Figure 50 6 shows Pedotheque is gone out.Small Figure 50 8 shows the homogeneous in test tube and/or dispersion sample.After small Figure 51 0 shows centrifugation Sample is divided into solvable fragment 512, cell 514,516 and of insoluble fragment518。

Fig. 6 is the flow chart for illustrating to separate and cultivate from sample according to some embodiments the method for cell.In step 600 In, obtain sample.In step 602, from least one cell of gained sample extraction.In step 604, in micromachining device or The cell of at least one extraction is loaded at least one high density microwell array of chip.Step 604 may include at least one The cell preparation cell concentration object of a extraction selects at least one nutrients/culture medium, and/or a selection at least film.In step In rapid 606, at least part of microwell array is sealed so that cell concentration object to be retained in micropore with the film of at least one selection. In step 608, it is incubated for chip.Step 608 may include selection temperature, determine atmosphere (such as aerobic or anaerobism), and/or incubate Educate the time.In step 610, according to method described herein, chip is separated and/or is replicated and (uses such as Chooser), obtained Obtain the culture cell of two parts.For example, an at least film can be removed, so that a part culture cell still adheres to, or stripping From or puncture to sample culture cell.In operating procedure 612, a part culture cell is put to death for identifying.Step 612 can be with It is analyzed including PCR, sequencing and/or various data.In step 614, aimed strain is identified.Can with such as aimed strain and/ Or the culture cell of remainder is further cultivated, tested and/or is identified.

Fig. 7 is the schematic diagram for illustrating to separate and cultivate from complex sample according to some embodiments the method for cell.Small figure 700 show the example of complex sample, especially micropopulation sample 702 and pedotheque 704.In small Figure 70 6, use example Scheme as shown in Fig. 5 A and 5B is from least one cell of sample extraction.In small Figure 70 8, by the cell of at least one extraction (and any environmental extract and/or diluent) is loaded into micromachining device or core at least one high density microwell array Piece 710.Chip 710 and kit 712 can be put into couveuse 714.Reagent can be used for adding liquid with maintain growth and/or The nutritional need of various screening purposes.Small Figure 71 6 shows output: cultivating the isolated bacterial strain of cell.

The type or hierarchical classification of the cell or microorganism that grow in identification micropore need skill including but not limited to below Art: DNA sequencing, nucleic acid hybridization, mass spectrum, infrared spectroscopy, DNA cloning and antibody combine, to identify genetic elements or other types Identifier.Many identification methods and processing step kill microorganism, to prevent the further culture and research of objective microbe. In order to identification of cell or microorganism, while subsequent culture, research and the further exhaustive for allowing specific objective to clone, Design further embodiment is used to sample each experimental considerations unit, hole or the micropore on substrate or chip, while keeping testing The locally completeness and separation of the micropopulation of unit, hole or micropore.

Substrate as described above can permit using further system, kit, device and method sampling cell mass.Example It such as, can be with based first surface application selecting device.The apparatus may include at least one protrusions towards first surface. The diameter of at least one protrusion is less than the opening diameter of each micropore, hole or experimental considerations unit.At least one protrusion can be inserted Enter to accommodate at least one micropore, hole or the experimental considerations unit of cell mass, so that one at least one micropore, hole or experimental considerations unit Divide cell mass adherency and/or is attached at least one described protrusion.It can be taken out by removing device from the first surface of substrate Cell mass sample at least one micropore, hole or experimental considerations unit, so that one at least one micropore, hole or experimental considerations unit Divide cell mass adherency and/or is attached at least one described protrusion.What each protrusion can be needle or multiple needles assembles object.

Fig. 8 A-8C is the schematic diagram for illustrating to be chosen according to some embodiments by a needle or multiple needles.Chip is provided 800 choose for observing via microscope 802, and via control device 804 is chosen.In fig. 8 a, control device 804 is chosen to wrap Containing the arm 806 with single needle.In the fig. 8b, the arm 808 with multiple needles is shown.Fig. 8 C is the saturating of chip during choosing View.

Fig. 9 A-9D is to show the image that hole is chosen according to some embodiments.In figure 9 a, Kong Shiman.In figures 9 b and 9, Needle is moved in position.In Fig. 9 C, hole is chosen.In Fig. 9 D, sample is taken out from hole.

Figure 10 A-10D is to show the schematic diagram for being used to choose the tool of chip according to some embodiments.In Figure 10 A, Tool comprising multiple needles is aligned with the chip with multiple holes.In fig. 1 ob, reduction tool immerses needle in hole.? In Figure 10 C, the needle with attachment sample is shown, and sample is transferred to new chip.Alternatively, in figure 10d, quickly overturning Tool is retained in sample with tool sheet.

Alternatively, when at least one protrusion is inserted at least one micropore, hole or experimental considerations unit, at least one described micropore, A part of cell mass volume upward displacement in hole or experimental considerations unit and at least one protrusion is surrounded, so that at least some volumes are moved The part of position is on the first surface of substrate and/or on the inner surface of at least one micropore, hole or experimental considerations unit.Method is also wrapped It includes and cell mass is sampled at least one micropore by the cell mass for collecting at least some volume displacement parts.

Similar selecting device can be applied to the second surface opposite with the first surface of substrate.The apparatus may include At least one protrusion towards second surface.The diameter of at least one protrusion is approximately equal to or less than at least one micropore, hole Or the diameter of experimental considerations unit.At least one described protrusion is pressed against on the second surface and is accommodated described in cell mass at least One micropore, hole or the corresponding position of experimental considerations unit, and/or insertion accommodate at least one described micropore, the Kong Huo of cell mass In experimental considerations unit, so that a part of cell mass at least one micropore, hole or experimental considerations unit is moved to the first surface of substrate And/or on the inner surface of at least one micropore, hole or experimental considerations unit.Then the cell mass of movable part can be collected.Cell mass It can be located on the plug (such as hydrogel or other soft materials such as agar) at least one experimental considerations unit, hole or micropore, make Proper when at least one protrusion being pressed against second surface and being inserted at least one micropore, plug is moved to move described Partial cell mass.

Cell mass sample from least one experimental considerations unit, hole or micropore can be placed in the second position.Further Before sampling, at least one protrusion can be cleaned and/or be sterilized.In order to be conducive to cell attachment surface characteristics, at least one At least part of a protrusion can be made of the material for being handled and/or being coated with surface chemical modification agent.At least one protrusion It can be array of protrusions.When based first surface application apparatus, the array of protrusions can be inserted corresponding experimental considerations unit, Hole or microwell array.Number of projection can correspond to the quantity of experimental considerations unit in the first array, one second in array of protrusions In microwell array in the quantity or substrate of micropore micropore sum.

Another device for sampling cell mass in substrate includes at least one needle and/or nanometer towards first surface Suction pipe.At least one needle and/or the overall diameter of nanopipette are less than the opening diameter of each micropore, and its interior diameter can accommodate target Cell dia.At least one needle and/or nanopipette insertion accommodate at least one experimental considerations unit, hole or the micropore of cell mass. A part of cell mass is taken out described at least one from least one described experimental considerations unit, hole or micropore introducing device using pressure Cell mass sample in a experimental considerations unit, hole or micropore.

Cell mass sample from least one experimental considerations unit, hole or micropore can be placed in the second position.Further Before sampling, at least one needle and/or nanopipette can be cleaned and/or be sterilized.At least one needle and/or nanopipette can To be needle and/or nanopipette array.When to the first surface application apparatus of micro Process substrate, the needle and/or nanopipette Corresponding experimental considerations unit, hole or microwell array can be inserted in array.Needle and/or nanopipette in needle and/or nanopipette array Quantity can correspond to the quantity of experimental considerations unit in the first array, in second microwell array in the quantity or substrate of micropore The sum of micropore.

Another method for sampling cell mass in substrate includes at least one reality for accommodating the cell mass in liquid The acoustic wave energy that verification certificate member, hole or micropore application focus.Can by effectively from least one micropore spray drop in a manner of application The acoustic wave energy of focusing, such as sound drop injection (ADE) is (see, for example, Sackmann etc., " Acoustical Micro-and Nanofluidics:Synthesis,Assembly and Other Applications,"Proceedings Of the 4th European Conference on Microfluidics (in December, 2014)).The drop may include The sample of cell mass at least one experimental considerations unit, hole or micropore.The drop can import second container or surface or substrate.

Substrate may include at least first and second, and described first includes at least part first surface, described Second includes at least part second surface.Described first and second along at least part and the first surface and The parallel plane of second surface is detachably connected.The plane divides experimental considerations unit, hole or micropore.Pass through disassembly first and the Two cell masses sampled at least one experimental considerations unit, hole or micropore, so that at least one experimental considerations unit, hole or micropore The cell mass of first part is still adhered to first, and the second part at least one experimental considerations unit, hole or micropore is thin Born of the same parents group is still adhered to second.

Figure 12 is the schematic diagram for illustrating the cross section of the chip 1200 according to some embodiments.Chip 1200 includes defining The substrate of 1202 array of hole filled with content 1204.Substrate includes first 1206 and second 1208.First 1206 With second 1208 along with 1202 array parallel of hole and divide the plane 1210 of 1202 array of hole equally and be detachably connected.When first 1206 and when second 1208 disassembly, hole 1202 and its content 1204 are separated, and two parts of contents 1204 are caused, and retain core The isolation of content 1204 and position on piece 1200.

Each micropore, experimental considerations unit or microchannel may include partial barrier, by micropore, experimental considerations unit or channel portion It is separated into first part and bottom part, cell is grown in both first part and bottom part.It is thin in sampling Before born of the same parents group, the above method may include the cell cluster in dispersion and/or reduction cell mass.In dispersion and/or reduction cell mass Cell cluster can include but is not limited to using ultrasonic wave, oscillation and dispersed with little particle.

The above method, which may further include, is deposited into from least one experimental considerations unit, hole or micropore for cell mass sample Two positions.The second position can be corresponding experimental considerations unit, hole or microwell array.The second position can be single container.It can be with Retain the cell mass sample from least one experimental considerations unit, hole or micropore for then cultivating.Alternatively, can retain from extremely The remaining cell of the cell mass of few an experimental considerations unit, hole or micropore is for then cultivating.

The above method may further include from the remaining cell of cell mass sample and/or cell mass and identify that at least one is thin Born of the same parents.This may include carrying out DNA, cDNA and/or RNA amplification, DNA and/or RNA sequencing, nucleic acid hybridization, mass spectrum and/or antibody In conjunction with.Or or it is additional, this may include experimental considerations unit, hole or the micropore for identifying at least one cell origin.Packet can be used The unique label of the nucleotide sequence containing location specific marks each experimental considerations unit, hole or micropore.For identification experiment unit, Kong Huo Micropore can identify location specific nucleotide sequence, and location specific nucleotide sequence in sequencing and/or amplified reaction It may be related at least one experimental considerations unit of at least one cell origin, hole or micropore.

Micromachining device as described above is able to use further system, kit, device and method culture from ring Cell in the sample in border.For example, can be with based first surface application sample, so that at least one cell occupies at least one A micropore, hole or experimental considerations unit.To at least part first surface (for example, at least part experimental considerations unit or the inner surface in hole) Using semi-permeable film, nutrients is allowed to diffuse at least one micropore, hole or experimental considerations unit.Meanwhile it preventing and/or subtracting Gently cell is occupied to flee from from least one micropore, hole or experimental considerations unit.Semi-permeable film can be such as hydrogel layer.Use example Such as lamination can be reversible by semi-permeable film or be irreversibly connected or fixed to substrate.Therefore, there can be at least one It is incubated at least one micropore of nutrients, hole or experimental considerations unit and occupies cell.It is exchanged using progressive part, cell can be Other at least one nutrients or nutritional preparation are gradually transitions from least one nutrients in a period of time, so that experience is tamed and dociled Change or adapts to.

