CN102281950B

CN102281950B - Programmable microfluidic digital array

Info

Publication number: CN102281950B
Application number: CN200980154761.XA
Authority: CN
Inventors: 布赖恩·福勒
Original assignee: Fluidigm Corp
Current assignee: Standard Biotools Corp
Priority date: 2008-12-08
Filing date: 2009-12-07
Publication date: 2015-05-06
Anticipated expiration: 2029-12-07
Also published as: SG10201404682WA; WO2010077618A1; CN105964316A; SG10201404683TA; EP2376227A1; CN104741159B; CN104741159A; CN102281950A; SG10201404685PA; SG172015A1; EP2376227A4

Abstract

A microfluidic device includes a pressure source and a control line in fluid communication with the pressure source. The microfluidic device also includes a plurality of valves operated via the control line and an independent valve positioned adjacent the control line and between the pressure source and the plurality of valves.

Description

Programmable microfluidic digital array

Background of invention

For analyzing, preparing, measure and other operating functions on the yardstick that microfluidic device still cannot be able to be imagined before soon.The advantage of microfluidic device comprises saving fluid accuracy hardly in visible level of precious reagent and sample, the high density of sample analysis or synthesis and high flux, naked eyes and the degree of accuracy and replacing the space that the corresponding device that operates on macrofluid yardstick brings and reduces.Reduce to microfluidic device size and density to increase relevant be that higher complexity and larger engineering and manufacturing cost and meticulousr device construct.

Although have these to be in progress in microfluid design and devdlop, reduce the complexity of micro-fluid chip and simplify their operation or useful.In addition, the ability of the correlated response process occurred in the flowing of the control fluid of enhancing and microfluidic device is needed.Therefore, this area needs the method and system of the improvement relevant to microfluidic device.

Summary of the invention

The present invention relates to microfluidic device.More specifically, the present invention relates to Programmable microfluidic digital array and method of operating thereof.Only as an example, described method and apparatus has been applied in the system providing asynchronous logic function in micro-fluid chip.In addition, check valve is integrated into digital array to provide lock-in control line by embodiments more of the present invention.But, will be appreciated that the present invention has the much wide scope of application.

According to one embodiment of the invention, provide microfluidic device.Described microfluidic device comprises pressure source and the control line with pressure fluid communication.Described microfluidic device also comprises multiple valve operated by described control line, also comprises separate valves, and described separate valves is near described control line and between described pressure source and described multiple valve.

According to another embodiment of the invention, provide the method that operation has the microfluidic device of valve and control line, described control line has coupled valve group.Described method comprises closes described valve and applies pressure to described control line.The valve of described cut out causes the valve group be connected with described control line to operate.

According to another embodiment of the present invention, provide microfluidic device.The control line that described microfluidic device comprises the first valve, the second valve and is communicated with the second valve fluid with described first valve.Described microfluidic device also comprises the check valve near the acute build up of pressure device (pressureaccumulator) that is communicated with described control line fluid and the described control line between described acute build up of pressure device and described second valve.

According to an alternate embodiment of the present invention, provide microfluidic device.Described microfluidic device comprises multiple reative cell arranged with array layout.Each of described multiple reative cell has the first valve be communicated with the fluid in multiple reative cell and the second valve be communicated with the fluid described in described multiple reative cell.Described microfluidic device also comprises the input line group that can operate to start described first valve and the first control line of described second valve and be communicated with described multiple reative cell fluid.Described microfluidic device also comprises the multiple sample inlet be communicated with described input line group fluid and the check valve be arranged in described first control line.

According to another alternate embodiment of the present invention, provide microfluidic device.Described microfluidic device comprises multiple reative cell and multiple first input port.Each one of more than first input line of passing through of described multiple first input port is communicated with one or more fluids of described multiple reative cell.Described microfluidic device also comprises multiple second input port.Each one of more than second input line of passing through of described multiple second input port is communicated with one or more fluids of described multiple reative cell.Described microfluidic device also comprises the first acute build up of pressure device be communicated with the first control line fluid.Described first control line is set to close described more than first input line.In addition, described microfluidic device comprises the second acute build up of pressure device be communicated with the second control line fluid.Described second control line is set to close described more than second input line.In addition, described microfluidic device comprises the first check valve in described first control line be arranged between described first acute build up of pressure device and described more than first input line, and is arranged in the second check valve in described second control line between described second acute build up of pressure device and described more than second input line.

According to another alternate embodiment, provide the method that operation has the microfluidic device of multiple valve and check valve.The control line that described method comprises to described microfluidic device applies first fluid pressure, and responds to the applying of described first pressure and close described multiple valve.Described method also comprises the applying that responds to described first pressure and closes described check valve, and applies second fluid pressure to the second control line of described microfluidic device.

According to a specific embodiments of the present invention, provide the method that operation has the microfluidic device of multiple input port.Described method comprises provides input fluid to one of described multiple input port, and starts valve group to close the Part I input line be connected with the subgroup of described multiple input port.Described subgroup does not comprise the one of described of described multiple input port.Described method also comprises makes described input fluid flow through the input line be connected with one of described in described multiple input port, makes described input fluid flow through described input line and arrives the Part II of described input line and close the second valve group to separate multiple reative cell.

According to another specific embodiments of the present invention, provide the method for operation programmable microfluidic device, described device has the reaction site array be communicated with the second input line group fluid with the first input line group.Described method comprises the first valve group of starting and can operate to block described first input line group, and starts the second valve group of the Part I that can operate the subgroup of blocking the second input line group.Described method also comprises and sample is loaded into described reaction site by the Part II of described second input line group and starts the 3rd valve group that can operate to separate described reaction site.

According to another specific embodiments of the present invention, provide microfluidic device.Described microfluidic device comprises the input port of predetermined quantity and multiple input fluid line, and each operation of described input port receives one of multiple input fluid, and the fluid of the input port of each and described predetermined quantity of described multiple input fluid line is communicated with.Described microfluidic device also comprises valve group, and each operation of described valve group closes one of described multiple input fluid line.The quantity of described valve group is less than described predetermined quantity.Described microfluidic device also comprises the manifold and the second valve group that are communicated with each fluid of described input fluid line, and each of described second valve group operates a part of closing described manifold.

According to one embodiment of the invention, provide the method for operation programmable microfluidic device, described device has reaction site array, its with the first input line group, the second input line group and the manifold fluid be connected with described second input line group be communicated with.Described method comprises the first valve group of starting and can operate to close described first input line group, start the second valve group of the Part I that can operate the subgroup of closing the second input line group, and start and can be stopped using the 3rd valve group of (deactivate) in order to make described manifold by operation.Described method also comprises cancelling starts (deactuate) described second valve group, multiple sample is loaded into described reaction site by the Part II of described second input line group, and start can by the four valve group of operation in order to separate described reaction site.

According to another embodiment of the invention, provide the method for operation programmable microfluidic device, described device has the reaction site array be communicated with the second input line group fluid with the first input line group.Described method comprises that start can by the first valve group of operation in order to block described first input line group, and start can by the second valve group of operation in order to separate described reaction site.Described method also comprises cancelling starts described second valve group, and multiple sample is loaded into described reaction site by the second input line assembling and is started described second valve group.

According to another embodiment of the present invention, provide microfluidic device.Described microfluidic device comprises multiple reaction site and the first input line group, and described first input line group provides the fluid between the first input port of predetermined quantity and described multiple reaction site to be communicated with.The quantity of described first group is described predetermined quantity.Described microfluidic device also comprises the second input line group, and it provides the fluid between the second input port of predetermined quantity with described multiple reative cell to be communicated with.Described each of second input line group comprises trunk portion and component, and the quantity of described second group is less than described predetermined quantity.Described microfluidic device also comprises programmable input unit, and it can operate to use described first input line group or described second input line assembling to fill out described reative cell.

According to a specific embodiments of the present invention, provide the method arranging and there is the microfluidic device of multiple control line.Described method comprises starts the first control line and valve is placed in the first state.Described method is started after also comprising can by second control line of operation in order to valve group to be placed in the second state.The valve being in described first state stops described valve group to be placed in the second state.

According to another specific embodiments of the present invention, provide the method arranging and there is the microfluidic device of multiple control line.Described method comprises by starting the first control line and then starting the first state that the second control line sets up described microfluidic device, and by starting described second control line and starting the second state that described first control line sets up described microfluidic device subsequently.

According to another specific embodiments of the present invention, provide a kind of microfluidic device.Described microfluidic device comprises the first valve and the second valve.Described microfluidic device also comprises the control line be communicated with described second valve fluid with described first valve, and the pressure source be communicated with described control line fluid.Described microfluidic device also comprises and is arranged in pressure source described in described control line and the check valve between described second valve.

According to another alternate embodiment of the present invention, provide microfluid system.Described microfluid system comprises carrier.Described carrier comprises multiple first input port and multiple first input line.The fluid that is each and described multiple first input port of described multiple first input line is communicated with.Described carrier also comprises multiple second input port and multiple second input line.The fluid that is each and described multiple second input port of described multiple second input line is communicated with.Described carrier also comprises the first pressure source and the second pressure source.Described microfluid system also comprises the microfluidic device be contained on described carrier.Described microfluidic device comprises multiple 3rd input line and multiple 4th input line.The fluid that is each and described multiple first input line of described multiple 3rd input line is communicated with, and the fluid that is each and described multiple second input line of described 4th input line is communicated with.Described microfluidic device also comprises the first control line with described first pressure fluid communication, can by operation in order to block described first control line check valve at least partially, and with the second control line of described second pressure fluid communication.

The present invention has the many advantages surmounting routine techniques.Such as, the technology of the present invention allows to customize microfluidic device after fabrication, enables user provide the setting of multiple panel able to programme.These and other embodiments of the present invention and its many advantage and feature contact hereafter describe in more detail with accompanying drawing.

Accompanying drawing is sketched

Fig. 1 is the rough schematic view of microfluidic device according to an embodiment of the invention.

Fig. 2 A is the rough schematic view of check valve according to an embodiment of the invention.

Fig. 2 B is the simplification top view of check valve shown in display Fig. 2 A.

Fig. 3 is the simplified flow chart of the method for a kind of operation microfluidic device according to an embodiment of the invention.

Fig. 4 is the simplified flow chart of the method for a kind of operation microfluidic device according to another embodiment of the invention.

Fig. 5 is the simplified flow chart of a kind of operation according to the method for the microfluidic device of another embodiment of the present invention.

Fig. 6 A is the sketch of reative cell in array according to an embodiment of the invention.

Fig. 6 B is the simplified perspective view of control line in array according to an embodiment of the invention, fluid input line and reative cell.

Fig. 7 shows a kind of method for simplifying to programmable microfluidic device programming according to an embodiment of the invention.

Fig. 8 shows a kind of method for simplifying to programmable microfluidic device programming according to another embodiment of the invention.

Fig. 9 shows a kind of method for simplifying to programming according to the programmable microfluidic device of another embodiment of the present invention.

Figure 10 is the rough schematic view of programmable microfluidic device according to an embodiment of the invention.

Figure 11 is the rough schematic view of microfluid system according to an embodiment of the invention.

