MICRO-PIPETTE TIP FOR FORMING MICRO-DROPLETS

Abstract

A micro-droplet-emulsifier to generate a micro-droplet-emulsion for application on digital polymerase chain reaction is provided. This device comprises of a micro-pipette to hold a dispersed-phase-liquid; a droplet generator that attaches to the micro-pipette and has a plurality of substantially flat micro-channels. The dispersed-phase-liquid is forced through the micro-channels to form micro-droplet-emulsion of dispersed-liquid-phase in the continuous-liquid-phase inside the chamber. The size of the micro-droplets is controlled by the shape and the aspect ratio of the micro-channels, the depth of micro-channel and the material of the micro-droplet-generator-head that dictates the contact angle of the droplet on the micro-channels.

Claims

1) A micro-droplet-emulsifier to generate a micro-droplet-emulsion for application on digital polymerase chain reaction, comprising: a) a micro-pipette having an inlet and an outlet; b) a micro-droplet-generator-head that has an open-top to attach to the outlet of the micro-pipette, and has a plurality of substantially flat micro-channels, wherein each of the substantially flat micro-channels has a length and a cross-sectional shape that has a height and a width; c) a chamber to contain a continuous-phase-liquid in which the microdroplet-generator is inserted in, whereby a dispersed-phase-liquid is forced through the micro-droplet-generator-head and flowing through the plurality of substantially flat micro-channels to generate a plurality of micro-droplets of the dispersed-phase-liquid in the continuous-phase-liquid inside the chamber and whereby a size of each of the micro-droplets is controlled by the shape, and the length of each of the substantially flat micro-channels, and a contact angle of the micro-droplets that is defined by a material of the micro-droplet-generator-head.

2) The micro-droplet emulsifier of claim 1, wherein each of the substantially flat micro-channels opens to a larger-pore, wherein the larger pore is sized to allow the formation of a micro-droplet having a predetermined diameter, and whereby the continuous-phase-liquid enters the larger-pore and expedites a pinch-off process of the micro-droplet that is formed inside the larger pore.

3) The micro-droplet emulsifier of claim 2, wherein the larger-pore is cylindrical or ellipsoidal or rectangular.

4) The micro-droplet emulsifier of claim 1, wherein each of the substantially flat micro-channels comprising of a primary-micro-channel connected to a secondary-micro-channel, and wherein the secondary-micro-channel is in a cross direction with respect to the primary-micro-channel.

5) The micro-droplet emulsifier of claim 1, wherein each of the substantially flat micro-channels comprising of a primary-micro-channel connected to a secondary-micro-channel and a tertiary-micro-channel, wherein the secondary-micro-channel and the tertiary-micro-channel form a star cross-sectional shape together with the primary-micro-channel.

6) The micro-droplet-emulsifier of claim 1, wherein the micro-droplet-generator-head has a substantially flat bottom-wall and wherein the plurality of substantially flat micro-channels are made in the substantially flat bottom-wall.

7) The micro-droplet-emulsifier of claim 1, wherein the micro-droplet-generator-head has a substantially flat bottom-wall and a plurality of side walls, and wherein the plurality of substantially flat micro-channels are made in the bottom-wall and in the plurality of side walls.

8) The micro-droplet emulsifier of claim 1, wherein the diameter of the outlet of the micro-pipette is at least 1 mm.

9) The micro-droplet emulsifier of claim 1, wherein the micro-pipette volume is in a range of 10 microliter to 1 milliliter.

10) The micro-droplet emulsifier of claim 1, wherein a number and a pattern of the plurality of the substantially flat micro-channels is predetermined to avoid a coalescence or an interruption of generation of the micro-droplets, wherein a spacing between two neighboring micro-channels is at least 2 times, and preferably 3 to 5 times, of a predetermined diameter of a micro-droplet.

11) The micro-droplet emulsifier of claim 1, wherein the chamber is a pipette cap that caps to the outlet of the micro-pipette, and wherein the pipette cap is filled with an oil solution, thereby generating the micro-droplet emulsion inside the pipette cap.

12) The micro-droplet emulsifier of claim 1, wherein the height is in a range of 10 to 200 microns, the width is in a range of 1-100 microns, and the length is in a range of 10-500 microns.

