Method and apparatus for concentrating molecules
A method and apparatus for continuously separating or concentrating molecules that includes flowing two fluids in laminar flow through an electrical field and capturing at one of three outputs a fluid stream having a different concentration of molecules.
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The invention relates generally to a method and apparatus for continuous separation or concentration of proteins (or other biological molecules) from aqueous solutions in a flow-through configuration.
BACKGROUND OF THE INVENTIONIn analytical chemistry, electrophoresis refers to methods that use electric fields to manipulate and separate charged molecules in solution. Capillary electrophoresis (CE) refers to a family of “traditional” techniques where the sample is loaded into a capillary and its components are separated into bands along the axial direction. The separation arises whenever components of the sample have unique electrophoretic mobility. These bands can be detected, for example, by a fixed optical system as they flow past. The apparatus may be relatively simple since high voltage electrodes at each end of the capillary serve to not only separate the sample but also to inject the sample into the capillary by taking advantage of electroosmotic flow. The art of CE involves identifying appropriate parameters, buffers, and additives for each specific analyte to maximize separation and resolution between bands
The limits of resolution in CE are partly determined by diffusion of separated analyte into the surrounding buffer, which broadens the bands. Two methods of countering this effect are called focusing and stacking. The object of stacking is to compress the sample molecules into a narrow band, prior to performing the separation, so that differences in mobility are made more distinct. One kind of stacking is achieved by using electrophoresis to drive molecules against an interface between two solutions of differing conductivity. If the sample is in a low-conductivity buffer, electric field strength across it will be high, and electrophoretic velocity will be high. When the sample ions reach a transition to a high-conductivity buffer, electric field strength will drop, as will their velocity, and they will collect at the interface. However, this technique is limited to batch processing. A need exists in the art for a method and apparatus for continuously separating or concentrating molecules using stacking. So-called “free flow electrophoresis” is another technique known in the art that permits continuous separations, but it is not conventionally used with stacking.
SUMMARY OF THE INVENTIONThe invention addresses the deficiencies in the prior art by using a free flow electrophoresis configuration in conjunction with discontinuous buffers to achieve bulk concentration of molecules.
In one aspect, the invention relates to a device for separating or concentrating molecules. The device includes a microfabricated flow channel having an upstream end and a downstream end. At the upstream end, the channel includes two input ports. One input port introduces into the channel a low-conductivity fluid stream containing a target molecule and potentially other molecules. The other input port introduces into the flow channel a high-conductivity fluid stream. The channel includes three outlet ports. One outlet port receives most of the low-conductivity fluid stream. A second output port receives most of the high-conductivity fluid stream. A third output port is located around the center of the end of the channel. This port receives a mix of both fluid streams. The device also includes a pair of electrodes, one on each side of the channel. When voltage is applied, the electrodes will create an electric field across the channel.
In one embodiment, an electrode interface includes an opening in the channel adjacent to a fluid stream. The opening is separated from a reservoir by an ion-permeable barrier, which permits ions to pass but does not permit bulk fluid flow. The reservoir interfaces with an external electrode. The external electrode can be electrically connected to an external power source for applying a voltage of various polarities and magnitudes to the electrode. When voltage is applied to both electrodes, an electric field forms across the width of the channel.
In one embodiment, the device is molded into a substrate. In another embodiment, the device is etched into a substrate.
In one embodiment, the device is approximately 5 mm wide, 10 μm to 100 μm deep, and about 2.3 cm to about 5 cm long.
In one embodiment, the device includes a third input port at the upstream end. In such an embodiment, the sample fluid and the low-conductivity buffer are introduced from separate input ports.
In another aspect, the invention relates to a device for separating or concentrating molecules. The device includes a channel, two input ports, and three output ports, like the device described above. In addition, the device also includes integrated electrodes. The electrodes are positioned proximate to the channel.
In one embodiment, the electrodes are separated from the channel by ion-permeable barriers.
In one embodiment, the ion-permeable barriers are comprised of arrays of smaller channels.
In one embodiment, the device includes a power source for applying voltage to the electrodes and controls for controlling the strength and polarity of voltage applied to the electrodes.
In another aspect, the invention relates to a method for separating or concentrating molecules. The method includes selecting the pH of the low-conductivity buffer so that the charge of the target molecule is non-zero, flowing the low-conductivity buffer, with sample, and the high-conductivity buffer through the channel in laminar flow, applying an electric field with appropriate polarity and strength to cause the target molecule to accumulate at the interface between the two fluids, and collecting portions of both fluid streams at the interface between the two fluids at the end of the channel.