The first nutrients incubation from environment can be used and occupy the thin of at least one first experimental considerations unit, hole or micropore Born of the same parents, and the second nutrients from environment can be used and be incubated for the cell for occupying at least one the second experimental considerations unit, hole or micropore. The above method may include compare occupy at least one first experimental considerations unit, hole or micropore cell and occupy at least one second The cell of experimental considerations unit, hole or micropore, to analyze the first nutrients and the second nutrients.

For example, method may include one or more following steps:

Chip is obtained, 1000 to 10,000,000 or more micropores are defined in a large amount of large holes or fluidic cell, respectively The diameter of micropore is about 1 μm to about 800 μm, and depth is about 1 μm to about 800 μm, and chip further has one or more settings To promote target microorganism to be moved to the surface chemistry of micropore；

To the chip application environment sample or the derivative of environmental sample, so that any target microorganism is located at micropore In；

One or more semipermeability filter paper, hydrogel layer or other barriers are placed on chip, so that generating allows Nutrients diffuses into micropore but prevents and/or mitigate the barrier that microorganism is fled from from micropore；

Chip is incubated for at least one nutrients (for example originating from environment)；

By the exchange of progressive part gradually at least one other nutrients (such as preparation) replacement nutrition come Source；With

Detect any growth of microorganism in micropore.

Target cell can be Archimycetes, bacterium or eucaryote.Target viral can be bacteriophage.When targeting virus, The micropore of chip can also include the host cell that can wherein grow virus.Detection occupies cell or the growth of virus and can wrap Include detection following aspect variation: biomass (such as DNA/RNA/ protein/lipid), the existence or non-existence of metabolin, pH, Nutrients consumption and/or gas consumption.It may include carrying out real time sequence imaging, showing that detection, which occupies cell or the growth of virus, Micro mirror, optical density (OD), fluorescence microscope, mass spectrum, electrochemistry, amplification (DNA, cDNA and/or RNA), sequencing (DNA and/or RNA), nucleic acid hybridization and/or antibody combine.

Figure 13 is the flow chart illustrated according to some embodiments for the method for screening.In step 1300, sample is obtained Product.In step 1302, from least one cell of gained sample extraction.In step 1304, in micromachining device or chip The cell of at least one extraction is loaded at least one high density microwell array.Step 1304 may include being extracted at least one Cell preparation cell concentration object, select at least one nutrients/culture medium, and/or a selection at least film.In step 1306 In, at least part of microwell array is sealed with the film of at least one selection cell concentration object to be retained in micropore.In step In rapid 1308, it is incubated for chip.Step 1308 may include selection temperature, determine atmosphere (such as aerobic or anaerobism), and/or be incubated for Time.It can carry out genetic screening and/or functional screening.In step 1310, to chip application genetic screening.In step 1312 In, according to method described herein, chip is separated and/or replicated and (uses such as Chooser), obtains the culture of two parts Cell.For example, an at least film can be removed, so that a part culture cell still adheres to, or removes or puncture to sample training Support cell.In operating procedure 1314, a part culture cell is put to death for identifying.Step 1314 may include PCR, sequencing And/or various data analyses.In step 1316, aimed strain is identified.Such as aimed strain and/or remainder can be used Culture cell is further cultivated, tested and/or is identified.Alternatively, being screened in step 1318 to chip application function. In step 1320, one or more variables are observed, aimed strain is identified in step 1316.

Figure 14 is the schematic diagram for illustrating the screening technique according to some embodiments.Small Figure 140 0 shows complex sample Example, especially micropopulation sample 1402 and pedotheque 1404.In small Figure 140 6, using for example shown in Fig. 5 A and 5B Scheme is from least one cell of sample extraction.In small Figure 140 8, cell (and any environmental extract that at least one is extracted And/or diluent) it is loaded into micromachining device or chip 1410 at least one high density microwell array.It can be by chip 1410 and kit 1412 be put into couveuse 1414.Reagent can be used for adding liquid to maintain growth and/or various screening purposes Nutritional need.Small Figure 141 6 shows output: the isolated bacterial strain of the selection result and culture cell.

Figure 15 is a series of image of explanations according to the screening example of some embodiments.Image show with film and Application has a part of the chip of acid-sensitive layer thereon, to screen low pH.In image 1500, it is seen that micro- more than 1800 50 μm Hole, 9 clear hits 1502.Image 1504 is the enlarged drawing of frame 1504, and image 1506 is a micropore with hit 1502 Enlarged drawing.

Figure 16 A-16C is the image illustrated according to some embodiments from screening recycling.In Figure 16 A, microscope is used At least one hole is chosen with the selecting device at least one needle.In fig. 16b, needle is removed and is incubated in the medium. In Figure 16 C, it is seen that growth.

Figure 17 A is the exploded view for illustrating the chip for screening according to some embodiments.In Figure 17 A, chip 1700 include the high density microwell array in micropore with such as edaphon.Film 1702 is applied to chip 1700.In film Direction chip 1700 applies gasket 1704 on 1702.Direction chip, which is applied, on gasket 1704 and film 1702 has fluorescence large intestine bar The agar 1706 of bacterium.Figure 17 B and 17C are the images for illustrating the screening example according to some embodiments.In this example, it screens Keep-out region.Figure 17 B is the fluorescent image of chip after screening, and the chip is prepared as the chip 1700 in Figure 17 A.Figure 17 C It is to show the image for the process for choosing sample by agar from the chip.

In some embodiments, the position in equipment may be moved with a part (or part of a part) sample from equipment Existing a part of sample is related at this location after removing.Equipment can be microarray or including microarray.Microarray can To include multiple positions for applying sample, wherein each position is marked with unique label, and the unique label is for identifying A part of sample from microarray removal after, the sample segment from position.

This disclosure relates to for identifying that described a part contains at least one core in a part of sample after microarray removal The method of which position of the sample of acid molecule on microarray, method includes the following steps: (a) is in the multiple of microarray The sample of one or more parts is applied in the one or more of position, wherein unique label of each position comprising nucleic acid molecules Label, the unique label includes: (i) location specific nucleotide sequence；(ii) first target specificity nucleotide sequence； (b) the target nucleic acids molecule found and the label of mark position in the sample of at least one portion is allowed to anneal；(c) to annealing Nucleic acid molecules group carries out primer extend, reverse transcription, single-stranded connection or double-strand connection, so that location specific nucleotide sequence is whole It is incorporated into each nucleic acid molecules generated by primer extend, reverse transcription, single-stranded connection or double-strand connection；(d) merge in step (c) The nucleic acid molecules group of generation；(e) combined nucleic acid molecules group is sequenced, to obtain one or more location specific nucleotides sequences The sequence of column；(f) by least one the location specific nucleotide sequence obtained from combined nucleic acid molecules group with comprising Position on the microarray of the label label of the location specific nucleotide sequence is associated, so that identification includes at least one Which position of a part of sample of nucleic acid molecules from microarray.In some embodiments, sample may include at least one A cell, and one or more nucleic acid molecules are discharged from cell after step (a) and before step (b).Sample may include At least one cell, and after the step (a) and at least one cellular replication of step (b) foregoing description or division.In step (b) Before, a part of a part of sample can be removed from least one position, and a part of of a part of sample can deposit It is stored in independent container relevant to the initial position of a part of sample described on microarray.The method of association or identification position can To further include steps of the combined nucleic acid generated in the nucleic acid molecules generated in amplification step (c) or step (d) point Subgroup.Amplification step may include polymerase chain reaction amplification, multiplex polymerase chain re-action amplification, nested polymerase chain Reaction amplification, Ligase detection reaction amplification, strand displacement amplification, the amplification based on transcription, is based on ligase chain reaction amplification Amplification, rolling circle amplification or the hyperbranched rolling circle amplification of nucleic acid sequence.Other primers can be added in the amplification reaction.For example, PCR Reaction needs both 5' and 3' primers.A primer can be complementary with the nucleotide sequence in sample used in amplified reaction.

It in some embodiments, can include thin with nucleic acid enzymatic treatment before applying composition to micromachining device The composition of born of the same parents and/or virus, so that contaminated nucleic acid molecule is not amplified in later step.

Used in disclosed method and apparatus sequencing can be obtain sequence information any method, including hybridization and It uses sequence specific protein (such as enzyme).Sequencing may include Sanger sequencing, by sequencing by hybridization, by connection sequencing, Quantitative increment fluorescent nucleotide addition sequencing (QIFNAS), gradually connection and cutting, fluorescence resonance energy transfer, molecular beacon, The digestion of TaqMan reporter gene probe, pyrosequencing, fluorescent in situ sequencing (FISSEQ), wobble frequency sequencing, multiple sequencing, polymerization (see, for example, U.S. Patent Application Publication No. 2012/0270740, full text is incorporated by reference into this for bacterium colony (POLONY) sequencing Text)；(see, for example, U.S. Patent Application Publication No. 2009/0018024, full text passes through for nanometer lattice rolling ring (ROLONY) sequencing Be incorporated herein by reference), the connection measurement sequencing of allele-specific oligomerization, or the sequencing on next-generation sequencing (NGS) platform. The non-limiting example of NGS platform includes coming from system below:(San Diego, California) (example Such as, MiSeq^TM/NextSeq^TM/HiSeq^TMWith HiSeq X^TM)、Life Technologies(Carlsbad,California) (such as Ion Torrent^TM), and Pacific Biosciences (Menlo Park, California) (such as RS II)。

Organism or type obtained from the nucleic acid sequence of the organism and can contain each of organism sequence by comparing Database is planted to identify.For example, ribosomal RNA sequences data can be from SILVA rRNA database project (Max Planck Institute for Marine Microbiology,Bremen,Germany(www.arb-silva.de)；See, for example, Quast etc., " The SILVA Ribosomal RNA Gene Database Project:Improved Data Processing and Web-Based Tools, " 41Nucl.Acids Res.D590-D596 (2013), and Pruesse Deng " SINA:Accurate High-Throughput Multiple Sequence Alignment of Ribosomal RNA Genes, " 28Bioinformatics 1823-1829 (2012), the two full text are both incorporated herein by reference) obtain. Other ribosomal RNA sequences databases include ribosomes database project (Michigan State University, East Lansing,Michigan(www.rdp.cme.msu.edu)；See, for example, Cole etc., " Ribosomal Database Project:Data and Tools for High Throughput rRNA Analysis"42 Nucl.Acids Res.D633-D642 (2014), is hereby incorporated herein by reference) and Greengenes (Lawrence Berkeley National Laboratory,Berkeley,California(www.greengenes.lbl.gov)；See, for example, DeSantis etc., " Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB, " 72 Appl.Environ.Microbiol.5069-72 (2006), full text is by quoting simultaneously Enter herein).Genetic sequence database includes publicly available almost 260,000 species formally described Nucleotide sequence (National Institutes of Health, Bethesda, Maryland (www.ncbi.nlm.nih.gov)；See, for example, Benson etc., " GenBank, " 41 Nucl.Acids Res.D36-42 (2013)。

Sequence for matching and identifying may include 16S ribose body region, 18S ribose body region or offer authentication information Any other region.Required variant can be genotype (such as single nucleotide polymorphism (SNP) or other kinds of variant) Or the type comprising specific gene sequence (such as sequence of codase or protein).Organism or type can also by by its Sequence is identified with customization internal sequence database matching.In some cases, if obtained from a part of sample of some position The sequence obtained is with known dna, cDNA or the RNA sequence obtained from the type or microorganism with the same of at least particular percentile Property (for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity), it can be concluded that and find the type or micro- in the position of microarray Biology.

