CN100458444C - Micro-pool array chip processing method capable of being as ultra microhole analytical plate - Google Patents

Abstract

This invention relates to a process method of ultramicropore analysis plate, especially relates to a process method of micro array core which can be used as ultramicropore analysis plate. The process method in this invention, its character is that it using the following steps: (1) flaking: choose transparence material, and make it into two slices in the same size, (2) slotting: fetch one piece of slice, slot several hole in it by using laser perforating machine, (3) enveloping: align and clamp the slices with hole and the one without hole, envelop them into one. Using the process method in this invention has the following merit: the size of the micropore is stable, the process method is fit for industrialization, and the cost is low.

Description

Can be used as the micro-pool array chip processing method of ultra microhole analytical plate

(1) technical field

The present invention relates to a kind of job operation of ultra microhole analytical plate, particularly a kind of micro-pool array chip processing method that can be used as ultra microhole analytical plate.

(2) background technology

At present, medically with the bioanalysis chemical field to biological sample carry out high flux screening particularly the screening of substrate for enzymatic activity mainly rely on 96 orifice plates (96-well plates), 96 holes are arranged on orifice plate, 96 samples are added in 96 holes, in each hole, add reagent again and carry out chromogenic reaction, generation has the product of color or photoluminescent property, the porous plate that adds sample is placed in the 96 orifice plate analysers again, once finishes the analysis to 96 samples.The orifice plate analyser is actually a tintmeter or photofluorometer, detects on 96 orifice plates in each hole the absorbance of liquid or fluorescence intensity the concentration of measured matter in the hole is carried out quantitatively.

At present, along with going deep into of life science, some new biological samples need check.These samples often quantity are few, are difficult to extract, so cost an arm and a leg.Each hole need add about 0.5mL sample on 96 orifice plates, and sample size is bigger, and this is disadvantageous for measuring the trace biology sample.In addition, medical scholar need filter out from hundreds of thousands kind compound and can need measure sample as much as possible in one batch for use as medicinal compound.In order to address this problem, state is extrapolated 384 orifice plates and 1536 orifice plates, sample size has dropped to respectively about 10 μ L and 1 μ L, but these valuable product, and need be from abroad stocking up, import one case Germany Greiner company's 384 orifice plates (40) need about 17000 yuan (in February, 2007 China market price), and 1536 orifice plate prices are just higher.

The technology of producing microwell plate at present in the world is as follows:

U.S. Matrical, 96 orifice plates of Inc and 384 orifice plates are to adopt mould with the polypropylene moulding, and fill the glass-ceramic microballon to strengthen rigidity in forming process.1536 orifice plates that the said firm produces adopt the polypropylene moulding, and are equipped with at the bottom of the glass or at the bottom of the transparent plastic.

These technologies have been applied for patent by external manufacturer, if China domestic manufacturer is according to this explained hereafter, will be to external businessman payment great number patent royalties, increased cost greatly, therefore these microwell plate China do not produce a large amount of dependence on import of the microwell plate that medical science and life science are used substantially.

(3) summary of the invention

The present invention provides a kind of micro-pool array chip processing method that can be used as ultra microhole analytical plate in order to overcome the deficiency of above technology, has the processing pore size and stablizes, and job operation is suitable for industrialization, and processing cost is low.

The present invention realizes by following measure:

The present invention relates to a kind of micro-pool array chip processing method that can be used as ultra microhole analytical plate, it is characterized in that adopting following steps:

(1) film-making: choose transparent material, make two measure-alike thin slices;

(2) punching: get 1 plate sheet, on thin slice, evenly stamp some through holes with laser-beam drilling machine;

(3) involution: the thin slice alignment of the thin slice of punching and not punching is clamped, and involution is an one, promptly.

Job operation of the present invention, described transparent material is preferably organic glass.Organic glass is preferably adopted to adding the method for heat sealing, and heating-up temperature is 106～110 ℃, and be 5-15min heat time heating time.

Adopt job operation of the present invention, have the processing pore size and stablize, job operation is suitable for industrialization, and processing cost is low.

(4) description of drawings

Accompanying drawing 1 is a processing synoptic diagram of the present invention

Among the figure, the thin slice of 1-punching, the thin slice that 2-does not punch, 3-microslide, 4-micro-pool array chip.

(5) embodiment

1. adopt job operation of the present invention, the preparation sample size save reagent more than external 1536 orifice plates, and cost is very cheap less than the ultra microhole analytical plate of 1 μ L.

2. manufacture craft

2.1 the selection of material and cutting.

