# Patent

## Title
Capillary electrophoresis device

## Abstract

This invention provides a capillary electrophoresis device in which capillaries are thermally regulated on a thermally responsive electrical path attached to an electrically insulating circuit board. This invention also provides an optical scanner useful for scanning an array of capillaries. A laser, optical detector and optical selector are in an arrangement that allows the optical detector to selectively detect an optical signal from any one or more of the plurality of electrophoresis capillaries.

## Background
CROSS-REFERENCE
This application claims the benefit of the filing date of corresponding provisional patent application 61/349,680, filed May 28, 2010, the contents of which are incorporated by reference in their entirety.


STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with government support under Contract No. 2004*H838109*000 awarded by the Central Intelligence Agency. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Capillary electrophoresis is used in biochemical analysis to detect analytes in a mixture. One popular application is analysis of nucleic acids that are the product of sequencing or amplification reactions. Modern capillary electrophoresis devices use arrays of capillaries to perform multiplex analysis. Such devices regulate temperature in the capillaries in a variety of ways. One method circulates air at a particular temperature around the capillaries, such as in U.S. Pat. No. 7,601,252. In another method the capillaries are in thermal contact with a heating plate, such as in U.S. Pat. Nos. 7,223,326 and 7,473,342.
Analytes in capillary arrays are detected using a variety of optical assemblies.
SUMMARY OF THE INVENTION
Thermal Apparatus
One aspect of the technology is an apparatus with an electrically insulating circuit board, at least one electrical path attached to the circuit board, and at least one electrophoresis capillary in thermal contact with at least one thermal area. The electrical path forms a thermal area. The electrical path is thermally regulated responsive to electrical current through the electrical path. The at least one electrophoresis capillary is thermally regulated responsive to electrical current through the electrical path.
One embodiment includes at least one temperature sensor in thermal contact with the electrophoresis capillary, and a controller of a temperature of the electrophoresis capillary. The temperature sensor provides temperature data of the electrophoresis capillary. The controller changes the electrical current through the electrical path responsive to the temperature data from the temperature sensor.
In one embodiment the electrical path has at least one resistance providing temperature data of the electrophoresis capillary in thermal contact with the electrical path. The apparatus further includes a controller of a temperature of the electrophoresis capillary, which changes the electrical current through the electrical path responsive to the temperature data from the resistance of the electrical path.
One embodiment further includes at least one thermal insulation member attached to the circuit board and positioned by the electrical path and the electrophoresis capillary. The thermal insulation member reduces heat transfer between a part of the circuit board attached to the electrical path and the electrophoresis capillary, and a remainder of the circuit board. An example of such a thermal insulation member is an aperture in the circuit board.
One embodiment has multiple electrical paths in thermal contact with different sections of the electrophoresis capillary. The different sections of the electrophoresis capillary are separately thermally regulated by different electrical paths. One embodiment further includes multiple temperature sensors in thermal contact with the different sections of the electrophoresis capillary, and a controller of temperatures of the different sections of the electrophoresis capillary. The temperature sensors provide temperature data of the different sections of the electrophoresis capillary. The controller changes the electrical currents through the multiple electrical paths responsive to the temperature data from the temperature sensors. In another embodiment the multiple electrical paths have resistances providing temperature data of the different sections of the electrophoresis capillary in thermal contact with the multiple electrical paths, and the apparatus further includes a controller of temperatures of the different sections of the electrophoresis capillary, which changes the electrical currents through the multiple electrical paths responsive to the temperature data from the resistances of the multiple electrical paths. In one embodiment the electrophoresis capillary is covered by a thermally insulating material.
In one embodiment the electrophoresis capillary is attached to the circuit board. In one embodiment the electrophoresis capillary is attached to the circuit board with adhesive material.
In one embodiment the electrical path runs back and forth in a thermal area of the electrically insulating circuit board. On one embodiment an electrical path is configured as two electrical nodes connected by a plurality of electrical paths. In one embodiment the thermal area has a width no less than 5 mm. In one embodiment the thermal area widens by a part of the electrophoresis capillary entering the electrically insulating circuit board.
In one embodiment the electrically insulating circuit board has an aperture through the electrically insulating circuit board. The aperture facilitates optical interaction with the electrophoresis capillary.
In one embodiment the electrical path has at least one bend. In one embodiment the electrical path overall has an S-shape.
System Apparatus
Another aspect of the technology is an apparatus, including an electrophoresis thermal assembly, at least one analyte injector, a voltage source, a laser device, and an optical detector. The electrophoresis thermal assembly includes an electrically insulating circuit board, at least one electrical path attached to the circuit board which is thermally regulated responsive to electrical current through the electrical path, and at least one electrophoresis capillary in thermal contact with the electrical path such that the electrophoresis capillary is thermally regulated responsive to electrical current through the electrical path. The analyte injector is coupled to inject at least one electrophoresis analyte into the electrophoresis capillary. The voltage source is coupled to opposite ends of the electrophoresis capillary, providing an electrophoretic voltage difference between the opposite ends of the electrophoresis capillary. The laser device is positioned to deliver a beam from the laser device to the electrophoresis capillary. The optical detector is optically coupled to receive an optical signal from the electrophoresis capillary.
Thermal Method
One aspect of the technology is a method, comprising steps of: electrophoretically moving analytes through at least one electrophoresis capillary; and thermally heating the electrophoresis capillary via thermal contact with at least one electrical path carrying electrical current through an electrically insulating circuit board.
One embodiment further comprises: generating temperature data of the electrophoresis capillary in thermal contact with the electrical path; and changing the electrical current through the electrical path, responsive to the temperature data of the electrical path.
One embodiment further comprises: generating, via at least one temperature sensor of the electrophoresis capillary, temperature data of the electrophoresis capillary in thermal contact with the electrical path; and changing the electrical current through the electrical path responsive to the temperature data from the temperature sensor.
One embodiment further comprises: generating, via at least one resistance of the electrophoresis capillary, temperature data of the electrophoresis capillary in thermal contact with the electrical path; and changing the electrical current through the electrical path responsive to the temperature data from the resistance.
One embodiment further comprises: reducing heat transfer between a part of the circuit board attached to the electrical path and the electrophoresis capillary, and a remainder of the circuit board.