The disclosure further to including method for the microarray for applying multiple positions of sample for manufacturing, wherein At least one position is marked with unique label, the described method comprises the following steps: multiple labels (a) is synthesized, wherein each label Comprising nucleic acid molecules, the nucleic acid molecules include: (i) location specific nucleotide sequence；(ii) target specificity nucleotide Sequence；(b) label is placed at least one position of multiple positions on microarray.In another embodiment, originally Open to be related to for manufacture including method for the microarray for applying multiple positions of sample, wherein at least one position is with solely Special label label, the described method comprises the following steps: (a) synthesizing multiple labels, wherein each label includes nucleic acid molecules, it is described Nucleic acid molecules include target specificity nucleotide sequence and do not include location specific nucleotide sequence；(b) label is placed in On at least one position of multiple positions on microarray.Target specific sequence on each position of microarray can be identical. In any the embodiment above, step (a) can carry out before step (b).Placing step (b) may include passing through liquid Label is placed in respectively by operation sequence (for example, liquid relief, with solid syringe needle point sample, with hollow pinhead point sample or with ink discharge device deposit) A position.At least one label may include a part of pre-synthesis nucleic acid molecules or nucleic acid molecules.Step (a) can be with Step (b) carries out simultaneously.In some embodiments, at least one label includes the nucleic acid molecules of synthesis, at various locations Pass through fabricated in situ.Synthesis step (a) may include inkjet printing synthesis or photoetching synthesis.

Each position on microarray can be set to receive a part of sample.Nucleic acid molecules (such as few nucleosides can be used Acid) label or tag position, the nucleic acid molecules include at least one: (i) location specific nucleotide sequence (bar code)；With (ii) target specificity nucleotide sequence.Target specificity nucleotide sequence can be complementary with the nucleotide sequence found in sample Or it is substantially complementary.The sequence of nucleotide sequence in nucleic acid molecules from the end 5' to the end 3' can be (i) location specific Nucleotide sequence；(ii) target specificity nucleotide sequence.Alternatively, the nucleotide in nucleic acid molecules from the end 5' to the end 3' The sequence of sequence can be (2) then (1).Nucleic acid molecules can be connected to microarray in its end 5'.Equipment (such as micro- battle array Column) on one or more positions can be without label or unmarked.

Term " complementation " or " being substantially complementary " can refer between nucleotide or nucleic acid, such as such as double chain DNA molecule Two chains between or Oligonucleolide primers and single-chain nucleic acid on primer binding site between hybridization, base pairing or formation Duplex.In general, complementary nucleotide is A and T/U or C and G.When optimal comparison and compare and have appropriate nucleotides inserted or Missing a chain nucleotide and another chain at least about 80%, typically at least about 90%-95%, more preferably from about 98- 100% oligonucleotide ligand clock synchronization, two single stranded RNAs or DNA molecular are known as being substantially complementary.Alternatively, when RNA or DNA chain will be When hybridizing under selective cross condition with its complement, there are basic complementarity.In general, when in one section of at least 14-25 nucleosides When there is at least about 65% complementarity, at least about 75% or at least about 90% complementarity on acid, selective cross will occur.

Term " selective cross " refers to detectable specific binding.Polynucleotides, oligonucleotides and its segment are big Amount and the detectable combination of non-specific nucleic acid minimum hybridize and wash conditions under with nucleic acid chains selective cross.It can make The selective cross condition known in the art being discussed herein is realized with " high stringency " condition.The example of " high stringency " condition is A kind of method for being incubated for another polynucleotides with polynucleotides, one of polynucleotides can at 42 DEG C of hybridization temperature Hybridization buffer (the piece that 6x SSPE or SSC, 50% formamide, 5x Denhardt reagent, 0.5%SDS, 100 μ g/ml are denaturalized Sectionization salmon sperm dna) in be fixed to the surface of solids (such as film) 12-16 hours, then with washing buffer (1x SSC, 0.5%SDS) washed twice at 55 DEG C.

Nucleic acid molecules as location tags a part may include at least one deoxyribonucleotide or at least one Ribonucleotide.Nucleic acid molecules can be single-stranded or double-stranded.Nucleic acid molecules can be the duplex molecule with single-stranded outstanding end.

In some embodiments, location tags can be used for expanding the nucleic acid molecules annealed with it.Therefore, location tags can To include the nucleic acid sequence further containing amplimer binding site.The length of amplimer binding site can be at least 16 It is a, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, extremely Few 25, at least 26, at least 27, at least 28, at least 29 or at least 30 nucleotide.From the end 5' in nucleic acid molecules Sequence to the nucleotide sequence of the end 3' can be for example: (1) amplimer binding site；(2) location specific nucleotide Sequence；(3) target specificity nucleotide sequence.

In some embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include position special Property nucleotide sequence.In certain embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include position Set specific nucleotide sequences or amplification binding site sequence.In a further embodiment, nucleic acid molecules can only include Target specificity nucleotide sequence.In even further embodiment, nucleic acid molecules can only include target specificity core Nucleotide sequence.Amplimer binding site can combine polymerase chain reaction primer, multiplex polymerase chain re-action primer, nest Formula polymerase chain reaction primer, Ligase detection reaction primer, strand displacement primer, is based on turning ligase chain reaction primer The primer of record, the primer based on nucleic acid sequence, rolling ring primer or hyperbranched rolling ring primer.It can be to microarray during amplified reaction Other primers are added.For example, PCR reaction needs both 5' and 3' primers.Target specificity nucleotide sequence can contain target The position amplification of nucleic acid molecules is marked, and can be come for example, by the dyestuff of qPCR, terminal PCR and/or detection amplifier nucleic acid molecule Detection.

It is expected that the nucleic acid molecules annealed with location tags or amplified production based on this nucleic acid molecules are sequenced.Position Setting label may include nucleic acid sequence further containing aptamer nucleotide sequence.In certain embodiments, it is adapted to daughter nucleus Nucleotide sequence is not present in location tags, but is added to sample nucleic acid molecule in secondary PCR reaction or through connection.Adaptation Daughter nucleus nucleotide sequence can be General adaptive or specific microarray dataset (such asOr Ion Torrent^TM) it is suitable Gamete.Aptamer nucleotide sequence may include sequencing primer binding site.Sequencing primer binding site can in conjunction with for Under primer: Sanger sequencing, by sequencing by hybridization, pass through connection sequencing, the sequencing of quantitative increment fluorescent nucleotide addition (QIFNAS), gradually connection and cutting, fluorescence resonance energy transfer, molecular beacon, TaqMan reporter gene probe digestion, coke phosphorus Sour sequencing, the sequencing of fluorescent in situ sequencing (FISSEQ), wobble frequency, multiple sequencing, polymerization bacterium colony (POLONY) sequencing are (see, for example, beauty State's patent application publication number 2012/0270740, is hereby incorporated herein by reference)；Nanometer lattice rolling ring (ROLONY) is sequenced (see, for example, U.S. Patent Application Publication No. 2009/0018024, being hereby incorporated herein by reference), allele specific Property oligomerization connection measurement sequencing, the sequencing on NGS platform or any suitable sequencing program.The non-limiting example of NGS platform Including coming from system below:(San Diego, California) (such as MiSeq^TM/NextSeq^TM/ HiSeq^TMWith HiSeq X^TM), Life Technologies (Carlsbad, California) (such as Ion Torrent^TM), With Pacific Biosciences (Menlo Park, California) (such as RS II)。

The length of location specific nucleotide sequence (such as bar code) is at least two, at least three, at least four, at least 5 A, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14 It is a, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, extremely Few 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30 nucleotide.

The length of target specificity nucleotide sequence be at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, At least 23, at least 24, at least 25 nucleotide, at least 26, at least 27, at least 28, at least 29, at least 30 A, at least 40, at least 50, at least 75 or at least 100 nucleotide.

At least one position on microarray can further be marked with unique molecular identifier tags.Unique molecular mark Symbol can be used for quantitative growth (such as the growth of microbe colony or cellular replication in the position).Unique molecular identifier can be Random nucleotides.The reality of method and unique molecular identifier using unique molecular identifier has been described in the prior art Example, the WO 2013/173394 being hereby incorporated herein by reference see, for example, it.For example, the core of unique molecular identifier tags Nucleotide sequence can be NNNANNNCNNNTNNNGNNNANNNCNNN (SEQ ID NO:1), and wherein (equivalent of ACGT is random by Ns Mixing) big coding space is generated, so that it (is 4 in the example that each molecule of amplification, which obtains unique (specific) DNA sequence dna bar code, ^N bar code, or 4^21~40 are hundred million).The sequence can count, without by the interference from amplification preference or other technologies problem. Fixation base (A, C, G, T) in SEQ ID NO:1 helps accurately to read bar code, such as processing indel.

The disclosure, which covers using location specific label, monitors more than one target specificity nucleotide sequence in sample Presence or content (for example, multiple).At least one position on microarray can be further with only comprising nucleic acid molecules second Special label label, the nucleic acid molecules are including, for example, (i) amplimer binding site: (ii) location specific nucleotide sequence； (iii) target specificity nucleotide sequence.

In some embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include position special Property nucleotide sequence.In certain embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include position Set specific nucleotide sequences or amplification binding site sequence.In a further embodiment, nucleic acid molecules can only include Target specificity nucleotide sequence.In even further embodiment, nucleic acid molecules can only include target specificity core Nucleotide sequence.The length of target specificity nucleotide sequence can at least ten, at least 11, at least 12, at least 13, At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 nucleotide, at least 26, at least 27, at least 28, at least 29, extremely Few 30, at least 40, at least 50, at least 75 or at least 100 nucleotide.In certain embodiments, in microarray The target specific sequence of each position can be identical.Other target specificity nucleotide sequences can be monitored.For example, can use At least ten, at least 25, at least 50, at least 75 or at least 100 unique labels mark one or more positions, wherein Each label includes the target specificity nucleotide sequence different from other target specificity nucleotide sequences in the location tags.

Any target gene position can provide target specificity nucleotide sequence.Such as following sequence: bacterial 16 S ribose Body RNA (rRNA), 18S rRNA, poly- (A) RNA, rna polymerase gene, DNA polymerase gene, RecA gene, transposase Gene, ribosome internal transcribed spacer (ITS) sequence, the gene of codase, control zone DNA sequence dna, binding site DNA sequence Column, or can be used as a part of any of these sequences of target specificity nucleotide sequence.Disclosed system, is set kit The one or more bacterial 16 S rRNA primers described in the following: Sundquist etc. can be used in standby or method, " Bacterial Flora-Typing with Targeted,Chip-Based Pyrosequencing,"7:108 BMC Microbiology (2007) and Wang etc., " Conservative Fragments in Bacterial16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies,"4: 10 PLoS ONE e7401 (2009), are hereby incorporated herein by reference.

The sample used in disclosed device and method may include multiple nucleic acid molecules.Sample may include at least one A DNA molecular or at least one RNA molecule.Sample may include at least one nucleic acid molecules formed by restriction Enzyme digestion. Sample may include at least one cell (such as archeabacterial cell, eubacteria cell, fungal cell, plant cell and/or animal Cell).Sample may include at least one microorganism.Sample may include one or more viral (such as bacteriophages), to this It may need to provide host cell.A part of sample of a position can be individual cells or raw from individual cells on microarray Long bacterium colony.For example, individual microorganism or cell can be placed in micropore and allow the individual microorganism or cell Division or duplication so that bacterium colony is grown in each micropore, and wherein have individual microorganism or cell.Therefore, on microarray Position may include single microbial type or the growth that supports one another microbial strains population mixture.Sample may include Any suitable diluent.In non-limiting example, sample includes soil, sewage, fecal matter, body cavity content, biology stream Body, living organism, dead organic matter, microbial suspension, natural fresh water, drinking water, seawater, waste water, overcritical titanium dioxide Carbon, mineral, gas, buffer, alcohol, organic solvent and/or oil.In some embodiments, it is placed in by a part of sample Before at least one position of microarray, the position will be placed in comprising nucleic acid molecules below: (a) (i) location specific nucleotide Sequence and (ii) one or more target specificity nucleotide sequences；Or (b) one or more target specificity nucleotide sequences (that is, not including location specific nucleotide sequence).In other embodiments, a part of sample is being placed in microarray extremely Behind a few position, the position will be placed in comprising nucleic acid molecules below: (a) (i) location specific nucleotide sequence and (ii) One or more target specificity nucleotide sequences；Or (b) one or more target specificity nucleotide sequences (that is, not including Location specific nucleotide sequence).In an example, sample or a part of sample are placed on microarray and are incubated for, then To be placed on microarray comprising at least one nucleic acid molecules below: (i) location specific nucleotide sequence and (ii) target are special Specific nucleotide sequence.In some embodiments, by a part of a part of sample from least one on microarray Position remove, and store in a separate container, or before nucleic acid molecules to be placed in at least one position on microarray or Microarray is separated later.