Specific embodiments describes in detail

Fig. 1 is the rough schematic view of microfluidic device according to an embodiment of the invention.In a specific embodiment, the microfluidic device shown in Fig. 1 comprises Programmable high-density digital array.Described microfluidic device 100 comprises the first pressure source 102 and the second pressure source 104.In FIG, first pressure source 102 is called as and holds accumulator (containment accumulator), as described in more detail below, described pressure source 102 is communicated with control line fluid, and described control line can operate in order to close the valve be associated with reative cell (not shown) in array 106.Starting of carrying out owing to using pressure source 102 makes these reative cell valves close and makes sample and/or reagent be contained in described reative cell, and therefore pressure source 102 is called accommodation accumulator in some embodiments.

In operation, be placed on by liquid in acute build up of pressure device (pressure accumulator) or pressure source, described acute build up of pressure device or pressure source are connected with outside malleation supply (as containing compressed-air actuated container) subsequently.Under stress, the liquid in described acute build up of pressure device is pushed control line by described compressed air or other fluids, thus starts valve.Therefore, described pressure source does not comprise pressure fluid during fabrication usually, but during being provided in operation microfluidic device, pressure fluid can accumulate and store to execute stressed container to control line upon activation wherein.As more complete description in the application's full text, described acute build up of pressure device can operate to maintain the pressure in control line upon activation.At CO-PENDING and common assigned U.S.Provisional Patent application No.61/044, provide the additional description of digital array being applicable to implementing embodiment of the present invention in 417, its whole disclosure to be quoted by entirety all objects and is incorporated to herein.

Fig. 6 A is the sketch of reative cell in array according to an embodiment of the invention.As an example, embodiment more of the present invention adopts the cell with reative cell, and its lateral dimension is 100 μm × 60 μm, is highly 125 μm.In this exemplary embodiment, the volume of described room is about 0.75nl.Room volume energy little like this uses less sample volume and decreases running cost.Through hole (via) the coupled reaction room that diameter is 50 μm and mensuration/sample input line.With the second spacing placing response room, side direction room of the first side direction room spacing of 110 μm and 200 μm.From containing measuring/the different layer of the layer of sample input line in the valve of 50 μm × 50 μm is provided, described valve can operate and flow through described input line in order to prevention.The particular geometric profile of this cell is not intended to limit embodiment of the present invention, and is only to provide the example of specific embodiments.In another embodiment, other device geometric shapes being suitable for embody rule are adopted.

According to embodiments more of the present invention, adopt that width range is about 5 μm to about 400 μm, depth bounds provides the fluid flowing through described microfluidic device for the input line of about 5 μm to about 75 μm.The control line that width range is about 5 μm to about 400 μm, depth bounds is about 5 μm to about 75 μm flows through the fluid of input line for closing (valve off) by valve.Adopt that width range is about 10 μm to about 500 μm, length is about 10 μm to about 500 μm in some embodiments, altitude range is about the reative cell of 5 μm to about 500 μm.The geometric shape of these devices provides by way of example, and is not intended to limit embodiment as herein described.

As shown in Figure 6A, provide multiple input line 620, make fluid can flow through described input line in the horizontal direction.Show three input lines, but embodiment more of the present invention adopts the input line more than three, such as, 11 input lines.Adopt many input lines then single sample can be distributed in multiple input line, this provide multiple copies of given sample.As described in more complete description that Fig. 6 B is carried out, described input line or input channel are included in the ground floor of microfluidic device at least partly.See Fig. 1, described input line is communicated with mensuration input line 132 fluid on the left of array 106, and is communicated with sample input line 142 fluid on the right side of array 106.Therefore, derive from the fluid measuring input 130 or sample input 140 all can be supplied to input line and and then be supplied to reative cell.

Multiple control line 610 is provided in vertical direction, makes it possible to the fluid controlled through input line and flow.Show two input lines, but embodiment more of the present invention adopts the control line more than two, such as, 70 control lines.In another embodiment, 71 control lines are had.Described many control lines form along the length of input line the reative cell be separated, and providing package is containing multiple reative cells of same sample.As described in the more complete description of Fig. 6 B, described control line or control channel are included in the second layer of microfluidic device at least partly.See Fig. 1,114 partial fluid communication of described control line and lock-in control line 110, described lock-in control line is communicated with accommodation accumulator 102 fluid.Form valve 615 at the intersection point of described control line and described input line, it responds to the fluid pressure in control line and actuated, and can operate to stop fluid to flow through described input line.Usually, described multilayer microfluidic device comprise multiple elastomer layer and valve 615 comprise can moveable diaphragm.Fig. 6 A and 6B show embodiment in, described valve can moveable diaphragm deflect to be arranged in the intersection point of control channel on fluid passage.Therefore, shown embodiment adopts " above pushing away " valve, wherein can moveable diaphragm upward deflect to fluid passage with in closedown fluid passage, valve position.For the valve shown in figures 6 a and 6b, the fluid pressure existed in release control passage causes moveable diaphragm to return non-inflection point thus opening the valve of closedown.

The fluid flowing through input line 620 also upwards flows into reative cell 630 along the direction with Fig. 6 A plane orthogonal through through hole 625, and as described in the more complete description to Fig. 6 B, reative cell 630 is included in the third layer of microfluidic device at least partly.Therefore, described through hole is included at least described second or third layer of microfluidic device at least partly.Usually, a part for laser ablation process removal second or third layer is adopted to form described through hole.Because described microfluidic device is that air is permeable, closed filling technique (blind fill technique) filling reative cell can be adopted and carry out number of chemical, biology or other experiments.Those skilled in the art understand, and after there is fluid in the reaction chamber, starting of control line will cause valve close and fluid is retained predetermined time section in the reaction chamber.

Fig. 6 B is the simplified perspective view of control line, fluid input line and reative cell in the array of one embodiment of the invention.Array (array 106 such as, shown in Fig. 1) is a part for multilayer microfluidic device.Every layer comprises the elastomer structure with one or more groove, passage, room etc. usually.As shown here, ground floor 601 comprises the multiple control channels 610 and other control channel 611 that are arranged to array of parallel channels.Described control channel 610 is communicated with one or more pressure source or acute build up of pressure device fluid with other control channel 611.Therefore in one embodiment, control channel 610 is communicated with acute build up of pressure device 102 fluid, and control channel 611 is being communicated with acute build up of pressure device 104 fluid.Although show single control channel 611 in fig. 6b, those of ordinary skill in the art should understand shown single passage and represent one or more control channel be associated with 601 layers.

In addition, described control channel is not restricted to the position relevant to layer 601.Described control channel can be adapted to embody rule and be placed in other layers.Such as, in the enforcement of a multiple control (control-on-control), in order to make the first control line (such as control channel 611) produce control to the second control line (such as control channel 610), described second control line is placed in both ground floor 601 and the second layer 602 according to the length of control channel.Adopt the through hole similar to through hole 625 that second control line is transformed into the second layer 602 from ground floor 601.By passing the second control line above the first control line, form valve at the intersection point of two control lines.Once start described first control line, then between described first control line and described second control line, the flexible membrane of valve position is upwardly arranged in second control line of 602 layers to block at valve position.Therefore, as more complete description hereinafter, " above pushing away " valve can be formed between different control line or between control line and fluid input line.See Fig. 1, the control line be connected with valve 128 is transitioned into upper strata by through hole from the lower floor of structure, then makes control line 122 through control line 122 thus can start separator valve 124.Then, the control line be associated with valve 128 is worn through another through hole and is got back in ground floor thus pass through and start the valve of fluid input line 132.Once control line 122 actuated, separator valve 124 blocks the flowing through the control line be connected with valve 128, and prevention valve 128 responds to the cut out that control line 110 starts usually.

The second layer 602 comprises also with multiple fluid inlet channel 620 that array of parallel channels is arranged.In the embodiment shown by Fig. 6 A and 6B, control channel 610 and input channel 620 arrange perpendicular to each other.Starting of the valve that pressure realizes existing in microfluidic device is applied by the liquid (being generally basic incompressible fluid) existed in control channel 610.Usually, liquid to be placed in accumulator (accumulator) or pressure source and the fluid (such as air, nitrogen etc.) of pressurization is provided to accumulator.In accumulator, the increase of pressure makes liquid enter control line under stress.Respond to institute's applied pressure, form moveable diaphragm can upward deflecting and enter fluid inlet channel 620 of 601 layers of top.Therefore, " above pushing away " valve is produced in control channel and fluid inlet channel intersection point.Some other embodiments adopt by reorientating various control and fluid layer " under push away ", " drop-down " or " pull-up " valve.

In embodiments more of the present invention that multiple control is provided, one or more other key-course (such as lower floor 601) can be provided, or by control line is arranged into 602 layers.One or more other key-course described can comprise other control line (not shown), and it is once start the control line by existing in valve closed layer 601.Therefore, by applying fluid pressure to the control fluid (such as liquid) existed in one or more other control line, flexible membrane deflects, to control line 610, stops fluid to flow through control line.Therefore, embodiments more of the present invention provide key-course, also referred to as multiple control.Those of ordinary skill in the art can understand many changes, improvement and replacement.

The described second layer also comprises multiple through hole 625, produces the fluid passage provided from described input channel to the flowing of the fluid of third layer 603.Third layer 603 comprises multiple reative cell 630, and it is communicated with described fluid inlet channel fluid by through hole.In the embodiment illustrated, reative cell 630 is formed as being connected with the bottom of layer 603, and in other words, described reative cell is open from bottom.Therefore, through hole is completely contained in 602 layers.In other embodiments, described through hole can be comprised in 602 and 603 layers in both to be adapted to concrete application.

In some embodiments, microfluidic device can comprise according to one or more layer rotated or prepared by cast fabrication scheme.Such as, rotation approach can relate to places polymeric material on figuratum dish or mould, and rotates described dish to produce polymeric layer on dish.Illustrative polymers draws together polymethacrylates, polystyrene, polypropylene, polyester, fluorinated polymer, polytetrafluoroethylene (PTFE), Merlon, polysilicon and dimethyl silicone polymer (polydimethylsiloxane, PDMS).Casting program can relate to as being cast on figuratum template or mould by PDMS material, and this can produce the PMDS layer that can intactly peel from mould or leave behind.Usually, thickness prepared by the layer ratio rotation manufacturing technology prepared by cast manufacturing technology.Elastomer block can comprise one or more cast or rotating layer of any required combination.

In some embodiments, ground floor 603 can be manufactured according to rotation approach.Such as, PDMS can be placed on the mould had corresponding to the bossing of multiple required control channel 610.Rotatable described mould to produce PDMS thin layer on whole mould.After hardening, ground floor 601 can be peeled off from mould and be attached on applicable rigid matrix (as glass, silicon) or plastics (polystyrene).Or ground floor 601 can remain and be attached on mould.Ground floor 601 can comprise opening, groove or form or limit other spaces of control channel 610 at least partly.

In order to generate the second layer 602, can adopt rotation approach, being placed on by PDMS on the second mould, described second mould has the bossing corresponding to multiple required fluid inlet channel.Second mould also can comprise, such as, form the projection or the wavelike segments that correspond to and arrange mark in the second layer 602.These arrangement marks can use in the laser ablation for the formation of through hole 625, point to described arrangement mark with laser ablation during making ablation processes.Rotatable second mould to provide PDMS thin layer on whole second mould.The second layer 602 can comprise opening, groove or form or limit other spaces of input channel 620 at least partly.In some cases, the second layer 602 can be exposed to the above-mentioned laser ablation of one or many.The ablating laser beam pointing to the second layer 602 can form through hole 625.After the second layer 602 fully solidification, ground floor 601 can be peeled off from the second mould, and it is alignd with ground floor and contacts.The second layer and ground floor can be pasted to make two-layer sticking on rigid matrix.