The foregoing discussion will be understood more readily from the following detailed description of the invention with reference to the following drawings.
The input ports 102 and 104 are positioned to introduce two fluid streams into the flow channel in laminar flow. The sample input port 102 introduces a fluid stream having a target molecule in it 116. The buffer input port 104 introduces a fluid stream 118 having a conductivity that is substantially different than the fluid stream 116 introduced through the sample input port 102.
The width and depth of the flow channel 106 are selected to allow the fluid streams from the two inputs 102 and 104 to be in laminar flow through the flow channel 106. For example, the flow channel may be 0.5 mm to 5 mm wide and 10 μm to 100 μm deep. The length of the channel 106 is selected to be sufficiently long to allow the target molecules to have sufficient time to travel from one wall 120 of the flow channel to about the interface 122 between the two fluids. For example, in various embodiments the channel is between about 2.3 cm long to about 5 cm long, though shorter or longer flow channels may also be suitable.
The pair of electrodes 108 are separated from the flow channel 106 by a pair of electrode interfaces 124. Each electrode interface 124 includes an ion-permeable barrier that permits the flow of electrical current between the electrodes 108 as ion transport across the barrier without permitting bulk fluid flow. By applying voltage to the electrodes 108, an electric field is established transverse to the flow channel 106.
As the fluids 116 and 118 flow through the flow channel 106, the electric field causes molecules to move towards the interface 122 of the two fluid streams. The polarity of the field is selected based upon the charge of the target molecule. The polarity is selected to cause the target molecule to move towards the interface 122 of the two fluid streams. The strength of the electric field is selected based upon the charge of the target molecule and the flow rate of the fluid streams through the flow channel 106. For example, in various embodiments, the field strength is between about 200 V/cm to about 1000 V/cm.
Preferably, the electrode interface 124 includes an array of channels, as described further in
In one embodiment, the electrode interfaces 124 electrically couple the flow channel 106 to electrodes integrated into the device. For example, the electrode may be formed on the substrate on which the channel is formed. In other embodiments, the electrode interfaces 124 electrically couple the flow channel 106 to external electrodes, as described further in
Because of the difference in conductivities between the two fluids 116 and 118, the electric field across the first fluid stream 116 is higher than the electric field across the second fluid stream 118. Once molecules from the first fluid stream 116 traverse the interface 122 between the first fluid stream 116 and the second fluid stream 118, they experience the weaker electric field of the second fluid stream 118. Because the electric field is weaker, the velocity of a molecule slows, resulting in an accumulation of molecules at about the interface 122 between the two fluids 116 and 118.
At the downstream end of the channel 106 are a sample output port 110, a buffer output port 112, and a target output port 114. The sample output port 110 collects most of the first fluid stream 116. The buffer output port collects most of the second fluid stream 118. The target output port 114 is located at about the interface 122 between the two fluid streams 116 and 118. As the fluids flow down the flow channel 106, the molecules accumulate at the interface 122 between the two fluids. Because the target output port 114 is positioned at about that interface 122, when the fluids 116 and 118 reach the end of the channel, the fluid containing those molecules continues flowing into the target output port 114. The target output port 114 therefore collects the target molecules from the first fluid 116. It is sized, however, to collect a smaller volume of fluid than the fluid streams 116 and 118 than the output ports. The increased concentration of target molecules in the fluid collected at the target output port is higher than in the first fluid stream 116.
To achieve a desired concentration factor, K, the target output port 212, at the end of the flow channel 204, has a width less than the flow channel 204, while sample output port 214 and buffer output port 216 maintain constant flow velocity. Assuming the outlet channel 212 is positioned to capture 100% of the target molecules, the ratio of inlet channel width, win, to the target output port 212 width gives the concentration factor of the device
K=win/wout
The length of the channel 204 is then determined by the electric field and the fluid velocity. In the worst-case scenario, a molecule entering at the inlet wall 220 must traverse the entire inlet width, win, in order to be captured at the target output port 212. The channel 204 length, L, can be determined by the distance that a molecule travels in the z-direction while traversing the inlet width, win.
L=νfwin/νE low,
where νE low is the electrophoretic velocity of the molecule in the low conductivity buffer.
In the ideal case, the width of the target output port 212, wout, can be similarly determined by considering the trajectory of a molecule that enters the channel 204 at around the fluid interface 206.
wout=νE hiL/νf
where νE hi is the electrophoretic velocity of the molecule in the high conductivity buffer.
From the above three equations, the concentration factor, K, can be reduced to the ratio of conductivities of the two buffers.
