The invention relates to devices and methods for isolating chambers of an assay card, and more particularly to devices and methods for isolating chambers of an assay card by softening and/or deforming at least a portion of the assay card. The invention also relates to a method of manufacturing a tool device that includes one or more tools for heating and deforming portions of an assay card.
The detection and monitoring of diseases commonly employs various types of biological testing. Since there are often a large number of tests that are required to be performed in this field, it is typically desirable to reduce the cost and time associated with these tests. A commonly employed technique for reducing such costs and time is to simultaneously test numerous, relatively small samples, e.g., during each run of a thermal cycling unit or other like device. Substrates having multiple wells, detection chambers or reaction chambers to which a fluid sample is distributed through one or more channels to which the chambers are connected have been employed for simultaneously testing a large number of analytes in such a sample. Such substrates, which are sometimes referred to as “microcards,” “assay cards” or “analytical cartridges,” allow relatively small sample volumes to be distributed to a large number of detection chambers, such as 96, 384, or more, which may be preloaded with different analyte-specific reagents. Such substrates, in addition to systems and method for their use, are described, for example, in U.S. Pat. No. 6,126,899, U.S. Pat. No. 6,272,939 and U.S. Patent Publication No. 2004/0157343.
In substrates in which channels interconnect the chambers or detection chambers there is a potential for fluid communication between chambers during sample processing, for example, during thermal cycling, which may cross-contaminate the reactions in the connected chambers, Various strategies are disclosed in the art to reduce the potential for cross-contamination. For example, U.S. Pat. No. 6,126,899 discloses filling the delivery channels with an additional fluid, such as a mineral oil or a viscous polymer solution, to segregate the chambers from each other. U.S. Pat. No. 6,068,751 discloses the use of a valve between processing chambers that is closed to isolate the processing chambers from each other. U.S. Patent Publication No. 2004/0157343 discloses sealing of each loading passage connecting each of a series of chambers to a common channel by deforming the substrate cover adjacent each loading passage. The cover is deformed by bringing the substrate into contact with a thermal transfer block having bosses or protrusions in locations corresponding to the loading passages. The bosses can be heated to facilitate deformation of the cover material.
However, these and other devices and method for preventing contamination between chambers or detection chambers, may not do so in a manner that is adequately safe, reliable, and fast. In light of the foregoing, there is a need for a system and method that overcomes the disadvantages of the previous methods.
The invention relates to an assay card and a method for isolating chambers on an assay card. In this embodiment, the assay card comprises a substrate formed of plastic having a softening temperature, the substrate defining a first channel in communication with a first chamber and a second channel in communication with a second chamber. The method comprises the steps of heating the assay card in a region of the first and second channels to at least the softening temperature; and simultaneously deforming, with a single tool, the assay card in the region of the first and second channels such that plastic of the substrate is caused to at least partially obstruct both the first and second channels. The method may also include the step of cooling the deformed plastic.
In an embodiment, the heating step includes contacting the substrate with a heated tool. Contacting the substrate with a heated tool may also include inserting the tool into the channel, and/or contacting a region of the substrate adjacent to the channel. The heating step may include applying a source of ultrasonic energy to the substrate. Alternatively, the heating step may include directing a light or laser beam or a heated air jet to the substrate.
Additionally or alternatively to the substrate being deformed by contact and/or pressure with a tool, the deforming step may also occur spontaneously due to the surface tension of the softened region of the substrate. In the same manner that a water droplet may spontaneously flow into a capillary because of the surface tension, material from the softened region of the substrate may spontaneously flow into the channel without the application of a mechanical tool. Channels with properly designed features in the softened region of the substrate will allow the surface tension to locally pull the material from the substrate to obstruct the channel and to automatically cease the filling action after the channel is completely obstructed. After the substrate material cools down, the channel is thus permanently obstructed. An advantage of this technique is that it employs a passive mechanism, hence not requiring the mechanical application of a tool.
Also, the deforming step may include permitting gravity to deform the softened region of the substrate. Furthermore, the deforming step may include the application of a pneumatic pressure or vacuum to the substrate. Still further, the deforming step may include moving the assay card so as to cause an inertial stress onto the substrate, which in turn will deform and flow to locally fill the channel. In the same manner that a surface of a liquid within a container may change its shape when the container is set in motion, the softened region of the substrate may be made to flow into the channel by subjecting the card to a motion that would cause inertial forces and thus stresses, such as a rotary motion (centrifuge) or an impulsive linear motion (slide).
Various spatial arrangements of the substrate and, e.g., the tools, may be possible. For example, the heating and deforming steps may include heating and deforming the assay card from a side of the substrate to which the channels are directly or most nearly adjacent. Additionally or alternatively, the heating and deforming steps may include heating and deforming the assay card from a side of the substrate that is opposite the side of the substrate to which the channels are directly or most nearly adjacent.