In order to adopt casting program to produce third layer 603, PDMS can be cast on the 3rd mould that has corresponding to the bossing of multiple required reative cell 630.After hardening, can third layer 603 be peeled off from the 3rd mould and align with the second layer 602 and contact.Third layer can be pasted on the second layer to make all three layers to stick on rigid matrix.The material that can manufacture microfluidic device includes but not limited to the combination of elastomer, silicon, glass, metal, polymer, pottery, inorganic material and/or these materials.

Again see Fig. 1, pressure source 102 is communicated with closed control line 110 fluid.Described lock-in control line comprises section and the check valve 112 (also referred to as check-valves (checkvalve)) of several hereafter more complete description.The other description about check valve is provided in CO-PENDING and the common international patent application No.PCT/US07/080489 (open with international publication number WO 2008/043046A2, its whole disclosure is quoted by entirety and is incorporated to herein) transferred the possession of.First section 114 receives from pressure source 102 and starts pressure and can operate to close the accommodation valve (containment valve) existed closed array 106.As understood by the skilled person, array 106 is suitable for carrying out multiple micro-fluid experiment.Thus close accommodation valve by responding to starting of lock-in control line 110, the sample, reagent etc. existed in the reative cell in array is retained in the reaction chamber.The section 114 of lock-in control line 110 without check-valves, this make accommodation valve remove pressure source 102 start pressure after reopen.

Second section 116 of lock-in control line 110 is in the downstream of check valve 112.Because check valve 112 can operate to stop fluid to flow to section 114 from section 116, starting of pressure source 102 will cause controlling fluid (being generally liquid) by check valve 112 also shutoff valve 118a-118e.The fine rule be associated with lock-in control line 110 shown in Fig. 1 represents " overhead (flyover) " section of control line, and wherein the design of control line prevents from measuring input line 132 (describing more completely hereinafter) closedown after lock-in control line 110 starts.The thick line be associated with valve 118a-118e represents and can be operated in order to close or the valve of inactive manifold 135 (fluid be provided between multiple mensuration input line 132 flows).Closing to stop to measure and flow through manifold 135 flows to other mensuration input line from a mensuration input line in the pass of valve 118a-118e, this is called stopping using of manifold.

After starting lock-in control line 110 by pressure source 102, valve 118a-118e can close and remove lock-in control line start pressure after, check valve 112 can the closed condition of maintaining valve 118a-118e.Contrary with valve 118a-118e, the accommodation valve started by section 114 can be opened after starting pressure described in removal.Therefore, one or more section that in lock-in control line, it is feature that the locus of check-valves provides with locking or non-blocked behavior.As those skilled in the art are clear and definite, in control line, adopt extra check-valves to provide to have the extra section of locking behavior.

Lock-in control line 110 also comprises the 3rd section 128, and it has can by operation in order to close the valve from the mensuration input line 132 measuring input 130 to array 106.Measure input 130 and also can be called as input port.Because the 3rd section 128 is also in the downstream of check valve 112, starts lock-in control line 110 by pressure source 102 and can close valve in the 3rd section 128 and stop and measuring the flowing between input line 132 and array 106 and reative cell that it is arranged.Remove lock-in control line 110 start pressure after the valve of section 128 keep closing.

There is provided the second pressure source 104 be communicated with the second control line 120 fluid, it is called as interface accumulator (interface accumulator).Described second control line also comprises several section 121,123 and 125.Section 121 comprises elevated region 122 to make it possible to start separate valves 124 when not closing and measuring input line 132.Provide the other description to valve 124 (being called independence or separator valve (independent or decoupling valve) herein) in this specification, and hereinafter it is more specifically described.Although specific independence described in FIG or separator valve 124 use the second pressure source 104 to start, this is optional in the present invention.In other embodiments, independence or separator valve 124 can be machinery, electrostatic, fluid, electromechanical, thermodynamic, piezoelectricity etc.Therefore, although adopt the second pressure source 104 shown in Fig. 1 to start separator valve 124, this is optional in some embodiments.In addition, although use single second pressure source 104 to control to comprise multiple valve groups of separator valve, the combination that other embodiments can adopt multiple pressure source fluid to start and electrostatic start.Those of ordinary skill in the art understand multiple change, improvement and replacement.

Section 123 comprises can by the valve 134 of operation in order to close in the mensuration input line 132 shown in 65.Owing to only providing 5 valves 134 in the embodiment depicted in fig. 1, in the section 123 undertaken by pressure source 104, starting of valve 134 still can make the mensuration input line of low order end be held open.Section 125 comprises check valve 127 and can by the valve 126 of operation in order to close sample input line 142, and described sample input line 142 inputs (i.e. input port) 140 and is communicated with array 106 fluid with sample.Although should note showing mensuration and sample input in FIG, the invention is not restricted to only have mensuration and sample input, other inputs are included in the scope of the present invention.

Unidirectional or check-valves 127 are placed on the section 125 of lock-in control line 120, allow 126 valves to close and keep closing after section applies pressure, stoping fluid by the flowing of sample input line dealing array 106.Because section 121 and 123 does not comprise check-valves, cancelling of these sections is started and will be caused reopening of valve 134 and 124.

Embodiments more of the present invention provide asynchronous logic function in microfluidic device 100.Such as, owing to using independent and independent of control line 110 shown in other control line 120 to start the independence shown in Fig. 1 or separator valve 124, embodiments more of the present invention provide multiple control.In FIG, before starting separator valve 124, pressure is applied to lock-in control line 110 and locking can hold valve 128.On the other hand, before applying pressure by accommodation accumulator 102 pairs of lock-in control lines 110, start lock-in control line 120 by interface accumulator 104 thus close separator valve 124 and will stop the closedown holding valve 128.In other words, if separator valve 124 cuts out when starting to apply pressure to valve 128, then the closed condition of separator valve stops institute's applied pressure to arrive valve 128.Therefore, the order of starting of control line 110 and 120 causes different valve to be operated, and this provides asynchronous logic.

The multiple control that embodiment of the present invention provides makes array 106 be " programmed ".Such as, due to array 106 close by multiple valve, locking or the sequential programming that reopens, array 106 can be used with multiple layout.In embodiments more as herein described, 2 shown pressure sources are used to provide three kinds of different layouts.Check valve and separator valve (it can be considered in stacking control valve group) is adopted to provide these multiple layouts.Separator valve can limit the control to valve 128, and described control starts realization by what respond to lock-in control line 110, and described separator valve is an example of stacking control valve, and the scope of the invention comprises other layouts.

Check-valves is used to provide the part locking of control line in the predetermined segment of control line.Such as, start locking-valve 118 and 128 by initial control line 110.But, initial control line 120 start the closedown that can cause separator valve.Subsequently control line 110 start the locking that can cause valve 118 but on valve 128 without impact.Cancel while retentive control line 110 starts subsequently start control line 120 (such as by release interface accumulator applied pressure) can cause separator valve reopen after the locking of valve 128.

In the microfluidic device 100 shown in Fig. 1,6 measure input line 132 and are divided into 4 input lines separately, provide 24 the mensuration input lines altogether through holding valve 128.In addition, 24 sample input lines 142 are shown.The concrete numeral of these input lines is example, and other embodiments provide other concrete input line numbers.Such as, in a specific embodiment, the schematic diagram shown in Fig. 1 illustrate only half microfluidic device (right side of such as device), has the input group matched in the left side of described device.Those of ordinary skill in the art can understand many changes, improvement and replacement.

Fig. 2 is the simplification sectional view of the check valve of one embodiment of the invention.Fig. 2 B is the simplification top view of check valve shown in Fig. 2 A.See Fig. 2 A, start fluid (being generally liquid) and flow into check-valves 112/127 through entrance 210, and flow through room 220 through through hole 215.By the flowing of starting fluid, film 230 mentioned or remain on the position of basic horizontal, making to start fluid and from left to right pass through entrance 210.Start fluid via through holes 240a-240d and through hole 242 flows through room 220 and flows out from outlet 250.Therefore, in the embodiment illustrated, for each check valve 6 through holes altogether, one is had to input through hole and 5 output through holes.As shown in the top view at Fig. 2 B, provide extra structural detail and assembly according to specific design.

When fluid pressure is started in removal, film 230 is collapsed upon on layer 260, stops and is refluxed by entrance 210.Therefore, check valve 124/127 provides the flowing from entrance 210 to outlet 250, but stops reverse flowing.Be described in CO-PENDING and the common above-mentioned application of quoting transferred the possession of about other of check valve and provide.

Embodiments more of the present invention provide the microfluidic device being integrated with unidirectional or check-valves.In one embodiment, described microfluidic device comprises the first valve and the second valve.Usually, described first valve is one in multiple valves of formation first valve group, and described second valve is one in multiple valves of formation second valve group.The control line be communicated with described second valve fluid with described first valve provides together with pressure source as a part for microfluidic device, and described pressure source is communicated with described control line fluid.See Fig. 1, starting control line 110 by applying pressure to accommodation accumulator 102, causing the valve 615 in array 106 and valve 118a-118e to start thus manifold 135 is stopped using.Described microfluidic device also comprises the check valve in the control line be arranged between described pressure source and described second valve.Such as, the valve 112 originally opened is holding on the control line 110 between accumulator 102 and valve 118a-118e.After the pressure reduced or remove on control line 110, check-valves 112 provides the locking of valve 118a-118e, and the valve (non-blocked valve) existed in array 106 can reopen and makes sample flow into reative cell.Therefore, by adopting the valve of predetermined number in check-valves locking microfluidic device, provide the control of fluid flowing and the separation of sample in irrealizable microfluidic device in design usually.

In the embodiment shown in Fig. 1 and 6A, valve 615 is set to the reative cell of spaced apart on microfluidic device, and valve 118a-118e is set to the first fluid input line of six lines of be connected with mensuration input port 130 (coupled) to separate mutually with the second fluid input line measuring six lines that input port is connected.As more complete in this manual description, close and locking-valve 118a-118e thus the ability that manifold 135 is stopped using makes microfluidic device programme in many ways, this makes the fluid in input line 132 flow can the separated or merging according to embody rule.

Except check-valves is integrated into there is array layout microfluidic device in except, some designs provide separate valves, also referred to as separator valve.Such as, independent of other valves in device, controlled separator valve can by operation in order to stop starting of one or more valve, and one or more valve described controls to be flowed by the fluid of the fluid input line be connected with the reative cell existed in microfluidic device.Such as, separator valve 124 can be closed before starting control line 110, thus prevention valve 128 responds to the cut out that control line 110 starts.

Figure 10 is the rough schematic view of the programmable microfluidic device of one embodiment of the invention.Element shown in Figure 10 can provide or provides in the microfluidic device being attached at carrier as shown in figure 11 in the carrier.As shown in Figure 10, the profile being loaded on the microfluidic device on carrier is represented by dotted line 1050.See Figure 10, provide two emission sources (vent source) at the opposite side of microfluidic device.The emission source provided usually used as a part for carrier can not adopt in all embodiments.48 sample input lines 1010 are shown above Figure 10, wherein arrange 24 sample input lines and sampling on the left of pair array 106 in the left side of microfluidic device, on the right side of microfluidic device, arrange 24 sample input lines and sampling on the right side of pair array 106.Article 48, sample wire usually provides in the carrier and passes through formed in microfluidic devices and be communicated with array 106 fluid with the through hole 1030 that sample wire end aligns.Sample input line 142 from through hole 1030 to array 106 is provided in microfluidic devices.Article 48, sample wire is by the pressurization of UP source (common pressure source) (not shown), and the sample part of the carrier that described pressure source can be placed on it with microfluidic device is connected.Therefore, the sample of 48 sample ports can be written into microfluidic device and push array 106 through sample input line.See Fig. 1, for the sake of clarity show the right part of array 106, wherein 24 sample input lines of Figure 10 right part are shown as sample input line 142.Therefore, embodiments more of the present invention provide 48 sample input lines with as many as 48 different samples.Other enforcements provide different numbers according to embody rule.