13) The micro-droplet emulsifier of claim 1, wherein the cross-sectional shape of each of the micro-channels are rectangular or oval.

14) The micro-droplet emulsifier of claim 1, wherein an aspect ratio for each micro-channel defined as the width divided by the height of the channel is in the range of 3-40.

15) The micro-droplet emulsifier of claim 1, wherein the dispersed-phase-liquid is an aqueous solution and the continuous-phase-liquid is an oil based solution.

16) The micro-droplet emulsifier of claim 1, wherein the micro-droplet-generator-head is made of a hydrophobic material.

17) A micro-droplet-emulsifier to generate a micro-droplet-emulsion for application on digital polymerase chain reaction, comprising: a) a micro-pipette having an inlet side and an outlet side, where the outlet side has a set of outlet openings cut on the walls of the micro-pipette; b) a set of membranes sized to fit in the set of outlet opening, wherein each membrane has a plurality of substantially flat micro-channels, wherein each of the substantially flat micro-channels has a length and a cross-sectional shape having a width and a height; c) a micro-pipette cap to cap the micro-pipette and to contain a continuous-phase-liquid, whereby the dispersed-phase-liquid is forced through the micro-pipette to form micro-droplet-emulsion of dispersed-liquid-phase in the continuous-liquid-phase inside the micro-pipette cap.

18) The micro-droplet emulsifier of claim 17, wherein each of the substantially flat micro-channels comprising of a primary-micro-channel connected to a secondary-micro-channel, and wherein the secondary-micro-channel is in a cross direction with respect to the primary-micro-channel.

19) The micro-droplet emulsifier of claim 17, wherein each of the substantially flat micro-channels comprising of a primary-micro-channel connected to a secondary-micro-channel and a tertiary-micro-channel, wherein the secondary-micro-channel and the tertiary-micro-channel form a star cross-sectional shape together with the primary-micro-channel.

20) The micro-droplet emulsifier of claim 17, wherein the set of membranes are made of a hydrophobic material for a large contact angle for each of the micro-droplets.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Embodiments herein will hereinafter be described in conjunction with the appended drawings provided to illustrate and not to limit the scope of the claims, wherein like designations denote like elements, and in which:

[0024] FIG. 1 is a micro-droplet emulsifier of the present invention;

[0025] FIG. 2A shows a micro-droplet-generator-head attached to a pipette;

[0026] FIG. 2B shows a set of flat micro-channels at the bottom-wall of the micro-droplet-generator-head;

[0027] FIG. 3A is a perspective view of the micro-droplet-generator-head;

[0028] FIG. 3B shows the bottom wall of the micro-droplet-generator-head with a pattern of flat micro-channels;

[0029] FIG. 4 shows a high throughput capillary tip;

[0030] FIG. 5A shows capillary tip structure for the membrane support on the profile surface;

[0031] FIG. 5B shows the flat micro-channel array on the profile membrane;

[0032] FIG. 6 shows the attachment method of the tube and the tip, either tube rotating or tip move up and down;

[0033] FIG. 7A shows the double face etching pattern with circle channels and circular pores of Pattern;

[0034] FIG. 7B shows the cross-sectional view of the circular pores;

[0035] FIG. 7C shows the isometric view of the cross section of the circular pores;

[0036] FIG. 8A shows the double face etching pattern with rectangle stepped pores;

[0037] FIG. 8B shows a cross sectional view of the rectangle pores;

[0038] FIG. 9A shows a Double Face Etching Pattern with Cross Stepped Pores;

[0039] FIG. 9B shows a Cross Section View from Crossed Pores;

[0040] FIG. 9C shows Cross Section View from Crossed Pores in a first plane;

[0041] FIG. 9D shows Cross Section View from Crossed Pores in a second plane;

[0042] FIG. 10A shows Top View of the Combo Crossed Pores of Pattern 6;

[0043] FIG. 10B shows Cross Section Isometric View from combo pores of pattern 6 at Plane 6;

[0044] FIG. 11A shows Top View of the Combo Crossed Pores of Pattern 7;

[0045] FIG. 11B shows Cross Section Isometric View from combo pores of pattern 7 at Plane 6;

[0046] FIG. 12A shows the process of droplet formation in the present device;

[0047] FIG. 12B shows the process of droplet formation in the present device, and

[0048] FIG. 12C shows the process of droplet formation in the present device.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0049] The Figures are not intended to be exhaustive or to limit the present invention to the precise form disclosed. It should be understood that the invention can be practiced with modification and alteration, and that the disclosed technology be limited only by the claims and equivalents thereof.