One embodiment further comprises: reducing heat transfer with at least one aperture between a part of the circuit board attached to the electrical path and the electrophoresis capillary, and a remainder of the circuit board.
In one embodiment, thermally heating includes: separately thermally heating different sections of the electrophoresis capillary via thermal contact with multiple electrical paths carrying electrical currents through the electrically insulating circuit board.
One embodiment further comprises: generating temperature data of the different sections of the electrophoresis capillary; and changing the electrical currents through the multiple electrical paths, responsive to the temperature data from the different sections of the electrophoresis capillary.
One embodiment further comprises: generating temperature data of the different sections of the electrophoresis capillary, via different temperature sensors of the different sections of the electrophoresis capillary; and changing the electrical currents through the multiple electrical paths, responsive to the temperature data from the different sections of the electrophoresis capillary.
One embodiment further comprises: generating temperature data of the different sections of the electrophoresis capillary, via resistances of the multiple electrical paths; and changing the electrical currents through the multiple electrical paths, responsive to the temperature data from the different sections of the electrophoresis capillary.
One embodiment further comprises: injecting at least one analyte into said at least one electrophoresis capillary.
One embodiment further comprises: optically exciting at least one analyte in the electrophoresis capillary; and detecting an optical signal from the excited analyte.
Optical Apparatus
Another aspect of the technology is an apparatus with multiple electrophoresis capillaries, a laser device, an optical detector, and an optical selector. The laser device is positioned to deliver a beam from the laser device to at least one electrophoresis capillary. The optical detector is optically coupled to receive an optical signal from at least one electrophoresis capillary. The laser device, optical detector, and optical selector are in an arrangement that allows the optical detector to selectively detect an optical signal from any one or more of the multiple electrophoresis capillaries.
In one embodiment, the capillaries are arranged as an array. In one embodiment, the optical selector is optically positioned between the laser device and the multiple electrophoresis capillaries. The beam from the laser device is delivered to a single electrophoresis capillary and not delivered to other electrophoresis capillaries. In one embodiment, the optical selector is a scanning objective directing the beam from the laser device to the single electrophoresis capillary and not to other electrophoresis capillaries. In one embodiment, the scanning objective is adapted to make a traversing motion relative to the beam from the laser device entering the scanning objective. In another embodiment, the optical selector is an aperture passing the beam from the laser device to the single electrophoresis capillary and not to other electrophoresis capillaries. One embodiment further includes a capillary alignment detector optically coupled to receive a reflection of the beam from the single electrophoresis capillary. The reflection indicates an alignment of the beam with the single electrophoresis capillary.
In one embodiment, the optical selector is optically positioned between the multiple electrophoresis capillaries and the optical detector. The optical signal from the multiple electrophoresis capillaries to the optical detector is limited to a single electrophoresis capillary.
Various embodiments further include a wavelength dependent beam combiner optically coupled between the laser device and the optical detector, or a spatial beam combiner optically coupled between the laser device and the optical detector.
Optical Method
Another aspect of the technology is a method, comprising the steps of: electrophoretically moving analytes through multiple electrophoresis capillaries; optically exciting at least one analyte in a first electrophoresis capillary of the multiple electrophoresis capillaries; receiving, at an optical detector, an optical signal from the optically excited analyte of the first electrophoresis capillary of the multiple electrophoresis capillaries; optically exciting at least one analyte in a second electrophoresis capillary of the multiple electrophoresis capillaries; and receiving, at the optical detector, an optical signal from the optically excited at least one analyte of the second electrophoresis capillary of the multiple electrophoresis capillaries.
In one embodiment, optically exciting includes: optically exciting at least one analyte in a single electrophoresis capillary.
In one embodiment, optically exciting includes: traversing a laser beam across the multiple electrophoresis capillaries such that the single electrophoresis capillary, with optically excited analyte, changes with time.
In one embodiment, the method further includes: detecting an alignment of the laser beam with a single electrophoresis capillary, based on a reflection of the laser beam from the single electrophoresis capillary.
In one embodiment, traversing further includes: traversing an optical objective to traverse the laser beam, while keeping the beam and the optical signal within a fixed beam combiner.
In one embodiment, traversing further includes: traversing an optical objective and beam combiner to traverse the laser beam.
In one embodiment, traversing further comprising: receiving the laser beam from a laser device at a first side of an optical objective; passing the laser beam out of a second side of the optical objective towards the multiple electrophoresis capillaries; and traversing the optical objective relative to the laser beam at the first side of the optical objective, causing the laser beam at the second side of the objective to traverse across the multiple electrophoresis capillaries.
In one embodiment, optically exciting includes: optically exciting at least one analyte in multiple electrophoresis capillaries, and the method further includes: passing the optical signal from the single electrophoresis capillary to the optical detector, and blocking the optical signal from other electrophoresis capillaries.
In one embodiment, optically exciting includes: traversing an optical selector across the optical signal from the multiple electrophoresis capillaries, such that the single electrophoresis capillary which originates the passed optical signal, changes with time.
Biochemical Thermal Method
Another aspect of the technology is a method, comprising the steps: separately regulating temperature in different sections of at least one capillary via thermal contact with different electrical paths carrying electrical currents through an electrically insulating circuit board; and moving analytes through the capillary supporting a biochemical activity in the separately thermally regulated different sections of the electrophoresis capillary.
An example of such biochemical activity is a polymerase chain reaction. The different sections of the electrophoresis capillary have different temperatures for different temperature cycles of the polymerase chain reaction.
INCORPORATION BY REFERENCE
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIG. 1 is a top view of a circuit board part of a thermal assembly.
FIG. 1A is a close-up view of an electrical path of the thermal assembly of FIG. 1.
FIG. 2 is a top view of a thermal assembly, with a circuit board, electrical paths on the circuit board, a bundle of capillaries, and temperature sensors.
FIGS. 3 and 4 are simplified diagrams of an optical subsystem of the electrophoresis system.
FIGS. 5, 6, 7, and 8 are various perspective views of an electrophoresis system.
FIG. 9 is a block diagram of an electrophoresis system.
FIG. 10 is a block diagram of a temperature regulation system.
FIG. 11 shows an alternative optical subsystem of this invention comprising a cylindrical lens configured to narrow a beam of light to fall substantially along a narrow detector.
FIG. 12 shows a side perspective of the optical train of FIG. 11, also comprising a lens between two mirrors to focus light on the capillary detector.
FIG. 13 shows an electrical path comprising two electrical nodes and a plurality of parallel electrical lines joining the nodes. Together, they form a thermal area.



DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 is a top view of a circuit board part of a thermal assembly.
The electrically insulating circuit board has a generally S-shaped path for placement of capillaries. The generally S-shaped path is broken up into 6 different sections, 12, 14, 16, 18, 20, and 22. These 6 different sections, 12, 14, 16, 18, 20, and 22, separately regulate the temperature in the portion of a capillary in thermal contact with the particular section. Each of the different sections, 12, 14, 16, 18, 20, and 22 is filled with an electrical path that runs back and forth, e.g. in a serpentine shape in that section's area to fill that section's area. This electrical path that runs back and forth is shown in detail in section 22. Although not shown for purposes of clarity in the illustration, the other sections 12, 14, 16, 18, and 20 also are filled with an electrical path that runs back and forth in that section's area to fill that section's area.
In another embodiment, the thermal area can be formed from electrical paths configured in parallel traces joined together, for example, at common traces that attached to a voltage source or a source of current. A version of this configuration is depicted in FIG. 13.
The circuit board also has a row of apertures 10 that run along both sides of the generally S-shaped path for placement of capillaries. The apertures reduce heat transfer between the generally S-shaped path of the circuit board, and a remainder of the circuit board. Because air is a good thermal insulator, heat transfer is reduced between the two parts of the circuit board. The circuit board itself is also a poor thermal conductor. In another embodiment, instead of rows of apertures, poor thermal conductive material is positioned between these two parts of the circuit board. Such reduction of heat transfer eases thermal regulation of the generally S-shaped path and the capillaries placed on the generally S-shaped path. The apertures serve to reduce the thermal mass of the thermally regulated region to substantially the generally S-shaped path and the capillaries placed on the generally S-shaped path. With less thermal mass, a desired temperature is reached more quickly for the generally S-shaped path and the capillaries placed on the generally S-shaped path. This embodiment requires less energy. Also, the S-shaped configuration occupies less space and renders the device more easily portable.
The circuit board also includes an aperture 8 along the generally S-shaped path toward the exiting end of the generally S-shaped path. Because of the absence of circuit board material, the aperture 8 facilitates optical interaction with a capillary which is placed over the aperture 8. The aperture 8 allows for fluorescence excitation and detection using an optical configuration such as epi-fluorescent, and various skew illumination schemes.
FIG. 1A is a close-up view of an electrical path of the thermal assembly of FIG. 1.
The electrical path in various embodiments is a patterned, or etched, conductive trace bonded onto the electrically insulating circuit board. The patterned electrical path may be defined by “subtractive” patterning that removes unwanted conductive material to leave the desired conductive paths, or by “additive” patterning that adds additional conductive material to form the desired conductive paths. The circuit board may have the conductive paths on a single layer circuit board or as part of a multi-layer circuit board.
Various examples of conductive material in the electrical path are metallic material such as copper, aluminum, silver, or nonmetallic conductive material such as graphite, or conductive ink, but may be any other conductive material.
In contrast with the conductive material of the electrical path, the circuit board material is nonconductive, commonly a dielectric material.
Each electrical path creates and defines a thermal area. The current implementation has six heating areas, each comprised of approximately 1 m of 150 um wide copper traces that is folded into the shape needed to generate the heater shapes shown below. Various embodiments vary the length of the trace to shorter or longer than 1 m, depending on a length adequate for electrophoretic separation of analytes. Various embodiments widen or narrow the width of the electrical paths, depending on an adequate resistance of the electrical paths to generate adequate heat for thermal regulation of the thermally coupled capillaries. Various embodiments increase or decrease the number of heating areas.
In some embodiments, an electrical path such as a trace has a width in the range between 0.0001 to 0.5 inches, and a length in the range between 0.25 to 750 inches.
Performing electrophoresis in a capillary allows the heat to be effectively dissipated through the capillary walls. This allows high voltages to be used to achieve rapid separations.
FIG. 2 is a top view of a thermal assembly, with a circuit board, electrical paths on the circuit board, a bundle of capillaries, and temperature sensors.
On a circuit board such as the circuit board shown in FIGS. 1 and 1A, electrophoresis capillaries are attached to the generally S-shaped path, such as by adhesive material. This shape is an optional arrangement of the capillaries. Other curves or linear arrangements also are contemplated. In the shown embodiment, a bundle of 8 capillaries are attached. Other embodiments have any other number of capillaries ranging from 1 to a higher number, depending on a particular electrophoresis application's requirements for parallel processing of analytes. The entering end 54 of the capillaries have fanned out ends, to facilitate injection of analytes into the different capillaries. The exiting end 56 of the capillaries remains bundled together in the figure.
In each of the separately thermally regulated areas or sections of the generally S-shaped path, a temperature sensor is in thermal contact. The temperature sensors shown are 32, 34, 36, 38, 40, and 42. Temperature sensor 42 is in thermal contact not with the capillaries, but the circuit board itself, or alternatively the ambient air. Examples of temperature sensors are thermistors or other temperature-varying resistance, or thermocouples or other temperature-varying voltage source. In another embodiment, the temperature data of the separately thermally regulated sections is not gathered by discrete temperature sensor, but by the electrical paths themselves such as by the resistances of the electrical paths.
In the shown embodiment, temperature sensors are thermistors that are attached to traces that terminate on a portion of the circuit board outside of the array of thermal insulation apertures. The thermistors are folded down across the capillary array and embedded in the adhesive that bonds the capillary array to the board, to ensure good thermal contact between the thermistors and the capillaries, while minimizing thermal loss from the heaters.
The temperature data generated by such temperature sensors help to thermally regulate the temperature of the capillaries in thermal contact with the electrical paths. Electrical current through the electrical path deposits thermal energy in the electrical path via Joule heating. The amount of deposited thermal energy varies with the amount of electrical current and resistance of the electrical paths.
Optical Detector
FIGS. 3, 4 11 and 12 are simplified diagrams of an optical subsystem of the electrophoresis system.
In FIG. 3, the excitation source of the excitation beam 171 is a solid state laser, the output of which is projected into the capillary 174 using a beam combiner 162 placed at a 45 degree angle in the optical path immediately above the objective 160. In various embodiments the beam combiner comprises a wavelength sensitive reflector or a spatial beam splitter such as a small reflective dot placed on a transparent sheet of glass. The beam combiner is wavelength dependent, which is easier to align than a spatial beam combiner.
The high numerical aperture objective is used both by the excitation beam 171 on its way to the capillary 174, and by the optical signal of emitted fluorescence from the capillary 174.