At least one location specific label may include pre-synthesis and be placed in the position by liquid operation sequence A part of nucleic acid molecules or nucleic acid molecules.For example, liquid operation sequence can be liquid relief, with solid syringe needle point sample, with hollow Syringe needle point sample is deposited with ink discharge device.Multiple nucleic acid molecules separately synthesized in advance can be used and generate label in position.Extremely A few label may include to be synthesized by fabricated in situ (such as being synthesized by inkjet printing synthesis or photoetching) in the position Nucleic acid molecules.

The digital counting of type

This document describes the superchip devices comprising the surface with high density micropore.It can will come from micropopulation The microorganism dilution of sample is simultaneously applied to device, so that hole includes about microorganism/occupy hole.Then, chip is incubated for make Microorganism is replicated in hole.Further, this document describes the location index systems based on DNA, and to measure, there are what in each hole Type.The directory system may relate to for PCR primer to be loaded previously into each hole, and the PCR primer contains the addressing item in identification hole The offer information of specific gene sequence (such as 16S) in code and targeted microorganisms genome targets required gene order Primer sequence.After incubation, releasing microbe DNA, PCR primer expands non-target bacteria region of DNA domain, and collects each on chip Then the amplicon in a hole is read by next-generation sequencing.

System as described herein, kit, device and method can be used for carrying out each microbe species in sample or variant The absolute counting of quantity.Each hole can represent a digital event, and single microbial deposits in expression original dilution sample ?.It is any bacterial species that location index system, which allows user to measure in hole,.Measurement unit can be that " there are bacterium kinds in hole Class ", and can be unrelated with the quantity of bacterium in hole.

In an example, the mixing sample of microorganism includes the type of the type 2 and 20% of 50% type 1,30% 3.Then dilute sample is applied to chip, so that the hole (major part) respectively occupied has a kind of microorganism.Microorganism duplication.Note Meaning, different types of reproduction speed may be different.Then, chip is processed, so that the DNA in hole from microorganism is discharged, and 16S or some other target sequences are expanded.The DNA cloning product from each hole can be collected, and is carried out with next generation's sequencing Sequencing.Next-generation sequencing data be can analyze to measure for each hole, be what type respectively occupying in hole.Many holes can It can not be occupied.Can pass through the various types of abundance of following measurement: the sum in the various types of hole occupied is divided by occupying hole Sum.Following measurement absolute abundance can be passed through: by the various types of abundance % from step multiplied by microorganism in primary sample Sum.Sequencing data can be compared with publicly available sequence database, what type is respectively occupied in hole with measurement is. For example, ribosomal RNA sequences data can be obtained from above-mentioned SILVA rRNA database.Other ribosomal RNA sequences databases are also Including above-mentioned ribosomes database project, Greengenes andGenetic sequence database.

Method currently used for the abundance of microbe species in estimation sample is related to using traditional technology, such as microscope, Dyeing, selective medium, metabolism/physiology screening and with culture dish culture.These methods are typically due to lack specific (micro- Mirror, dyeing, selective medium) or lack the ability (selective medium, culture) of all kinds in count samples without essence Really, and many types grown with conventional method it is bad or do not grow.

Molecular method currently used for microbe species relative abundance in measurement micropopulation sample is related to from sample extraction Microbial DNA carries out PCR amplification to the 16S or some other region of DNA domains that provide type or other information, then to gained PCR Product carries out next-generation sequencing (NGS).Speculate in primary sample from the relative frequency of species specificity DNA sequence dna in NGS data Various types of relative abundance.For document about many examples of this alanysis, this method is that base is laid in the research of many micropopulations Plinth.

It is following the problem of method to be at present that it is not controlled: the quantity for being present in the 16S gene in different microorganisms may not Together, PCR preference (wherein the sequence of different microorganisms type may be expanded with different rates) and sequencing preference (wherein different micro- lifes The sequence of species may be sequenced with different rates).As a result, the accuracy of the relative abundance data obtained with current method There are many uncertainties.

Different types of counting can be based on the presence of type in single hole.This be divided into during load in hole from original The single microbial of beginning sample is directly related.Only PCR/NGS can be used for identifying that there are what microorganisms in each hole.The sequence of identification The quantity of column is not from a part of calculating.Therefore, PCR, NGS or the variation of target sequence copy number or preference do not weigh in method It wants.

Some embodiments can have below application: micropopulation research, microniological proudcts are developed and are developed, clinical Diagnosis and any other field for wherein needing the microbe species in accurate metering sample.

Therefore, some embodiments can provide the measurement that various types of relative abundance is much more accurate in micropopulation sample with And by the relative abundance measurement to convert the various types of ability directly counted in absolute abundance or primary sample (dilute by calculating Release and/or in conjunction with microbial count in primary sample measurement).Some embodiments can provide for high density micro Process chip New opplication (in addition to cultivating and screening microorganism).

Figure 18 is the flow chart for illustrating the method for counting according to some embodiments.In step 1800, sample is obtained.? In step 1802, from least one cell of gained sample extraction.In step 1804, at least the one of micromachining device or chip The cell of at least one extraction is loaded in a high density microwell array.Step 1804 may include the cell extracted at least one It prepares cell concentration object, select at least one nutrients/culture medium, and/or a selection at least film.In step 1806, use The film of at least one selection seals at least part of microwell array so that cell concentration object to be retained in micropore.In step 1808 In, it is incubated for chip.Step 1808 may include selection temperature, determine atmosphere (such as aerobic or anaerobism) and/or incubation time. In step 1810, a part culture cell is put to death for identifying.Step 1810 may include PCR, sequencing and/or various data Analysis.In step 1812, it can be estimated that and/or information (for example, microbial community structure) of the measurement about sample.

Figure 19 is the schematic diagram for illustrating the method for counting according to some embodiments.Small Figure 190 0 shows complex sample Example, especially micropopulation sample 1902 and pedotheque 1904.In small Figure 190 6, using for example shown in Fig. 5 A and 5B Scheme is from least one cell of sample extraction.In small Figure 190 8, at least one cell extracted (and any environmental extract And/or diluent) it is loaded into micromachining device or chip 1910 at least one high density microwell array.It can be by chip 1910 and kit 1912 be put into couveuse 1914.Reagent can be used for adding liquid to maintain growth and/or various screening purposes Nutritional need.Small Figure 191 6 shows output: the relative abundance of sequence and culture cell.

Platform based on drop

Platform based on discrete droplets can be used for separating, cultivate and/or screen, with the basic phase of mode for using chip Together.Drop is micropore analog, is used as nanometer or picoliters container.Drop forming method, especially when with Cell Sorter on chip When type instrument combination, it can be used for isolating microorganism from complex environment sample.Drop addition can be used for feeding microorganism.Drop point It splits and can be used for sequencing or other destructive testings, while leaving sample living.All preparations needed for sequencing can also be with drop Form carries out.

Some embodiments can be used for taking out microorganism from complex environment, become drop.For example, be used to prepare containing The regulator control system of the drop of cell suspending liquid may include one or a small amount of cell.It can be by water drop suspension in unmixing liquid Body makes them keep separation each other, and prevents from contacting or polluting any surface.It can be generated in each microchannel in such as 30Hz Drop is converted into millions of daily.

Microfluidic system based on drop can encapsulate, manipulate and/or be incubated for droplet (for example, about 30pL).It notices Cell survival and proliferation are similar with the control experiment in bulk solution.Drop can be generated in hundreds of Hz, it is meant that can be several Millions of drops are generated in a hour.The simply device based on chip can be used for generating drop, and drop can be engineered with Include individual cells.

Some embodiments can be used for screening the cell in drop.It can be with 500 drops for example per second on chip Drop after the incubation of rate fluorescent screening.Drop, which can flow through, can be set to show for the epi-fluorescence of a variety of different measurements The channel of the focal point of micro mirror.This may be the screening particularly effective method of metabolin, because due to being confined to very small liquid Drop, so local concentration is quite high.

Some embodiments can be used for sorting drop.Once cell can sort them by separation, growth and/or screening So that obtaining useful sample.Can by with as common FACS machine type in a manner of drop is sorted.

Some embodiments can be used for dividing drop.Some embodiments may need the energy for obtaining sample and being divided Power so that a part is sent to sequencing (disruptive method), and is retained as another part of culture living.There are many points at present The distinct methods of drop are split, is including but not limited to intersected with the size configurations T shape carefully calculated, is caused in flowing or electrowetting It generates drop breakup (being careful not to cause cell cracking with too high voltage).

Some embodiments can be used for merging drop and/or add reagent to drop.For example, the long-time of cell is incubated for (such as a few weeks) needs the ability of adding liquid, to maintain the nutritional need of growth.Reagent is added for various screening purposes It is useful.Drop screening is dependent on the drop containing compound password and capable of containing the droplet coalescence of individual cells.So After can be incubated for drop, and/or return it into analysis chip with by its password authentication compound.This may be needed as needed The ability of precise merging drop.

Some embodiments can be used for carrying out PCR in drop.PCR can be used for finally being sequenced specific genetic elements (such as The region 16S), to identify microorganism.It is what kind of microorganism that this, which can be used to measure grow in each hole,.It is being based on In the system of drop, it is any microorganism present in each drop that this method, which can be used for measuring, as long as devising amplification gene group The correct primer sequence in correct region.

Some embodiments can be used for being sequenced from drop DNA (such as generating in a PCR step), and/or preparation DNA text Library.

Location specific label for superchip

The superchip device on the surface with high density micropore can be used.It can will be from micropopulation sample Microorganism dilution is simultaneously applied to device, so that hole includes about microorganism/occupy hole.Chip, which can be incubated for, makes microorganism exist It is replicated in hole, gained group represents single type.Location index system based on DNA can be used for measuring in each hole there are what plant Class.The directory system may relate to for PCR primer to be loaded previously into each hole, and the PCR primer contains the addressing bar code in identification hole With the primer sequence (such as 16S of microbial genome) for the specific genetic elements of targeting for providing information.After incubation, release Microbial DNA, PCR primer expand non-target bacteria region of DNA domain, the amplicon in each hole on chip are collected, under then passing through Generation sequencing is read.

Above-mentioned location index system may relate to each hole that different location password is added to microchip, or by multiple positions Each hole is added in password, so that password sum needed for encoding certain amount of hole is reduced.For example, as having 100 on fruit chip Hole, if one, each hole password, needs 100 passwords.If reading two passwords from each hole, same chip can be only With 20 cipher codings (that is, the x-axis of 10 coding grids, 10 coding y-axis).

Table 2 provides the example that the PCR strategy of two passwords is added in each hole.

Table 2

Table 3 provides the example that the PCR strategy of three passwords is added in each hole.

Table 3

Three oligomer primers are for manufacturing single PCR product.The system is put using two oligonucleotides in one end of molecule The advantages of setting multiple slitting codes may include the maximum length or manufacture overlength bar code of oligonucleotides needed for for example reducing.

