HIGH PERFORMANCE DISPENSING APPARATUS
FIELD OF THE INVENTION
The present invention is related to separation technology. In particular, the present invention is related to high performance capillary electrophoresis, chromatography and extraction.
BACKGROUND OF THE INVENTION
In chemical and biological research and routine analysis, a large number of samples need to be analyzed at the same time and under similar conditions. Since microfabricated capillary electrophoresis (CE) chips were introduced in 1992 (J. Chromatogr. 1992, 593, 253-258) Many experiments have been done on them and results have shown that these devices can increase the speed of CE separation by an order of magnitude.
In some previous experiments with CE chips (J. Chromatogr. 1992, 593, 253-258; Anal. Chem. 1992, 64, 1926-1932; Anal. Chem 1994, 66, 3472- 3476; Proc. Natl. Acad. Sci. USA 1994, 91 , 11348-1 1352) and capillary array electrophoresis chip (CAE) chips (Anal. Chem, 1997, 69, 2181 - 2186), plastic pipet tips was pushed into sample holes or tubing glued to the substrate to form reservoirs along with manual electrical contacts. These systems, however, are impractical for large numbers of samples.
To simplify sample handling and electrode introduction, and to increase the volume of buffer in the cathode and anode reservoirs, an elastomer (Sylgard 184, Dow Corning) reservoir array and electrode array were developed. (Proc. Natl. Acad. Sci. USA 1998, 95, 2256-2261). However, almost all steps in these experiments were performed manually, including loading of materials in reservoirs and channels, washing and flushing. Such manual operations is not only time-consuming but also error prone. Furthermore, they are not conducive for handling large numbers of samples.
With the development of CAE on chips, the possibility of analyzing multiple samples in parallel moves one step closer to reality. However, numerous problems remain to be solved, including multi-sample loading, injection and electrophoresis. Furthermore, the system needs to be washed and flushed effectively after use. Similarly, for microfabricated liquid chromatography system, effective methods for sample and mobile phase introduction are required. In addition it is often necessary to perform sample preparation or extraction prior to chromatography or electrophoretic separation. In particular, for samples which are available only in minute quantity, a microscale liquid dispensing system is required to prevent sample loss during sample preparation and extraction.
OBJECT OF THE INVENTION
It is therefore an object of the present invention to provide a dispensing or loading system to overcome the shortcomings as stated above.
It is another object to provide as high performance capillary electrophoresis system which can handle a plurality of samples.
It is another object to provide a capillary array electrophoretic system which can perform parallel loading of multiple buffers and samples.
It is a further object to provide a dispensing system embodiment which may be adapted for microscale liquid chromatography.
It is a further object to provide a dispensing system embodiment which may be adapted for microscale sample preparation and extraction.
SUMMARY OF THE INVENTION
The above objects are generally accomplished by providing an automatic loading or dispensing system for various applications, for example separation techiques such as chromatography and electrophoresis, and biochemical and biological reactions. For electrophoresis, the electrophoretic system includes an electrophoretic chip having a plurality of electrophoretic channels each connected to a sample reservoir and a buffer reservoir. At the heart of the system is an automated loading system for the parallel loading of multiple channels. The system is further equipped with a power source, electrically
connected to the channels, for electrophoresis of the samples therein. A controller, connected to each element, is provided to control the loading and electrophoresis operations.
In the preferred embodiment the loading system comprises a buffer loading module and a sample loading module, which may have similar structures and functions. The buffer loading module, connecting at least one buffer container to each of the buffer reservoirs by channel connecting means, is provided for the parallel loading of multiple buffer reservoirs and channels. The sample loading module, connecting the sample vials to the sample reservoirs, is provided for sample loading. In another embodiment, a single loading module is provided for loading both the buffer and the sample. This is accomplished by a switch in the connection, in which the loading module is first connected to the buffer reservoir such that buffer loading can occur, followed by switching over of the channel connecting means to the sample reservoir and replacement of the buffer vials with sample vials, such that sample loading can occur.