The optical signal of fluorescence emitted from the analytes of the capillary 174 is collimated by the objective 160. The optical signal passes through the wavelength sensitive reflector 162 and impinges on a long pass filter 164 that rejects the portion of the optical signal including the excitation beam 171.
The fluorescence detection scheme is prism spectrometer based. The optical signal is then projected onto a dispersive prism 166, which serves to change the angle of the rays according to wavelength. This dispersed optical signal is then focused on the plane of the detector 170 using an image forming lens 168, causing different wavelengths of the dispersed optical signal to focus at different locations in the plane of the detector 170. An example of the detector 170 is a CCD camera. An alternative is a CMOS camera or other optical sensors.
In one embodiment, the optical subsystem described above is a point detector, to detect optical signal of analyte from a single capillary. In other embodiments, the optical subsystem further includes additional components to excite and detect the fluorescence of an array of capillaries.
In a first embodiment a shaped excitation beam illuminates the entire array of capillaries simultaneously. This creates an image in the plane of the detector which is comprised of the spectra of all the capillaries in parallel. This arrangement can result in cross talk between channels. In one embodiment, after the shaped excitation beam illuminates the entire array of capillaries simultaneously, a filter such as an aperture between the array of capillaries and the detector eliminates the optical signal from extra capillaries, thereby addressing crosstalk.
In another embodiment to capture information from all capillaries in the array, the objective is scanned across the array. In this embodiment, the objective is moved relative to a laser beam entering the objective, so that as the objective moves, the point at which the laser beam exiting the objective strikes the capillaries traverses, thereby allowing a selected capillary to be excited. In this configuration, cross talk between capillaries is eliminated because only one capillary is illuminated at a time.
With an array that comprises 200 um diameter capillaries, the scan range for the detection device covers +/−0.8 mm for an eight channel array. This limited scan range minimizes the number of moving parts. Other embodiments widen or narrow the scan range to accommodate a different number of capillaries and/or different number of capillaries. As only the objective 160 moves, the excitation laser beam 171 remains very close to the center of the objective 160, even when the beam 171 is located at the top of the end capillary in the array. The excitation beam 171 impinges on the capillaries at different angles depending on the location of the capillary in the array.
In one embodiment, the objective 160 is moved continuously, or continuously for a scan interval. With the input of the capillary detector 172 described below, the software and/or electronics of the instrument predicts that the objective will pass over a selected capillary. The detector 170 is turned on as the objective passes over a selected capillary. Alternatively, the detector 170 can remained turned on regardless of whether the objective passes over a selected capillary, and the data from the detector 170 is discarded as the detector 170 is not passing over a selected capillary, and collected or processed as the detector 170 is passing over a selected capillary.
In another embodiment, the objective 160 is moved discontinuously, such that the objective moves quickly as the objective passes over a space between capillaries, and then stops over a selected capillary sufficiently long for the detector 170 to collect optical signal from the selected capillary. This can be accomplished, for example, by using a stepper motor.
Similarly, the optical signal of fluorescent emission moves across the face of the prism 166 and the lens behind the prism 168, but the image of the spectrum remains in the same location regardless of the objective location, because the prism 166 is located in collimated optical space.
The capillary detector 172 receives a reflection of the excitation beam 171 from the capillaries by way of capillary detection beam splitter 114/beam element 178 as the scanning objective 160 passes across each capillary. The reflection varies in intensity as a function of the position of the scanning objective 160 relative to the top of each capillary. This results in a distinct intensity profile that is used by software and/or electronics embedded into the instrument that determine the locations of the capillaries, and alignment of the laser beam relative to the capillaries. That information is then used to trigger data acquisition of the optical signal striking the detector 170.
In FIG. 4, the ray trace shows the effect of the objective being off by 1 mm from mechanical center on the emission path.
FIG. 4 shows the “end view” of the optical path. FIG. 4 illustrates the effect of scanning the objective 160, on the image of the capillaries 176. Because the prism 166 is located in collimated optical space, the spectrum remains in the same location of the detector 170 regardless of the location of the objective 160.
There are various embodiments directed to alternatives of arranging the optical path around the beam combiner.
In one embodiment, a stationary beam combiner uses a dichroic mirror that reflects the excitation beam from the laser device to the capillaries, and transmits the emitted fluorescence from the analyte in the capillary to the detector. This embodiment is advantageous in that, with less mass to move, the motion mechanism is simpler. However, some embodiments with a fixed beam combiner limit the number of scannable capillaries.
In another embodiment, a beam combiner is rotated 90 degrees relative to the vertical axis in FIG. 4. In such a configuration, the beam combiner moves with the objective, similar to a CD or DVD player. Such a geometry scans larger arrays of capillaries without being limited by the boundaries of a fixed beam combiner.
In another embodiment with a beam combiner, the laser beam is transmitted and the emitted fluorescent optical signal from the sample is reflected. In such a system, the excitation and emission paths change places.
Another embodiment implements a system with a spatial beam combiner rather than a wavelength dependent beam combiner. The spatial beam combiner is implemented as a small mirror that covers a fraction of the arc of the emission path. The excitation laser is aligned to reflect off that mirror. The physical implementation of the mirror is alternatively a small reflective area on a piece of optical glass or a small physical mirror that is held in the proper location.
In another embodiment the excitation laser beam passes through a small opening in a solid mirror that reflects the majority of the emission towards the detector. Again the opening could be either a physical hole in a mirror or simply a non reflective area on a glass substrate that is otherwise coated with a mirror coating.
In various embodiments the mirror/aperture in the two cases above is located on or off of the optical axis of the system.
In another embodiment, depicted in FIGS. 11 and 12, the optical path comprises a cylinder lens in the detection path to reduce alignment sensitivity of the emission path of the fluorescence detection path and allow for use of detectors with geometries that have a large length to width ratio (that are designed for line or spectrum detection purposes).
Another embodiment optionally comprises a cylindrical lens in the excitation path that produces an oblong excitation spot in the capillaries to excite a larger volume of the labeled molecules inside the capillary without affecting the spectral resolution. This improves the signal-to-noise ratio of the detected optical signal, particularly when taking into account potential photo bleaching of the dye.
FIGS. 5, 6, 7, and 8 are various perspective views of an electrophoresis system.
