WO2000005435A2

WO2000005435A2 - Array electrophoretic apparatus

Info

Publication number: WO2000005435A2
Application number: PCT/SG1999/000080
Authority: WO
Inventors: Sam Fong Yau Li
Original assignee: Ce Resources Pte Ltd.
Priority date: 1998-07-24
Filing date: 1999-07-16
Publication date: 2000-02-03
Also published as: AU5311599A; WO2000005435A3

Abstract

A multi-channel array electrophoretic apparatus comprising a separation platform wherein a plurality of separation channels (20) are longitudinally aligned. Each separation channel comprises an inlet end connected to an inlet reservoir with an inlet electrode (72) and an outlet end connected to an outlet reservoir with an outlet electrode (76). A plurality of sample channels (10) are provided, each sample channel being connected to a corresponding separation channel. A sample injection reservoir with a sample loading electrode (74) is connected to the separation channels via connecting means; the connecting means allowing electrical connections between the sample injection reservoir and the separation channel under operating conditions.

Description

ARRAY ELECTROPHORETIC APPARATUS

FIELD OF THE INVENTION

The present invention is related to electrophoretic apparatus. In particular, the present invention is related to high throughput array electrophoretic apparatus and method of using the same.

BACKGROUND OF THE INVENTION

Microfabricated capillary electrophoresis (CE) chips were introduced in 1992, and separations of fluorescent dyes, fluorescence labeled amino acids and metal ion complexes have shown that these devices can increase the speed of CE separations by an order of magnitude. However, the utility of these highspeed miniaturized separation devices will be limited unless multiple samples can be analyzed in parallel, just as capillary array electrophoresis (CAE) was needed to make CE a practical method for high-throughput DNA analysis. The current method for CAE was based on the design for the single- channel CE chips. In one example (Anal. Chem., 1997, 69, 2181 -2186), thirty seven electrodes were used for injection and separation in 12 channels. We could imagine for 1024 channels (this number is based on the number of the elements already available in modern solid state imaging devices for detection), at least 3072 electrodes will be needed . In another example (Proceedings of the National Academy of Sciences of USA, 1998, 95, 2256- 2261 , March), an array of platinum electrodes was used for injection and separation in 48 channels. The system required for making these devices work correctly could be quite complicated. In addition, the current cross- design for introducing samples into the channels requires quite large spacing in order to prevent current leakage between channels (for example, the apparatus for the 48 channels was fabricated on a 10 cm wafer sized substrate). The whole apparatus could become quite large, which is contrary to the purpose of miniaturization.

Linhares and Kissinger (Analytical Chemistry, 1991 , 63, 1342-1346) describe an on-column fracture on capillary which can be used for sample introduction in capillary electrophoresis. In this publication, a single capillary is fractured with the inlet end immersed in running buffer or sample. During injection, a potential is applied between the fracture and the outlet of the capillary. Electroosmotic flow (EOF) pulls sample ions into the capillary through the inlet of the capillary. The amount of sample introduced is proportional to the EOF. In addition, the on-column fracture can be used to isolate the column end from the high voltage for reducing the noise in electrochemical detection (for example, as described in Analytical Chemistry, 1992, 64:2461 -2464 and in Electrophoresis, 1997, 18:2024-2029). The fracturing process, however, is unpredictable, and therefore difficult to apply to array electrophoretic systems in which uniformity and ease of manipulation of the capillaries are prerequisites for a reliable system.

OBJECT OF THE INVENTION

It is therefore an object of the present invention to provide a system to overcome the shortcomings as stated above.

It is another object of the present invention to provide a simple electrophoretic system for the simultaneous injection of multiple samples.

It is another object of the present invention to provide a simple electrophoretic system with reduced number of electrodes.

It is another object to provide one embodiment of the present array electrophoretic apparatus which requires only three electrodes for sample loading and separation.

It is a further object of the present invention to provide a rugged and efficient embodiment which prevents high voltage from interfering with electrochemical detection for array electrophoresis.

SUMMARY OF THE INVENTION

The present invention is a multi-channel array electrophoretic apparatus comprising a separation platform wherein a plurality of separation channels are longitudinally aligned. Each separation channel comprises an inlet end connected to an inlet reservoir with an inlet electrode and an outlet end connected to an outlet reservoir with an outlet electrode. A plurality of sample channels are provided, each sample channel being connected to a corresponding separation channel. A sample injection reservoir with a sample loading electrode is connected to the separation channels via connecting means; the connecting means allowing electrical connections between the sample injection reservoir and the separation channel under operating conditions.