Show control line 1004 in Fig. 10, the interface accumulator 104 that itself and Fig. 1 show is connected.In addition, show several check-valves (check valve, CV) in Fig. 10, this provides the one-way fluid flow through control line in microfluidic devices.See the bottom of array 106, for the control line being connected to this part of array provides other CV.Aquation line is provided in the outside of array 106.These aquation lines are that aquation fluid provides source, and the effect of aquation fluid is the evaporation reducing or prevent fluid in array.

Comprise 12 in 48 input lines of Figure 10 bottom display and measure input lines (on the left of array 106 6, on the right side of array 106 6).Article 12, measure line by the pressurization of UP source (not shown), the mensuration part of the carrier that described pressure source can be placed on it with microfluidic device is connected.Therefore, 12 measure the mensuration of mouths and can be written into microfluidic device and input line pushes 2 arrays 106 after measured.See Fig. 1, for the sake of clarity show right array 106, it has 6 the sample input lines being shown as Figure 10 right part measuring input line 132.Therefore, embodiments more of the present invention provide 12 the mensuration input lines having as many as 12 difference and measure.Measure input line to pass through between central authorities' two arrays 106 of microfluidic device, 6 lines are branched off into right array 106,6 lines and are branched off into left array 106.The use of shown manifold can provide and be less than 12 mensuration, such as, few to single mensuration.Other enforcements provide different numbers according to embody rule.

In 36 input lines of other shown in Figure 10 bottom, 2 lines are used for aquation, and in this specifically implements, 34 is untapped.Measure input line 1020 and the input of the single input line entering array 106 is provided, and measure input line 1022 and be branched off into the input line (such as, 4 lines) that many enter mensuration 106.

Fig. 6 A shows the example of every sample 3 input lines, but in the embodiment depicted in fig. 10, has 11 input lines.In addition, in fig. 6,2 control line displays in fig. 6, but in the embodiment depicted in fig. 10, have 71 control lines to be operated to be formed 70 reative cell/input lines.Therefore, 770 reative cells (11 input line × 70 reative cell) are had for each sample in the present embodiment.Along with 48 sample dispense enter 770 reative cells, provide highdensity integrated fluid loop (integrated fluidic circuit, IFC).In one embodiment, microfluidic device energy one-time detection reaches 48 independently samples.Described nearly each of 48 samples is assigned with into 770 reative cell independent groups, thus sends as add up to 36, and the synchronous digital PCR of 960 reacts.In another embodiment, remove some programmability of microfluidic device, then for identical device surface sum size of components, add the reaction number of chambers/sample.Such as, an embodiment uses nearly 48 samples, and it has 814 reative cell/samples.

Use can obtain the product from assignee of the present invention, can complete whole digital pcr process being less than in 4 hours.In addition, microfluidic device as herein described is the part of complete gene alaysis system.This system can comprise microfluidic device, the controller of microfluidic device, BioMark ^tMsystem or integral type thermal cycler, EP1 reader and related software.The distribution design of microfluidic device as herein described and finished product reagent and cellular form is compatible.Those of ordinary skill in the art understand many changes, improvement and replacement.

Figure 11 is the rough schematic view of the microfluidic device of one embodiment of the invention.The microfluidic device 1108 that described microfluid system comprises carrier 1100 and is loaded on described carrier.Microfluidic device 1108 incorporates the element described in Fig. 1.Described carrier comprises and is arranged in multiple first input port on platform (bank) 1106a or hole 1105.Described carrier also has multiple first input lines 1115 (such as, 48 input lines) be communicated with multiple first input port fluid.Around described multiple first input port, there is outer peripheral edge, allow to UP source described first input port pressurization.In an exemplary application, 48 samples are loaded into the first input port, from the top of carrier, pressure are applied to described mouth and cause 48 samples to be pushed over multiple first input line and finally enter the fluid line microfluidic device.

Described carrier also comprises the second input line 1115 being arranged in multiple second input ports 1105 on platform 1106b and multiple correspondence.Be similar to multiple first input port, described multiple second input port by outer peripheral edge around, enable the fluid being dispensed into the second input port be pushed over the second input line and enter the fluid line in microfluidic device.

In order to provide fluid to be communicated with between described carrier and the microfluidic device being placed on described carrier, microfluidic device has the through hole 1114 formed in microfluidic device bottom, and it aligns with the end section of described first input line and described second input line.The fluid flowing through input line upwards enters through through hole the fluid line that microfluidic device provides.As shown in Figure 10, connect the fluid line of described sample input port through entering the offside of microfluidic device with the through hole 1030 of Right vertical arrangement on the left of microfluidic device in figure, and flow to the central authorities of microfluidic device.In fig. 6, these sample input lines may correspond to the input line 620 that level in Fig. 6 A flows through.Use this 48 sample input lines, in the reative cell often can gone at microfluidic device, fill different samples.

Through hole 1040 is additionally provided and it aligns with multiple second input line 1020 in the bottom of microfluidic device.As shown in Figure 10, the fluid (as measured) being loaded into the second input port flows through the second input line 1020, enters manifold 1060 through through hole 1040.Described microfluidic device comprises can valve crew-served with described manifold, is provided reative cell in two arrays to cause 1 fluid (sample or mensuration) or 12 different fluids.If application need single input fluid, manifold be unlocked and input fluid flow in Figure 10 through array centre perpendicular through all fluid lines.Fluid line is branching out through array centre, and the through hole 1030 finally flowing through array side along with them finally flows through reative cell.See Fig. 6 A, these measure the input line 620 that input line may correspond to process under reative cell.In this example, the flowing of left array side is from central authorities to left side, and the flowing of right array is from central authorities to right side.

Or, manifold 135 can be closed to introduce the different fluid (such as measuring) (mensuration that namely each array 6 is different) of 12 reative cells to be introduced.As shown in Figure 10, fluid is perpendicular flow between array, and then branch goes out, in many rows of wherein each inflow array of 12 fluids, flatly from central authorities through arriving the contiguous through hole 1030 in edge.

Carrier provide acute build up of pressure device 1106c and 1106d to make it possible to start control line and check-valves that microfluidic device exists.In the mode similar with fluid line, the control line 1002 in carrier is communicated with 120 fluids with the control line 110 on microfluidic device by the through hole that microfluidic device is formed with 1004.Therefore, although Fig. 1 shows the interface of carrier and the control line held in accumulator and microfluidic device, should understand this schematic diagram is be clear and conveniently sketch.Other details relevant to the carrier can placing microfluidic device thereon provide in U.S. Patent Application Publication No.2005/0214173, and its disclosure to be quoted by entirety for all objects and is incorporated to herein.

Carrier 1100 has the acute build up of pressure device hole 1101 and 1102 of integration, has the dry hole 1103,1104 for holding valve during it is each, and described valve is preferably and covers the check-valves be connected.Carrier 1100 also comprises one or more hole platform 1106a, b, c and d, and it is each has and be positioned at one or more hole 1105 (also referred to as input port).The hole 1105 of each carrier 1100 has the passage being placed in the microfluidic device 1108 of carrier positions 1107 from hole 1105 guiding.Hole platform 1106c and 1106d is generally used for the pressure provided for starting control line existing in microfluidic device 1108.The fluid line controlling fluid is provided, the hole of Kong Taizhong is connected with the valve existed in microfluidic device or other control appliances.Microfluidic device is preferably the elastomer block formed by two-layer or more layer elastomeric material, and it has groove or the passage of the micro-manufacture formed wherein.

The one or more passages be communicated with one or more through hole 1114 fluid are had in described microfluidic device, it provides the fluid between the passage in microfluidic device and the passage in carrier to be communicated with then, then the hole 1105 in its pilot hole row 1106a-d, thus provide the hole 1105 in carrier 1100 to be communicated with the fluid between the passage in microfluidic device 1108.Accumulator bore top 1109 and 1110 is connected to accumulator bore 1101 and 1102 to form accumulator room 1115 and 1116.Accumulator bore top 1109 and 1110 comprises valve 1112 and 1111 respectively, and it is preferably introduces accumulator room 1115 and 1116 and the check-valves retaining gas under stress by gas.When being present in accumulator room 1115 and 1116, valve 1111 and 1112 be positioned at dry hole 1102 with 1104 inside do not contact valve 111 and 1112 to make liquid.Preferably by the mechanically open valve 1111 and 1112 such as pressing thin slice (shave), pin in preferred check-valves, its automatic closing forces overcoming check-valves with from accumulator room release pressure, thus reduces fluid pressure contained in accumulator room.

Carrier 1100 and its associated component can by polymers manufacturings, as polypropylene, PEF, Merlon, high density polyethylene (HDPE), polytetrafluoroethylene PTFE or Teflon (R), glass, quartz, metal (such as aluminium), transparent material, polysilicon etc.Accumulator bore top 1109 and 1110 also comprises adjustment screw, and it is removable to introduce from accumulator room 1115 and 1116 or to remove gas or liquid.Preferably, valve 1112 and 1111 can be started to discharge fluid pressure in accumulator room 1115 and 1116, otherwise fluid pressure then retains wherein.Notch 1117 is used to help to be placed on by microfluidic device in other instruments suitably, such as, for the instrument of reaction operating or analyze microfluidic device or wherein carry out.

Fig. 3 is the simplified flow chart of the method for display operation microfluidic device according to an embodiment of the invention.In the embodiment depicted in fig. 3,48 panel layouts of digital array are provided.First pressure is applied to pressure source 102 (accommodation accumulator), which results in starting of lock-in control line 110 (310).In a specific embodiment, described first pressure is 30psi.In other embodiments, other pressure is adopted according to embody rule.See Fig. 1, all valve (not shown)s in array 106 can be closed due to the pressure of section 114.Pressure is applied to section 116 by check valve 112, and this can cause closedown and the locking of valve 118a-118e.The overhead portion of section 116 can stop mensuration input line to be closed due to starting of section 116.Close and close the flowing of input fluid through manifold 135 in the pass of valve 118, thus separate by being connected to 6 the mensuration input lines measuring input 130.

Should note, in the control flow check shown in Fig. 3, not using pressure source 104.Therefore, starting or the closedown of separator valve 124 of control line 120 is not had.Therefore, respond to starting of lock-in control line 110, hold valve 128 and close and locking.Therefore, after applying the first pressure (such as, 30psi) by accommodation accumulator, described array valve cuts out and valve 118 and 128 cuts out and locking.

The first pressure (312) reducing pressure source 102 makes array valve (not shown) reopen.In some embodiments, applied pressure is zero to make in process (312), to remove pressure.As noted above, valve 118 and 128 can keep locking in off position.Supply the reative cell be loaded into by sample input line 142 from sample input or the sample of mouth 140 in (314) array.The design of chip provides by sample input line 142, measures input line 132 or both loadings.Because in this embodiment, measure input line and closed by locking-valve 128, load and carry out through sample input line.After completing sample loading, the second pressure (316) is applied to pressure source 102 again, thus close closed array valve and the sample separated in described reative cell.In some embodiments, the second pressure equals the first pressure (such as, 30psi), although this is optional in the present invention.Other are suitable for the pressure of pass closed array valve within the scope of the present invention.In addition, although remove the first pressure in some embodiments, this is optional in the present invention, still provides the sufficient fluid flowing of load sample because some embodiments Pressure Drop can be low to moderate nonzero value.