In the most preferred embodiment, the loading module is deployed with a pneumatic system of interconnecting fluid conduits to supply simultaneously the forces required to load multiple reservoirs/channels. In order to allow different receptacles (e.g. reservoirs or channels) to be loaded with different liquids, tubings are provided to directly connect the liquids to the corresponding receptacle. When the fluid pressure is switched on, the different liquids would be loaded simultaneously.
The power source may be a plurality of high voltage power supply with one or more output outlets for maintaining voltage differences between at least two points. The power supply is connected to the two ends of each channel for maintaining a voltage difference required for electrophoresis of samples along the channels. In the preferred embodiment, a third electrical connection is provided in the sample reservoir to prevent a continuous leaching of samples during electrophoresis.
Other optional features include a vacuum source, connected to a buffer waste container, which is used to facilitate the removal of excess buffer during loading, and the removal of washing fluid during the washing and rinsing cycles. The vacuum source may also be connected to another optional sample waste container to facilitate the removal of excess sample.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A is a system diagram of one modular electrophoretic system according to the present invention.
Figure 1B is the top plan view of an electrophoretic module of the same system according to the present invention.
Figure 1C is the longitudinal cross-sectional view along a channel of a capillary chip of an electrophoretic module of the same embodiment.
Figure 1 D is the bottom plan view of the loading end interface of the same embodiment.
Figure 1 E is the bottom plan view of the running end interface of the same embodiment.
Figure 1 F is the cross sectional view of part of the buffer loading module of the same embodiment.
Figure 1G is the cross sectional view of a waste collector module of the same embodiment.
Figure 2A is a system diagram for a second embodiment according to the present invention.
Figure 2B is a top plan view of an electrophoretic module according to the second embodiment.
Figure 2C is the longitudinal cross-sectional view along a channel of a capillary chip of an electrophoretic module of the second embodiment.
Figure 2D is the bottom plan view of the loading end interface structure of the second embodiment.
Figure 3 is the system diagram for a third embodiment according to the present invention.
DESCRIPTION OF THE INVENTION
In the following description, numerous specific details are set forth such as the specific connections between the various module, in order to provide a thorough understanding of the present invention. In other instances, well known elements such as pressure and vacuum pumps, power supply and pneumatic valves are not described in detail in order not to unnecessarily obscure the present invention.
Figure 1A is a diagrammatic illustration of the various modules of the and how they are connected to each other according to one embodiment of the present invention. At the center is the capillary array chip 20 with a plurality of electrophoretic channels 22. Each channel has a loading end 28 and a running end 30, with a buffer reservoir 24 and a sample reservoir 26 connected to the loading end. For ease of illustration, only one channel and corresponding reservoirs are shown in these figures. It should be understood that a CAE chip may contain numerous channels, and the following description to one channel and its corresponding reservoirs apply to all other channels as well.
Electrical connections are provided at the two ends (loading end 28 and running end 30) of each channel, allowing the channel to be connected to a power source 32 which may include several high voltage power supplies. Another optional electrical connection can be made between the running end 30 and the sample reservoir at point 34 for initial sample injection into the channel. During electrophoresis, the voltage at point 34 is set slightly higher than the voltage at point 28 to prevent leaching of the sample continuously into the channel.
The loading of buffer in the buffer vial 36 into the buffer reservoir 24 is performed by the buffer loading module. In this embodiment, a pneumatic system is employed to force the liquid buffer through tubing 38 into reservoir 24. Pressure source 40 is connected to the buffer vial via fluid conduits 41 , and provides the fluid pressure, which is controlled by valve 42. The fluid pressure source may be a conventional air pump with a preset air pressure and flow rate. The flow rate used depends on the number and size of the channels and connecting tubings, and may be determined without undue experimentation. Residual pressure after loading is released by opening release valve 44. Only one buffer container or vial is shown in Figure 1 A for ease of illustration. It should be understood that numerous buffer vials each connected by a separate tubing to a different channel may be provided. Furthermore, through air- tightly interconnected fluid conduits 41 , a plurality of different buffers in different buffer vials can be loaded into different buffer reservoirs as described in a later section.