Therefore, to achieve the desired concentration factor, K, the buffers are selected based on their conductivities (σ):
Kideal=σhi/σlo
The device 300 includes a sample input port 302, a buffer input port 304, and a high-conductivity buffer input 306. The device 300 also includes a flow channel 308, a pair of electrode interfaces 310, a sample output port 312, a buffer output port 314, and a target output port 316.
The sample enters through the sample input port 302. The buffer fluid enters from the buffer input port 304. The combined fluid stream then flows in laminar flow with a second fluid stream introduced through the high-conductivity buffer input 306. A pair of external platinum wire electrodes 318 are wetted in buffer reservoirs 320 positioned on either side of the flow channel 308. The buffer reservoirs 320 are in fluid and electrical communication with the flow channel 308 through an ion-permeable membrane 310, such as a cast gel or porous glass. The external electrodes 318 generate an electric field transverse to the flow channel 308.
The sample output port 312 and the buffer output port 314 carry away the bulk of the first and second buffer fluid streams. The target output port 316 carries away a portion of both fluid streams containing a higher concentration of molecules than the sample.
While in
In one embodiment, the input ports 508 and 510 are 1200 μm in diameter and 2.5 mm wide at the channel 504. The flow channel 504 is 2.3 cm long and 10 μm high. The ion-permeable barriers 506 are 1800 μm long, 6 μm wide, and composed of 420 channels along the length of the main flow channel 504. The target output channel 516 width is 250 μm. The bond pads 518 for interfacing with the integrated electrodes are 3640 μm long by 1190 μm wide. The bond pads 518 provide an interface to an external power source.
More specifically, the pH of the sample buffer is selected such that the target molecules 816 will have some non-zero charge (step 802), whether positive or negative. Without some amount of charge, the target molecules 816 will have a low electrophoretic mobility and will therefore not move towards the fluid interface 813 with a sufficient electrophoretic velocity in response to the electric field. Thus, the magnitude of the charge must be above a lower limit, below which the electrophoretic velocity is insufficient to move the target molecule to the fluid interface 813 before reaching the end of the channel 814. However, the magnitude of the charge can also be too large. In that case, the electrophoretic velocity of the target molecule does not decrease sufficiently upon reaching the fluid interface 813, allowing the target molecule to continue to move towards the wall of the channel 815. These molecules would be collected by a waste outlet 822 rather than the target output port 818. The upper and lower limits will be different for different types of target molecules. Ideally, the pH of the sample buffer fluid is selected such that the electrophoretic velocity of a target molecule is large enough for the target molecule to reach the fluid interface 813 before reaching the end of the channel 814, but not so large that the molecule travels beyond the target output port 818 by the time it reaches the end of the channel 814.
Once an appropriate pH for the sample buffer is selected (step 802), two buffers are simultaneously flowed through the channel such that they establish laminar flow within the channel (step 804). The sample buffer (i.e., the buffer containing the target molecule) has a low conductivity. The other buffer has a high conductivity. Because the flows are laminar, the mixing between the two fluid streams is minimal. Absent an external force, the molecules in the first fluid stream remain largely in the first fluid stream. Once the flows are laminar, an external force is applied in the form of an electric field. The polarity and magnitude of the electric field are chosen such that the field will cause the target molecules 816 to move towards the fluid interface 813. Once the polarity and magnitude of the desired field are selected, voltage is applied to the electrodes (step 806).
As the fluid streams flow through the channel, the target molecules move laterally across the channel in response to the electric field. As they flow through the channel 814, the high-conductivity buffer and the low-conductivity buffer diffuse into each other. Therefore the conductivity of the fluid surrounding a target molecule becomes higher near the fluid interface 813. As the conductivity of the surrounding fluid becomes higher, the electric field becomes weaker and the electrophoretic velocity of the target molecule becomes slower. Therefore, around the fluid interface 813, the target molecules 816 lose velocity and begin to accumulate at about the fluid interface 813.
At the end of the channel 814, the fluid streams exit through three output ports 818 and 820 and 822. The target output port 818, positioned at about the fluid interface 813, collects portions of both fluid streams and the target molecules 816 that have accumulated at about the fluid interface 813 (step 808). The first output port 820 collects the sample buffer, which contains a substantially reduced concentration of the target molecules 816. The second output port collects the high-conductivity buffer 822. The fluid stream collected by the target output port 818 can then be collected or routed for further separation, concentration, or analysis.
More specifically, the pH of the sample buffer is selected (step 902) such that the target molecule is at its isoelectric point. At its isoelectric point, the target molecule has zero charge. Because the target molecules 924 have zero charge, their distribution within the first fluid stream is largely unaffected by the electric field. However, with the sample buffer at the selected pH, interferents within the sample will have a positive or negative charge.