[0050] The technology disclosed herein, in accordance with one or more various embodiments, is described in detail with reference to the following Figures. The drawings are provided for purposes of illustration only and merely depict typical or example embodiments of the disclosed technology. These drawings are provided to facilitate the reader's understanding of the disclosed technology and shall not be considered limiting of the breadth, scope, or applicability thereof. It should be noted that for clarity and ease of illustration these drawings are not necessarily made to scale.

[0051] This invention presents a method to realize micro-droplet emulsions using a micro-pipette. FIGS. 1-3 show a micro-droplet generator 100 that is attached to the tip of a micro-pipette 102. A dispersed phase liquid 104 is forced through the micro-pipette 102 and the micro-droplet generator 100 to form micro-droplets inside a pipette cap 106 or any chamber containing the continuous phase liquid.

[0052] In order to make this device, a standard 200 l pipette 102 is cut off from the end orifice to form a circular cross section of at least 3 mm in diameter. Any other size pipettes, including 10 l, 20 l, 50 l, 500 l and 1 ml in volume, can also be used. Then, a micro-droplet-generator-head 310 (FIG. 3A) is attached to the tip of the micro-pipette 102 with an attachment system, such as bonding, press fit, etc. The micro-droplet-generator-head is basically a microfluidic socket head or a membrane with specific structure that has a plurality of flat micro-channels 206. The micro-channels have a large aspect ratio (height/width) with a height in a range of 10 to 200 microns, a width is in a range of 1-100 microns, and the length is in a range of 10-500 microns. The number of micro-channels might be from one to several hundred according to the required droplet diameter. There are no other requirements for the micro-channel pattern except that the spacing of micro-channel need to satisfy the minimal distance to avoid the coalescence of the adjacent droplets or interruption of droplet formation.

[0053] FIG. 2A shows the micro-droplet-generator-head 200 that comprises of a socket 202 that is open from the top inlet side 204 and has a plurality of channels at its outlet side 206. The channels 206 are shown in FIG. 2B. Front views of the channels are shown in FIGS. 3A and 3B. The channels are generally elongated to force the liquid out of its equilibrium spherical shape. When a liquid is forced through a non-circular channel, it exits as a noncircular liquid mass. A non-circular mass of liquid is unstable and quickly deforms under the action of the surface tension forces. Since the surface tension forces are inversely related to the curvature of the jet (i.e., /r, where is the coefficient of the surface tension of the liquid and r is the curvature of the liquid ligament), the narrower the liquid mass, the faster the deformation. Therefore, micro-channels with large cross-sectional aspect ratio are provided.

[0054] Another embodiment of the present invention with high throughput is shown in FIGS. 4A, 4B, and 4C. In this case, the tip 400 has several walls: a bottom wall 410, and several side walls 420. Orifices are constructed on all surfaces to allow for high rate of droplet generation.

[0055] FIG. 5 shows another embodiment of the present invention. The micro-pipette 500 is cut to have a bottom opening 510 and several side openings 520. A bottom membrane 530 with micro-channels and several side membranes 540 with micro-channels are inserted into the micro-pipette openings. This provides an integrated micro-pipette with micro-droplet generator. Once the micro-channels are installed onto the micro-pipette, a pipette tip 610 is inserted onto the micro-pipette 500 and turned to lock-in position as shown in FIG. 6. By injecting the aqueous liquid through the micro-pipette, emulsified droplets are formed inside the pipette tip.

[0056] Another embodiment of the present invention is a step emulsification based Pattern as shown in FIGS. 7 and 8. FIG. 7 shows that at the end of each of the flat micro-channels 710, a slightly larger cylindrical channel 720 is designed, which helps in the droplet pinch-off. The step-structural micro-channels on the membrane can be etched to reach the instability effect making the droplets fall off from the tip spontaneously. In order to make the stream pinch off, the cross section area of the larger channel shall be at least 2 times than the area of the smaller one. FIG. 8 shows a larger rectangular channel 820, at the end of each micro-channel 810.