The invention, in accordance with another embodiment thereof, relates to a method for isolating a reaction chamber of an assay card. In this embodiment, the assay card comprises a substrate formed of plastic having a softening temperature, the substrate having a first surface and a second surface opposite the first surface, the substrate defining the chamber, the substrate further defining a channel adjacent the first surface and in communication with the chamber, the substrate further defining in the second surface a depression that is aligned with at least a portion of the channel. The chamber and the channel may be sealed by a second layer attached to the first surface of the substrate. The method may comprise the steps of: heating the assay card in a region of the depression and the channel to at least the softening temperature; and deforming the assay card in the region of the depression such that plastic of the substrate is caused to at least partially obstruct the channel. The method may also include the step of cooling the deformed plastic.
Also, the heating and deforming steps may include contacting and applying pressure to a surface of the depression of the substrate with a heated tool. Additionally or alternatively, the heating step may include applying a source of ultrasonic energy, a light or laser beam or a heated air jet to a surface of the depression in the substrate. In an embodiment, the substrate defines two or more chambers and two or more channels, each chamber having a respective channel in communication with it, and wherein the heating and deforming steps simultaneously heat and deform the assay card in a region that includes the two or more channels. The region that includes the two or more channels may be simultaneously heated by contact with a single heated tool.
The invention, in accordance with another embodiment thereof, relates to a method for isolating a chamber of an assay card. In this embodiment, the assay card comprises a substrate formed of a first material having a first softening temperature and a second material having a second softening temperature, the substrate defining a channel in communication with a chamber, the second material being adjacent to the channel. The method comprises the steps of: heating the assay card in a region of the channel to at least the second softening temperature; and deforming the assay card in the region of the channel such that the second material at least partially obstructs the channel.
The first and second materials may be first and second types or grades of plastic, wherein the first softening temperature is greater than the second softening temperature. The heating step may include heating the assay card in a region of the channel to a temperature that is greater than the second softening temperature but less than the first softening temperature. Also, the channel may be located adjacent to a first surface of the substrate, a second surface of the substrate being opposite the first surface and including a depression, wherein the depression is aligned with at least a portion of the channel. Advantageously, the second material may be disposed between respective bottom surfaces of the depression and the channel, such that, upon the second material being heated to at least the second softening temperature, the second material may be deformed to obstruct the channel. In an embodiment, the method may further comprise the step of cooling the deformed plastic. Also, the heating and deforming steps may include contacting and applying pressure to a surface of the depression of the substrate with a heated tool. Alternatively, the second material may be a thin section bonded to the first surface of the substrate to complete the formation of the channel. In this embodiment, the heating and deforming step may including contacting the second material over the channel with a heated source causing the second material to at least partially obstruct the channel.
The invention, in accordance with another embodiment thereof, relates to an assay card. The assay card may include a substrate formed of plastic having a softening temperature, the substrate having a first surface and a second surface opposite the first surface. The substrate may define a chamber, the substrate further defining a channel adjacent the first surface and in communication with the chamber. The chamber and the channel may be sealed by a second layer attached to the first surface of the substrate. The substrate may also define in the second surface a depression that is aligned with at least a portion of the channel, wherein, upon heating the assay card in a region of the depression and the channel to at least the softening temperature, the assay card in the region of the depression is configured to be deformed such that plastic of the substrate may at least partially obstruct the channel. The deformed plastic may be cooled so as to maintain isolation of the chamber.
The assay card may be configured to be deformed by heat and pressure applied by a heated tool. Additionally or alternatively, the substrate may be configured to be heated by the application of a source of ultrasonic energy, a light or laser beam or a heated air jet. The substrate may define two or more chambers and two or more channels, each chamber having a respective channel in communication with it, and the two or more channels may be arranged so as to be simultaneously heated and deformed. The two or more channels may be arranged so as to be simultaneously heated by contact with a single heated tool. The plastic may include a first region having a first softening temperature and a second region having a second softening temperature, wherein the first softening temperature is greater than the second softening temperature. The assay card may be configured to be heated in a region of the channel to a temperature that is greater than the second softening temperature but less than the first softening temperature. In an embodiment, the second region of the plastic may be disposed between the depression and the channel, such that the second region is configured to be heated to at least the second softening temperature and to be deformable for obstructing the channel.
The invention, in accordance with still another embodiment thereof relates to a method of manufacturing a tool device, the tool device including pins for heating and deforming an assay card. The method may comprise the steps of providing a rigid insulator defining through-holes; applying a resist layer to the insulator, wherein the resist layer is patterned with holes which match the through-holes of the insulator; plating pins through the resist layer and the rigid insulator; stripping the resist layer to expose the pins; and forming a conductive pathway linking the pins. The method may also include the step of smoothing and rounding the pins, e.g., by performing an isotropic wet etch process. Also, the step of forming a conductive pathway linking the pins may include patterning a metal layer on a back side of the tool device.