The layout of the electrophoresis system is generally divided into two areas: i) the laser 112 or other excitation optics 104, the capillary detection sub-system, and the actuator used to move the objective across the capillaries; and ii) the electrophoresis area 102 of the unit which includes the heaters, the capillary array, and the anode and cathode assemblies under the circuit board. The two areas are divided by a vertical wall.
In FIG. 5, a bread board implementation is shown. The detector 110 is a CCD camera. Another embodiment uses a one dimensional detector array. The figure also shows detector focus adjustor 136.
Replacement of the heater assembly can easily be accomplished by folding the locking lever 108 and pulling out the slide mounted assembly of the circuit board 106 for complete top access.
The folding mirror mount 130, 134 and the Penta prism 132 provide optical alignment of the system. The Penta prism 132 is replaced by a mirror in another embodiment.
In FIG. 6, physical access to the electrophoresis circuit board is implemented.
The locking lever is in a service access position, and the capillary array assembly is in service position.
FIG. 7 shows a detailed view of the scanning mechanism of the objective.
There are many embodiments that generate accurate linear motion for the scanner. The shown embodiment is a cam driven system implemented as an eccentric disk 122, but there are many other embodiments, such as linear solenoid actuators, galvanometer mechanisms, and piezo electric actuators.
FIG. 8 shows another detailed view of the scanning mechanism of the objective.
The objective mount 118 is attached to a crossed roller slide 120 that controls its motion. A ball bearing is mounted at the end of the objective mount 118 and is held against an eccentric disk 122 by a spring (not shown). The linear back and forth motion of the objective across the capillaries is generated by rotating the eccentric disk 122 using a motor 116 (FIG. 7).
The printed circuit board heater is held against a hard stop defining scanner focus 124 which is adjusted to align the capillaries to the focal plane of the scanning optics. The focal plane is adjustable with the adjusting screw 138.
Many different embodiments of this apparatus exist. Mounting the objective to a flexure eliminates the need for the crossed roller bearing slide. A voice coil or similar actuator can also generate the linear motion.
FIG. 9 is a block diagram of an electrophoresis system.
An electrophoresis thermal assembly 210, such as the one shown in FIG. 2, has one or more electrophoresis capillaries in thermal contact with electrical paths on a circuit board. A voltage source 208 facilitates the capillary electrophoresis by applying a voltage difference across the capillary inlet 204 and the capillary outlet 206 of the capillary 52. In one embodiment, the cathode of the array is located under the circuit board below the triangle shaped electrical path corresponding to the entering end of the capillaries, and the anode of the array is located under the circuit board below the exiting end of the capillaries.
An analyte injector 205 adds analyte to the capillary inlet 204. The injected analytes are electrophoretically moved through the capillary 52. Examples of injector types are gravity injection, pressure or hydrodynamic injection, and electrokinetic injection. The sample can be isolated by boluses of gas upstream and downstream to the sample. Electrophoresis buffer can also enter the capillary.
An example sample injection procedure is to dip the capillary and electrode into the sample solution vial and to apply a voltage. If the sample is ionized and the appropriate voltage polarity is used then sample ions will migrate into the capillary. This type of injection is known as electrokinetic sampling. The capillary is filled with electrolyte solution which conducts current through the inside of the capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte.
Alternative embodiments use capillary gel electrophoresis with physical gel that entangles polymers, or chemical gels with covalent structure.
In an embodiment generating temperature data from discrete temperature sensors or from the electrical paths themselves, a controller 220 raises or lowers the electrical currents to achieve a desired temperature of the capillary, or to achieve a desired temperature of a particular portion of capillary which corresponds to the electrical path in thermal contact with the particular portion of capillary. The temperature controller 220 runs current through the paths or traces on the board, causing them to heat, due to the resistance of the traces. The software running in the controller utilizes the temperature information collected by the sensors to control the temperature of the individual electrical paths using any of a variety of control algorithms to achieve a uniform temperature along the path of the capillaries. The temperature controller in one implementation is housed on a separate printed circuit board and is based on a microcontroller that controls the temperature using a PID type control algorithm to manage the temperature of each electrical path. Thermal imaging of the board in operation shows that a thermal uniformity of 2 degrees C. peak to peak is achievable over the entire length of the capillaries.
The laser device 212, optical detector 216, and one or both of optical selector #1 214 and optical selector #2 218 are arranged to limit optical signal to a single capillary. In the case of optical selector #1 214 between the laser device 212 and the capillary 202, the optical selector #1 214 limits the beam from the laser device to exciting analyte in a single capillary. In the case of optical selector #2 218 between the capillary 202 and the optical detector 216, the beam from the laser device 212 may excite analyte in one capillary or multiple capillaries, but the optical selector #2 216 limits the beam from the laser device to exciting analyte in a single capillary 202.
In various embodiments the capillary tubing has an outer diameter of about 150 to 500 microns and an inner diameter of about 10 to 100 microns. In various embodiments the capillary is polyimide or polytetrafluoroethylene clad. The capillary can be about 2 to 100 cm long, depending on the electrophoretic separation requirements.
Migration time (tm) is the time it takes to move from the beginning of the capillary to the detector window. Electrophoretic mobility, mu (cm2/Vs), is the electrophoretic velocity vep (cm/s), divided by the electric field strength, E (V/cm).
Velocities are measured by dividing the migration time by the length of the capillary to the detector, Ld. Mobilities are highly dependent on the buffer type and pH as well as temperature. As the temperature increases, the viscosity decreases, and the electrophoretic mobility increases as well. Accordingly, higher temperature accelerates the electrophoresis process.
FIG. 10 is a block diagram of a temperature regulation system. One embodiment of the temperature regulation system is a thermal cycling system.
Certain biochemical reactions require appropriate temperature ranges. With a biochemical reaction performed in capillary tube, a sample is moved into a segment of the capillary at a particular temperature. Then the temperature of the sample can be changed, such as by changing the temperature of the capillary segment, or having a sequence of capillary segments and moving the sample into a subsequent segment, or some combination.
Some embodiments perform biochemical reactions requiring changes in temperature, e.g., thermal cycling reactions such as polymerase chain reaction, and subsequent product analysis (such as via the electrophoresis system of FIG. 9). This device can be used for thermal cycling of different temperatures in different electrical paths, or isothermal reactions along the different electrical paths.
A temperature regulation assembly 310, such as the one shown in FIG. 2, has one or more electrophoresis capillaries in thermal contact with electrical paths on a circuit board. A capillary 302 has a capillary inlet 304 and a capillary outlet 306.
An analyte injector 305, e.g., a DNA fragment injector, adds analyte to the capillary inlet 304.
In an embodiment generating temperature data from discrete temperature sensors or from the electrical paths themselves, a controller 320 raises or lowers the electrical currents to achieve a desired temperature of the capillary, or to achieve a desired temperature of a particular portion of capillary which corresponds to the electrical path in thermal contact with the particular portion of capillary. The temperature controller 320 runs current through the paths or traces on the board, causing them to heat, due to the resistance of the traces. The software running in the controller utilizes the temperature information collected by the sensors to control the temperature of the individual electrical paths using any of a variety of control algorithms to achieve a uniform temperature along the path of the capillaries. The temperature controller in one implementation is housed on a separate printed circuit board and is based on a microcontroller that controls the temperature using a PID type control algorithm to manage the temperature of each electrical path. Thermal imaging of the board in operation shows that a thermal uniformity of 2° C. peak to peak is achievable over the entire length of the capillaries.
PCR typically involves the following steps and temperatures: Initialization step—94-96° C. for 1-9 minutes. Denaturation step—94-98° C. for 20-30 seconds Annealing step—50-65° C. for 20-40 seconds. Extension/elongation step—around 72° C. Final elongation—70-74° C. for 5-15 minutes. Final hold—4-15° C. for an indefinite time.
These steps can be repeated as needed to perform sufficient amplification.
The capillary contains a reaction mixture and an analyte, e.g., a nucleic acid enriched from a sample (collectively referred to as the PCR reaction sample). An optical assembly can be used to monitor or control the reaction. The optical assembly can introduce or detect light. For example, an optical assembly can be used for performing real-time PCR or other real-time or end point measurements.
In one embodiment a sample preparation device can be used in conjunction with a temperature modulator as a flow-through thermal cycler. Driving force for moving the fluid can be an external pressure source or an internal pressure source. A flow-through thermal cycler can be used when highly sensitive or high throughput temperature change reaction, such as PCR, is desired. There are many situations in which one might want to sample air, blood, water, saliva, a cellular sample, or other medium in a sensitive PCR assay. This can be used to look for a variety of biological contaminants including influenza, bacterial pathogens, and any number of viral or bacterial pathogens. Flow-through PCR can allow PCR to be practiced in an automated manner without the need for human interaction. A flow-through PCR system can also serve as an early warning system in HVAC systems of buildings, airplanes, busses, and other vehicles, and can be used in the monitoring of blood, water, or other sample sources for the presence of an infectious agent or a contaminant.
The flow-through PCR device takes a sample from a collection device, such as a buccal swab, a syringe, an air sampler, fluid sampler or other sampler and delivers it to a sample preparation device. The sample is prepared in the preparation device, which in some embodiments may include cell lysis, DNA, RNA, or micro RNA enrichment or purification, filtration, or reverse transcription. In one embodiment at least one nucleic acid is enriched. In another embodiment at least one enriched nucleic acid is prepared for PCR by adding the nucleic acid to PCR reagents (such as at least one DNA polymerase, RNA polymerase, dNTPs, buffer or a salt) and primers, (such as assay-specific primers or broadly applicable primer sets for multiple target pathogens). These primers may be chosen to selectively amplify at least one nucleic acid isolated from a specific pathogen (such as a mold, virus, bacteria, parasite or amoeba), gene, other desired nucleic acid, or any combination thereof. The composition comprising at least one nucleic acid enriched from a sample, PCR reagents and primers is called a PCR reaction sample. In one embodiment, the flowthrough PCR can be used as a continuous flow device while in other embodiments samples are moved into the thermal cycling region and stopped.
The PCR reaction sample then flows through a reaction channel and circuit board with the temperature controlled electrical paths. In some embodiments the reaction channel is clear or transparent. In another embodiment the reaction channel is opaque. In one embodiment the reaction channel is a cylinder. In another embodiment the reaction channel's cross section comprises one or more planes forming a shape such as a triangle, square, rectangle, pentagon, hexagon, heptagon, octagon, nonagon, decagon, or other polygon. In one embodiment the volume of PCR reaction sample is such that it takes up a small discrete length of space in the reaction channel, the rest of which is occupied by air, gas, or a non-reactive liquid, such as mineral oil. Air, gas, or a non-reactive liquid can be used to separate individual PCR reaction samples from each other.
In one embodiment a detection module measures fluorescence, luminescence, absorbance or other optical properties to detect a signal emitted from a PCR reaction sample while it is located with a temperature control region, or after it has left a temperature control region. A detection module can comprise a light source (such as a coherent light source or incoherent light source) used to excite a fluorescent dye (such as an intercalating dye, including but not limited to ethidium bromide or Syber green) in a PCR reaction sample, and the excitation light is sensed with a photodetector (such as a CCD, CMOS, PMT, or other optical detector). Detection electronics can evaluate the signal sent from the detection module.
In one embodiment, after the desired number of thermal cycles are complete, the PCR reaction sample is pumped or pushed further down the reaction channel, using pressure or vacuum, exiting the temperature controlled region. In one preferred embodiment, a downstream device is an analytical devices that can be used for performing electrophoresis, mass spectroscopy, or other analytical techniques.
Multiple reaction channels may be used in parallel to increase sample throughput. In yet another embodiment the system may alert the user when amplification has occurred (a positive result), indicating that the target sequence is present. In one embodiment a reaction channel is used for a single use only, then disposed of. In an alternative embodiment a reaction channels can be used to amplify and detect the presence or absence of PCR amplification products in multiple samples. More than one PCR reaction samples can be loaded at intervals and interspaced with a barrier bolus of gas or liquid to prevent intermixing. In one embodiment samples are spaced apart in a manner so that as one is undergoing thermal cycling another sample is in the detection region undergoing interrogation. The PCR amplification can be replaced by other nucleic acid amplification technologies which may use thermal cycling or be isothermal reactions.
In other embodiments, the device can perform isothermal reactions such as sandwich assays using affinity reagents such as antibodies or aptamers to determine if cells, proteins, toxins, or other targets are present with the detection module providing a reading of the amount of target present. In these applications, the an affinity purification may be performed such as an IMS purification and then add a secondary antibody that may have a fluorescent label attached. The sample can then move into a thermally controlled region set to optimize the reaction. A detection module can then monitor the reaction.
While the present invention is disclosed by reference to the preferred embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than in a limiting sense. It is contemplated that modifications and combinations will readily occur to those skilled in the art, which modifications and combinations will be within the spirit of the invention and the scope of the following claims.

## Claims

1. An apparatus, comprising:
an electrically insulating circuit board;
at least one electrical path comprising at least one conductive trace attached to the circuit board, said at least one electrical path generating Joule energy responsive to electrical current through said at least one electrical path, wherein the at least one electrical path forms at least one thermal area; and
at least one electrophoresis capillary in thermal contact with said at least one thermal area, such that said at least one electrophoresis capillary is thermally regulated responsive to electrical current through said at least one electrical path.

2. The apparatus of claim 1, further comprising:
at least one temperature sensor in thermal contact with said at least one electrophoresis capillary, such that said at least one temperature sensor provides temperature data of said at least one electrophoresis capillary; and
a controller of a temperature of said at least one electrophoresis capillary, the controller changing the electrical current through said at least one electrical path responsive to the temperature data from said at least one temperature sensor.

3. The apparatus of claim 1, wherein said at least one electrical path has at least one resistance providing temperature data of said at least one electrophoresis capillary in thermal contact with said at least one electrical path, and the apparatus further comprises:
a controller of a temperature of said at least one electrophoresis capillary, the controller changing the electrical current through said at least one electrical path responsive to the temperature data from said at least one resistance of said at least one electrical path.

4. The apparatus of claim 1, further comprising:
at least one thermal insulation member attached to the circuit board and positioned by said at least one electrical path and said at least one electrophoresis capillary, said at least one thermal insulation member reducing heat transfer between a part of the circuit board attached to said at least one electrical path and said at least one electrophoresis capillary and a remainder of the circuit board.

5. The apparatus of claim 1, further comprising:
at least one aperture in the circuit board attached to the circuit board and positioned by said at least one electrical path and said at least one electrophoresis capillary, said at least one aperture reducing heat transfer between a part of the circuit board attached to said at least one electrical path and said at least one electrophoresis capillary and a remainder of the circuit board.

6. The apparatus of claim 1, wherein said at least one electrical path includes a plurality of electrical paths in thermal contact with different sections of said at least one electrophoresis capillary, such that the different sections of said at least one electrophoresis capillary are separately thermally regulated by different electrical paths of the plurality of electrical paths.

7. The apparatus of claim 6, wherein the apparatus further comprises:
a plurality of temperature sensors in thermal contact with the different sections of said at least one electrophoresis capillary, such that the plurality of temperature sensors provides temperature data of the different sections of said at least one electrophoresis capillary; and
a controller of temperatures of the different sections of said at least one electrophoresis capillary, the controller changing the electrical currents through the plurality of electrical paths responsive to the temperature data from the plurality of temperature sensors.

8. The apparatus of claim 6,
wherein said plurality of electrical paths have resistances providing temperature data of the different sections of said at least one electrophoresis capillary in thermal contact with the plurality of electrical paths, and
the apparatus further comprises:
a controller of temperatures of the different sections of said at least one electrophoresis capillary, the controller changing the electrical currents through the plurality of electrical paths responsive to the temperature data from the resistances of the plurality of electrical paths.

9. The apparatus of claim 1, wherein said at least one electrophoresis capillary is attached to the circuit board.

10. The apparatus of claim 1, wherein said at least one electrophoresis capillary is attached to the circuit board with adhesive material.

11. The apparatus of claim 1, wherein said at least one electrical path includes an electrical path that runs back and forth in said at least one thermal area or an electrical path configured as two electrical nodes connected by a plurality of electrical paths.

12. The apparatus of claim 1, wherein said at least one thermal area has a width no less than 5 mm.

13. The apparatus of claim 1, wherein the thermal area widens by a part of said at least one electrophoresis capillary entering the electrically insulating circuit board.

14. The apparatus of claim 1, wherein said electrically insulating circuit board has an aperture through the electrically insulating circuit board, the aperture facilitating optical interaction with said at least one electrophoresis capillary.

15. The apparatus of claim 1, wherein said at least one electrical path has at least one bend.

16. The apparatus of claim 1, wherein said at least one electrical path overall has an S-shape.

17. An apparatus, comprising:
(a) an electrophoresis thermal assembly, including:
an electrically insulating circuit board;
at least one electrical path comprising at least one conductive trace attached to the circuit board, said at least one electrical path generating Joule energy responsive to electrical current through said at least one electrical path;
at least one electrophoresis capillary in thermal contact with said at least one electrical path, such that said at least one electrophoresis capillary is thermally regulated responsive to electrical current through said at least one electrical path;
(b) at least one analyte injector coupled to inject at least one electrophoresis analyte into said at least one electrophoresis capillary;
(c) a voltage source coupled to opposite ends of said at least one electrophoresis capillary providing an electrophoretic voltage difference between the opposite ends of said at least one electrophoresis capillary;
(d) a laser device positioned to deliver a beam from the laser device to said at least one electrophoresis capillary;
(e) an optical detector optically coupled to receive an optical signal from said at least one electrophoresis capillary.

18. A method, comprising:
providing an apparatus comprising:
an electrically insulating circuit board;
at least one electrical path comprising at least one conductive trace attached to the circuit board, said at least one electrical path generating Joule energy responsive to electrical current through said at least one electrical path, wherein the at least one electrical path forms at least one thermal area; and
at least one electrophoresis capillary in thermal contact with said at least one thermal area, such that said at least one electrophoresis capillary is thermally regulated responsive to electrical current through said at least one electrical path;

electrophoretically moving analytes through at least one electrophoresis capillary; and
thermally heating said at least one electrophoresis capillary via thermal contact with said at least one thermal area with Joule energy generated by passing electrical current through said at least one electrical path attached to said electrically insulating circuit board.

19. The method of claim 18, further comprising:
generating temperature data of said at least one electrophoresis capillary in thermal contact with said at least one thermal area; and
changing the electrical current through said at least one electrical path, responsive to the temperature data of said at least one electrical path.

20. The method of claim 18, further comprising:
generating, via at least one temperature sensor of said at least one electrophoresis capillary, temperature data of said at least one electrophoresis capillary in thermal contact with said at least one thermal area; and
changing the electrical current through said at least one electrical path responsive to the temperature data from said at least one temperature sensor.

21. The method of claim 18, further comprising:
generating, via at least one resistance of said at least one electrophoresis capillary, temperature data of said at least one electrophoresis capillary in thermal contact with said at least one thermal area; and
changing the electrical current through said at least one electrical path responsive to the temperature data from said at least one resistance.

22. The method of claim 18, further comprising:
reducing heat transfer between a part of the circuit board attached to said at least one electrical path and said at least one electrophoresis capillary, and a remainder of the circuit board.

23. The method of claim 18, further comprising:
reducing heat transfer with at least one aperture between a part of the circuit board attached to said at least one electrical path and said at least one electrophoresis capillary, and a remainder of the circuit board.

24. The method of claim 18, wherein said thermally heating includes:
separately thermally heating different sections of said at least one electrophoresis capillary via thermal contact with a plurality of electrical paths carrying electrical currents through the electrically insulating circuit board.

25. The method of claim 24, further comprising:
generating temperature data of the different sections of said at least one electrophoresis capillary; and
changing the electrical currents through the plurality of electrical paths, responsive to the temperature data from the different sections of said at least one electrophoresis capillary.

26. The method of claim 24, further comprising:
generating temperature data of the different sections of said at least one electrophoresis capillary, via different temperature sensors of the different sections of said at least one electrophoresis capillary; and
changing the electrical currents through the plurality of electrical paths, responsive to the temperature data from the different sections of said at least one electrophoresis capillary.

27. The method of claim 24, further comprising:
generating temperature data of the different sections of said at least one electrophoresis capillary, via resistances of the plurality of electrical paths; and
changing the electrical currents through the plurality of electrical paths, responsive to the temperature data from the different sections of said at least one electrophoresis capillary.

28. The method of claim 18, further comprising:
injecting at least one analyte into said at least one electrophoresis capillary.

29. The method of claim 18, further comprising:
optically exciting at least one analyte in said at least one electrophoresis capillary; and
detecting an optical signal from said excited at least one analyte.

30. A method, comprising:
separately regulating temperature in different sections of at least one capillary via thermal contact with different thermal areas formed from different electrical paths carrying electrical currents through an electrically insulating circuit board, wherein each of said different electrical paths comprise different conductive traces attached to the circuit board, said different electrical paths generating Joule energy responsive to electrical current through said different electrical paths, wherein the different electrical path forms said thermal areas; and
moving analytes through said at least one capillary supporting a biochemical activity in the separately thermally regulated different sections of said at least one electrophoresis capillary.

31. The method of claim 30 wherein the biochemical activity is polymerase chain reaction or a sandwich assay using affinity reagents.

32. The method of claim 31 wherein the capillary contains at least one DNA polymerase dNTPs and primers.

33. The method of claim 30 comprising moving the analytes through thermal areas adapted for PCR including thermal areas adapted for a denaturation step; an annealing step and an extension/elongation step.

34. The method of claim 33 wherein the thermal area adapted for a denaturation step has a temperature between 94-98° C., the thermal area adapted for an annealing step has a temperature between 50-65° C.; and the thermal area adapted for extension/elongation step has a temperature around 72° C.

35. The method of claim 30 comprising moving the analytes through thermal areas adapted for PCR including a thermal area adapted for an initialization step having a temperature between 94-96° C.; a thermal area adapted for a denaturation step having a temperature between 94-98° C., a thermal area adapted for an annealing step having a temperature between 50-65° C.; a thermal area adapted for extension/elongation step has a temperature around 72° C.; a thermal area adapted for final elongation having a temperature between 70-74° C. and a thermal area adapted for final hold having a temperature between 4-15° C.