Selecting thickness is the poly (methyl methacrylate) plate of 1.4mm, and being cut into length with digital-controlled carving machine is 5.0cm, and the rectangular sheet of width 2.5cm cuts the thin slice 1 of several punchings like this;

2.2 rectangular sheet punching

Select the thin slice 1 of the punching of 1 well cutting, with the laser boring on sheet of industrial numerical control laser cutter, bore dia 0.80mm, hole centre distance all is 2.5mm, forms 17 * 6 array, and 102 micropores are arranged on each chip.

2.3 the heat sealing in microarray pond

The thin slice 1 of punching is sticked together with the thin slice 2 that does not punch, they are clamped (as shown in Figure 1) with two sheet glass microslides 3 (microscope is used), 106 ℃ of insulation 10min in baking oven, two organic glass rectangular sheets just are closely linked.Porose on a slice, another sheet does not have the hole, has just obtained micro-pool array chip 4.

3. the index of micro-pool array chip

3.1 master data:

Chip length 50.0mm, width 25.0mm, thickness 2.80mm

102 little ponds, diameter 0.80mm, degree of depth 1.40mm

The volume that calculates each little pond is: 0.70 μ L

3.2 the consistance of bottom, little pond transparency

For the needs of colorimetric and fluorescence analysis, the bottom in little pond needs to keep transparent on the chip, and transparent degree is identical at the bottom of each pond.In process, the end in little pond is a same poly (methyl methacrylate) plate, and thickness, transparency all are identical.In the involution process, be not capped as the part at the bottom of little pond, kept original transparency.Therefore the transparency at the bottom of each little pond keeps highly consistent.

3.3 the consistance of little pond volume

Little pond that we obtain is a columniform space, and the height of cylinder (degree of depth in little pond) is exactly the thickness of perforated rectangle poly (methyl methacrylate) plate, because the thickness of poly (methyl methacrylate) plate is even, so the degree of depth in all little ponds all is consistent.

In laser drilling process, because beam diameter is constant, it is constant that the aperture of ablating out also keeps, the constant diameter in little pond.The volume in little pond can be used cylindrical volume formula V=π r ²H is calculated, and wherein r is little pond radius, and h is little tank depth.And the radius in each little pond is consistent with the degree of depth, and then the volume in each little pond has very high consistance.

4. discuss

4.1 the selection of chip material

We need select material transparent to make chip.The hard transparent material that is easy to get has glass, organic glass etc.In the optical wavelength range of 287-2600nm, the transmittance of glass is 89-92%, and the transmittance of organic glass (PMMA) is greater than 92%.Therefore, we select for use organic glass to process chip at decision.

4.2 organic glass selection of processing technology

4.2.1 whether the poly (methyl methacrylate) plate cutter is worn

Process micro-pool array chip with organic glass, two kinds of selections are arranged.A kind of is to dig out pit on an organic glass, but the organic glass cutter is not worn, and stays a part as at the bottom of the pond.Another kind of organic glass cutter is worn, do the pond with a transparent plexiglass plate more in addition at the bottom of.Preceding a kind of method, the degree of depth of restive cutting is difficult to keep up to a hundred pits that the identical degree of depth is arranged.A kind of method in back can guarantee that the degree of depth of all pits is identical.

4.2.2 the mode of punchinging

We need dig out the circular hole of diameter generally thick (0.80mm) up and down, and require these circular hole proper alignment to become matrix-like.Owing to require these hole proper alignment, need to use the digital control processing mode.The cutting stylus of common digital-controlled carving machine is a sharp cone distal, can't dig out general up and down thick circular hole, can only leave bellmouth, so we select the Laser Processing means.

4.3 the selection of ultra microhole analytical board size

We select for use the organic glass thin plate as the material of making analysis plates.We wear a poly (methyl methacrylate) plate with the laser cutter, its thickness of slab is exactly little tank depth, because we wish to obtain little little pond volume to reduce the sample consumption as far as possible, need poly (methyl methacrylate) plate thin as much as possible.In addition, another piece poly (methyl methacrylate) plate is as the bottom in little pond, and the thin more then light transmission of base plate is good more.That the poly (methyl methacrylate) plate that we have bought from the market is the thinnest is 1.4mm, so our ultramicropore plate thickness is 2.8mm.

The sheet material area size of ultra micro orifice plate is big more, and little pond that can process in the above is just many more, and is just many more at the sample of a batch of energy measurement.But because we have adopted thin poly (methyl methacrylate) plate to process, material is hard inadequately, and is crooked easily if poly (methyl methacrylate) plate is oversize, is difficult to guarantee the smooth of the ultra micro orifice plate that obtains.And, in the heat sealing process, two poly (methyl methacrylate) plates need be clamped together, in clamping process,, just be difficult to guarantee on whole area stressed even if chip area is excessive.We select long 50mm, and the size of wide 25mm is convenient to use the microslide clamping.