Under operating conditions, running medium such as ionic buffer is provided in the separation channel, the inlet reservoir and the outlet reservoir; sample solution is provided in at least one sample channel; and the electrodes are connected to a power supply. Electrical conducting medium is added into the sample injection reservoir, such that electrical connection is made between the sample loading electrode and the inlet and outlet electrodes. A voltage pulse is applied between the sample loading electrode and the outlet electrode such that a current pulse flows from the sample injection reservoir to the outlet channel and sample is drawn into the separation channel due to electroosmotic flow. The amount of sample injected into the separation channel is proportional to the injection voltage and injection time, and may be determined and varied without undue experimentation. This is followed by the application of a running voltage between the inlet electrode and the outlet electrode for electrophoresis of the test sample in the separation channel.

The connecting means of the sample injection reservoir may be any structure which allow electrical current to pass through under operating conditions while preventing the free flow of bulk fluid. For example, small pores may be provided which are filled with an electrically conducting medium such as ionic buffer during operation, such that electrical current can pass from the sample injection reservoir into the separation channel while reducing bulk flow of the medium. Alternatively, the connecting means may be plugs or strips of electrically conducting material such as semi-solidified or solidified agar with ionic buffer in direct electrical contact with the sample loading electrode and the separation channels. This results in electroosmotic flow which causes the sample loaded in the sample channel to be pumped into the separation channel. The separation channels may be longitudinally aligned tubes or open channel etched into the separation platform. In the preferred embodiment, the connecting means is a porous layer at the bottom of the sample injection reservoir adapted for direct contact with open separation channels. The porous layer may be made from glass, silicon, quartz, ceramics or polymers.

The inlet and outlet reservoirs may be provided separately for each separation channel, in which case more electrodes are required (one electrode per inlet and outlet), and the distance between the separation channels should be lengthened to prevent sparking due to the high voltages applied between each set of outlet and inlet electrodes. The electrical connections may be simplified by having one common connecting electrode for the inlet electrodes and one common electrode for the outlet electrodes for connection to the power supply for electrophoresis. Preferably, the inlet reservoirs and outlet reservoirs are joined to form one inlet channel and one outlet channel respectively. This reduces the number of required electrodes to three.

In another embodiment, another reservoir and porous layer may be added peφendicularly across the separation channels before the outlet end such that an electrochemical detection system disposed at the outlet end would be electrically isolated from the high voltage used for electrophoresis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pattern of a glass plate of a multichannel CAE chip according to one embodiment of the present invention. FIG. 2A and B are schematic drawings of the cover plate of the CAEC showing the top and bottom views respectively according to one embodiment of the present invention.

FIG. 3 is a traverse cross-sectional view of the CAEC taken along the sample injection reservoir after bonding the two glass plates in FIG. 1 and FIG.2 (not to scale).

FIG. 4 is a schematic diagram of the side view of a longitudinal cross-section of the CAEC showing how sample introduction and separation may be performed

FIG. 5 is a schematic diagram of the top view of the glass plate of a CAEC with detection electrodes for electrochemical detection.

DETAILED DESCRIPTION

The preferred embodiment of the present invention is based upon the fact that a porous section or a fracture on a capillary can be used for sample introduction in capillary electrophoresis. Unlike the prior designs which require a cross on the channels and many electrodes for sample introduction and separation, the most preferred embodiment of the present invention involves making a perpendicular porous layer across all the channels and therefore simplifying the whole system. Advantageously, the electrophoretic apparatus of the present invention eliminates the needs to connect many electrodes and to develop complicated software to control the voltages for injection. It also saves space. In operation, an injection voltage pulse is applied between the porous layer and the outlet end of the separation channel through an electrode in the buffer solution which is in contact with the porous layer and another electrode in the buffer solution at the outlet reservoir. After injection, a separation voltage is applied between the inlet end and outlet end of the separation channel, as normally done in capillary electrophoresis separation.