This method may be summarized to be each reaction and n bar code be added.This method can also have different implementations, so that bar code exists The same side in target sequence region.NGS sequencing aptamer can be used, and read bar code PCR product group using next-generation sequencing Full sequence.

In another kind is implemented, fixed bar code can be added to indicate sample size or plate quantity, and allow in two items Collect multiple sample/plates in the operation of code, as shown in table 4.

Table 4

In another kind is implemented, fixed bar code can be added to indicate sample size or plate quantity, and allow in three items Collect multiple sample/plates in the operation of code, as shown in table 5.

Table 5

Note that in all cases, information, therefore CODE1, CODE2 and CODE3 item are conveyed in the position of bar code in the sequence Code is not necessarily different from each other in particular bore.

Prepare oligonucleotide and with solid size coded system printing chip higher cost.For example, the chip in 10,000 holes In the hole for needing 10,000 single bar codes and 10,000 independent print cycles so that these bar codes to be placed on chip.If made With dicode system, only 200 bar codes and 200 print cycles may be needed to manufacture chip.This means that substantial saved widow Nucleotide cost, print time and printing capital input.

Using dual bar code PCR primer, amplification and sequencing analysis are then carried out, is assigned randomly to micro Process chip to provide On DNA or the position data of the part containing DNA may have very high practicability and relatively low cost.

Figure 20 is the schematic diagram for illustrating the directory system according to some embodiments.Microwell chips 2000 have N row and M Column, to generate NxM unique index.It is considered that the position of micropore has coordinate (N, M) in chip 2000.Each column has normal Reverse primer sequences (for example, R1, R2, R3..RM) and every row have common forward primer sequence (for example, F1, F2, F3..FN).For example, the unique label of specific genetic elements may include just in target such as 16S ribosomes nucleotide (rRNA) To primer sequence F515 and reverse primer sequences R806.It, can be based on forward primer sequence and anti-after chip 2000 is carried out PCR The presence of target genetic elements is mapped back unique original micropores by the presence to primer sequence.For example, F515's and R806 deposits In the micropore that will there are coordinate (515,806) in user guiding chip 2000.

The variability of pcr amplification product containing germy micropore reduces

The location index system based on DNA can be used, and to measure, there are what types in each hole.The directory system may It is related to for PCR primer being loaded previously into each hole, the PCR primer contains the addressing bar code and targeted microorganisms genome in identification hole Specific genetic elements (such as 16S) offer information primer sequence.After incubation, releasing microbe DNA, PCR primer Non-target bacteria region of DNA domain is expanded, the amplicon in each hole on chip is collected, is then read by next-generation sequencing.

Some embodiments of the variation of the amount of PCR product may include the limiting holes during manufacturing chip between limiting holes The amount of middle PCR primer, so that the amount of PCR primer is in DNA amplification reaction for most of possible sample DNA concentration It is limited, so that difference of the amount of the PCR product generated between each hole will be smaller.

Some embodiments of the variation of the amount of PCR product may include by the recurring number of PCR on chip between limiting holes It is limited to less than 3 circulations or less than 5 circulations or less than 10 circulations or less than 15 circulations or less than 20 circulations, Or less than 25 circulation or less than 30 circulation so that relative to complete alternation PCR amplification scheme, the amount of the PCR product of generation exists Difference between each hole will be smaller.

Some embodiments of the variation of the amount of PCR product may include the core in limited reactions mixture between limiting holes The amount of thuja acid, so that the quantity of PCR amplification of generation and the amount of nucleotide are more relevant compared with the amount of DNA in primary sample. Micropore with a large amount of target DNA will exhaust nucleotide in cyclic process early stage, and the micropore with a small amount of target DNA will follow The ring process later period exhausts nucleotide, but generates the amplified production of nearly identical amounts.

Some embodiments of the variation of the amount of PCR product may include being limited in the micro- life grown in hole between limiting holes The amount of the available nutrients of object, so that cellular replication, until exhausting culture medium and then stopping replicating.

Some embodiments of the variation of the amount of PCR product may include that identification PCR is put into each hole between limiting holes The dyestuff of product, so that the PCR product generated is more, signal is brighter.It can monitor the intensity of dyestuff during each PCR cycle, one Denier observes required signal strength, and sample is taken out from hole.

Some embodiments of the variation of the amount of PCR product may include the mixture using hybridized bead between limiting holes With with the DNA amplification selective cross from each hole, the hybridized bead is covered with the few core complementary with each hole specificity bar code Thuja acid.Once globule is saturated, uncombinating DNA is washed off, combines DNA from globule release.It then can be by the amount normalizing of the DNA in each hole Change to the saturation limit of globule.

Some embodiments of the variation of the amount of PCR product may include being incubated for chip for a long time between limiting holes, make Fast microorganism, which must be grown, could quickly fill up hole and stops replicating, and grows slow microorganism and gradually fills up hole, once and have in hole The cell of about the same quantity then stops growing.

It in some embodiments, can be with using bar code primer and next-generation sequencing (NGS) in chip format and method For identifying which type is grown in which hole on high density microchip.When the bacterium of approximately equivalent quantity occupies on chip Each micropore when, the signal from each hole is about the same in NGS data.

For example, being generated at one in the typical NGS operation of 12,000,000 sequence reads, if 24 cores are sequenced in the operation Piece, every has bacterial number in 10,000 micropore and each hole identical, then 50 reads of average individual hole.

However, the different bacterium speed of growth is different, therefore a some holes may have seldom bacterium, and a some holes has very More bacteriums.This may make the operation of NGS become bad, because not detecting the seldom hole of bacterium in NGS analysis.

Therefore, in the example of typical NGS operation that 12,000,000 sequence reads are generated at one, each run is sequenced 24 Chip, every have 10,000 micropore, more 100 times than the other half of the bacterium that wherein half has.It grows thin in slow hole The probability that bacterium is detected significantly reduces.In this case:

(10,000x 24)/2x 100=12,000,000 (1)

(10,000x 24) 72=120,000 (2)

Low frequency hole accounts for the 1% of sum.Therefore, in 12,000,000 read of NGS operation, 120,000 Low frequency hole, i.e. 1 read of average individual hole will be come from.

To minimize this phenomenon, need to develop new method to keep the amount of the PCR product between hole equal, thus NGS operation detects all holes.

Silicon substrate microwell chips for microorganism separation, growth, screening and analysis

It substitutes or in addition to plastics, glass and/or polymer, micromachining device or chip can be by least part silicon structure At to allow electrical measurement on the basis of hole-specifically.For example, the wall in each hole can be separated to generate micro- capacitor.At another In example, FET makes a surface be exposed to the content in hole in each hole.Instead of pure silicon base chip, plating, gas can be passed through Mutually deposition, and/or electric arc/flame-spraying thin metal layer of upper generation at the top of existing chip.This can be more for chip addition Functionality, using cheaper and/or more clean another manufacturing method, and/or allow hand-held or portable device small Type.

Some embodiments, which can permit to monitor by electrical measurement, to be grown.Microorganism can be measured using impedance monitoring The growth of (such as bacterium).For example, in the Ur etc. that it is hereby incorporated herein by reference, " Impedance Monitoring of Bacterial Activity, " in 8:1 J.Med.Microbiology 19-28 (1975), by the test tube containing Escherichia coli Impedance compared with its cell count.It can also be to other kinds of bacterium (including pseudomonas, Klebsiella and chain Coccus) to measure with proving effect be universal.Hole can be filled with different culture medium to test the growth of different preparations Condition.

Some embodiments can permit is screened by electrical measurement.Electrical measurement can be carried out on the basis of hole-specifically, To be screened.One example will be pH.PH dependence is obtained on the door of mono- equipment of Kong Zhongcong there are many different modes Reaction, including but not limited to ISFET and pH meter.It includes to produce that hole array with embedded pH sensor can measure those with electricity The hole of the microorganism of raw acid or alkaline metabolin.One simple example is the generation from lactose screening lactic acid.Bacterium is existed It dilutes, grow in hole, be then fed with lactose.Record pH decline hole contain can by lactose metabolism be lactic acid microorganism.

Some embodiments can permit electrical measurement redox probe.Another kind is to see using the method for electrical measurement Examine how the bacterium in hole influences known redox probe.Substantially, measurement there can be clear reaction in the presence of bacterium System, and the deviation of expected property can be attributed to bacteria samples.Typical redox probe is similar to the iron cyanide: [Fe (CN)6]3-/4-.The iron cyanide is reduced to ferrocyanide by detailed characterizations, so that qualitative small variation, especially The electronics transfer of surrounding them can be found.The system is " no label ", because it does not need to be directly changed bacterium itself It can be detected.

It can will identify that the antibody of microorganism (such as Escherichia coli) is fixed in ITO electrode.It can be surveyed from electrode Measure the resistance of the electronics transfer of the solution containing the iron cyanide.Escherichia coli are bound to electrode surface, with Escherichia coli on surface Concentration proportionally increase resistance.This is that redox probe can be used to detect certain types of microorganism or metabolin A kind of measurement method an example.

Work provides the advantage that including but not limited in silicon (or at least metal or metal plastic): production chip is more just Preferably (for example, by improving the prior art)；Allow small-sized and/or portable versions comprehensive detection abilities；In other materials (example Such as LCR, CV) in the additional measurement capability that does not have；The newfound detection method based on chip is integrated in existing apparatus； And the combination with electrical measurement and sequencing.These advantages are beneficial for any consumer for using Interference Detection.

Protect the releasable barrier of the hole specific chemical object on chip

Figure 21 A-21E is the schematic diagram for illustrating the chip for having hole specific chemical object according to some embodiments.Figure 21 A Show micromachining device or chip with multiple micropores.In Figure 21 B, it is special in each micropore of chip to joined micropore Property chemicals.In Figure 21 C, sealant is applied on the micropore specific chemical object in each hole of chip, to prevent chemicals It interacts with other additives in hole.In Figure 21 D, sample is added, sample is tested in micropore.In Figure 21 E In, triggering agent (such as hot) discharges micropore specific chemical object, interacts with the sample in hole.

Microwell chips can be manufactured or be cleaned, and/or handle its surface.Specific chemical object can be prepared separately, then Such as using allowing to import a group-specific chemicals in specific one or the method and/or device adding hole in multiple holes. Then can be with application sealant to protect various chemicals not by environmental pollution, and/or it will with some specific external trigger agent Its removal/release/granting.

It can be particular design by micromachining device or chip manufacturing, such as clean and/or surface treatment to promote Wetting.PCR primer can be printed or pin mark enters particular bore.It is dry to can permit chip, then by evaporating from ethanol solution Deposit wax layer.Optium concentration is about 1%v/v.Molten wax, or spraying aqueous or alcohol wax solution can directly be applied.Alternatively, can be with Use spin coating or vapor deposition.Various waxes can be used, including but not limited to: containing and the glycerol stearate without polyethylene glycol Ester, cetostearyl alcohol, Cetyl OH, tristerin, glycerin monostearate, (the CAS registration of ceteareth -10 Number 68439-49-6) and some commercial products, including but not limited to Lotionpro^TM165 (can be from Eastsound, Washington are obtained) and Polawax^TM(can be obtained from Croda, Inc., Edison, New Jersey).It Afterwards for example, by heating until wax thawing discharges following chemicals.It will be 50 DEG C -70 DEG C for these compositions.It is important Be its sufficiently low aqueous solution without damaging any chemical component or boiling us.