Pressure source 40 is also connected via a second series of interconnected fluid conduit 46 to a series of sample vials 49 (only one shown in Figure 1A). Operating under the same principle as the buffer loading module, an air-tight system allows fluid pressure, controlled by valve 48, to force liquid samples from sample vial 49 into the corresponding sample reservoir 26 via tubing 50. Release valve 52 is opened after loading is complete to release any residual pressure within fluid conduit 46.
The running end 30 of the electrophoretic channel is also connected via tubing 54 to buffer waste container 56. In this embodiment, buffer waste container 56 is further connected to a vacuum source 58. When control valve 60 is opened, the suction generated from vacuum source 58 sucks excess liquid in the channel into buffer waste container 56. This feature is particularly useful for more viscous buffers, but is optional for non-viscous buffers. In such cases, the excess buffers can passively drain into buffer waste container 56.
The entire electrophoretic system may be controlled by a computer or processor 62 for precision loading and electrophoresis.
Figures 1 B and C show in greater detail the electrophoretic module. A chip tray 66 holds an array chip 20 with channels 22 etched into it. A upper plate 70 is bonded together with the bottom plate 71 to form the array chip such that the individual channels are isolated from each other to prevent seeping and mixing of liquid. A loading end interface structure 72 and a running end interface structure 74 are provided to allow
connection to the other modules. Sample reservoir 26 and buffer reservoir 24, located below the loading end interface structure, are shown as dotted lines in Fig. 1B. Pneumatic interface 76 with an elastomeric sealant 78 provides an air-tight connection to the buffer reservoir 24 via buffer access holes 68 and tubings 38. The tubings may be made of silica, silicone or plastic materials or any other chemically inert material. The sample reservoir is also connected in an air-tight manner to tubing 50 via sample access hole 67 using a second elastomeric sealant 79 (Tubing 50 is shown in Fig. 1C as the vertical double dotted lines as it falls in front of the plane of the cross-section). Running end interface structure 74 includes pneumatic interface 80 with elastomeric sealant 82 providing an air-tight connection to the running end of channel 22, which is in turn connected to waste vial 56 via tubing 54 and waste access hole 69.
Figures 1 D and E shows the underside of the loading end and running end interface structures respectively in this embodiment. High voltage interfaces 84 and 86 are also provided for electrical connection between the two ends of the channel for electrophoresis. In this example, each end of each channel is provided with three electrical contacts 88. At high voltage interface 86 as shown in Fig. 1E, two of the electrical contacts are optional and may be used for conductivity detection after separation of the samples by electrophoresis, while the third electrical contact is meant for the running voltage. For high voltage interface 84 as shown in Fig. 1 D, all three electrical contacts may be connected to the high voltage power source, with one connection used to deliver voltage for separation at the electrophoretic channels and other one or two connections to maintain a
voltage for the sample reservoir. Fig. 1 D and E also show one embodiment of the elastomeric sealants 78, in which rectangular sheets of silicone are used to provide an air-tight seal for the entire row of buffer access holes 68 or sample access holes 67. In the same manner, elastomeric sealant 82 seals off the entire set of waste access holes 69. Silicone sheets such as Sylgard 184 by Dow Corning may be used.
Figure 1 F shows part the buffer loading module, with rack 90 acting as a vial holding means for receiving and securing the vials of buffer solution. The vials of buffer 36 are attached to rack 90 via elastomeric gaskets 92 which gives an air-tight seal along the rim of each vial. Fluid conduit 41 as shown in this embodiment comprises manifold-like fluid channels which interconnect all the buffer vials to the pressure source 40, and control valves 42 and 44. Tubing 38 is provided as channel connecting means to connects the content of buffer vials 36 to buffer reservoir 24. The sample loading module (not shown) is very similar to the buffer loading module, and operates under the same pneumatic principles. The fluid from the pressure source may be any non-reactive gas, such as compressed purified air or nitrogen.