[0057] FIGS. 9A-D and 10A-B show another embodiment of the present invention using two crossed channels. The first channels 910 and 1010 start from the insider of the micro-droplet-generator-head to form large aspect ratio liquid flows. These larger aspect ratio aqueous liquid flows suddenly enter into secondary channels 920 and 1020 that is in the cross direction with respect to the first channel 950 and 1050. Therefore, the central part of the liquid is forced into a cross direction forming a cross-shaped liquid flow. At the exit of the nozzle, a liquid flow having a cross-shape cross-section is generated. Therefore, the surface tension forces push the liquid inward from the larger curvature regions tending to pinch off the droplet. Since there are now four corners in the liquid attachment point, the pinch off occurs rapidly and a small droplet is formed.

[0058] FIG. 11A-B show another embodiment of the present invention using a star shaped channel 1100. In this configuration the liquid exits with a star shape, having six high curvature corners. Each micro-channel 1110 crosses two other micro-channels 1120 and 1130 that form a star shaped cross section together with the original micro-channel 1110. Therefore, the pinch-off process is further expedited, forming even smaller droplets.

[0059] The process of droplet formation in the present microchannels are shown in FIG. 12A-C. FIG. 12A shows the aqueous flow 1210 inside the microchannel 1212. Because of the large aspect ratio of the channel, a parabolic flow 1214 is formed inside the channel. Because of the continuous phase is oil, which has a higher viscosity than water, once oil 1216 enters the channel, it may remain there forming a spindle type deformation zone 1218 on the liquid, from the inside of the channel and extending to the outside of the channel. Therefore, the aqueous flow becomes parabolic. It is also possible that no oil enters the channel, and the channel is filled with aqueous liquid. Once aqueous liquid enters the continuous oil base liquid chamber 1220, the surface tension forces tend to make the pendent drop 1222 to breakup and become a spherical micro-droplet 1224. Since the neck region 1226 of the attached drop is small, the drop can easily detach from the nozzle forming a droplet inside the oil.

[0060] In another embodiment of the same device, as depicted in FIG. 12B, it is found that by having a slightly larger channel 1230 at the exit of the smaller channel 1232, the droplet shape is forced to remain ellipsoidal 1234 until it become about the size of the larger channel 1230. The reason for this that the oil inside the larger channel 1240 prevents the droplet to become spherical. As the top of the drop, which is open to the oil reservoir of the pipette tip, becomes more spherical, the bottom and the attached part remains ellipsoidal, since the oil is trapped under the drop and prevents growth of the drop in all directions. This double chamber system expedited droplet separation, and therefore, results in the formation of smaller droplets in oil. FIG. 12C shows another embodiment of the same invention using an ellipsoidal or oval shaped larger chamber 1250. Similar effects as described for FIG. 12B results in the rapid breakup of the micro-droplets from the core liquid.

[0061] Aspect ratio of the micro channel is defined as the height of channel over the width of the channel, if the height of the channel is 140 microns, and the width of the channel is 4.3 microns, the aspect ratio will be 32.6. the range of aspect ratios are greater than 3.0, they may be in the range of 3 to 40.

[0062] The size and the shape of the channels are designed to facilitate the breakup of the liquid into droplets as soon as the liquid exits the channels. The number and spacing's of the channels are also determined to prevent the coalescence of the droplets as they form. If the channels are too close to each other the droplets will touch and coalesce. The spacing in between micro pores is determined by the droplet diameter, and it is greater than 2 times of the droplet diameter, and preferably 35 times of the droplet diameter. Also the number of droplets generated per unit area is in the range from 1020,000 per square centimetre for the droplet diameters in the range of 5 microns to 200 microns.

[0063] Because of small size of the micro-channels, external liquid cannot be drawn back into the tip. Therefore, the continuous phase liquid is injected into the tip from the other opening end 105 to fill the pipette cap or the chamber.

[0064] While a dispersed phase liquid is injected into the tip, the air lock resists the injection of the liquid to fill the tip. Therefore, an external pressure is required to drive the liquid flow and venting out the remaining air in the tip and making the liquid reach the inner surface of micro pores of the socket head. It is noted that the depth of the micro channels are far less than the depth of the tip, and the volume of the remaining air in the micro channel is negligible.