In the most preferred embodiment, a plurality of open separation channels, inlet, outlet and sample channels are made on a glass plate as the separation platform through etching by the normal microfabrication method. A glass plate is provided as a separate cover plate. Access holes are provided on the cover plate for access to the corresponding channels and reservoirs. After bonding the separation platform and the cover plate, a microfabricated CAEC is produced which needs only three electrodes for the entire process of sample injection and separation. While those skilled in the art will recognize that there exists a multitude of variations and applications, the present invention is described in the context of making a CAEC.

Additional features, aspects and advantages of the invention will be more fully understood when considered in connection with the accompanying drawings and the detailed description which follows.

A microfabricated CAEC according to the preferred embodiment of the present invention is made of a glass plate as a separation platform and a glass cover plate. Referring to Fig. 1 , sample channels 10, separation channels 20 inlet channel 30 and outlet channel 31 are produced through etching onto the separation platform 22. Referring to Figure 2, access holes 40 to the inlet channel 30, access holes 41 to the outlet channel 31 , access holes 50 to the sample channels 10, and sample injection reservoir 60 are provided on the cover plate 24. A porous glass strip 70 at the bottom of the sample injection reservoir 60 is also provided on the cover plate 24 as shown in Figure 2B. The support plate and the cover plate are adapted to be heat bonded together. After heat bonding, the porous glass strip 70, and the inlet and outlet channels 30 and 31 are disposed perpendicularly above the parallel array of separation channels 20. This is further illustrated in Figure 3.

In operation, the separation channels 20, the inlet 30 and outlet channels 31 are filled with running buffer via access holes 40 and 41 as shown in Figure 4. Sample solutions are filled in the sample channels 10 through the access holes 50. The running buffer is also filled in the sample injection reservoir 60. Three platinum electrodes, which are connected to a power supply, are placed into the system, with the inlet electrode 72 being placed into inlet channel 30 via access hole 40, the sample loading electrode 74 into sample injection reservoir 60, and the outlet electrode 76 into outlet channel 31 via access hole 41 . Alternatively, the various electrodes may be made from any metallic element or alloy, and may be pre-fabricated onto the separation platform, for example by vacuum deposition as illustrated by the sample loading electrode 74 in FIG. 3. FIG. 4 shows how the sample introduction and separation is done using the present invention. For injection, an injection voltage pulse is applied between the sample loading electrode 74 in the reservoirs 60 and the outlet electrode 76 in outlet reservoir 31 . Because the porous glass layer 70 can let current pass through but prevent the bulk flow of the running buffer from the sample injection reservoir 60 into the separation channels 20, the sample solutions in the sample channels 1 0 and the running buffer in the side channel 30 will be drawn into the separation channels 20 by electroosmotic effects. The duration of the injection voltage pulse may be 1 -30kV for 0.1 -5 seconds. For separation, a separation voltage of 1 -30kV is applied between the inlet electrode 72 in inlet reservoir 30 and the outlet electrode 76 in outlet reservoir 31.

In another embodiment, a CAEC with additional electrodes adapted for electrochemical or conductivity detection can be made by introducing another detection porous layer 80 and a corresponding detection reservoir (not shown) on top of layer 80 before the outlet end of the separation channels 20, as indicated in Figure 5. In this embodiment, the outlet electrode 76 is fabricated by vacuum deposition onto the detection reservoir. The structure and fabrication method of the porous layer 80 and the detection reservoir are the same as that of layer 70 and reservoir 60 as described above. For injection using an apparatus according to this embodiment, a voltage of 1 - 30kV pulse for 0.1 to several seconds may be applied between the sample loading electrode 74 and the outlet electrode 76. After injection, a separation voltage of 1 -30kV is applied between the inlet 72 and outlet 74 electrodes. The components of the sample in the separation channels 20 will be carried along by the EOF in the channels to pass the porous layer 80, and be detected through the detection electrodes 100. For best results, the outlet end of each sample may be broadened into a reservoir (not shown in the embodiment in Fig. 5) for the accumulation of the fluid flowing into the outlet end due to EOF. Various designs are useful for the detection electrodes. The embodiment in Figure 5 shows two detection electrodes connected to each separation channel. These electrodes may be fabricated for example by vacuum deposition and may be used for conductivity, potentiometric or amperometirc detection by connecting to the appropriate measuring instrument such as a potentiostat. One detection electrode may serve as a reference electrode while the other one serves as the measuring electrode. Other designs, such as a three-electrode detection system for every separation channel may also be possible, if the separation channels are spaced sufficiently far apart, and is within the knowledge of one of ordinary skill in the art based on the teaching disclosed herewith, which allows one in the art to design a simplified microfabricated CAEC with or without electrodes for electrochemical detection with high signal to noise ratio.