Fig. 1G shows one embodiment of a waste collector module according to the present invention. In this example, multiple tubings 54 connected to the running end of the electrophoretic channels are drained into waste vials 56. Fluid conduits 96 are connected to vacuum source 58 to facilitate the removal of waste buffers or wash buffer.
In operation, buffer vials and sample vials are connected to the buffer and sample loading modules respectively. Valve 42 is opened to allow air pressure to build up within the fluid conduit 41 of the buffer loading module, which results in buffer being forced via tubing 38 into buffer reservoir 24 and channel 22. At the same time, valve 60 may be opened to facilitate the removal of air inside the channels, such that no air bubbles would be trapped in the system. After buffer has been loaded, valves 42 and 60 are closed and valve 44 opened to release any residual pressure within fluid conduit 41 . Then valve 48 is opened and pressurized air increases the fluid pressure inside fluid conduit 46, forcing liquid samples from sample vial 49 into sample reservoir 26 via tubing 50. Once sample loading is complete, valve 52 is closed and valve 48 opened to allow the release of residual air pressure. To perform the electrophoretic separation, the power supply 32 is switched on, and a sample injection voltage generated at the electrical connection between the loading end 28 and the running end of the channel 30. A portion of the sample migrates into the channel in the presence of the electrical potential. To prevent further leaching of the sample, a voltage higher than that at point 28 is generated at point 34. For conductivity or electrochemical measurements, the appropriate electric connections 88 are set up beside the high voltage interfaces 84 and 86. After electrophoresis and analysis, the system may be washed by replacing the sample and buffer vials with cleaning solutions, and opening the appropriate valves to flush the system. To remove all cleaning solution, air or other drying gas may be flushed through the system.
Figure 2A shows a second embodiment of the present invention in which an additional outlet is provided in the sample reservoir in order to facilitate sample loading. All other elements of the system remain the same as the first embodiment. Sample reservoir 100 is subdivided into inlet reservoir 130 and outlet reservoir 134. Inlet reservoir 130 functions in the identical manner as in the previous embodiment while outlet reservoir 134 has an additional connection via tubing 106 to a sample waste vial 108. Sample waste vial 108 may in turn to be connected vial fluid conduits 1 10 to the vacuum source 112 and controlled by valve 114. Pressure source 160 is connected to the sample vial 162 via fluid conduit 164 which is controlled by valves 166 and 168. Sample vial 162 is connected to the sample inlet reservoir via tubing 170. Buffer in buffer vial 174 is connected to buffer reservoir 126 via tubing 176.
Figure 2B and C show in greater detail the arrangement of the electrophoretic module. The chip tray 116 and the array chip 1 18 with its electrophoretic channels 120 are the same as that of the previous embodiment. The running end pneumatic and high voltage interfaces 1 22 and 124 respectively also remains unchanged. However, besides the buffer reservoirs 126 and the buffer access holes 128, the sample reservoir is provided with a sample inlet reservoir 130 and a sample outlet reservoir 134. The loading end pneumatic interface 138 is further provided with an inlet access hole 132 and an outlet access hole 136.
Tubings 170 and 106 form air-tight connections with sample access holes
132 and 136 respectively. As they are not located along the cross- sectional plane, their positions are shown in dotted lines in Figure 1C.
Figure 2D shows the bottom view of the loading end pneumatic and high voltage interfaces 138 and 140 respectively. As in the previous embodiment, a sheet of elastomeric sealant 142 is provided such that the row of sample access holes are airtight to the environment. Similar sealants 144 and 146 are used to isolate the rows of inlet access holes and outlet access holes respectively from the environment. In this way, the entire system is rendered airtight. The running end interface remains the same as the previous embodiment. The dotted lines show the orientation of the buffer reservoir, the sample inlet reservoir, the sample outlet reservoir and the electrophoretic channel for ease of understanding.