In a first stage, two fluid streams are flowed through the channel 921 (step 904), as described for
The second stage is a second device 946, according to an illustrative embodiment of the invention. The fluid stream from the first waste outlet 930 of the first device 944 enters the sample input port 934 of the second stage 946. For the second stage, the polarity of the electric field is the opposite of the polarity of the electric field of the first device 944. Again, two fluid streams are flowed through the channel 937 of the second stage, as described for
A continuous microfluidic protein concentrator would have applications in fieldable, rapid response instruments for clinical diagnostics, pathogen/toxin detection, and environmental monitoring. In these systems it would enhance overall sensitivity, reduce system size and make a critical contribution to the vision of automated “droplet in—answer out” instruments. These applications are not limited to liquid samples, since many strategies for air, soil, or food sampling involve first dissolving or suspending the sample in liquid before performing the analysis.
Currently sample preparation for microsensors typically involves a series of batch processes on multiple bench top instruments. In addition to reducing the size of equipment required to make a measurement, the invention offers the advantage of continuous operation, as opposed to batch processes such as centrifugation. This feature is essential for automated continuous monitoring.
A microfluidic scale is appropriate and advantageous to this design. Preliminary calculations suggest that a concentration factor of at least 20 is feasible in a 5 cm channel, and that higher factors can be readily obtained by using a series of concentrators. Sample throughput for a single concentrator 0.5 cm wide is estimated to be on the order of 30 uL/min. Therefore, an array of devices on a board measuring approximately 0.5×10×10 cm could potentially process 1 ml of sample in under 2 minutes, delivering the captured protein in a volume of less than 3 microliters.
The invention may be embodied in other specific forms without departing form the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative, rather than limiting of the invention.
Claims
1. A device for separating molecules contained in a first fluid comprising:
- a) a channel having an upstream end and a downstream end, the channel comprising a first inlet at the upstream end to introduce a first fluid containing molecules into the channel at a first concentration;
- b) a second inlet to the channel at the upstream end to introduce a second fluid into the channel in laminar flow with the first fluid;
- c) a first electrode interface associated with the first fluid along a first linear portion of the channel;
- d) a second electrode interface associated with the second fluid along a second linear portion of the channel;
- e) a first outlet at the downstream end for receiving said first fluid;
- f) a second outlet at the downstream end for receiving said second fluid; and
- g) a third outlet at the downstream end positioned at about the interface between the first fluid and the second fluid for receiving an output stream with a concentration of molecules greater than the first concentration.
2. The device of claim 1 where the first electrode interface is an opening in the channel in fluid communication with the first fluid flow.
3. The device of claim 1 where the second electrode interface is an opening in the channel in fluid communication with the second fluid flow.
4. The device of claim 2 where the first opening is separated from a first electrode reservoir by a first ion-permeable barrier.
5. The device of claim 3 where the second opening is separated from a second electrode reservoir by a second ion-permeable barrier.
6. The device of claim 4 where the first electrode reservoir interfaces with a first external electrode.
7. The device of claim 5 wherein the second electrode reservoir interfaces with a second external electrode.
8. The device of claim 7 comprising a power source for applying a voltage to the electrodes.
9. The device of claim 7 comprising a control for changing the polarity of the electric field.
10. The device of claim 7 comprising a control for changing the strength of the electric field.
11. The device of claim 7 wherein the electrodes generate an electric field across the channel transverse to a length of the channel.
12. The device of claim 11 wherein the electric field is sufficient to cause at least a portion of the molecules in the first fluid to move towards the second fluid.
13. The device of claim 12 where the movement of the molecules in the first fluid towards the second fluid results in an accumulation of the molecules at the interface.
14. The device of claim 1 wherein the channel is molded in a substrate.
15. The device of claim 1 wherein the channel is etched into a substrate.
16. The device of claim 1 where the width of the channel is approximately 0.5 mm to 5 mm.
17. The device of claim 1 where the depth of the channel is from about 10 μm to about 100 μm.
18. The device of claim 1 where the length of the channel is from about 2.3 cm to about 5 cm.
19. The device of claim 1 where the width of the third outlet is determined by the following formula: (width of channel)/(2*desired concentration factor)
20. The device of claim 1 comprising a third input port placed at the upstream end to introduce a buffer solution into the first fluid stream.
21. The device of claim 1 where the device contains flowing streams of first and second fluids.
22. The device of claim 1 where the electrophoretic mobility of a target molecule in the first fluid is substantially different from its electrophoretic mobility in the second fluid.