Although the specific embodiment described above is for the use of a porous glass strip to create the connecting means on the surface of the capillary tube or channel, it should be understood that the connecting means may be created from other material. For example, a laser beam may be used to create small connecting pores below sample injection reservoir 60 of Figures 2A and 3 to substitute for the porous glass strip. These pores provide the necessary electrical connection for the injection current to pass through. The size of the pores should be large enough for electrical current to pass through without allowing a free flow of buffer, such that an electroosmotic effect causes a net flow of buffer into the separation channel via the sample channels. Besides holes, other connecting pores such as fractures or agar strips of ionic buffer may also be provided to create the necessary electroosmotic effects. The ratio between the flows of the sample solutions and the running buffer will depend on the dimensions of the separation channels and the sample channels, the size of the connecting pores, and the viscosity of the running buffer and the sample solutions, and may be determined by experimentation by one or ordinary skill in the art based on teachings disclosed herein. Those skilled in the art will recognize that the foregoing description provides a design for microfabricated CAEC which in its most preferred embodiment need only three electrodes for sample injection and separation. In addition, the foregoing description of the specific embodiment of this invention have been presented for the purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, as many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

CLAI MS

1. An electrophoretic apparatus comprising :

a separation platform with a plurality of separation channels aligned longitudinally and at selected intervals, each channel having an inlet end and an outlet end; each of said inlet end connected to an inlet reservoir with an inlet electrode, each of said outlet end connected to an outlet reservoir with an outlet electrode; said inlet and outlet electrodes adapted for connection to a power supply;

a plurality of sample channels, each sample channel connected to a corresponding separation channel at a position between said inlet end and said outlet end; and

a sample injection reservoir connected via connecting means to each of said separation channels between the inlet end and the outlet electrode, said sample injection reservoir having a sample loading electrode adapted for electrical connection to said power source, said connecting means adapted for electrical connection and preventing free fluid flow between the sample injection reservoir and the separation channel under operating conditions.

2. An electrophoretic apparatus according to claim 1 wherein

a detection reservoir is provided for retaining electrically conducting medium; the detection reservoir connected via detection connecting means to each of said separation channels between said injection reservoir and said outlet end; said outlet electrode is disposed within said detection resevoir; said detection connecting means adapted for electrical connection and preventing free fluid flow between the detection reservoir and the separation channel under operating conditions; and

each of said separation channel is further connected to at least two detection electrodes at a position between the detection reservoir and the outlet end, each electrode adapted for connection to a measuring instrument for electrochemical or conductivity detection of samples flowing towards the outlet end.

3. An electrophoretic apparatus according to claim 1 or 2 wherein

the separation channels are aligned in a planar, parallel array;

running medium is provided in the separation channels, the inlet reservoirs and the outlet reservoirs;

electrically conducting medium is provided in the sample injection reservoir and the connecting means;

sample solution is provided in at least one sample channel; and the inlet, outlet and sample loading electrodes are connected to a power source.

4. An electrophoretic apparatus according to claim 3 wherein the inlet reservoirs of the separation channels are connected to form at least one common inlet channel each with one common inlet electrode.

5. An electrophoretic apparatus according to claim 3 wherein the outlet reservoirs of the separation channels are connected to form at least one common outlet channel each with one common outlet electrode.

6. An electrophoretic apparatus according to claim 3 wherein the inlet reservoirs of the separation channels are connected to form at least one common inlet channel each with one common inlet electrode, and the outlet reservoirs of the separation channels are connected to form at least one common outlet channel each with one common outlet electrode.

7. An electrophoretic apparatus according to claim 6 wherein the inlet electrode, the outlet electrode and the sample loading electrode are connected to the power source.

8. An electrophoretic apparatus according to claim 3 further comprising a cover plate for covering said separation platform, said cover plate being bonded to the separation platform and having sample access holes adapted for accessing the corresponding sample channels; at least one inlet reservoir access hole for accessing the inlet reservoir; and at least one outlet reservoir access hole for accessing the outlet reservoir.