Key concept is hole specific chemical object, is isolated on chip and discharges until experimenter triggers.This method can With the coding for hole, but it can also be more broadly used for a series of different problems.It can be used for not assimilating for encapsulating chip Learn the various metabolism that object includes but is not limited to antibiotic, fluorescein, dyestuff, PCR primer, dissolution accelerator, antibody and/or test Product.Although wax is to seal the good method of thing and can be discharged later by heating, other materials sealing can be used, work as It is released when being exposed to light, ultrasound and/or some other triggering agent.Reagent is simply added compared to chip, this method Advantage first is that based on the control on hole-specifically (well-by-well).By printing chemicals into hole after progress Experiment on Microbiology In may be implemented similar effect, but this can lead to the problem of (printing operation can be up to one day) with the time, in the known integrated circuit it is a fact that, if Microorganism on chip after fill each hole respectively, each hole can not at the same time in exposure.Using releasing mechanism, often It a hole can be in same time exposure.In an example, wax can be deposited by solvent cast.

Simple and/or control relative abundance separation micropore

Superchip device may include the surface with high density micropore.It can will be from the micro- of micropopulation sample Biology or other cell types dilutes and are applied to device so that hole comprising about microorganism or cell/occupy hole.It can use Semi-permeable film seals these micropores, and the semi-permeable film allows nutrients to diffuse into micropore, but prevents whole or at least one Biology or cell are moved from micropore slightly.

Microorganism or cell sample can be prepared, then with impermeable or only gas-permeable film be sealed against into Chip.Only have the nutrients in hole to can be used for supporting micro- life in micropore when there is no liquid reservoir on chip or film, therefore sealing The growth of object or cell.Two reasons of this feature include: that (1) building and workflow are simple, because device does not need half infiltration Permeable membrane or storage cavern do not need addition nutrients, and possibility of pollution is smaller；(2) by limitation fast-growing type obtain nutrients come Check its relative abundance.For the sample comprising some fast-growing types and some slow raw types, fast-growing type will very in its respectively micropore It fastly by limitation resource, stops growing or slow growth, while slow raw type continued growth.With wherein fast-growing type not by limitation nutrition The case where amount of object, is compared, this to account for higher relative abundance in micropopulation of the raw type on chip slowly.When to chip On all when being sequenced, this is extremely important for downstream processing.This also provides better detectable limit for rare type, because It will not be surmounted by the fast type of growth to put rare type at some, to limit their ability of system detection.

Current method attempts that all kinds is allowed to grow, no matter they substantially grow it is fast or slow.It is inevitable in this way As a result, the leading group of fast-growing type and only relative abundance are increase with time.The downstream analysis of many types, such as sequencing or fluorescence Screening can not differentiate each type in given group, but be only capable of differentiating and be higher than those of a certain limitation relative abundance.If Target is to retain diversity and detect rare type, needs to limit fast-growing type in some way.

For example illustrate the idea, consider a simple situation, sample includes two types: it is a kind of double daily, It is another double weekly, as shown in table 6.If raw type is rare (relative abundance 5%) when starting slowly, with two kinds The growth of class, it becomes very rare quickly.

Table 6

It is slow raw after a period of time if limiting fast-growing type by the nutrition of growth and/or the competition of physical space The relative abundance of type will start to increase, as shown in table 7.

Table 7

Limitation	0th day	1st day	2nd day	3rd day	7th day	14th day
							Fast-growing type	19	38	50	50	50	50
Slow raw type	1	1	1	1	2	3
							It amounts to	20	39	51	51	52	53
The relative abundance of fast-growing type	0.950	0.974	0.980	0.980	0.962	0.943
							The relative abundance of slow raw type	0.050	0.026	0.020	0.020	0.038	0.057

High density micro Process array for biological library cell

Biological library is designed so that researcher obtains the great amount of samples from big group, to carry out certain form of grind Study carefully, such as biomarker relevant to disease discovery.The sample that the prior art of biological storage art provides is stored in test tube Or in low-density plank format, such as 96 holes or 384 orifice plates.When sample size to be stored is relatively fewer and sample itself be from It is useful in this way when scattered segregating population.When storing following sample (such as micropopulation sample): wherein sample size may The quantity of variety classes or variant may be from each sample number hundred to thousands of or millions of a, current lifes in higher and each sample Object storage method becomes very cumbersome.Use current method, it is necessary to carry out laborious separation scheme, so as to storing step it It is preceding or isolate single type or variant later, to obtain desired type or variant.

System as described herein, kit, device and method can be used for biology storage (biobank) cell, microorganism, disease Poison and other biological entity.There may be every chip-count thousand by the superchip device of the surface composition with high density micropore A, hundreds of thousands of or millions of a micropores.For example, can (or another biology be real by the microorganism from micropopulation sample Body, such as different types of cell or virus) device is diluted and is applied to, so that hole includes about 1 microorganism/occupy hole.Then, Being incubated for chip replicates microorganism in hole, and gained group represents single type.Location index system based on nucleic acid is available In measuring in each hole, there are what types.The directory system may relate to for PCR primer to be loaded previously into each hole.The PCR draws Object contains the addressing bar code for identifying hole and provides in the targeted microorganisms genome of information or targeting target genetic sequence The primer sequence of specific genetic elements (such as 16S).After incubation, releasing microbe DNA, PCR primer expands non-target bacteria region of DNA Domain.The amplicon in each hole on chip is collected, is then read by next-generation sequencing.

Using high density micro Process chip to carry out biological storage enables many types in each sample or variant as singly Only group's storage, without carrying out laborious separation scheme before storing or later.Use the location index based on DNA System or custom analysis allow simpler general method to identify the heredity signature or feature of each micropore content, to provide Information, such as information.In addition, chip apparatus provides a kind of method of effective torage cell isolate in very space.Example Such as, the significant spatial that the chip of the single microslide size with 100,000 hole occupies is lower than corresponding conventional store Format.Chip format can also be by filing a chip suitably comprising many different samples from single subject With planning sample and/or management subject (for example, patient) information database.

Cell is stored to use the equipment, cell can be added and/or be placed in the micropore of the device.It can handle Then cell in micropore places it in suitable storage condition to mitigate the influence of storage.For example, can be for example sweet with reagent Oil processing cell is to mitigate the influence of freezing.It is then possible to which chip is placed in suitable storage condition such as refrigerator.It can incite somebody to action Cell dehydration, freeze-drying, and/or freeze-drying, and chip can be placed in suitable condition to protect dry cell.It can be to Additional structure is added further to promote it as the purposes of storage device in chip.For example, can be placed on chip film or Another structural detail, to seal at least some holes before storing.

Chip can be mounted with cell, so that a part of micropore on chip is respectively occupied by each about cell. Chip is incubated for allow cellular replication.Chip is replicated, each Kong Zhongcun of authentication chip is carried out using the chip of original chip or duplication Cell or type or gene signature (by using above-mentioned location index system).By chip processing, and/or storage Under suitable conditions.Copy step and/or authentication step can after storing rather than carried out before storage.

One or more cells of each bacterial strain from one group of pre-existing isolated strains can be placed on chip Isolated micropore in.Can recorde the micro well locations that each bacterial strain or cell type are placed, and handle chip, and/or by its Storage is under suitable conditions.

A kind of version of chip can be manufactured, wherein anti-corrosion chemicals are isolated under the wax barrier in each hole.Permit Perhaps isolate seals growth in the chip, discharges preservative by thermal induction before storage later to save.

Such equipment can be used for storing/mixed micropopulation the sample of biology storage, such as from soil, people's enteron aisle, sea The micropopulation sample of water, oral cavity, skin etc..Chip can be used for storing other kinds of biological entities, such as fungi, Gu are carefully Bacterium, people's cell (including proliferative cell), zooblast and virus.

DNA location index system is suitable for all biological entities types, to generate the information about each hole content.It can be with Entire chip is screened using custom analysis (such as analysis based on antibody or substrate) to obtain expectation activity.For example, can sieve Select the chip with the T cell group of storage to obtain specific immunocompetence.

It, can be from research participant collector fecal microorganism group's sample in an illustrative example.It can will come from The fecal microorganism biology of each individual is stored on chip, to retain the record of the individual micropopulation and later as target Mark microbe-derived micropopulation sample.In another example, during clinical or research or as treatment workflow A part (for example, using the cell therapy of ex vivo treatment cell), can be from individual sampling T cell or other immunocytes Population mixture.Also in another example, edaphone can be stored together with seed bank.

High-resolution is chosen

High-precision/accurate selected equipment or system can be designed with executed on above-mentioned micro Process chip from and/or to The various selection functions of micropore.Target substrate or chip can be the microscope glass on a surface with injection moulding feature Piece format (about 25mm x 75mm x 1mm).Micropore can be arranged with mesh model, have about 4- in the perimeter of chip 8mm without bore edges.Size and the interval in hole can be determined based on the ability of Chooser.Micropore can be is along each side size About 25 μm to about 200 μm of square, between bore edges between be divided into about 25 μm to about 100 μm.Micropore can be circle or six Side shape.The depth in hole can be about 25 μm to about 100 μm.For example, edge of the slide of 75mm x 25mm with 7mm, 100 μm Square hole, while while between be divided into 100 μm, have about 16,775 micropores.

High-precision/accurate selecting system can be designed executed on above-mentioned micro Process chip from and/or to corresponding to The various selection functions of a part of film of micropore.Film is used to for the bacterium of growth being encapsulated into the thin slice of micropore before can be.When When removing from chip, film can retain the trace and bacteria samples of microwell array on the surface thereof after separating from chip.Cause This, the film of removing can be used as the duplicate of the bacterium grown in the chips.

High-resolution Chooser can receive data input from user.Input may include that at least a pair of of chip micropore is sat Mark, so that Chooser is from least a pair of coordinate inputted and/or chooses the coordinate at least a pair of of input.It between cycles can be with Carry out sterilizing program.High-resolution Chooser can operate in anaerobic box.

High-resolution Chooser system can receive chip, and will for example be chosen using fiducial marker and/or reference bore Device is aligned with chip.For example, Chooser can choose 96 of the cell grown in the micropore on chip extremely containing growth medium Hole or 384 orifice plates.Chooser may include single selection needle or multiple selection needles.

High-resolution Chooser system can receive film, and for example using the reference bore marker on film by Chooser and film Alignment.For example, Chooser can choose cell to 96 holes containing growth medium or 384 orifice plates from film.Choosing needle can have There are different shape (such as mushroom-shaped) and/or surface (such as texture) to choose from film.System can also include one or more The mechanism (such as floating needle and/or vacuum) of a holding and/or flattening film.

High-resolution Chooser system can be via the transfer replication chip of chip to chip.Based on fiducial marker and/ Or reference bore, Chooser can receive and be aligned the first chip.Based on fiducial marker and/or reference bore, Chooser can be with It receives and is aligned the second chip.For example, the cell grown in the micropore on the first chip can be transferred to second by Chooser The micropore of chip.

For being chosen at the target type of the multiple types for at least one biological entities cultivated in micromachining device automatically High throughput system may include interface for receiving micromachining device.Micromachining device defines high density microwell array.It is high Each micropore of density microwell array is equipped with to separate and cultivating at least one type of at least one biological entities, and including more At least one label of a unique label.Each label of the multiple unique label include nucleic acid molecules and with high density micropore The relevant location specific nucleotide sequence of at least one micropore of array, the nucleic acid molecules include and at least one life The target specificity nucleotide sequence of object entity annealing.System further includes that the high-resolution selection at least one protrusion is set It is standby, it is used to choose at least one biological entities from least one micropore of high density microwell array.System further comprises using It is set in the input unit for receiving at least one target specificity nucleotide sequence instruction, and with input unit and high-resolution selection At least one processor of standby communication coupling.It is special that at least one described processor obtains at least one target from input unit The instruction of specific nucleotide sequence, by least one described target specificity nucleotide sequence compared with multiple unique labels, base At least one micropore of the high density microwell array comprising target type is determined in comparison result, and is controlled high-resolution selection and set It is standby to choose at least one biological entities from least one micropore determined of high density microwell array.