[0065] Once the micro-pipette is filled with the dispersed phase liquid, the tip is immersed into a chamber, such as a pipette cap, that contains the continuous phase liquid, after air is vented out. Keeping the pressure to drive the dispersed phase into the flat micro-channels, the liquid will be self-broken into micro-droplets to form a emulsified droplet when in contact with the continuous phase. The micro-droplets may flow to the bottom of the tube by gravity.

[0066] Micro-droplets can be generated at a wide range of flow rates, varying from 1 to 100 microliter/min. The flow rate of the dispersed phase can be easily changed by changing the pumping rate of a pump, and without affecting the drop size. The number and generation rate of the micro-droplets depends on the emulsification performance of the continuous phase, droplet size and number of the micro-channel. For example, a single micro channel of aspect ratio in the range of 3.0 to 20, can generate droplet diameters in the range of 50300 microns with frequencies in the range of 530 Hz. Usually, with the same channel size and the same time, the stepped combo channel generates more droplets than simple micro channel, and the star shape combo channel generates more droplets than the stepped combo channel.

[0067] The size of the micro-droplets that are formed depend on the following factors: (i) The material of the droplet generator that dictates the contact angle of the droplet at the exit of the channel, preferably hydrophobic; (ii) the shape of the micro-channel, preferably flatten shape such as rectangle or ellipse; (iii) the aspect ratio of the cross section of micro-channel, preferably greater than 3:1; (iv) the depth of micro-channel, enough for the self-breakup in the channel.

[0068] The table below shows the range of nozzles that can provide proper droplets.

TABLE-US-00001 Depth Droplet Depth m Droplet Diameter Length Width m (rec- Contact Diameter (Rectangle) m m (oval) tangle) Angle (Oval) m m 120 15 160 200 127.5 75 90 120 10 130 180 120 60 75 120 18 170 200 132 80 90 120 20 170 200 135 85 95 120 16 160 200 129 75 90 130 10 200 280 115 75 90

[0069] The main principals of the droplet formation in the present micro-channel device are as follows: By forcing a liquid through a straight through micro-channel, droplets are formed at the exit of the pores. This is referred to as Edge Based Droplet Generation. Droplets may fall to the bottom of the pipette tip by the force of gravity (since aqueous droplets are heavier than the surrounding oil). Since droplets may stick to the exit of the pores, an external flow may be needed to separate the droplets from the pore surfaces or dispersed them in the continuous phase. This can be achieved, by simply shaking the pipette, which make the droplets fall off from the tip.

[0070] After droplets are formed, the tube containing the droplets can be heated and amplified in thermal cycling machine. Then the amplified emulsion will be poured into a reader chip. The reader apparatus is usually composed of air pressure control system, optical imaging capture and mono-layer chip in which all the droplets are introduced into the observe area under the control of air pressure of inlet and outlet. In order to keep the fluid at the edges of the system and the center line moving in a perpendicular line, the shape of the edge is modified as a curve edge to slower the edge flow rate.

[0071] The detail operation for optical observation is that the tube is firstly placed in a holder and then the cover is opened after the temperature returns room temperature. Taking a reader chip to cover the tube completely and assemble the holder and chip together.

[0072] The combined chip is inclined inversely and the emulsion in the tube will flow into the chip reader. With the control of the air pressure at the outlet, all emulsion will pave in the mono-layer observation area. An optical image camera scans whole observation area and gives an absolute quantitative analysis report. Based on such a capillary tip, regular quantitative PCR can be easily upgraded into absolute quantitative PCR.

[0073] Replacing the traditional pipette tip with capillary tip, the sample will be dispersed into a standard tube and thermal cycling in traditional qPCR device, just with the utilization of a unique mono-layer imaging process, an absolute quantitative PCR is simply realized.

[0074] This invention provides a feasible way to upgrade a regular quantitative PCR into a droplet digital PCR, only adding an extra droplet reader unit. This invention will lower the user's investment and make an effective use of the existing instruments.

[0075] The foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.

[0076] With respect to the above description, it is to be realized that the optimum relationships for the parts of the invention in regard to size, shape, form, materials, function and manner of operation, assembly and use are deemed readily apparent and obvious to those skilled in the art, and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention.