WO2005040331A1

WO2005040331A1 - Integrated bio-analysis and sample preparation system

Info

Publication number: WO2005040331A1
Application number: PCT/US2004/035406
Authority: WO
Inventors: Varouj Amirkhanian; Ming-Sun Liu
Original assignee: Egene, Inc.
Priority date: 2003-10-24
Filing date: 2004-10-25
Publication date: 2005-05-06
Also published as: JP4736056B2; JP2007510407A; CN1898374B; CN1898374A

Abstract

An integrated bio-analysis system incorporates built-in sample preparation capabilities. In one aspect of the present invention, a bio-analysis instrument is provided with a built-in sample preparation device based on PCR (or thermal cycling block/module). In one embodiment of the present invention, a peltier unit in the sample preparation device provides thermal cycling of samples supported in a multi-well tray. In another aspect of the present invention, a CE instrument is provided with a built-in sample preparation capability, which may comprise a sample preparation (bio-molecular reaction) device based on thermal cycler type. In another aspect of the present invention, a PCR device is provided with a built-in analysis device, such as a CE device, for verifying the results of the PCR (bio-molecular reaction) process.

Description

INTEGRATED BIO-ANALYSIS AND SAMPLE PREPARATION SYSTEM

BACKGROUND OF THE INVENTION

This application claims the priority of U.S. Provisional Patent Application No. 60/514,381,

filed on October 24, 2003. This Provisional Patent Application is folly incorporated by reference herein, as if folly set forth herein. This application is a Continuation-in-Part of U.S. Patent

Application No. 10/059,993 entitled "Multi-Channel Bio-Separation Cartridge," filed on January

28, 2002; and U.S. Patent Application No. 10/060,052, entitled "Optical Detection In A Multi-

Channel Bio-Separation System", filed on January 28, 2002; and U.S. Patent Application No.

10/319,803, entitled "Optical Detection Alignment Coupling Apparatus", filed on December 13,

2002; and PCT Application No. PCT/US03/39971, entitled "Optical Detection Alignment Coupling Apparatus", filed on December 15, 2003; and U.S. Patent Application No. 10/823,382,

entitled "Multi-Capillary Electrophoresis Cartridge Interface Mechanism", filed on April 12,

2004, which are all commonly assigned to BioCal Technology, Inc., the assignee of the present

invention.

The above-mentioned applications, and all other applications, documents and references

noted in the disclosure herein below, are folly incorporated by reference as if folly set forth herein.

1. Field of the Invention The present invention relates to bio-analysis, and more particularly a bio-analysis system

integrating sample preparation process, and more particularly to a multi-channel bio-analysis system integrating sample preparation process.

2. Description of Related Art Bioanalysis, such as DNA analysis, is rapidly making the transition from a purely scientific quest for accuracy to a routine procedure with increased and proven dependability. Medical

researchers, pharmacologists, and forensic investigators all use DNA analysis in the pursuit of

their tasks. Yet due to the complexity of the equipment that detects and measures DNA samples

and the difficulty in preparing the samples, the existing DNA analysis procedures are often time- consuming and expensive. It is therefore desirable to reduce the size, number of parts, and cost of

equipment, to ease sample handling during the process, and in general, to have a simplified, low

cost, high sensitivity detector.

One type of DNA analysis instrument separates DNA molecules by relying on

electrophoresis. Electrophoresis techniques could be used to separate fragments of DNA for

genotyping applications, including human identity testing, expression analysis, pathogen

detection, mutation detection, and pharmacogenetics studies. The term electrophoresis refers to

the movement of a charged molecule under the influence of an electric field. Electrophoresis can

be used to separate molecules that have equivalent charge-to-mass ratios but different masses. DNA fragments are one example of such molecules. There are a variety of commercially available instruments applying electrophoresis to

analyze DNA samples. One such type is a capillary electrophoresis (CE) instrument. By applying

electrophoresis in a fused silica capillary column carrying a buffer solution, the sample size requirement is significantly smaller and the speed of separation and resolution can be increased

multiple times compared to the slab gel-electrophoresis method. These DNA fragments in CE are

often detected by directing light through the capillary wall, at the components separating from the

sample that has been tagged with a fluorescence material, and detecting the fluorescence emissions induced by the incident light. The intensities of the emission are representative of the

concentration, amount and/or size of the components of the sample. In the past, Laser-induced

fluorescence (LLF) detection methods had been developed for CE instruments. Fluorescence

detection is often the detection method of choice in the fields of genomics and proteomics because of its outstanding sensitivity compared to other detection methods.

Heretofore, CE instruments are designed to work with samples first prepared at other

devices, and then loaded onto a sample tray in the CE instruments. Some of the sample

preparation procedures could be quite involved, requiring manual and/or automatic procedures.

Dedicated devices and systems have been designed to handle only sample preparation, involving

steps such as sample extraction, purification, amplification, stabilization, etc., to produce samples

that are suitable for separation by the CE instruments. For example, DNA samples may have to be

prepared by a polymerase chain reaction (PCR) process, to amplify sufficient quantities of

samples from a trace amount of DNA samples. The product of the PCR process may be subject

to a CE process to verify the integrity or state of the result of the PCR process. The transfer from

separately prepared samples to CE separation/analysis instruments require significant manual

interventions, which affect overall throughput. It would be desirable to develop a folly integrated bio-analysis system including built-in sample preparation process capabilities, to avoid user intervention during sample preparation and s ep ar ation analysis .

SUMMARY OF THE INVENTION The present invention provides an integrated bio-analysis system with built-in sample

preparation process capabilities. In one aspect of the present invention, a bio-analysis instrument

is provided with a built-in sample preparation device. In one embodiment, the sample preparation process include a bio-molecular reaction process. In a particular embodiment, the bio-molecule

reaction process is based on PCR. In a further embodiment of the present invention, a peltier unit

in the sample preparation device provides thermal cycling of samples supported in a multi-well

tray. h another aspect of the present invention, a CE instrument is provided with a built-in sample preparation capability, which may comprise a bio-molecular reaction process, for example,

a sample preparation device based on PCR. i another aspect of the present invention, a bio-molecular reaction system is provided

with a built-in analysis device, such as a CE device, for verifying the results of the reaction

products, which may be modified bio-molecular samples, i a particular embodiment, the bio- molecule reaction system prepares samples based on PCR. i a forther embodiment of the present

invention, a thermal cycling unit is provided in the sample preparation device. The samples

prepared by the PCR device may be used for other analysis in other systems.

BRIEF DESCRIPTION OF THE DRAWINGS For a fuller understanding of the nature and advantages of the invention, as well as the

preferred mode of use, reference should be made to the following detailed description read in

conjunction with the accompanying drawings. In the following drawings, like reference numerals

designate like or similar parts throughout the drawings.

Fig. 1 is a schematic representation view of a capillary electrophoresis system that

comprises a sample preparation device in accordance with one embodiment of the present

invention.

Fig. 2 is a perspective view of a capillary electrophoresis system that comprises a sample

preparation device in accordance with one embodiment of the present invention.

Fig. 3 is a block diagram of the control system for the capillary electrophoresis system in

accordance with one embodiment of the present invention.

Fig. 4 is a perspective view of the sample preparation device.

Fig. 5 is a sectional view of the sample preparation device taken along line 5-5 in Fig. 4. Fig. 6 is a schematic block diagram illustrating the relationship of various sample

processing stages.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

This invention is described below in reference to various embodiments with reference to

the figures. While this invention is described in terms of the best mode for achieving this

invention's objectives, it will be appreciated by those skilled in the art that variations may be accomplished in view of these teachings without deviating from the spirit or scope of the

invention.

The present invention provides for a bio-analysis system integrated with sample

preparation. In one aspect of the present invention, a bio-analysis instrument is provided with a

built-in sample preparation device. In one embodiment, the sample preparation process include a

bio-molecular reaction process. In a particular embodiment, the bio-molecule reaction process is

based on PCR. In another aspect of the present invention, a CE instrument is provided with a

built-in sample preparation capability, which may comprise a bio-molecular reaction process, for

example, a sample preparation device based on PCR. h another aspect of the present invention, a

bio-molecular reaction system is provided with a built-in analysis device, such as a CE device, for

verifying the results of the reaction products, which may be modified bio-molecular samples. In a

particular embodiment, the bio-molecule reaction system prepares samples based on PCR. hi a

forther embodiment of the present invention, a thermal cycling unit is provided in the sample

preparation device. The samples prepared by the PCR device may be used for other analysis in

other systems. For purpose of illustrating the principles of the present invention and not by limitation, the

present invention is described by reference to embodiments directed to CE analysis and PCR

sample preparation. In the illustrated embodiment, the invention provides a folly integrated system of PCR sample preparation in an automated multi-channel CE system for genetic analysis. Biocal Technology, hie, the assignee of the present invention, developed a CE-based

automated instrument (HDA-GT12 DNA Analyzer System). The illustrated embodiment of the inventive automated instrument is based on Biocal's CE instrument, which incorporates low-cost

and sensitive optical detection technology, integrated reagents cartridge and micro-fluidic

electrophoresis principle for a real-time fluorescent analysis, to form a sensitive and accurate

bioagent detection (genetic analysis) system. The system is designed to be high-throughput, easy-

to-use, portable, inexpensive, very robust and for field operation / applications. The cartridge is designed to be supported by the instrument, with all essential cartridge

elements aligned and coupled to support elements in the instrument. The cartridge is held with

respect to sample trays that can be moved in relation to the capillary separation channels in the

cartridge. The instrument is improved with the provision of a PCR based station to prepare

samples from raw samples loaded into the system for separation and analysis, all within the same

instrument, thereby minimizing forther user intervention once raw samples (e.g., extracted DNA)

have been loaded into the automated instrument, throughout the processes of sample preparation

(DNA amplification / PCR), separation and analysis. It is noted that in the context of the present invention, the raw sample (e.g., extracted

DNA) may be an intermediate state or form of a sample taken in the field, which may have

undergone prior preliminary preparation processes before being loaded into the sample

preparation station in the automated system. In the context of the present invention, the raw

sample undergoes a significant sample preparation process (e.g., PCR) in the automated

instrument, which transforms the sample into a significantly different and/or modified form and/or

state, beyond simply subjecting the raw sample to dilution, or other processes which merely

presents the raw sample in a format that facilitates handling in subsequent processes. For example, the raw sample is subject to a bio-molecular reaction process in the sample preparation device. Fig. 6 is a schematic block diagram illustrating the relationship of the various stages of

processing discussed here, i reference to the subsequent separation and/or analysis processes, the

resultant sample prepared by the sample preparation device is the input to the subsequent

separation and/or analysis processes, and which is the sample subject to separation and/or

analysis. The raw sample is the sample that is input to the sample preparation device, which is the

sample subject to processing by the sample preparation device. The raw sample may be the

product of prior preliminary processing.

For example, for a biological sample taken in the field, such as a saliva or blood of a

subject, it is subject to preliminary processes, including without limitations extraction and purification, to obtain a DNA sample representative of the subject. Such extracted DNA fragment

is the "raw sample", along with necessary chemicals (e.g., primers, polymerase, etc.), that is

loaded into the integrated system 200 of the present invention (disclosed below), and more

specifically the sample preparation device, such as for PCR amplification. The output of the PCR

process is the sample for subsequent separation and/or analysis.

Overview of CE System

The system is designed and built for field robustness weighing no more than 40 lbs.

The portable system also incorporates a built in modular and integrated PCR thermocycler

with peltier cooler device for DNA amplification.

Fig. 1 is a schematic representation of a capillary electrophoresis (CE) system 200 in

accordance with one embodiment of the present invention. The CE system 200 generally

comprises a capillary separation column 22 (e.g., 200-500μm O.D.), which defines a separation channel 36 (e.g., 25-200 μm ID.). The capillary column 22 maybe made of fused

silica, glass, polyimide, or other plastic/ceramic/glassy materials. The inside walls of the

separation column 22 (i.e., the walls of the separation channel 36) may be coated with a

material that can build up an electrostatic charge to facilitate electrophoresis and/or electrokinetic migration of the sample components. The separation channel 36 is filled with a

separation support medium, which may simply be a running buffer, or a sieving gel matrix

known in the art. For radiation induced fluorescence detection, the gel matrix includes a

known fluorophore, such as Ethidium Bromide. One end of the capillary column 22 is submerged in a reservoir 28 of running buffer/gel

34. The other end of the capillary column 22 is coupled to the sample vial 26. It is understood that

the detection configurations shown in the other embodiments can be equally implemented in a

system similar to the CE system 200. Also, the separation channel 36 may be one straight

capillary or micro-channel with a section of the detection window closest to the gel-reservoir at

the exit end being the detection zone, which is the current preferred mode of our invention. A

radiation detector 24 is positioned outside a transparent section of the capillary walls at detection

zone 30. An excitation fiber 16 extends from a radiation source 18 (e.g., LED or laser) and is

directed at the detection zone 30 outside the walls of the column. Electrodes 12 and 14, that are

part of the cartridge assembly are coupled to the buffer reservoirs 26 and gel reservoir 28 to

complete the electrophoresis path. hi accordance with the present invention, the system 200 includes a sample preparation

device 250. The illustrated embodiment in Fig. 2 shows a sample preparation device suitable for

DNA amplification by PCR. In particular, the sample preparation device 250 comprises a thermal

cycler for PCR (which comprises a heating/cooling element and a controller to effect the required heating and cooling conditions). The extracted biological molecules/DNA samples from the field

(soil, air and water) are loaded onto the micro titer plate 72 along with the necessary chemistry

and/or protocol, and amplified (e.g., in a bio-molecules reaction process of the sample preparation

device) to allow easy detection during capillary electrophoresis. After the PCR process has been

completed, the micro titer plate 72 is moved to under the capillary columns 140 and to move the cartridge 100 vertically to allow the capillary columns 140 to access the wells in the micro titer

plate 72. The amplified DNA samples (PCR products) are then automatically manipulated and

introduced by electro-kinetic injection through the micro-channels of the gel-cartridge for separation and fluorescence detection of molecules.

Overview of CE Separation and Analysis hi operation, a raw sample taken from the field is prepared (e.g., DNA is extracted,

purified and then amplification by PCR) into a sample suitable for CE. For example, a prepared biological sample (e.g., a DNA sample) in the sample vial 26 prepared by the sample

preparation device 250 (i.e., a DNA amplification device in the illustrated embodiment) is

introduced into the far end of the capillary column 22 away from the detection zone 30 by any

of a number of ways (e.g., electrokinetic injection from the sample reservoir). The sample

binds to the fluorophore in the gel matrix supported in the capillary column 22.

When a DC potential (e.g., 1-30 KV) is applied between electrodes 12 and 14, the sample migrates under the applied electric potential along the separation channel 36 (e.g. DNA that is

negatively charged travels through the sieving gel with an integrated dye matrix/fluorophore

toward a positive electrode as shown in Fig. 1) and separates into bands of sample components

(DNA fragments). The extent of separation and distance moved along the separation channel 36 depends on a number of factors, such as migration mobility of the sample components, the mass

and size or length of the sample components, and the separation support medium. The driving

forces in the separation channel 36 for the separation of samples could be electrophoretic, pressure, or electro-osmotic flow (EOF) means. When the sample reaches the detection zone, excitation radiation is directed via the

excitation fiber 16 at the detection zone. The sample components fluoresce with intensities

proportional to the concentrations of the respective sample components (proportional to the

amount of fluorescent tag material). The detector 24 detects the intensities of the emitted

fluorescence at a wavelength different from that of the incident radiation. The detected emitted radiation may be analyzed by known methods. For an automated system, a controller 32 controls

the operations of the CE system 200.

Overview of PCR

The purpose of a PCR is to make a large number of copies of a gene. This is necessary

in order to have enough starting template for DNA fragment analysis by CE. Below is a brief

explanation of the PCR thermal cycling reactions.

1. The Cycling Reaction:

There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool sample vials with the reaction mixture in a very short time. As will be described later below, a peltier cooler/heater type mechanism maybe integrated as part of the transport carriage frame of the X-Y transport mechanism of the multi-channel capillary electrophoresis instrument for a complete solution of DNA amplification and analysis. DNA amplification by PCR is well known in the art. Details of the protocol and chemistry (e.g., primers, polymerase, etc.) are omitted from the discussion herein. Further references may be made to any of the well documented literatures in the field. In accordance with one embodiment of PCR, the three major steps in a PCR include: 1. Denaturation at 94°C:

During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).

2. Annealing at 54°C: The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.

3. Extension at 72°C:

This is the ideal working temperature for the polymerase. The primers, where there are a few

bases built in, already have a stronger ionic attraction to the template than the forces breaking

these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment.

The bases (complementary to the template) are coupled to the primer on the 3' side (the

polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template).It is understood that the foregoing steps may be modified but

nonetheless provides a PCR or DNA amplification, without departing fro the scope and

spirit of the present invention.

Multiple Capillary Cartridge Based CE System

In accordance with the present invention, for nucleic acid detection, PCR amplification is

integrated into a micro-fluidic electrophoresis system. Raw samples of DNA or RNA extracted

from the field are purified, then loaded (using standard PCR plate preparation steps) into sample vials (e.g., a 96-well micro titer plate) in the automatic micro-fluidic electrophoresis system for

PCR amplification. Primer pairs for specific gene markers will be used for PCR analysis. Further

in accordance with the present invention, the finished PCR products in the sample vials are

automatically introduced to the multi-channel gel cartridge via electro-kinetic sample injection for

high-resolution separation and fluorescence detection.

Fig. 2 shows an overall perspective view of the CE system 200 (e.g., an DNA

Analyzer). The CE system 200 incorporates an interface mechanism 300, in accordance with

one embodiment of the present invention. The interface mechanism 300 supports a multi¬

channel cartridge 100 in accordance with the one embodiment of the present invention, which

provides easy handling of multi-channel separation columns, and allows easy optical coupling

of the detection zones to the detection optics of the CE system 200. Details of the interface

mechanism 300 may be referenced to U.S. Patent Application No. 10/823,382, which has been

folly incorporated by reference herein. The fully automated DNA analysis system 200 has a base 74, supporting a modular X-Z mechanism 80 having a sample tray support frame 81. The

X-Z mechanism 80 moves the PCR sample preparation device 250 (details disclosed later below in reference to Figs. 4 and 5) that supports a 96-well micro-titer plate 72, and a buffer

plate 70 in relation to the multi-capillary cartridge 100 supported by the interface mechanism 300. Specifically, the mechanism 80 comprises an X mechanism 82 for moving the support

frame 81 along the X-direction relative to the cartridge 100, and a Z mechanism 83 for moving

the cartridge in the Z direction relative to the support frame 81. The PCR sample preparation

device 250 is controlled by a PCR thermoelectric controller 68.

PCR Sample Preparation Device

Referring to Figs. 4 and 5, the PCR sample preparation device includes a heating unit

such as a peltier thermoelectric unit 251, which supports on it top an thermal interface structure 252 having complementary wells 255 that are sized to receive the bottom of the wells

73. The peltier thermoelectric unit 251 can be controlled by the controller 68 to heat or cool

the wells 73, to subject the contents in the wells 73 to thermal cycling as required by PCR

process. At the bottom of the peltier thermo electric unit 251 , a heat sink is provided to

remove heat from the peltier thermoelectric unit 251 during the cooling cycle of the unit

and/or to remove excess heat during the heating cycle. The PCR sample preparation device

250 is supported on the transport frame 81 of the transport mechanism 80.

Control of the Automated System 200

The system 200 provides easy handling of multi-channel separation columns, and

allows easy optical coupling of the detection zones to the detection optics of the CE system 200. The operations of the CE system 200, including the interface mechanism 300, are

controlled by a controller 32 interfacing with an external user control interface (e.g., athe PC

918). The PCR thermoelectric controller 68 may also be operatively couple to the controller

32 and/or the PC 918, so that the controls of the PCR sample preparation device and the rest of the system 200 are coordinated to achieve the functions described herein are achieved.

Referring also to Fig. 3, in accordance with one embodiment of the present invention,

the block diagram of the controller 32 for the CE system 200 is illustrated. The controller 32

comprises a processor as part of the A/D Board (LED Processor PCBA) 912 with CPU 910 for converting detection signals received from the detector 24 (e.g., a PMT) to corresponding

digital signals, coming from LEDScan PCBA interface 914 for transferring and receiving

signals to and from respective parts of the CE system 200 by instructions from the CPU 910.

The A/D (LED Processor PCBA) interface 912 is coupled to the various actuators in the

interface mechanism 300 to control and connect (using the interface mechanism 300) at least

high voltage power supply 76, pneumatics 78 (hidden from view in the interface mechanism

300 in Fig. 2), motor controls (X-Z sample/buffer tray) 80 and interlocks (cartridge and

transport doors) 61 and 62 (details of these are not shown in the interface mechanism 300 in

Fig. 2). The A/D or LED Processor PCBA 912 also controls the high- voltage power supply 76

for sample injection and electrophoresis functions of the CE system 200, a circuit 914 (LEDScan Board) for modulating the excitation radiation source (e.g., LEDs) 921 and the

detector module 24 of the CE system 200. Details of the modulation of the excitation

radiation source may be referenced to copending U.S. Patent Application No. 10/060,052,

which had been folly incorporated by reference herein. The A/D (LED Processor PCBA) 912 may be further coupled to an external personal

computer 918, which in turn performs data processing or additional control function for the CE system 200, e.g., using BioCal's BioCalculator Software to control various features and

functions of the automated multi-channel CE system 200 (including the integrated PCR sample preparation device).

The PCR sample preparation device 250 is controlled by the controller 68 to perform the sequence of thermal cycle reaction discussed above. The thermoelectric unit 251, which is

supported by the X-Z transport mechanism 80 is designed for 96-well micro titer plate 72. The

thermoelectric unit 251 is controlled by the thermal cycling controller module 68, which

directly is controlled by the A/D or microprocessor board 912 and the control firmware is

controlled by the user interface (BioCalculator software) at the PC 918 via an RS232 cable as

shown in Fig. 3. The thermoelectric controller 68 may be incorporated in controller 32, in an

alternate embodiment.

The components of the controller 32, with the exception of the PC 918, maybe

packaged as an electronic board 64 (Fig. 2) and cooling fans 63, on board the CE system 200

and electrically coupled to the PC 918 via a serial port (not shown), or they may be part of a

separate controller module outside of the CE system 200. The CPU 210 and/or the PC 218 are

programmed to accomplish the various control functions and features for the CE system 200. i one embodiment, the PC 218 can be configured to provide the user control interface for the

CE system 200 (e.g., user initiation of the connection sequence of the interface mechanism 300). It would be within a person skilled in the art to implement the program code given the

functions and features disclosed herein. In an alternate embodiment, the controller 32 or

components thereof may be incorporated as part of the PC 918. Capillary Cartrid e

The multi-channel capillary cartridge 200 includes twelve detection zones (schematically

represented as 30 in Fig. 1), defined by capillaries 140 held in a cartridge body. The cartridge 100 includes a twelve-channel fosed silica capillary array that is used for separation and detection of

the samples as part of a disposable and/or portable, interchangeable cartridge assembly 100. The

cartridge 100 shown in Fig. 2 holds up to 12 capillaries 140, 12-18 cm long. The cartridge 100 is

integrated with a top, outlet buffer reservoir 130 common to all capillaries 140, which is directly

coupled by the interface mechanism 300 to a modular compressed gas source 78, such as a replaceable pressurized gas cartridge of an inert, compatible or non-reactive gas (e.g., Nitrogen,

CO₂, etc.) or a pressure pump. Appropriate pressure plumbing, including tubing, pressure valve

and solenoid controls, is provided. (Details of such plumbing are omitted, since it is well within

one skilled in the art to configure such plumbing given the disclosure herein of the functions,

features and operations of the system 200.) The pressure source 78 provides the required gas

pressure to fill-up all the 12-capillaries with the sieving gel contained in the reservoir 130 and to

purge the gel from the previous run from the capillaries during the refilling process. Depending

on the viscosity of the gel, pressures of up to 40 PSI may be applied to the capillaries 140 through

the gel-filled reservoir 130. The cartridge gel-reservoir 130 is equipped with a built in common

electrode anode 134 for all 12-capillaries, which is automatically connected by the interface mechanism 300 to the high voltage power supply 76 (Fig. 2) for electrophoresis when installed

inside the system 200. A fan or Peltier cooler (not shown) on the adjacent structure to the

cartridge 100 may be provided to provide temperature control of the cartridge, hi addition or in

the alternate, the cartridge may have vent holes (input and output) for air circulation (temperature controlled air to be introduced to the cartridge from the instrument side). Depending on the heat generated during CE separation, the cartridge may simply be exposed to ambient temperature,

with no auxiliary cooling features. A power supply 66 (Fig. 2) provides DC power to the CE

system 200. Further details of the cartridge may be referenced to the copending application no.

10/059,993, which had been folly incorporated by reference herein.

Detection System

U.S. Patent Application No. 10/060,052, which had been folly incorporated by reference

herein, is more specifically directed to the time staggered/multiplexed detection scheme that can

be adopted in the CE system 200.

Interface Mechanism

The structure and operation of the interface mechanism of the CE system 200 may be

referenced to the copending U.S. patent application No. 10/823,382, which had been folly incorporated by reference herein. The cartridge interface accomplishes quick and reliable

interface connections to the disposable gel contained capillary cartridge 100. These interface

connections include a gas pressurization connection, high voltage connections, and precision

optical connections. The interface also provides precise and repeatable mechanical positioning of

the cartridge, to accurately position the components of the cartridge in relation to the support

elements in the CE system 200, including positioning the capillary tips in relation to external

sample or buffer reservoirs, found on 96-well titer plate, for example. Additionally, the interface

provides separate electrical, optical and pneumatic comiections to each separation channel, thus providing channel-to-channel isolation from cross talk both electrically and optically and

insulation to the rest of the instrument from high voltage.

Operation of CE System In operation, the sample handling tray transport mechanism 80, with a 96-well plate (8x12)

72 and 70, is used to introduce the amplified DNA samples (or analytes) to each capillary 140.

The X-Z transport mechanism 80 indexes a row of sample carrying wells 73 in the micro titer

plate 72 under the row of capillary tips 140 and dip the tips into the well. By applying a voltage,

electrokinetic injection moves a known amount of the DNA sample to the beginning of the

separation column 140. After injection, the DNA samples from sample tray 72 may be replaced

with a running buffer from tray 70. Alternatively, after injection, the transport mechanism 80 may

index to move a row of 12 wells 73 in the titer plate 72 containing buffer solution into position under the cartridge to replace the twelve wells containing DNA samples.

By applying high voltage across the total length of the capillary 140, separation of the DNA

sample into DNA fragments is achieved. As the fragments approach the end of the capillaries 140

and enter into the detection zone, the excitation light energy (e.g., from twelve LEDs delivered by

optical fibers) is directed at the detection zone, illuminating the migrating DNA fragments. The

detection scheme may be in a time-staggered manner as disclosed in copending U.S. Application Serial No. 10/060,052. To prepare for the next run with a different sample, the old gel from the previous run is

purged from the capillaries by pressuring the reservoir to refill the capillaries with fresh gel. The

trays 70 and/or 72 carry cleaning solutions, waste collection, and samples. The purged gel is

collected by one of the trays 70 and 72 by positioning the tips of the capillaries at a row of waste collecting wells in one of the trays. The tips of the capillaries may be cleaned with water or a cleaning solution by positioning and dipping the tips of the capillaries in such solution in the

appropriate tray wells. When the capillaries are refilled and ready for the next run, the tips of the

capillary are dipped into the samples by repositioning the tray 72. The above mentioned sequence

of process maybe programmed as one of the automated functions of the controller 32. The

interface mechanism 300 provides the interfacing of support elements in the CE system 200 to the cartridge, such as high voltage, gas pressure, LED radiation source, and detection optics, as

described above. Compared to the conventional methods, which utilizes separate PCR machines

and electrophoresis (Slab gel or Capillary Electrophoresis) instruments, the new portable

automated system is designed to incorporate the combined functions of sample preparation and

analysis (e.g., PCR and electrophoresis) as one instrument. The inventive portable micro-fluidic

electrophoresis based instrument that comprises an integrated PCR device with a multi-channel

and multi-color fluorescence detection system is a reliable tool for a faster and lower in cost

genetic analysis type applications. The inventive system as shown in the Fig. 2 could be a bench top system designed for

high throughput biological agent (DNA) detection analysis. The 12-channel

separation/detection cartridge can analyze multiple samples simultaneously. The 12-channel

separation/detection cartridge combined with the integrated PCR plate can complete a 96-well

sample plate amplification and electrophoresis in much reduced time with predictable results.

The system is simple, reliable and fast. The entire process from PCR to DNA separation can

be completed in 2 hrs. The analysis time for 12 PCR samples is targeted to be within several

minutes. The cost of the proposed instrument would be a factor of 10 lower than the current

multiple-channel high-end capillary electrophoresis instruments, i average, the chemistry cost per analysis is estimated to be less than significantly less that the costs for prior art CE systems.

The inventive automated instrument includes the sample handling tray mechanism with an

integrated PCR device for the direct nucleic acid molecule amplification, electrophoresis /

separation, fluorescence detection, with improved detection for any conjugated dye for

immunoassays. The folly automated and portable system is controlled by a general computer.

Data analysis for the detected results is automated to give on-the-spot amplification (PCR), peak

identification and semi-quantitative analysis. With this new, inexpensive and high-throughput

analyzer, the DNA laboratories will have the capability to perform high throughput and rapid bioagent identification with lower in cost, less labor intensive and high accuracy all in one unit.

The detection capability of the system can be designed for dedicated detection of certain

potential biological agents and pathogens.

In one embodiment of the present invention, the raw samples are introduced to the 96-

well titer plate in the thermal cycler, to be subjected to different temperatures (hot and cold

thermal cycling process) before, during or after the injection of the samples to the gel-

cartridge. The thermal cycler has several purposes or functions during the sample preparation

prior to separations and analysis of bio-molecules. One function is for straight PCR

amplification of bio-molecules and the other function would be for different temperature

cycling of the samples/bio-molecules (sample preparation step) to change the state/condition

of the sample prior to electrophoresis with the gel-capillary cartridge. The 12-Channel

cartridge could also function as a micro-dispenser. For example one could apply electrokinetic

at the first position of the sample tray (position A on 96-well plate) to inject the negatively

charged DNA into the capillaries or micro-channels of the cartridge and then the system can reverse the HV Supply polarity to be able to go to position B of 96-well plate to mix the DNA samples (or bio-molecules) from well A with well B (bio-molecule reactions). The cartridge

can function as a sample dispenser (liquid dispenser). The heat block could be used to heat or

cool any bio-molecules and then electrophoresed and analyzed by the CE cartridge. The bio-

molecules (i.e. DNA, RNA, Proteins, Antibody and Antigen) could all be analyzed with the

present intergrated sample preparation and electrophoresis mechanism in a very flexible manner. For example one could heat the samples up to 90 degree C and then drop it to 80 or

70 degree C and quickly analyze it with the CE cartridge/device in between, before or after the

temperature cycling to verify the binding effects of probes with DNA. Or similar to microarray

concepts of hybridization, one can manipulate the sample temperatures to wash away the point

mutations and then do a quick electrophoresis to quantify it or do a simple quality check of the

sample. This approach/process will allow one to check the position of the detected peaks

quickly for qualitative work, which is useful in point mutation type detection. The detection

sensitivity can be increased by improving the optical detection system of the current fluorescence detection mechanism (i.e. from LED excitation to Laser excitation) to be able to

achieve higher detection sensitivity during electrophoresis by reducing to a less number of

thermal cycling (PCR) steps with the heat block. With this thermoelectric module one could

also store the PCR-ed samples at a lower set temperature for storage or preservation purposes.

With the inventive process, the PCR steps could be simplified to detect the right or wrong

detected fragments (mutation analysis) or single strand analysis or analysis of proteins due to

temperature change. This system of thermal cycling block/process for sample preparation

could also be used as a PCR step/process of injected samples inside the capillary tubes. One

could simply inject the samples electrokineticaly from one sample well, then go to another well, which has oil in it and dip the capillary tips inside the oil and start the temperature

cycling of the injected samples inside the micro-channels (inside capillary PCR) and then do

electrophoresis. This system of multi-channel capillary electrophoresis combined with thermal cycling features (sample plate heat block) of the sample plate for sample preparation

will provide flexibility similar to microarrays or real-time-PCR type devices for complete and

an one stop solution of bio-molecules analysis.

While the invention has been particularly shown and described with reference to the

preferred embodiments, it will be understood by those skilled in the art that various changes in

form and detail may be made without departing from the spirit, scope, and teaching of the

invention. The sample preparation station may be configured to undertake processes other than PCR

to produce samples in a form suitable for CE analysis. The automated system 200 may be

configured to conduct other types of analysis different or in addition to CE separation and

analysis. For example, for protein or bioagent detection, immunoassays combined with the micro-

fluidic electrophoresis system could also be used. Protein extract from cultures is used for

immunoassays. The amplification signals via interaction of antigen and antibody conjugated

fluorescence dye is automatically applied to a multi-channel cartridge for high-resolution detection

within several minutes.

Interface mechanisms maybe adapted to receive capillary cartridges of other structural

designs. A person skilled in the art will recognize that the instrument incorporating the essence of this invention can also be used for bio-molecular analysis other than DNA analysis. For example,

by altering the separation gel or buffer, the instrument can also be modified to analyze

biomolecules like proteins, carbohydrates, and lipids.

By way of example and not limitation, the detection scheme of the present invention is described in connection with capillary electrophoresis and radiation induced fluorescence

detection. It is understood that the present invention is also applicable to detection of analytes

separated based on bio-separation phenomenon other than electrophoresis, and detection of

radiation emissions other than fluorescence emissions, including other types of emissive radiation,

such as phosphorescence, luminescence and chemiluminescence, as well as absorbance based detection.

Furthermore, while the separation channels in the described embodiments are defined by

cylindrical columns or tubes, it is understood that the concepts of the present invention is equally

applicable to separation channels defined by channels, for example micro-channels (such as

square, rectangular or essentially semicircular cross sections) defined by etching in a substrate (micro-fluidics type devices or bio-chips).

The transport mechanism can be configured to move the trays in a horizontal plane, and an

additional transport mechanism may be provided to move the trays vertically to access the trays.

The sample output from the sample preparation device may be input to an analysis devices

without necessarily being subject to separation. Accordingly, the disclosed invention is to be considered merely as illustrative and limited

in scope only as specified in the appended claims.

Claims

CLAIMSWe Claim:

1. A bioanalysis system, comprising: a sample preparation device, receiving a raw sample to be processed into a sample in a form

suitable for subsequent analysis; and a sample analysis device, receiving the sample from the sample preparation device to be

subject to analysis; wherein the sample preparation device and the sample analysis device are operative coupled

within an integrated automated system, whereby sample output from the sample analysis device is

input to the sample preparation device without forther user intervention.

2. The bioanalysis system as in claim 1, wherein the sample analysis device comprises separation

capability that separates the sample into components.

3. The bioanalysis system as in claim 2, wherein the sample analysis device forther comprises

detection capability providing analysis of the separated components.

4. The bioanalysis system as in claim 3, wherein the sample analysis device comprises capillary

electrophoresis that provides separation and detection capabilities.

5. The bioanalysis system as in claim 1, wherein the sample preparation device is structured and configured to subject the raw sample to a bio-molecular reaction.

6. The bioanalysis system as in claim 5, wherein the sample preparation device is structured and

configured to provide thermal cycling processing of the raw sample to output a sample that is

amplified from the raw sample.

7. The bioanalysis system as in claim 1, wherein the sample preparation device comprises a

thermoelectric unit that is controller to cycle the temperature of the raw sample to cause a bio-

molecular reaction.

8. The bioanalysis system as in claim 7, wherein the sample preparation device is structured and configured to effect DNA amplification by PCR in the bio-molecular reaction.

9. The bioanalysis system as in claim 8, wherein the analysis device comprises a capillary

electrophoresis system.

10. The bioanalysis system as in claim 9, wherein the capillary electrophoresis system comprises

mufti-channel separation and analysis.

11. The bioanalysis system as in claim 10, wherein the raw sample comprises DNA extracted from

a field sample.

12. The bioanalysis system as in claim 9, wherein the raw sample is loaded into a sample well in

the sample preparation device, and further comprising an integrated transport mechanism to

position the sample well to be accessible by the capillary electrophoresis system.

13. The bioanalysis system as in claim 1, wherein the raw sample is loaded into the sample preparation device, and forther comprising an integrated transport mechanism to position the

sample preparation device to be accessible by the sample analysis system.

14. The bioanalysis system as in claim 1, wherein the sample preparation device comprises a device for effecting theπnal cycling.

15. A PCR system, comprising: a thermal cycler performing PCR, receiving a raw DNA sample to be amplified into a DNA

sample by PCR, in a form suitable for subsequent analysis; and a capillary electrophoresis system, receiving the DNA sample from the thermal cycler to be

subject to capillary electrophorsis to analyze result of the PCR; wherein the capillary electrophoresis system and the thermal cycler are operative coupled

within an automated integrated system, whereby DNA sample output from the thermal cycler is

input to the capillary electrophoresis system without further user intervention.

16. A process for analyzing a raw sample, comprising the steps of: providing a sample preparation device, which receives a raw sample to be processed into a

sample in a form suitable for subsequent analysis; and providing a sample analysis device, which receives the sample from the sample preparation device to be subject to analysis; wherein the sample preparation device and the sample analysis device are operative coupled

input to the sample preparation device without further user intervention.