Therefore, the embodiment of Fig. 3 display provides the loading (that is, the sample input line × device both sides shown in 24) of 48 samples.As discussed below, programmable digital array as herein described also provides other sample layout.

A kind of ad hoc approach that the concrete steps shown in Fig. 3 provide the microfluidic device of operation one embodiment of the invention should be understood.Other sequence of steps also can be implemented according to some alternate embodiments.Such as, alternate embodiments more of the present invention can implement step listed above with different orders.In addition, single step shown in Fig. 3 can comprise multiple sub-step, and it can be implemented with multiple order according to the requirement of single step.In addition, can add according to concrete application or remove other step.Those of ordinary skill in the art can understand many changes, improvement and replacement.

Fig. 4 is the simplified flow chart of the method for a kind of operation of display microfluidic device according to another embodiment of the invention.Different from 48 panel layouts described by Fig. 3, provide single sided board layout in the embodiment depicted in fig. 4.Apply the first pressure (410) to pressure source 104, this causes starting of lock-in control line 120.Pressure can apply to close and the valve 126 in locking section 125 by check valve 127.Therefore, the flowing through sample input line 142 is not had in this layout.Elevated bridge section 122 can transmit controlled pressure to close separator valve 124.

Controlled pressure in lock-in control line 120 can be passed to and measure 5 valves be associated 134 in input line 132 with 6 by section 123.Therefore, valve 134 will be closed, and make to flow to only pass through the mensuration input line of low order end.

Mensuration input line through low order end loads single sample (412).Because the section 116 of lock-in control line 110 does not actuated in this layout, manifold 135 is opened, and during low order end sample is inputted, contained single sample is provided to whole sample input lines 132.Therefore, use the single sample from the input of low order end sample determination to input after measured and load described array.Second pressure is applied to acute build up of pressure device 102, starts lock-in control line 110 and also close the array valve be connected with section 114.Therefore, use different control flow check, the multiple layout using single programmable digital array is possible, such as, and single sided board layout.

A kind of particular methods of operation that the concrete steps shown in Fig. 4 provide microfluidic device according to another embodiment of the invention should be understood.Other sequence of steps also can be implemented according to alternate embodiment.Such as, alternate embodiments more of the present invention can implement step listed above with different orders.In addition, single step shown in Fig. 4 can comprise multiple sub-step, and it can be implemented with multiple order according to the needs of single step.In addition, can add according to concrete application or remove other step.Those of ordinary skill in the art can understand many changes, improvement and replacement.

Fig. 5 is that a kind of operation of display is according to the simplified flow chart of the method for the microfluidic device of another embodiment of the present invention.In the embodiment depicted in fig. 5,12 panel layouts of digital array are provided.First pressure (510) is applied to pressure source 104.In a specific embodiment, described first pressure is 45psi, although this is optional in the present invention, can adopt the pressure that other are applicable to.As the discussion of the process (410) about Fig. 4, start lock-in control line 120.Pressure is applied with the valve 126 in closedown also locking section 125 by check valve 127.Therefore, the flowing through sample input line 142 is not had in this layout.Elevated bridge section 122 can transmit described controlled pressure to close separator valve 124.As discussed below, the Temporarily Closed of separator valve can stop accommodation valve 128 to be closed under the effect of starting of control line 110 subsequently.The controlled pressure of lock-in control line 120 can be passed to and measure 5 valves be associated 134 in input line 132 with 6 by section 123.Therefore, valve 134 can by temporary close.

Second pressure (512) is applied to pressure source 102, starts lock-in control line 110.In a specific embodiment, described second pressure is 30psi, is less than the first pressure (such as, 45psi).As discussed below, described second pressure is enough to close predetermined valve and be not open at the separator valve 124 of closing in process (510).Other pressure can be used according to embody rule.Section 114 start the valve that can close in closed array 106.Pressure can be applied to section 116 through check valve 112, and this can cause closedown and the locking of valve 118a-118e.The overhead portion of section 116 can stop mensuration input line to be closed due to starting of section 116.Closing and close the flowing of input fluid through manifold 135 in the pass of valve 118, thus measures that input line is each to be separated by with 6 that measure that input 130 is connected.

Start different from control line 102 initial, separator valve cuts out by initial the starting of control line 104, thus provides programmable sequences, wherein control line 102 start valve 128 locking do not caused with measuring input line 132 and being associated.This multiple control characteristic provides the asynchronous logic function adopting some embodiments of the present invention, uses conventional microfluidic device can not obtain this characteristic.

The Pressure Drop being applied to pressure source 102 is low to moderate such as zero (514).Because section 116 is in check valve 112 downstream, section 116 cancel start after valve 118 continue to be latched in closed position, this stops 6 fluids flowings measured between input line.The cancelling of section 114 starts the valve opened in described array, makes load sample in loading process subsequently.As discussed above, valve 128 is non-latching in process (512).

The Pressure Drop being applied to pressure source 104 is low to moderate such as zero (516).Valve 134 is reopened by the pressure reducing section 123.As discussed above, use and measure input 130 and provide input for the reative cell in array.Separator valve 124 is reopened by the pressure reduced in section 121 in elevated bridge section 122.Because this stage at programming process does not apply pressure to control line 110, so valve 128 remains on opening.Although show for Pressure Drop is as low as zero, these concrete pressure are not essential to the invention, can adopt other be applicable to pressure.

Measure input from 6 and be loaded into sample (518).Keep measuring the separation between input by the functionalization of going of the manifold 135 caused due to the locking of valve 118 before.Therefore, in this layout, 12 panel layouts (2 sides of 6 mensuration input × devices) are provided.3rd pressure (520) is applied to close the array valve be associated with section 114 to pressure source 102.In addition, because separator valve 124 is switched on again, so valve 128 is blocked cut out.

A kind of concrete grammar that the concrete steps shown in Fig. 5 provide the microfluidic device of another embodiment of operation the present invention should be understood.Other sequence of steps also can be implemented according to alternate embodiment.Such as, alternate embodiment of the present invention can implement step listed above with different orders.In addition, single step shown in Fig. 5 can comprise multiple sub-step, and it can be implemented with multiple order according to single step.In addition, can add according to concrete application or remove other step.Those of ordinary skill in the art can understand many changes, improvement and replacement.

Table 1 provide programmable digital array microfluidic device as herein described multiple may one of panel layouts start pressure and loading process.Pressure source 102 and 104 applied pressure exemplary pressure is described, although other press packet be applicable to are contained in the scope of embodiment of the present invention.

Programming step	48 panel layouts	1 panel layout	12 panel layouts
				1	102＝30psi	104＝30psi	104＝45psi
2	102＝0psi	Be loaded into sample	102＝30psi
				3	Be loaded into sample	102＝30psi	102＝0psi

4	102＝30psi	104＝0psi
			5	Be loaded into sample
6		102＝30psi

Table 1

The integration of check valve and separator valve and digital array provides and uses the multi-user of single high-density digital array chip to programme layout.Therefore, embodiments more of the present invention provide the saving of cost and the raising of experiment flux.Serial described herein or asynchronous logic provide the apparatus function of enhancing, and this is because the state of particular valve not only depends on starting of the control line be communicated with particular valve fluid, also depend on the state of the separator valve that independently control line starts.Therefore, the order of pressurization determines the logic output of panel layout.Therefore, embodiments more of the present invention provide the device layout and function that adopt conventional microfluidic body device not obtain.

According to one embodiment of the invention, provide the method that the microfluidic device with multiple control line is arranged.Serial or asynchronous logic is implemented according to the method.Described method comprises starts the first control line and valve is placed in the first state.Valve (can be in multiple valve) can respond to described first starting of control line and be placed in closed condition.Such as, separator valve 124 can respond to the applying pressure of interface accumulator 104 and the pressurization of control line 120 and cut out.More specifically describe following as more complete description in this specification, be placed in valve 128 due to separator valve and hold between accumulator 102, separator valve can provide multiple control function, this provide serial logic operation.

Described method also comprises, and after valve is placed in the first state, starting can by second control line of operation in order to valve group to be placed in the second state.The situation that valve is in the first state (namely cutting out) stops valve group to be placed in the second state (such as, closed condition).As shown in Figure 1, before starting control line 110, closing separator valve 124 stops valve 128 respond to starting of control line 110 and close (and locking).Therefore, embodiments more of the present invention provide serial logic function, and the order that wherein valve starts creates the Different outcomes being selected from several end-state.In this example, the initial of control line 110 starts the closedown and locking that cause valve 128.On the other hand, the initial of control line 120 starts the closedown causing separator valve 124, and when remaining on closed condition, it stops closedown and the locking of valve 128.Although Fig. 1 shows apply to independently pressure source 102 and 104 control line 110 and 120 that pressure starts, other start method and comprise within the scope of the present invention.

According to another embodiment of the invention, provide the another kind of method adopting microfluidic device to perform serial logic.The first state setting up microfluidic device by starting the first control line being comprised to the method that the microfluidic device with multiple control line is arranged, then starting the second control line.First state of microfluidic device can comprise and responds to starting of many control lines and to be set to by valve open or closed condition.Such as, as mentioned above, before starting control line 110, start the closedown that control line 120 causes separator valve 124.Therefore, valve 128 does not respond to starting of control line 110 and locking and cuts out.

Described method also comprises the second state setting up microfluidic device by starting the second control line, then starts described first control line.The programmable features of microfluidic device as herein described provides and depends on that valve starts the different end-state of the microfluidic device of order.Therefore, in this example, before starting control line 120, start the closedown that control line 110 causes the closedown of valve 128 and locking and separator valve 124 thereafter.After locking-valve 128, close separator valve is adiaphorous, because check-valves 112 keeps the constant pressure of the control line to check valve downstream, stops the closedown of separator valve to affect the state of microfluidic device.

The example of serial logic is implemented, the first input line group (input line 142 that such as valve 126 blocks) that the first state of microfluidic device can comprise closedown or blocked state and the second input line group (such as input line 132) be communicated with reative cell fluid multiple in described microfluidic device as microfluidic device.Described second state can comprise the described first input line group (such as input line 142) be communicated with reative cell fluid multiple in described microfluidic device, and the second input line group (input line 132 that such as valve 128 blocks) of closedown or blocked state.Second state also can comprise the valve group (such as valve 118a-118e) of closed condition, thus stops fluid through connecting the manifold flow of the second input line group.

Although should notice that separator valve is started by control line 120, other embodiments can adopt the another kind of separator valve started by control line 110, using substituting or supplementing as shown separator valve.Therefore, such as, overhead route can lead to extra separation valve between interface accumulator 104 and check-valves 127 from holding accumulator 102.Before applying pressure to the section 125 of the control line being connected to interface accumulator 104, close this extra separator valve valve 126 can be stoped to respond to starting of control line 120 and close and locking.Therefore, although Fig. 1 shows concrete enforcement, the invention is not restricted to this and specifically implement, the design of other microfluidic devices within the scope of the present invention.