9. An electrophoretic apparatus according to claims 8 wherein the separation platform and the cover plate are bonded to form a microfabricated capillary array electrophoresis chip.

10. An electrophoretic apparatus according to claim 8 wherein the sample injection reservoir is disposed in said cover plate.

1 1. An electrophoretic apparatus according to claim 10 wherein the detection reservoir is disposed in said cover plate.

12. An electrophoretic apparatus according to claim 10 wherein the sample injection reservoir is an elongated recess in said cover plate, and disposed horizontally between the sample channel connection and the outlet reservoir, said recess further having a longitudinal axis traversing the longitudinal axes of the separation channels.

13. An electrophoretic apparatus according to claim 12 wherein the connecting means comprises a porous layer disposed at the bottom of said sample injection reservoir, the porous layer adapted for direct contact with the separation channels under operating conditions.

14. An electrophoretic apparatus according to claim 1 1 wherein the detection reservoir is an elongated recess in said cover plate, and disposed horizontally between the sample injection reservoir and the outlet reservoir, said recess having a longitudinal axis traversing to the longitudinal axis of the separation channel.

15. An electrophoretic apparatus according to claim 14 wherein the connecting means comprises a porous layer disposed at the bottom of said detection reservoir, the porous layer adapted for direct contact with the separation channels under operating conditions

16. An electrophoretic apparatus according to claim 13 wherein the porous layer is made from agar, glass, silicon, quartz, ceramics or polymer.

17. An electrophoretic apparatus according to claim 15 wherein the porous layer is made from glass, silicon, quartz, ceramics, polymer or agar.

18. An electrophoretic apparatus according to claim 8 wherein the microfabricated capillary array electrophoresis chip is made from glass, quartz, silicon or polymers.

19. An electrophoretic apparatus according to any one of claims 3 wherein the electrodes are made of metal, metal alloys, conducting inorganic material, conducting organic material or conducting polymers.

20. An electrophoretic apparatus according to claim 3 wherein the running medium is an ionic buffer, gel, polymer solution or electrically conducting medium.

21. An electrophoretic apparatus according to claim 3 wherein the electrically conducting medium is an ionic buffer, gel or polymer solution.

22. An electrophoretic apparatus according to claim 3 wherein the connecting means is connected to said separation channels at a position proximate the interconnection with the sample channel.

23. An electrophoretic method using an electrophoretic apparatus having

a separation platform with a plurality of separation channels each having an inlet end and an outlet end;

an inlet channel with an inlet electrode connecting said inlet ends and an outlet channel with an outlet electrode connecting said outlet ends;

a plurality of sample channels each connected to a corresponding separation channel at a position between said inlet end and said outlet end;

a sample injection reservoir with a sample loading electrode; said sample injection reservoir connected to said separation channels via connecting means; and

a power source

said method comprising the steps of :

a) adding running medium into the separation channels, inlet channel and outlet channel;

b) adding electrically conducting medium into the sample injection reservoir; c) connecting said inlet electrode, outlet electrode and sample loading electrode to said power source;

d) adding at least one test sample into at least one sample channel;

e) applying a voltage pulse between the sample loading electrode and the outlet electrode such that a current pulse flows from the sample injection reservoir to the outlet channel via the separation channel, and sample is drawn into the separation channel due to electroosmotic flow;

f) applying a running voltage between the inlet electrode and the outlet electrode for electrophoresis of the test sample in the separation channel.

24. An electrophoretic method according to claim 22 wherein said electrophoretic apparatus further includes

a detection reservoir connected via detection connecting means to each of said separation channels between the injection reservoir and the outlet end; said outlet electrode disposed within said detection reservoir;

at least two detection electrodes connected to each separation channel at a position between the detection reservoir and the outlet end; and

a measuring instrument for electrochemical or conductivity detection said method further comprising the steps of :

connecting said detection electrodes to the measuring instrument for electrochemical or conductivity detection in step (c); and

adding electrically conducting medium into the detection reservoir before step (e).

25. An electrophoretic method according to claim 23 or 24 wherein the voltage pulse and the running voltage are constant, stable but variable or ramped.