The operations of the second embodiment is the same as the first, except that the samples have an escape route at the sample outlet reservoir 134 via the outlet access hole 136. During sample loading, valve 166 is opened such that the air pressure from pressure source 1 60 forces samples from sample vial 162 into sample inlet reservoir 130. At the same time, valve 172 connected to vacuum source 112 may also be opened. As sample inlet reservoir 130 is connected to the sample outlet reservoir 134 sample is loaded into the sample reservoir, and any air originally present would be removed via tubing 106. As a result, air bubbles are less likely to be trapped in the system. Furthermore, this additional feature also improves the washing process, particularly of the sample reservoir, after electrophoresis.
Figure 3 shows a third embodiment of the present invention, in which one of the loading modules have been removed compared to the first embodiment. In this case, only one inlet control valve 180, one outlet
control valve 182, and one fluid conduit 184 are provided. In this case, one vial holding rack (not shown) would have to act as the receiving means for both the buffer vials and the sample vials (both shown as reference numeral 184 for ease of illustration) sequentially as they are loaded. This may be done by first attaching buffer vials to the vial holding rack, and connecting tubing 186 to the buffer reservoir 188. Then buffer loading may be initiate by opening valve 180. In this embodiment, valve 192 may also be optionally opened to facilitate buffer loading, with or without vacuum source 194 activated. After buffer loading is complete, excess buffer in the tubing is removed, valve 180 closed, and tubing 1 86 switched either manually or automatically, to connect to sample reservoir 190. This is followed by the replacement of the buffer vials with the sample vials. Then valve 180 is opened again to load the samples from the sample vials to the sample reservoir. Electrophoresis will then proceed according to the standard protocols.
While the present invention has been described particularly with references to Figs 1 to 3 with emphasis on an automated loading or dispensing system for capillary electrophoresis, it should be understood that the figures are for illustration only and should not be taken as limitation on the invention. In addition it is clear that the method and apparatus of the present invention has utility in many applications where parallel loading of multiple samples is required. It is clear that the features of the various embodiments may be combined to form additional embodiments. It is contemplated that many changes, combinations and
modifications may be made by one of ordinary skill in the art without departing from the spirit and the scope of the invention described.
For example, it would be clear that other configurations of the sealing sheet or other sealing means may be used to render the system air-tight, e.g. a single larger sheet of silicone may be used to seal both the row of sample access holes and buffer access holes. As the pneumatic means is preferably detachable to facilitate cleaning, a single larger sheet improves the ease of operations.
A similar configuration may be used for the dispensing of liquid in other applications, such as for micro-solid phase extraction and liquid chromatography. In these applications, the electrophoretic channels described in Figures 1-3 would be replaced by columns. The tubings which connect the buffers or samples to the channels would be connected to the columns instead. Additional applications of the loading system include liquid dispensing for microwells and microtiter plates, in which case the channels would be replaced by wells and the tubings leading directly into the wells. It is clear that although buffer and aqueous samples are the examples of liquids to be dispensed, other liquids, such as solvents and salt solutions are equivalents, and are intended to be covered by the scope of the claims.
The vial holding rack for buffers may be adapted to receive a single or small number of containers of buffers, if the running buffers of the various channels are the same. This can be easily accomplished by modifying the holding rack such that the end of the fluid conduit is wide enough to
accommodate a larger-mouthed container, and at the same time allow buffer tubings connected to different electrophoretic channels to be submerged inside the buffer in the larger buffer container. A suitable sealing gasket may be used to ensure that they system is air-tight.
Release valves 44, 52, and 182 are useful for the quick release of residual pressure inside the system. However, a low-cost system without these release valves may also function, albeit with a lag time for the residual air pressure to escape.