In addition, although the embodiment shown in Fig. 1 and replaceability geometric shape discussed above have employed the separator valve by being started to the row replacement plus-pressure in two control lines and provide two levels of multiple control, embodiment of the present invention provide other levels of multiple control.Such as, can other separate valves (such as, electrostatic starts) be integrated in the design of microfluidic device, to provide the control of the 3rd level.Except the starting of the control line shown in 2, this other separate valves initial is started and can the logic of drive unit state be exported.

Fig. 7 shows the method for programming to the programmable microfluidic device of one embodiment of the invention.In the embodiment depicted in fig. 7, described microfluidic device is programmed for the reative cell of described microfluidic device is separated into 48 independently panels.The concrete number of panel depends on the actual design of microfluidic device, and can be different numbers in the embodiment of replaceability, as 12 or 192 panels.The reative cell of each separate panels can fill different samples, detects while providing as many as 48 different samples in the present embodiment.

As shown in fig. 1, microfluidic device has and is arranged in reaction site in array 106 or reative cell array.Described reaction site is communicated with the second input line group 142 fluid with the first input line group 132.Described method comprise start (710) can by operation in order to block the first valve group of (712) first input line groups.Such as, the first valve group is valve 128, also referred to as being operated in order to prevention or the accommodation valve of closing input fluid line 132.Such as fluid is stoped to be flowed by input line 132 by applying pressure closes valve 128 to accommodation accumulator 102.Because existence that is unidirectional or check-valves 112 makes control line 110 become lock-in control line, after accommodation valve 128 cuts out, they keep closing.

Described method also comprise start (714) can by operation in order to separate the second valve group of (716) described reaction site.As shown in Figure 6B, reative cell 630 is limited in the elastomer layer 603 of microfluidic device.Described second valve group is arranged in array, and is shown as valve 615 in fig. 6.Except the first valve group 128 and the second valve group 615, the 3rd valve group 118a-118e and described first valve group are started simultaneously.The existence of check-valves 112 also makes valve 118a-118e in closedown i.e. locking afterwards.In the embodiment depicted in fig. 1, described first valve group and described second valve group respond to and start simultaneously control line 110 applied pressure or close.

After described first, second, and third valve group is closed, described second valve group is cancelled and is started (718), such as, by reducing or eliminating by pressure source 102 pairs of control line 110 applied pressures.Described valve cancel the unlatching of starting and causing described valve, this allows fluid flow the input line be connected with valve.Multiple sample is loaded into reaction site (720) by the second input line group 142.In FIG, sample is included in sample input or mouth 140.The both sides of described microfluidic device respectively comprise 24 different input ports, provide totally 48 input ports, although this concrete numeral can change according to the specific design of microfluidic device.Sample port can comprise 48 different samples or when providing single sample in multiple input port, can comprise the sample that sum is less.Therefore, although table 1 relates to 48 panel layouts, in fact programmable microfluidic device provides as many as 48 independently panel according to the sample provided at 48 sample ports.After sample is loaded into reaction site, second time starts the second valve group to make reative cell separate (722).

The concrete steps shown in Fig. 7 should be understood and provide a kind of concrete grammar that the microfluidic device of one embodiment of the invention is programmed.Also can implementing according to alternate embodiment of other sequence of steps.Such as, alternate embodiment of the present invention can implement step listed above with different orders.In addition, single step shown in Fig. 7 can comprise multiple sub-step, and it can be implemented with multiple order according to single step.In addition, can add according to concrete application or remove other step.Those of ordinary skill in the art can understand many changes, improvement and replacement.

Fig. 8 shows the method for programming to the programmable microfluidic device of another embodiment of the invention.The method of Fig. 8 display provides having single panel and the method that operates of the programmable microfluidic device that all reative cells comprise same sample.As shown in Figure 1, described programmable microfluidic device has the reaction site array be communicated with the second input line group fluid with the first input line group.In the exemplary shown in Fig. 8, described first input line group is input the 140 input fluid lines 142 be connected with sample, and described second input line group is the input fluid line 132 be connected with mensuration input 130.First input line group is started for closed position (810), blocks the flowing (namely closing) (812) through the first input line group.Therefore, in an example, interface accumulator 104 pressurization is caused to the closedown of valve 126, stop fluid to flow through input line 142.The existence of check-valves 127 causes the first valve group to be latched in closed position.

Described method also comprise start (814) can by operation in order to block the second valve group of Part I of (816) second input line group subgroups.See Fig. 1, the Part I of described second input line group is the part of the input line 132 under valve 134, and described second valve group is valve 134, can operate it to block or to close and 65 of measuring in 6 relevant input lines 132 of input 130.Therefore, in the present embodiment, the subgroup of input line is 5 in 6 input lines, adopts the mensuration input port (being connected with the input line of low order end) of the low order end shown in Fig. 1 to provide the sample being loaded into reative cell or site.Be not included in input line (i.e. the input line of low order end) in subgroup for open and for sample being loaded into reative cell as hereafter more complete description.In this is specifically implemented, do not use other to measure input port.In the embodiment depicted in fig. 1, interface accumulator 104 applied pressure starts valve 126 and valve 134 simultaneously.

See Fig. 1, do not implement starting of the valve undertaken by the use of control line 110.Therefore, mensuration input port sample being loaded into low order end can be caused sample to flow through section 116 and be flow to the Part II (part of the input line 132 namely on valve 134) of described second input line group by manifold 135.Sample is by the reative cell in described second input line group loading array 106 or site (818).Should note making manifold stop using (flowing namely through manifold is prevented from) by the starting of section 116 through uninflated control line 110 in the present embodiment.Therefore, manifold is opened and is connected all 6 input fluid lines in the preliminary sectors of the Part II of input line 132.Through manifold 135, sample can be assigned to the component of input line 132, finally flows through 24 input lines, through the valve 128 opened.Therefore, single sample is supplied to all reative cells in array 106.In order to separate the sample in reative cell (822), start the 3rd valve group (820), such as, accommodation valve 615 shown in Fig. 6 A.The pressure that pressure can provide the valve 615 closed in closed array 106 is applied by using accommodation accumulator 102 pairs of control lines 110.

The concrete steps shown in Fig. 8 should be understood and provide a kind of concrete grammar that the microfluidic device of another embodiment of the invention is programmed.Other sequence of steps also can be implemented according to alternate embodiment.Such as, alternate embodiment of the present invention can implement step listed above with different orders.In addition, single step shown in Fig. 8 can comprise multiple sub-step, and it can be implemented with multiple order according to single step.In addition, can add according to concrete application or remove other step.Those of ordinary skill in the art can understand many changes, improvement and replacement.

Fig. 9 shows the method for simplifying of programming to the carrying out of the microfluidic device of another embodiment of the present invention.Described programmable microfluidic device has the reaction site array be communicated with the first input line group, the second input line group and the manifold fluid that is connected described second input line group.Described method comprise start (910) can by operation in order to block the first valve group of (912) first input line groups.See Fig. 1, the first valve group can be the valve 126 in check-valves 127 downstream.The existence of check-valves 127 makes valve 126 be latched in closed position after starting.Start the second valve group (914) to block the Part I (916) of the second input line group subgroup.Apply cause starting of valve 126 and valve 134 from the pressure of interface accumulator 104 to control line 120.

See Fig. 1, the Part I of described second input line group is input line 132 part under valve 134, and the second valve group is valve 134, and it can by operation in order to block or to close 5 that to measure with 6 in 6 input lines that input 130 is connected.Therefore, in the present embodiment, the subgroup of input line is 5 in 6 input lines.Because valve 134 is not by unidirectional valve blocking, when reopening these valves, the Part I that fluid can flow through all 6 input lines 132 flows to manifold 135.In the embodiment depicted in fig. 1, apply to start valve 126 and valve 132 from the pressure of interface accumulator 104 simultaneously.

Described method also comprises starts (918) and can be stopped using the 3rd valve group of (920) in order to make manifold by operation.In an exemplary embodiment, the pressure source (such as, holding accumulator 102) comprised being communicated with the second valve group fluid that starts of the 3rd valve group applies predetermined pressure.See Fig. 1, start valve 118a-118e by applying pressure to the section 116 of control line 110 and can block flowing through manifold, to provide the separation to the fluid flowing through input line 132.The existence of check-valves 112 can cause valve 118a-118e to be latched in closed position.As following more complete description, inactive 12 samples (microfluidic device 6, every side sample) that make of manifold 135 can be loaded into reative cell from 6 mensuration input ports 130.As in the embodiment of Fig. 7 discuss, can be loaded into be less than 12 samples by identical sample being supplied to more than one mensuration input.Those skilled in the art will appreciate that many changes, improvement and replacement.

Again see Fig. 1, should notice that the initial of control line 120 starts the closedown and locking that not only cause valve 126 and valve 134 before starting control line 110, also causes the closedown of separator valve 124.Hold between accumulator 102 and valve 128 because separator valve is arranged in, the closedown of separator valve can stop valve 128 to respond to control line 110 to start and close.In the embodiment depicted in fig. 9, be predetermined pressure for starting the pressure of control line 120, it is enough to stop starting of the control line 110 causing valve 128 to be closed.As shown in table 1, the pressure of 45psi is enough to, when applying the pressure of 30psi to control line 110, valve 128 is remained on enable possition.Concrete value 45psi and 30psi is not essential to the invention, and other pressure can be adopted to implement this multiple control function.

Described method also comprises cancelling starts the second valve group (922).Usually, starting the second valve group by being reduced such as to cancel to 0psi by the first pressure being applied to the first pressure source, taking enable possition to make it.The reduction being applied to the pressure of control line makes the flexible membrane being deflected into described input fluid line return to the position substantially do not deflected, and the fluid flowing flowing through input fluid line is recovered.After reopening valve 134, the sample provided in mensuration input 130 is written into reaction site (924).As shown in Figure 1, each Part I (part of the input line 134 such as under valve 134) of the second input line group is communicated with mensuration input port fluid, and described mensuration input port is set to receive one of multiple sample.The first and second parts that sample flows through the second input line group enter reaction site.As shown in Figure 6 A and 6B, described reaction site can multiple reative cell form formed, wherein each reative cell is communicated with the Part II fluid of input fluid line 132 by through hole, described through hole arrives described reative cell through input fluid line, and described reative cell can be arranged in the elastomer microfluidic device layers on containing the layer of fluid line.

As shown in Figure 1, the element branches being positioned at the input line 132 on valve 134 becomes four input line groups, and it is communicated with the reaction site in array 106 or room fluid.By providing 6 different samples in each mensuration input port, 12 panels (because Fig. 1 illustrate only the first side of microfluidic device) altogether can be defined in an array.As apparent to those skilled in the art, in more than one input port, use a sample that generation can be caused to be less than 12 panels.In order to separate reaction site, start the 4th valve group (926), such as valve 615.

In some implementations, described method also comprises starts valve to stop the closedown of the 5th valve group, wherein can operate the 5th valve group to close the Part II of the second input line group.As shown in Figure 1, the closedown of separator valve 124 stops valve 128 to respond to starting and closing of control line 110.In the embodiment depicted in fig. 9, the pressure being applied to the 45psi of control line 120 time initial causes the closedown of separator valve, makes the Part II of input line 132 be held open manifold simultaneously and is stopped using by the closedown of valve 118a-118e.Separator valve starts by applying pressure to control line or can otherwise start, and includes but not limited to machinery, electrostatic etc.Using in the application based on the control line of pressure, single control line can be used to start multiple valve and/or valve group simultaneously.Therefore, when pressurizeing to interface accumulator 104, separator valve 124 and valve 126 and valve 134 respond to starting and actuateding of control line 120 simultaneously.