23. The device of claim 1 where the conductivity of the first fluid is substantially different from the conductivity of the second fluid.
24. The device of claim 1 where the solubility of a target molecule in the first fluid is substantially different from its solubility in the second fluid.
25. A device for separating molecules contained in a first fluid comprising:
- a) a channel having an upstream end and a downstream end, the channel comprising a first inlet at the upstream end to introduce the first fluid containing molecules into the channel;
- b) a second inlet to the channel at the upstream end to introduce a second fluid into the channel in laminar flow with the first fluid;
- c) a pair of electrodes positioned proximate to the channel for applying a voltage to produce an electric field across the channel transverse to a length of the channel;
- d) a first outlet at the downstream end for receiving said first fluid;
- e) a second outlet at the downstream end for receiving said second fluid; and
- f) a third outlet at the downstream end positioned at about the interface between the first fluid and the second fluid for receiving an output stream with a concentration of molecules greater than the first concentration.
26. The device of claim 25 where a first ion-permeable barrier separates a first electrode from the channel and a second ion-permeable barrier separates a second electrode from the channel.
27. The device of claim 26 where the ion-permeable barriers form opposite walls of the channel.
28. The device of claim 26 where the first and second ion-permeable barriers are comprised of first and second arrays of channels.
29. The device of claim 25 comprising a power source for applying a voltage to the electrodes.
30. The device of claim 25 comprising a control for changing the polarity of the electric field.
31. The device of claim 25 comprising a control for changing the strength of the electric field.
32. The device of claim 25 wherein the electrodes generate an electric field across the channel transverse to a length of the channel.
33. The device of claim 25 wherein the electric field is sufficient to cause at least a portion of the molecules in the first fluid to move towards the second fluid.
34. The device of claim 25 where the movement of the molecules in the first fluid towards the second fluid results in an accumulation of the molecules at the interface.
35. The device of claim 25 wherein the channel is molded in a substrate.
36. The device of claim 25 where the channel is etched in a substrate.
37. The device of claim 25 where the width of the channel is approximately 0.5-5 mm.
38. The device of claim 25 where the depth of the channel is from about 10 μm to about 100 μm.
39. The device of claim 25 where the length of the channel is from about 2.3 cm to about 5 cm.
40. The device of claim 25 where the width of the third outlet is determined by the following formula: (width of channel)/(2*desired concentration factor)
41. The device of claim 25 also comprising a third inlet placed at the upstream end to introduce a buffer solution into the first fluid stream.
42. The device of claim 25 where the device contains flowing streams of first and second fluids.
43. The device of claim 25 where the electrophoretic mobility of a target molecule in the first fluid is substantially different from its electrophoretic mobility in the second fluid.
44. The device of claim 25 where the conductivity of the first fluid is substantially different from the conductivity of the second fluid.
45. The device of claim 25 where the solubility of a target molecule in the first fluid is substantially different from its solubility in the second fluid.
46. A method for separating molecules from a fluid comprising:
- flowing a first fluid into a channel, the first fluid having a first conductivity;
- simultaneously flowing a second fluid into the channel in laminar flow with the first fluid, the second fluid having a second conductivity substantially different from the first conductivity;
- applying an electric field transverse to a length of the channel, whereby at least a portion of molecules in the first fluid are caused to migrate towards the second fluid;
- flowing a portion of the first fluid from the channel through a first outlet placed to receive the first fluid;
- flowing a portion of the second fluid from the channel through a second outlet placed to receive the second fluid; and
- flowing a portion of both fluids containing a greater concentration of molecules than the first fluid through a third outlet placed at about the interface between the first fluid and the second fluid.
47. The method of claim 46 where the pH of the first fluid is selected to be either higher or lower than the isoelectric point of the target molecule.
48. The method of claim 46 where the voltage applied is from about 200 to about 1000 V/cm
49. The method of claim 46 where the first conductivity is below about 20 mS/cm.
50. The method of claim 46 where the second conductivity is from about 10 to about 1000 times that of the first conductivity.
51. The method of claim 46 comprising accumulating molecules at about the interface between the first fluid and the second fluid.
52. The method of claim 46 where the flow rate of the first fluid and the second fluid through the channel is from about 0.001 to 1 mL/min.
Type: Application
Filed: Mar 28, 2007
Publication Date: Oct 2, 2008
Applicant: The Charles Stark Draper Laboratory, Inc. (Cambridge, MA)
Inventors: Jason Fiering (Boston, MA), Mark Keegan (Littleton, MA)
Application Number: 11/729,046
International Classification: B01D 61/42 (20060101);