Disclose the target of multiple types for being chosen at at least one biological entities cultivated in micromachining device automatically Mark the high throughput system of type.Micromachining device defines high density microwell array, each micropore of high density microwell array and more At least one unique primers of a unique primers are related.System includes the interface for receiving the film removed from micromachining device, Each micropore of film sealing high density microwell array at least one biological entities to be retained in high density microwell array, So that a part of at least one biological entities corresponding to high density microwell array is still adhered on film after removing film.System System further includes high-resolution selected equipment comprising: at least one protrusion, for from correspond to high density microwell array at least At least one film location of one micropore chooses at least one biological entities；Input unit is related to target type for receiving At least one target specificity nucleotide sequence instruction；With communicated with input unit and high-resolution selected equipment coupling At least one processor.At least one described processor obtains at least one target specificity nucleotides sequence from input unit The instruction of column, by least one described target specificity nucleotide sequence compared with multiple unique labels, based on comparative result really Surely at least one film location of at least one micropore corresponding to high density microwell array comprising target type, and control high score Resolution selected equipment chooses a part of at least one biological entities from least one film location determined.

The embodiment above can be embodied in various ways.It is, for example, possible to use hardware, software, or its combinations to realize embodiment party Case.When implemented in software, software code (either can individually calculated in any processor appropriate or processor sets There is provided, be also distributed across in multiple computers in machine) on execute.

In addition, it should be understood that computer can embody in a variety of forms, such as frame type computer, desktop computer, pen Remember this computer or tablet computer.It is not generally regarded as computer in addition, computer can be embedded in but there is proper treatment In the device of ability, including personal digital assistant (PDA), smart phone or any other suitable portable or fixed electronics are set It is standby.

Also, computer can have one or more and output and input device.These devices can be used for presentation user circle Face.Can be used for providing the output device of user interface example include printer or for output vision present display screen and Loudspeaker or other flexible piezoelectric sound-generating devices of the sound presentation for output.It can be used for providing the reality of the input unit of user interface Example includes keyboard, indicator device such as mouse, touch tablet and digitizer tablet.As another example, computer can pass through sound Sound identification or other form of sound receive input information.

This computer can in any appropriate form by one or more network interconnections, including such as enterprise network, The local area network or wide area network of intelligent network (EST) or internet etc.This network can be based on any technology appropriate, and can be with It is operated according to any scheme appropriate, and may include wireless network, cable network or fiber optic network.

The various methods or process listed herein can be encoded to software, can use various operating systems or platform One of any one or more processors on execute.In addition, this software can use any various suitable programming languages And/or programming or wscript.exe are write, and the executable machine language code executed on a framework or virtual machine can also be compiled as Or intermediate code.

Combined culture base strategy

Above system, kit, device and method can be used together with one or more Combined culture base strategies, described Strategy uses extensive parallel laboratory test (different culture medium on microwell array being related on the micromachining device of the disclosure) to look for To one or more suitable or desired culture mediums for a specific purpose.For example, Combined culture base strategy can be used for from suitable In specified microorganisms type or bacterial strain fast-growth or inhibit a large amount of of different culture medium of specified microorganisms type or bacterial strain can A kind of culture medium can be determined or selected in property.

Culture medium used may include common growth medium, difference culture medium and/or for certain biological entities or The selective medium of biological entities with certain characteristics (such as synthesis of antibiotic resistance or certain metabolin).Culture medium It may include promoting the nutrients or preparation of cell growth, or inhibit/delay cell to grow or even kill cell (such as antibiosis Element) component.As used herein, when composition difference or when content or concentration difference, it is believed that two be loaded into different micropores Kind culture medium is different.When completing the load of different culture medium, at least some single micropores of micromachining device are wherein It is different in terms of the culture medium for including.The variation of culture medium content can cross over one or more dimensions of multiple micropores, be used for Test or find preparation for a specific purpose or condition.In some embodiments, one or more components of culture medium can Comprising report analysis object, such as the reagent of colorimetric or fluorescent marker, when there are target analyte (such as enzyme, metabolin, gene marks Note) when, signal is generated in micropore.

According to some embodiments of the disclosure, a kind of method for selecting culture medium from a variety of different culture mediums is provided.It should Method includes: to obtain the micromachining device comprising multiple micropores；A variety of different culture mediums are loaded into multiple micropores, so that institute Each micropore for stating multiple micropores includes a kind of culture medium, and the multiple micropore includes a variety of different culture mediums；It will come from At least one cell of sample is loaded into each micropore of the multiple micropore；Micromachining device is incubated in predefined conditions Educate the scheduled duration；Content on more multiple micropores；With based on comparing, from a variety of different culture mediums determinations or select At least one culture medium.

In some embodiments, culture medium is loaded into multiple micropores can be used can to each micropore load it is small In the printer of the liquid volume of micro pore volume, the control with sufficient accuracy or resolution ratio is individually to each micropore It is addressed.For example, printer can have the resolution ratio or even higher for the single micropore that diameter is 100 μm or 50 μm Resolution ratio.Such printer can be drop printer, can be on multiple chips by 200pL or the solution of smaller volume Printing is into the thousands of micropores of each chip, and it addresses each micropore with enough accuracy.Volume can be low To tens picoliters, allow more components in smaller micropore and/or each micropore.This allows rapid preparing in an automated manner A large amount of different culture media compositions.It can be in print procedure by the way that the different base solns of various concentration are printed to micropore In come the cultivation of editors base test library, or can prepare in advance before the printing culture medium test library.The small physics of chip Size and low cost make thousands of or even millions of experiments become feasible (relative to thousands of flat plate/discs).In an example In, drop print system can extract liquid from 96 orifice plates, then with the as low as capacity distribution of 30pL they.It can be to printing Machine is programmed, and to pick up different liquid from source plate, and is allocated them on microwell array with different combinations.

In order to which cell or other biological entity (or mixture of they and cell) to be loaded into micropore, also can be used Drop printer as described above.In some embodiments, the cell loading method of low accuracy can be used.For example, such as The cell of only one type of fruit is the object of research to determine its preferred culture medium in multiple selections, or if all Multiple micropores will load the part of same sample, then the lower spraying system of accuracy, needle or pipette can be used.But It is, if using these less accurate loading methods, it should be noted that as far as possible minimize the dislocation for having loaded culture medium in micropore.

Figure 22 A and 22B be in the microwell array printed to according to some embodiments of the present disclosure on micro Process chip not With the matrix image of solution.Figure 22 A shows the culture medium drop for the printing for being partially filled with micropore.Figure 22 B shows and fills out completely Fill the printed droplets of micropore.

In some embodiments, culture medium can be loaded prior to, concurrently with, or after cell is loaded into micropore.? In some embodiments, it has been preloaded in micropore on the micromachining device of different culture medium and has loaded cell or other target organisms Entity.In other embodiments, the micro Process of the cell or other target biological entities that require study has been preloaded in micropore A variety of different culture mediums are loaded on device.

In culture medium to be printed to some embodiments in micropore first, it can permit them and dry.It can store The micromachining device so handled.Later, the solution of cell (for example, microorganism or other cells) to be studied can be loaded The upper surface of the dehydrated medium of (for example, liquid relief or printing) into micropore.Then it can for example be sealed by film or other materials Then micropore can carry out including the down-stream for being incubated for and observing/comparing.

Cell sample solution is being loaded into (such as printing or liquid relief) to some embodiments on micromachining device first In, allow sample drying.The upper surface of cell is loaded in micropore it is then possible to which a variety of culture mediums are printed to.Then it can apply With sealing material (such as film) to seal micropore, then can carry out including the subsequent step for being incubated for and observing/comparing.This In method, if loading cell using the load (such as liquid relief) compared with low accuracy, cell sample is initially loaded to micro- Consistency (for example, the amount for the cell being loaded into each micropore) in hole is extremely important, to ensure to incubate from different culture medium The result educated can be analyzed meaningfully.In order to improve as a result, more many cells are loaded on micropore and will be helped when starting In the opposite variation on reduction micropore.

In some embodiments, it can be loaded to micropore containing there are many culture mediums of nutrients or component.For example, beating During print, a variety of different base solns are printed in micropore respectively by multiple channels of printer, thus in micropore Interior composition preparation.

After both culture medium and cell are loaded on micromachining device and before incubation, it can seal or not close Seal micropore.In some embodiments, micromachining device is loaded with sample and is packaged, molten to allow while limiting sample Liquid flows in and out micropore.In some embodiments, micromachining device can be by allowing to observe (for example, being supervised with inverted microscope Survey growth) optically transparent material be made.In some embodiments, micropore is sealed with film, the film can be ventilative, liquid Body is permeable or impermeable.In some embodiments, using the lamination being arranged with appropriate pressure, temperature and speed Machine, with the micropore of the thin layer PDMS of polypropylene backing sealing load, micropore is isolated, at the same allow they in microscope at Picture.

The culture medium being placed in the micropore on chip can indicate in one or more dimensions of chip between micropore Content and/or preparation gradually or other variations.With find two or more culture dishes content between small difference Different difference, variation in the microwell array of chip in identical relative time section and identical external condition (for example, temperature, Humidity, atmosphere etc.) under occur, thus allow more meaningful comparison is carried out between micropore.

In incubation period and/or later, it can be estimated that in micropore content one or more observable properties (for example, Biology, chemistry and/or phenotype) variation.Assessment can carry out one or many on scheduled timetable or time course. For example, this assessment can be with view-based access control model inspection and/or technology including but not limited to below: measurement (compares discoloration for example, generating Change, fluorescent marker etc.), real time sequence imaging, microscopy, optical density, fluorescence microscopy, mass spectrum, Raman spectrum, thermal imaging, electricity Chemistry, amplification (DNA, cDNA and/or RNA), sequencing (DNA and/or RNA), nucleic acid hybridization and/or antibody combine.In some realities It applies in example, this assessment can carry out digital image analysis based on the image of the content to multiple micropores.

In some embodiments, the content in micropore can be assessed by following: to micro-pore imaging (whenever need When wanting), and any other index based on the variation of optical density, color, fluorescence or micropore content carries out digital picture to image Analysis.In order to be imaged, the film previously sealed can be removed so that liquid evaporation, and microscope photographing micro Process can be used Device.

In some embodiments, a variety of different culture mediums are loaded into multiple micropores can be by training a variety of differences Feeding base is loaded into the micropore of individual region to complete.In each individual region, all micropores all include identical culture medium, But the micropore of different zones includes different culture medium.Individual region can with but do not need include identical quantity micropore.One In a example, four duplicate culture medium groups are printed on micro Process chip.As shown in fig. 23 a, each group includes 9 × 9 Micropore is divided into three regions 2310,2320,2330,3 × 9, each region micropore.It is 100 that all micropores, which contain concentration, The Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) of a cells/well.First area 2310 only receives PBS (phosphate buffered saline (PBS)) retains the cell of printing as control.Second area 2320 receives R2A, slowly gives birth to known to one kind The minimal medium of long acinetobacter calcoaceticus.Third region 2320 receives TSB, a kind of abundant culture of known fast-growth acinetobacter calcoaceticus Base.Micro Process chip is incubated at room temperature one day with growth.If Figure 23 B-23D (is each region after incubation 2310, the micro-image of the content of 2320 and 2330 representative micropore) shown in, it is thin to bacterium which show different culture mediums The influence of intracellular growth rate.As expected, the micropore for loading TSB is grown with maximum Acinetobacter calcoaceticus, and load pair It is not grown then according to the micropore of PSB.