The programmable nature of microfluidic device provided herein be fact proved by following: the order that change valve starts step can cause the valve in array to be in different states, and thus makes array be in different states.Such as, in the embodiment depicted in fig. 9, by following steps, 12 samples are loaded into array, described step is: close separator valve 124, then apply pressure to control line 110, and this makes manifold 135 stop using, then reduce the pressure to accumulator 102, then reduce the pressure to accumulator 104.In this example, the closedown of starting to the control line 110 that the pressure of accumulator 104 causes to stop valve 128 to respond to use accumulator 102 applied pressure is kept when pressure is applied by accumulator 102.On the other hand, the closedown that the pressure being applied to accumulator 104 can cause valve 128 is discharged when being applied pressure by accumulator 102.Therefore, the order of starting by changing valve achieves two different conditions of microfluidic device, is loaded into the ability of sample or closed input line 132 by input line 132.This programmable features provides and adopts the not obtainable benefit of conventional design.

A kind of concrete grammar that the concrete steps shown in Fig. 9 provide the microfluidic device of another embodiment of programming the present invention should be understood.Other sequence of steps also can be implemented according to some alternate embodiments.Such as, alternate embodiments more of the present invention can implement step listed above with different orders.In addition, single step shown in Fig. 9 can comprise multiple sub-step, and it can be implemented with multiple order according to single step.In addition, can add according to concrete application or remove other step.Those of ordinary skill in the art can understand many changes, improvement and replacement.

Should understand and the invention is not restricted to concrete grammar as herein described, scheme and reagent etc., it will be understood by those skilled in the art that these change.Will also be understood that term used herein is just in order to for describing specific embodiment, is not intended to limit the scope of the invention.Should also be noted that as in this article with claims in used, unless the context clearly indicates, when not indicating quantity, comprise plural form.Therefore, such as, one or more cell when mentioning " cell ", is mentioned, those skilled in the art's its equivalent known.

Except as otherwise noted, all scientific and technical terminologies used herein have the identical meaning that the technical field of the invention those of ordinary skill is understood usually.The details of embodiment of the present invention and various features and advantage carries out more complete explanation with reference to the non-limiting embodiments described in detail at accompanying drawing and following description and/or explain and embodiment.Should note what feature shown in accompanying drawing was not necessarily drawn to scale, and as understood by a person skilled in the art, even if do not explicitly point out in this article, the feature in an embodiment can be used for other embodiments.Can be omitted to avoid to produce embodiment of the present invention unnecessary fuzzy to the description of known assemblies and treatment technology.Example used herein just can effective mode for the ease of understanding the present invention, and makes those skilled in the art implement embodiment of the present invention further.Therefore, example herein and embodiment should not be construed as limiting the scope of the invention, and the scope of the invention is only limited by claims and applicable law.In addition, should notice that similar Reference numeral represents similar part in several views of accompanying drawing.

Therefore, following present " definition " part, wherein in order to clearly specifically define some terms related to the present invention, but all definition are consistent with the understanding of those skilled in the art to these terms.Describe concrete method, equipment and material, although can be used to implement or test the present invention with those similar or any methods of being equal to as herein described and material.The all bibliography related to herein are quoted by entirety and are incorporated to herein.

Definition

PNA is peptide nucleic acid (peptide nucleic acid)

LNA is lock nucleic acid (locked nucleic acid)

DA is dynamic array (dynamic array)

PCR is PCR (polymerase chain reaction)

BSA is bovine serum albumin(BSA) (bovine serum albumin)

FRET is FRET (Fluorescence resonance energytransfer)

GT is Genotyping (genotyping)

PEG is polyethylene glycol (polyethylene glycol)

PLP is padlock probe (padlock probe)

Term used herein " vicinity " typically refers to the position of primer relative to probe on target nucleic acid analyte complementary strand.Primer and probe can interval about 1 to about 20 nucleotides, more specifically, about 1 to about 10 nucleotides, or can be directly adjacent one another are.

The inventive method to be used that typically refers to term used herein " analysis thing " detects or quantitative with undefined nucleic acid molecules or nucleic acid molecules mixture.Term " target nucleic acid analyte " and " nucleic acid analyte " can exchange with term " analysis thing " in the present invention and use.

Term used herein " complementation " or " complementarity " can be included under the salt of permission and temperature conditions polynucleotides by the natural combination of base pairing.Such as, sequence " A-G-T " is in conjunction with its complementary series " T-C-A ".Complementation between two single chain molecules can be " part ", wherein only has some nucleic acid to combine, or is completely when the complementation between two single chain molecules exists overall complementary between single chain molecule.Complementarity between nucleic acid chains plays an important role to the hybridization efficiency between nucleic acid chains and intensity.This is even more important in the amplified reaction of the design and use of the combination depended between nucleic acid chains and molecule.

Term used herein " covalently bound " typically refers to the connection of a molecular structure by covalent chemical bond and another molecular structure.

Term used herein " dyestuff " typically refers to any organic or inorganic molecule absorbing electromagnetic radiation when wavelength is more than or equal to 340nm.

Term used herein " fluorescent dye " typically refers to any dyestuff being sent the electromagnetic radiation of longer wavelength after being irradiated by electromagnetic radiation source (such as, lamp, photodiode or laser) by Fluorescence Mechanism.

Term used herein " GT sample buffer " typically refers to the buffer solution binding site of reaction channel and chamber surface in DA chip can closed.Described buffer solution prevents the loss of reactive component when chip is loaded into process or reaction.For reagent cost, the use of other Taq-Gold polymerase reduction also can be less than about 80% by it.20 × GT buffer solution can comprise following combination: betaine (FW 117.15), BSA, Superblock t20 (in PBS) (Thermo Scientific, Rockford, IL), Superblock (in PBS) (Thermo Scientific, Rockford, IL), Superblock (in TBS) (Thermo Scientific, Rockford, IL), Superblock t20 (in TBS) (Thermo Scientific, Rockford, IL), glycerine, PEG 20,000, PEG MME550, PEG MME5000 and Tween 20.

Term used herein " homogeneity measures (homogenous assay) " typically refers to the method for detection or quantitative nucleic acid analysis thing, and it is without the need to measuring post processing to record measurement result.Homogeneity measures can carry out in the pipe closed or microfluidic arrays, and after mensuration is initial, does not need to add further reagent or supplementary chemicals to record result.Homogeneity measures the result that energy real time record measures, and this means that measurement result can be recorded continuously when measuring and being in progress in time.

Term used herein " hydrolysis probes " in U.S. Patent No. 5,210, carried out general description in 015, it is quoted by entirety and is incorporated to herein.Hydrolysis probes make use of the 5 '-nuclease (TaqMan existed in heat endurance Taq polymerase used in PCR reaction probe technique, Applied Biosystems, Foster City CA).With fluorescent detection dye (as fluorescein) and receive dyestuff or quencher mark hydrolysis probes.Usually, fluorescent dye is covalently attached to 5 ' end of probe, and quencher is connected to 3 ' group of probe, and when probe is complete, the fluorescence detecting dyestuff is quenched due to FRET (FRET).Probe can at the anneals downstream of a primer, and described primer limits PCR and reacts the end that nucleic acid target analyzes thing amplification target position.Utilize the polymerase activity of Taq enzyme, the amplification of target nucleic acid analyte by probe upstream a primer be positioned at probe downstream but second primer of annealing on the relative chain of target nucleic acid guides.Along with the extension of upstream primer, Taq polymerase arrives the region of label probe annealing, and probe-template crossbred is identified as substrate, and the phosphodiester bond of hydrolysis probes.Hydrolysis irreversibly discharges the cancellation effect of quencher dyes to reporting dyes, thus causes along with each continuous print PCR circulates, and detector fluorescence increases.Specifically, hydrolysis probes of the present invention can high T at common 8-mer or 9-mer motif in people and other transcript profile and by adopting LNA analog to have about 70 DEG C _m.

Term used herein " mark " refers to can be used for providing and can to detect and/or can any atom of quantifiable signal or molecule.Specifically, described mark can be connected to nucleic acid or protein.Mark can provide the signal detected by fluorescence, radioactivity, colorimetric, X-ray diffraction or absorption, magnetic, enzymatic activity etc.

Term used herein " nucleic acid " typically refers to cDNA, DNA, RNA, its strand or double-strand and any chemical modification, as PNA and LNA.LNA in U.S. Patent No. 6,794,499,6,670,461,6,262,490 and 6,770, describe in 748, and be incorporated to herein by quoting in full.Nucleic acid can be any size.Nucleic acid is modified can comprise interpolation chemical group, and it adds extra electric charge, polarity, hydrogen bond, electrostatic interaction and function to independent nucleic acid base or nucleic acid entirety.This modification can comprise that the base of modification is as sugar-modified in 2 '-position, 5-position pyrimidine is modified, 8-position purine is modified, the modification of cytimidine exocyclic amine, 5-bromouracil replacement, backbone modification, methylate, unusual base combinations of pairs cytidine as different in different base and different guanidine etc.Nucleic acid can come from complete chemical building-up process (chemical synthesis as solid phase mediation) or come from biogenetic derivation (as by being separated from almost any species that can provide nucleic acid) or come from and relate to by the biology tool operation process (such as DNA replication dna, pcr amplification, reverse transcription) of nucleic acid or the combination from these processes.

Term used herein " nucleic acid probe " is the nucleic acid carrying at least one covalently bound dyestuff (as fluorescent dye).Specifically, probe does not comprise and the sequence for causing the complementary that PCR reacts.

Term used herein " padlock probe " or " PLP " typically refer to the linear oligonucleotide with about 100 base pairs length.Contiguous complementary in the sequence of PLP 3 ' and 5 ' end and target nucleic acid analyte.Have " sequence label " that can be used for identifying specific PLP in the central incomplementarity region of PLP.The flank of described sequence label can be universal primer site or unique and/or Auele Specific Primer site, and it allows sequence label described in pcr amplification.In hybridization to target, 5 ' of PLP and 3 ' end closely adjacent and can be connected subsequently.Products therefrom is the cycling probe molecule connecting target nucleic acid analyte.Such as TAQMAN can be used pCR in real time is to the label area amplification of cyclisation PLP and quantitative and/or detection.Existence and the amount of amplicon can be associated with the existence of target sequence in sample and amount.To the description of PLP see such as Landegren etc., 2003, Padlock and proximityprobes in situ and array-based analyses:tools for the post-genomic era, Comparative and Functional Genomics 4:525-30; Nilsson etc., 2006, Analyzing genes using closing and replicating circles Trends Biotechnol.24:83-8; Nilsson etc., 1994, Padlock probes:circularizing oligonucleotidesfor localized DNA detection, Science 265:2085-8.Above-mentioned bibliography is incorporated to herein by quoting in full.

Term used herein " PCR " typically refers to for increasing, detecting or the method for quantifying analytes specific region.Those skilled in the art will appreciate that basic PC R technical foundation has several change, as allele specific pcr, set PCR (assembly PCR) or polymerase cycle set (polymerase cycling assembly, PCA), colony PCR, helicase dependent amplification, heat start PCR, specificity (intersequence-specific between sequence, ISSR) PCR, inverse PCR, ligation-mediated PCR, methylation status of PTEN promoter, multidigit point join dependency probe amplification, Multiplex PCR, nest-type PRC, Overlap extension PCR, quantitative PCR, quantitative PCR in real time, RT-PCR, hot asymmetric interlaced (thermal asymmetric interlaces, TAIL) PCR, touchdown PCR and PAN-AC.In addition, it will be understood by those skilled in the art that how by these change application on the round pcr on basis.