4.4 the diameter in little pond

With regard to processing technology, it is more easy to process large diameter micropore, and the minor diameter micropore of processing uniformity is more difficult.Adopt laser cutting machine to punch on the poly (methyl methacrylate) plate that we purchase, the micropore minimum diameter that laser beam can be got is through being measured as 0.80mm.

4.5 the selection of chip involution means

Organic glass is being commonly called as of polymethylmethacrylate (PMMA), and people utilize acetone and other organic solvent can dissolve the characteristics of PMMA usually, utilizes organic solvent to carry out bonding to organic glass.The organic glass bonding agent principal ingredient of Xiao Shouing also is an organic solvent in the market.Zhou etc. utilize mixed organic solvents to realize the low-temperature atmosphere-pressure sealing-in of PMMA micro-fluidic chip.Because organic solvent can be partly dissolved PMMA, the PMMA that separates out after the solvent evaporates can influence transparency attached to the poly (methyl methacrylate) plate surface, so we do not prepare to use the solvent sealing-in.Equally, for fear of introducing the impurity effect transparency, we do not prepare to adopt other bonding agent yet.

PMMA is a kind of thermoplastics, and softening temperature is 105 ℃, and we add heat sealing to orifice plate and base plate under 106 ℃, obtained good effect.Such involution mode is not introduced any impurity, and little pond shape uniformity has kept original transparency at the bottom of the pond.

4.6 the distortion of micropore reaches the influence to assay determination in the involution process

We have measured the micro-pore diameter and the degree of depth before and after the involution, find behind the involution that micro-pore diameter changes and change in depth all in 1%, as calculated as can be known involution front and back volume change 3% with interior ((1.01) ³-1 ≈ 3%).

Need to prove, the light analytical approach, no matter be colourimetry or fluorescence method, it all is the relative measurement method, the measured value that the obtaining of its measured value depends on standard solution and the measured value of detected solution are relatively, therefore if behind the involution on the same chip shape, volume, the size in each little pond be consistent, little pond is exactly identical to the influence of standard model and sample, just can not influence final measured value.By measuring, we find, the degree of depth in little pond and diameter uniformity all behind the involution.

4.7 little pond density determines on the chip

People wish that little pond as much as possible is arranged on a block analysis plate, can measure sample as much as possible a batch of analysis like this.But we have adopted and have added the heat sealing means, if little pond density is excessive, then the space area occupied is big on the orifice plate, and that organic glass takes up space is little, may produce deformation in clamping heating process, are unfavorable for the maintenance of micropore shape.So, should keep enough intervals between little pond, we select little pond center distance of 2.5mm by experiment.

4.8 comparison with existing micropore analysis plates

The sample consumption: the ultra microhole analytical plate that we make, the total volume in its little pond is 0.70 microlitre (sample cell is not generally filled it up with, and can measure 0.2-0.6 microlitre sample easily), it consumes sample size and is less than 1536 present orifice plates, 384 orifice plates and 96 orifice plates.

Analysis throughput: on the micropore analysis plates that we make 102 little ponds are arranged, analysis throughput still is lower than 384 orifice plates and 1536 orifice plates a little more than 96 orifice plates.

Transmittance: because the transmittance of the PMMA that we adopt is higher than glass and polypropylene, and polypropylene and glass are the materials of commercialization micropore analysis plates, therefore the micropore analysis plates transmittance of our making is a little more than present commercial 96 orifice plates, 384 orifice plates and 1536 orifice plates.

5. conclusion:

We find out the manufacture craft of micropore analysis plates, and the micropore analysis plates that makes has higher analysis throughput (102 samples/piece) and low-down sample consumption (less than 0.70 microlitre).Utilize this technology, can produce the product of specification and commercially produced product same size.

Claims (1)

1, a kind of micro-pool array chip processing method that can be used as ultra microhole analytical plate is characterized in that adopting following steps:

(1) film-making: choose transparent material, make two measure-alike thin slices, described transparent material is an organic glass;

(2) punching: get 1 plate sheet, on thin slice, evenly stamp some through holes with laser-beam drilling machine;

(3) involution: the thin slice alignment of the thin slice of punching and not punching is clamped, and involution is an one, promptly; Described involution is for adding heat sealing, and heating-up temperature is 106～110 ℃, and be 5-15min heat time heating time.

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