Except the simple visual inspection of eyes, it can be obtained further from the analysis to micropore content object image Information.The digital picture of examined micropore can be run by image analysis software to select micropore, and calculated in micropore Pixel attribute, such as the intermediate value gray value of each micropore (this is the value for indicating that region is dark or bright).Figure 24 shows above-mentioned The result of this micropore image analysis.Then it can calculate in the different micropores being loaded in the same area of same medium It is worth gray value, to obtain another statistic (such as average value) for indicating micropore group entirety.For example, empty micropore, load PBS's is micro- The average median gray value of the micropore in hole, the micropore for loading R2A and load TSB is respectively 204,199,189,125.By including These intermediate value grayvalue transitions from image can be photo densitometry by the standard of known optical density on micromachining device Value, to be measured together with other micropores.

In addition to cell (for example, from microorganism such as bacterium or mammal), above-mentioned strategy and program can also be used for finding Other biological entity (such as mixture or association of cellular component, cellular products, virus or different plant species or entity composition) Culture medium or growth conditions.Some embodiments can be used for optimizing chemical reaction condition or need for goal approach or output Another situation of a large amount of conditions of parameter testing.

For cell, loading the culture medium in micropore may include the expected one or more nutrients for promoting cell growth Or preparation.In other embodiments, culture medium may include predicted delay or cytostatic one or more antibiotic. For example, the antibiotic of different antibiotic and/or different content or concentration can be placed in different micropores.For example, for 600 The different test condition of kind, each micropore can load one of 30 kinds of different antibiotic with one of 20 kinds of various concentrations, or even The test condition different for 6000 kinds, each micropore can be loaded with one of 20 kinds of various concentrations 300 kinds of different antibiotic it One, all on single micro Process chip.It is then possible to chip be loaded, so that some microorganisms are (for example, such as from clinical sample The isolate or pure culture of the microorganism of blood, urine or abscess separation) it is located in each hole.It can be used previously described Tool and program are incubated for chip and monitor its growth.Microorganism it is to antibiotic therein resistant or in which antibiotic In the micropore that content is not enough to suppress growth of microorganism, microorganism will grow.In the inhibition concentration that wherein there is microbe-sensitive Antibiotic micropore in, microorganism will grow relatively slow or not grow.Therefore, these embodiments can determine microorganism The antibiotics sensitivity of isolate, and for example instruct the antibiotic treatment of subject.

Conclusion

While characterized as and illustrate various invention embodiments, those skilled in the art are readily apparent that various other sides Formula and/or structure execute function as described herein and/or obtain result as described herein and/or one or more advantages, this Each of kind variant and/or modification are considered in the range of invention embodiment as described herein.More generally, this field skill Art personnel are it will be readily understood that all parameters, size, material and construction as described herein are intended to be exemplary, actual parameter, ruler Very little, material and/or construction will depend on using the specific one or more application of the teachings of the present invention.Those skilled in the art will It recognizes or is able to use no more than routine experiment and determine many equivalents of specific invention embodiment as described herein. It will thus be appreciated that foregoing embodiments only indicate by way of example, and in the range of the following claims and their equivalents Interior, invention embodiment can carry out in addition to specifically describing and is claimed.The invention embodiment of the disclosure is claimed Each individually feature, system, object, material, kit and/or method as described herein.In addition, if this feature, being System, product, material, kit and/or method do not have conflicting, two or more this features, system, product, material, examination Any combination of agent box and/or method includes in the invention scope of the disclosure.

Also, various concept of the invention, which can be embodied in, provides one or more methods of example.One as method The movement that part carries out can sort in any suitable manner.Therefore, it can construct and wherein be executed with the sequence for being different from diagram The embodiment of movement, including some movements are performed simultaneously, although being in an exemplary embodiment sequentially-operating.

The full text of all publications, patent application, patent and other bibliography that are mentioned above is incorporated by reference into Herein.

Being defined for defining and use herein should be understood as that control dictionary defines, in the document being incorporated by reference into Definition and/or the ordinary meaning for defining term.

The indefinite article " one " used in the specification and in the claims unless otherwise clear explanation and "one" should manage Solution is "at least one".

The phrase "and/or" used in the specification and in the claims is construed as referring to element " any one of connection Or both ", that is, element combines in some cases to be existed, and is respectively present in the case of other.The multiple members enumerated with "and/or" Element should understand in the same manner, that is, in conjunction with " one or more " element.In addition to the element particularly pointed out with "and/or" attribute Outside, other elements are optionally present, no matter it is related or uncorrelated to the element that those are particularly pointed out.Therefore, as non-limiting Example, when with open language for example " comprising " is used together when, " A and/or B " in one embodiment, only refer to A (optionally Include the element in addition to B)；In another embodiment, only refer to B (optionally comprising the element in addition to A)；Also at another In embodiment, refer to both A and B (optionally comprising other elements)；Etc..

As used in the specification and in the claims, "or" should be understood as thering is identical meanings with above-mentioned "and/or".Example Such as, when separating the project in list, "or" or "and/or" should be construed as include, that is, include a large amount of or series of elements At least one, but also comprise more than one, and other optional unlisted projects.Only when term is explicitly indicated it is opposite when, Such as " only one " or " only one " or " by ... form " refer to (when used in a claim) only comprising a large amount of or One element of series of elements.In general, be exclusiveness term (such as " any one ", " one of them ", " only one " before Or " only one ") when, the term as used herein "or" should be understood merely as indicating exclusiveness selection (that is, " one or another Both it is a, but be not ").When used in a claim, " substantially by ... form " should have it in Patent Law field Common meaning when use.

As used in the specification and in the claims, when referring to the list of one or more elements, phrase " at least one It is a " it is understood to refer at least one element selected from any one or more elements of the element list, and it is different It surely include each element specifically enumerated in element list, and any combination for the element being not excluded in element list.This is fixed Justice also allows to be optionally present the element in addition to the element particularly pointed out in the element list of phrase "at least one" meaning no matter Its to those of to particularly point out element related or uncorrelated.Therefore, as non-limiting examples, " at least one of A and B " (or equally, " at least one of A or B ", or equally, " at least one of A and/or B ") in one embodiment, Refer at least one (optionally includes more than one) A and (and optional includes element in addition to B) is not present in B；In another reality It applies in scheme, refers at least one (optionally includes more than one) B and (and optional includes element in addition to A) is not present in A；Also In another embodiment, refer at least one (optionally including more than one) A and at least one (optionally includes more than one It is a) B (optionally including other elements)；Etc..

In claim and description above, all transition phrases for example "comprising", " comprising ", " carrying ", " having ", " containing ", " being related to ", " receiving ", " by ... constitute " etc. be interpreted as it is open, that is, refer to including but not limited to.Only cross Cross phrase " by ... form " and " substantially by ... form " closed or semi-closed transitional phrase should be interpreted as respectively, such as U.S. Patent Office, patent examining procedure handbook, shown in the 2111.03rd trifle.

Claims

1. a kind of method, comprising:

Obtain the micromachining device comprising multiple micropores；

A variety of different culture mediums are loaded into multiple micropores, so that each micropore of multiple micropores includes a kind of culture medium, And multiple micropores include a variety of different culture mediums；

At least one cell from sample is loaded into each micropore of multiple micropores；

Micromachining device is incubated for the scheduled duration in predefined conditions；

The content of multiple micropores on more multiple micropores；With

Based on comparing, at least one culture medium is determined from a variety of different culture mediums.

2. method described in claim 1, wherein culture medium is loaded into each micropore using drop printer.

3. method described in claim 1, wherein at least one cell is loaded into each micropore using drop printer.

4. method described in claim 1, wherein at least one cell is loaded into each micropore using pipette.

5. method described in claim 1, wherein the content of more multiple micropores includes the content for assessing multiple micropores At least one observable property.

6. method described in claim 1, wherein the content of more multiple micropores includes the figure to the content of multiple micropores As carrying out digital image analysis.

7. method described in claim 1, wherein load culture medium carries out before loading at least one cell.

8. method described in claim 1, wherein load culture medium carries out after loading at least one cell.

9. method described in claim 1, wherein loading at least one cell includes that multiple cells are loaded into each micropore In.

10. method described in claim 1, wherein it includes by a variety of differences that a variety of different culture mediums, which are loaded into multiple micropores, Culture medium is loaded into the micropore of the individual region of micromachining device, and wherein each individual region includes more than one micropore, It so that including identical culture medium in micropore in each individual region, and include different trainings in the micropore of different zones Support base.

11. method described in any one of claim 10, wherein the content of more multiple micropores includes the micro- of each individual region of comparison The statistic of the property of content in hole.

12. method described in claim 1 further comprises sealing multiple micropores before being incubated for micromachining device.

13. method described in claim 12 further comprises sealing off multiple micropores before the content of more multiple micropores To allow liquid evaporation.

14. method described in claim 1, wherein the content of more multiple micropores is included in multiple time point assessment contents At least one property.

15. method described in claim 1, wherein the culture medium being loaded at least one of multiple micropores includes nutrients.

16. method described in claim 1, wherein the culture medium being loaded at least one of multiple micropores includes antibiotic.

17. the micromachining device includes to be loaded onto using the method for the micromachining device Selective agar medium comprising multiple micropores A variety of different culture mediums on multiple micropores, this method comprises:

At least one cell from sample is loaded into each of multiple micropores；

The content of more multiple micropores；With

Based on comparing, at least one culture medium of a variety of different culture mediums is determined.

18. the micromachining device includes to be loaded onto using the method for the micromachining device Selective agar medium comprising multiple micropores At least one cell in each of multiple micropores, this method comprises:

A variety of different culture mediums are loaded on multiple micropores；

Multiple cells on more multiple micropores；With

19. a kind of method, comprising:

Obtain the micromachining device comprising multiple micropores；

A variety of different culture mediums are loaded into multiple micropores, so that each micropore of the multiple micropore includes a kind of culture Base, and the multiple micropore includes a variety of different culture mediums；

Target biological entities are loaded into each micropore of multiple micropores；

The content of multiple micropores on more multiple micropores；With

20. method described in claim 19, wherein the target biological entities include at least one cell.

21. the described in any item methods of claim 1-20, wherein the superficial density of multiple micropores of micromachining device is at least 150 micropore/cm², at least 250 micropore/cm², at least 400 micropore/cm², at least 500 micropore/cm², at least 750 Micropore/cm², at least 1000 micropore/cm², at least 2500 micropore/cm², at least 5000 micropore/cm², at least 7500 it is micro- Hole/cm², at least 10000 micropore/cm², at least 50000 micropore/cm², at least 100000 micropore/cm²Or at least 160000 micropore/cm²。

22. the described in any item methods of claim 1-21, wherein each micropore of multiple micropores of micromachining device is straight Diameter is about 5 μm to about 500 μm, about 10 μm to about 300 μm or about 20 μm to about 200 μm.

23. a kind of kit, comprising:

Micromachining device comprising multiple micropores；With

The a variety of different culture mediums being loaded into multiple micropores, each of the multiple micropore include a variety of different culture mediums A kind of culture medium.

24. kit described in claim 23, wherein a variety of different culture mediums to be loaded into the individual region of micromachining device Micropore in, wherein each individual region comprises more than a micropore, so that including in the micropore in each individual region Identical culture medium, and include different culture mediums in the micropore of different zones.

25. the described in any item kits of claim 23-24, wherein the superficial density of multiple micropores of micromachining device be to Few 150 micropore/cm², at least 250 micropore/cm², at least 400 micropore/cm², at least 500 micropore/cm², at least 750 A micropore/cm², at least 1000 micropore/cm², at least 2500 micropore/cm², at least 5000 micropore/cm², at least 7500 Micropore/cm², at least 10000 micropore/cm², at least 50000 micropore/cm², at least 100000 micropore/cm²Or at least 160000 micropore/cm²。

26. the described in any item kits of claim 23-25, wherein each micropore of multiple micropores of micromachining device Diameter is about 5 μm to about 500 μm, about 10 μm to about 300 μm or about 20 μm to about 200 μm.