Term used herein " purifying " typically refers to according to electric charge, molecular size or the binding affinity any process by protein, polypeptide or nucleic acid and other compositions or compound separation.

Term used herein " quencher " typically refers to the dyestuff of the fluorescent emission reducing another dyestuff.

Term used herein " inquiry (querying) " typically refers to determines target specific probe whether relevant to nucleic acid analyte (such as, connecting or link), and optionally quantitatively sample hit and mark the amount of specific probe.

" sample " used herein typically refers to tissue from human or animal or fluid sample, include but not limited to blood plasma, serum, spinal fluid, lymph liquid, the outer section of skin, respiratory system, intestines and urogenital tract, tears, saliva, haemocyte, tumour, organ, the sample of tissue and Cell culture invitro component.Specifically, sample can be unicellular, paraffin-embedded tissues and acupuncture biopsies.In addition, sample can comprise environmental sample, as lake water and foodstuff samples.

Word used herein " basic purifying " or " basic separation " generally include the nucleic acid or amino acid sequence that obtain from their natural surroundings, be separated or separate, they at least containing about 60%, especially at least about 75%, the most especially at least about 90% other components associated with it, and comprise the separate nucleic acid thing of restructuring or clone and the analog of chemical synthesis analog or system biological synthesis.

Consider the very big diversity of polymer chemistry, precursor, synthetic method, reaction condition and available additive, have a large amount of useful elastomers systems to can be used for manufacturing elastic module, layer, film, miniature valve, pump etc.The change of material therefor in some cases can due to the specific material behavior of needs, and namely solvent resistance, rigidity, gas permeability or temperature stability decide.There is very eurypalynous elastomeric polymer.Provide the simple description of modal elastomer type herein, even if object is the polymer showing to adopt " standard " relatively, also there is the multiple of combination may.Conventional elastomeric polymer comprises polyisoprene, polybutadiene, polychlorobutadiene, polyisobutene, poly-(s-B-S), polyurethane and silicone or polysiloxanes.

Polyisoprene, polybutadiene and polychlorobutadiene are polymerized by diene monomers, and therefore when being polymerized, each monomer has a double bond.This double bond makes polymer be converted into elastomer by sulfuration (usually, sulphur is used for being formed crosslinked between double bond by heating).This makes the incomplete vulcanization by treating binder course carry out the soft printing of easy homogeneous multilayer (homogeneous multilayer softlithography); Photoresist encapsulation (photoresistencapsulation) can be carried out by similar mechanism.

Pure polyisobutene does not have double bond, but is undertaken being cross-linked thus being used as elastomer by the isoprene introducing a small amount of (about 1%) when being polymerized.This isoprene monomer provides pendency double bond on polyisobutene skeleton, and it can cure subsequently as described above.

Poly-(s-B-S) is produced by active anionic polymerization (namely in the reaction without natural chain termination step), therefore can there is " activity " polymer ends at the polymerization species of solidification.This is the natural material standed for (wherein having a large amount of unreacted monomer in the liquid layer being cast in cured layer top) of photoresist package system.Incomplete solidification makes it possible to carry out the soft printing of homogeneous multilayer (A and A combination).This chemical property is also beneficial to and makes one deck have extra butadiene (" A ") and coupling agent, and another layer (" B ") lacks butadiene (for the soft printing of homogeneous multilayer).SBS is " thermo-setting elastomer ", means its fusing on uniform temperature and plastic (relative with elasticity); Reduce temperature and produce elastomer again.Therefore, layer combines by heating.

Polyurethane is made up of diisocyanate (A--A) and glycol or diamines (B--B); Because diisocyanate and glycol/amine have a variety of, the enormous amount of dissimilar polyurethane.But the A of polymer makes them as RTV 615 for the soft printing of heterogenous multilayer relative to the characteristic of B: by using excessive A-A in one deck, use excessive B-B in another layer.

Siloxane polymer has the change of great structure, and provides multiple commercial preparation.Discussed the vinyl of RTV 615-to (Si--H) crosslinked (the soft printing of heterogenous multilayer and photoresist encapsulation can be carried out), but this is just for one of several cross-linking methods of siloxane polymer chemistry.

Except adopting above-mentioned simple " pure " polymer, crosslinking agent can be added.Some reagent (as having the monomer of the pendency double bond for sulfuration) are suitable for the soft printing of homogeneous (A to A) multilayer or photoresist encapsulation; Identical reagent is mixed in the method in two elastic layers.Complementary reagents (namely a kind of monomer has pendency double bond, and another kind has pendency Si-H group) is suitable for the soft printing of heterogeneous (A to B) multilayer.Complementary agent is added in the method to adjacent layer.

Be below can with the elastomeric non-exhaustive list of conbined usage of the present invention: polyisoprene, polybutadiene, polychlorobutadiene, polyisobutene, poly-(s-B-S), polyurethane and siloxane polymer, or poly-(two (fluoroalkyl) phosphine nitrile) (PNF, Eypel-F), poly-(carborane-siloxanes) (Dexsil), poly-(acrylonitrile-butadiene) (acrylonitrile-butadiene rubber), poly-(1-butylene), poly-(chloro trifluoro ethylene-difluoroethylene) copolymer (KeI-F), poly-(ethyl vinyl ether), poly-(difluoroethylene), poly-(difluoroethylene-hexafluoropropene) copolymer (Viton), polyvinyl chloride elastic composition (PVC), polysulfones, Merlon, polymethyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE) (Teflon).

Allcock etc., Contemporary Polymer Chemistry, elastomer is described as being present in the polymer at the temperature between glass transition temperature and condensing temperature by the second edition.Elastomer display elastic characteristic is because the motion that is easy to twist of, polymer chain makes skeletal chain untie under force, the curling shape returned originally of skeletal chain when unable effect.Usually, distortional elastomer when force is applied, reverts to original shape when removal force.The elasticity of elastomer display can characterize with modulus of elasticity.The modulus of elasticity of the material that embodiment more of the present invention uses is about 1Pa to about 1TPa or about 10Pa to about 100GPa or about 20Pa to about 1GPa or about 50Pa to about 10MPa or about 100Pa to about 1MPa, although also can adopt these extraneous moduluss of elasticity according to the needs of embody rule.In some cases, the modulus of elasticity of material can be about 100MPA (MPa) or less.In other embodiments, the modulus of elasticity of material be about 75MPA or less, about 50MPA or less, about 25MPA or less, about 10MPA or less, about 8MPA or less, about 5MPA or less or about 2MPA or less.

Embodiment of the present invention provide microfluidic device, and it comprises assembly such as passage, valve and room, described assembly at least partly by fast one or more layer of springform or level comprise, embed, or formed by it or formed wherein.A kind of exemplary microfluidic body device has and is formed at reagent circulation road in elastomer ground floor or reagent line.Described reagent circulation road comprises and holds valve and room conduit.Microfluidic device also can have and is formed at control channel in the elastomer second layer adjoining with described ground floor or holds line.In addition, microfluidic device can comprise and is formed at sample flow channel in the elastomer third layer adjoining with the described second layer or sample wire.Described sample flow channel can comprise and hold valve and room conduit.Described control channel can be held valve and sample flow channel with reagent circulation road and hold valve operability and be connected.Described microfluidic device can comprise the reagent chamber be communicated with reagent linear flow body, and the sample room be communicated with sample wire fluid.Described reagent chamber and sample room are by being formed at reaction stream passage in described elastomer third layer or response line and fluid communication with each other.Described response line can comprise interface valve.Described microfluidic device also can comprise the interface channel be formed in the 4th layer, the elastomer adjoining with described third layer.Described interface channel can be connected with described reaction stream Entry Interface valve operability.

Embodiment of the present invention also contain the method manufacturing and use microfluidic device disclosed herein.Such as, the operation of microfluidic device can comprise one or more separate valves of unlatching, closes one or more interface valve and make material flow through separate valves and flow into one or more room, optionally carries out under stress.Technology also can comprise and changes pressure in potted line to close separate valves, and with closed separate chambers, and the pressure of change parting line is to open interface valve.The interface valve that first material of the first Room can flow through unlatching enters the second Room, and wherein the first material mixes with the second wherein contained material or reacts.

Should understand example as herein described and embodiment just in order to example object, those skilled in the art can advise multiple improvement or change according to it, and these are included in the spirit of the application and authority and in the scope of claims.

Claims

1. a microfluidic device, it comprises

First pressure source;

First control line, itself and described first pressure fluid communication;

More than first valve, it is communicated with described first control line fluid, and operates described more than first valve by making fluid arrive described more than first valve from described first pressure source by described first control line;

Second pressure source;

Second control line, itself and described second pressure fluid communication;

Separator valve, it is communicated with described second control line fluid, and arrive described separator valve from described second pressure source by described second control line by making fluid and operate described separator valve, the position of wherein said separator valve between described first pressure source with described more than first valve and described first control line crossing, described separator valve can be operated thus obstruct fluid flow flow to described more than first valve from described first pressure source, thus make cannot through described more than first valve of described first control line operation.

2. the microfluidic device of claim 1, it also comprises the first check valve be communicated with described first control line fluid.

3. the microfluidic device of claim 2, wherein said first check valve between described first pressure source and described more than first valve, and can be operated to stop fluid to flow from described more than first valve to described first pressure source.

4. the microfluidic device of claim 3, it also comprises multiple rooms valve, described multiple rooms valve is communicated with described first control line fluid, and operates described multiple rooms valve by causing fluid to arrive described multiple rooms valve from described first pressure source by described first control line.

5. the microfluidic device of claim 4, wherein said multiple rooms valve is positioned between described first pressure source and described first check valve.

6. the microfluidic device of claim 4, wherein said first check valve is between described first pressure source and described separator valve.

7. the microfluidic device of claim 1, it also comprises more than second valve, described more than second valve is communicated with described second control line fluid, and operates described more than second valve by causing fluid to arrive described more than second valve from described second pressure source by described second control line.

8. the microfluidic device of claim 7, it also comprises the second check valve, described second check valve is communicated with described second control line fluid, and between described second pressure source and described more than second valve, wherein said second check valve can be operated to stop fluid to flow from described more than second valve to described second pressure source.

9. operation has the method for the microfluidic device of the first control line, the first valve group, the second control line and separator valve, described first valve group is communicated with described first control line fluid, described separator valve is communicated with described second control line fluid, wherein said separator valve is crossing with described first control line, and described method comprises:

Close described separator valve; With

First pressure is applied to described first control line; Wherein cut out separator valve makes described first valve group response stay open described first pressure of described first control line applying.

10. the method for claim 9, wherein close described separator valve and comprise:

Second pressure is applied to described second control line; With

Respond to described second control line apply described second pressure and close described separator valve.

The method of 11. claims 9, wherein said microfluidic device has the second valve group, and described second valve group is communicated with described first control line fluid, and described second valve group respond to described first control line apply the first pressure and close.

The method of 12. claims 11, wherein said second valve group is blocked.

The method of 13. claims 11, wherein said microfluidic device has the second valve group, and described second valve group is communicated with described first control line fluid, and described method also comprises:

Keep described second pressure being applied to described second control line; With

Apply to be less than the 3rd pressure of described second pressure to described first control line, wherein said second valve group responds to described 3rd pressure that applies described first control line and closes.

The method of 14. claims 13, wherein said second valve group is blocked.