Additional features of the device and methods of the invention are discussed in greater detail below.
a) is an assembled top view of the assay card, according to an embodiment of the invention;
b) is an assembled perspective view of the assay card, according to an embodiment of the invention;
a) through 5(d) illustrate the steps that may be performed, according to an embodiment of the invention, to isolate the reaction chambers of an assay card;
a) through 6(d) illustrate schematically a method of isolating a reaction chamber by heating and deforming an assay card with a heated tool from a side of the substrate to which channels are directly or most nearly adjacent, according to an embodiment of the invention;
a) through 9(d) illustrate schematically a method of isolating a reaction chamber by heating and deforming an assay card with a heated tool from a side of the substrate that is opposite the side of the substrate to which channels are directly or most nearly adjacent, according to an embodiment of the invention;
a) through 10(d) illustrate schematically a method of isolating a reaction chamber by heating and deforming an assay card with a heated tool in a region of the substrate that is not aligned with a channel to be obstructed, according to an embodiment of the invention;
a) and (b) illustrate an arrangement in which each one of one or more tools may be employed to heat and deform a region of the substrate that includes two or more channels, according to an embodiment of the invention;
a) and (b) are perspective top and bottom views, respectively, of a tool device, formed in accordance with the steps set forth in the flowchart of
a) through 16(d) are side cross-sectional views, and
a) and (b) are graphs that provide test results, namely Delta Rn curves, for illustrating the effect on temperatures within a reaction chamber when the method of the invention, in accordance with an embodiment, is employed.
a) and (b) illustrate the PCR of Flu B with a concentration of 1 k copies/μL in an assay card spotted with two different assays—Flu B and Mycoplasma pneumoniae. FIG. 19(a) illustrates the spotting pattern in the 24 wells.
The invention, according to various embodiments thereof, is directed to devices and methods for isolating reaction chambers of an assay card.
The assay card 10 may include an inlet port 12 through which a sample is introduced. A bus channel 14 extends from the inlet port 12. Feeder channels 16 branch off of the bus channel 14 and lead into reaction chambers 18. Vent channels 20 extend from the reaction chambers 18 to a vent port 22. In the example embodiment shown in
In addition to the varied arrangement of the reaction chambers, channels and ports, the assay card 10, according to various embodiments of the invention, may be formed of various different layers. For example,
a) and 4(b) provide additional views of the assay card 100. Specifically,
The invention also includes, according to an embodiment, a method to isolate the reaction chambers of an assay card.
As set forth above, there are various ways in which the assay card 10 may be heated in the region of the channel 16 to a predetermined temperature. For example, the heating step may include contacting the substrate 41 with a heated tool.
The tool 43 may be any suitable shape or size, some of which are described in further detail below. Also, the temperature at which the tool 43 may be heated, the duration of contact between the tool 43 and the assay card 10, and the amount of pressure applied may be predetermined or determined by an operator during the heating process. Advantageously, any or all of the factors of tool shape and size, temperature, duration and applied pressure may be varied to insure that the assay card 10, at least in a region of the channel 16, is heated to a predetermined temperature, e.g., at least a softening temperature of the substrate 41 in the region of the channel 16. The heating of the assay card 10 in this manner may provide that the region of the channel 16 is more easily deformed than would be the case had the region not been heated.
c) illustrates the assay card 10 being deformed by the tool 43 applying a pressure to the assay card 10. In the embodiment shown, the tool 43 is positioned directly over the channel 16, and the motion of the tool 43 (shown as arrow A) is perpendicular to the surface 42 of the assay card 10. It should be recognized that the manner in which the assay card 10 may be deformed in the region of the channel 16 may be varied by varying the position of contact of the tool 43 relative to the channel 16, the direction of motion of the tool, the amount of pressure applied, and other factors. In the embodiment shown in
Again, it should also be recognized that the relative positions of the heat source or tool that heats the assay card 10 may be varied in the invention, depending on a number of factors. For example, in an embodiment, the heat source or tool acts on a first surface of the substrate that is opposite the surface that is directly or most nearly adjacent to the channel 16 (which may be the top or the bottom surface depending on the location of the channel).
c) illustrates the assay card 10 being deformed by the tool 43 applying pressure to the assay card 10. In the embodiment shown, the tool 43 is positioned directly over the depression 191 of the channel 16, and the motion of the tool 43 (shown as arrow A) is perpendicular to the surface 42 of the assay card 10. In the embodiment shown in
There are various other ways that heat may be applied to the substrate. For example, in an embodiment, the heating step includes applying a source of ultrasonic energy.
Additionally or alternatively, the heating step may include applying heat to the substrate with a light or laser beam, or other source of radiant energy.
a) through 10(d) illustrate schematically a further embodiment of the invention in which a reaction chamber 18 is isolated by heating and deforming an assay card 10 with a heated tool in a region of the substrate 41 that is not aligned with a channel 16 to be obstructed. For example,
Again, the tool 43 may be any suitable shape or size, examples of which are described in further detail below. Also, the temperature at which the tool 43 may be heated and the duration of contact between the tool 43 and the assay card 10 may be varied and may be predetermined or determined by an operator during the heating process. Advantageously, the assay card 10, at least in a region of the channel 16, is heated to a predetermined temperature that is, for example, at least a softening temperature of the substrate 41 in a region that includes at least some portion of the substrate immediately adjacent to the channel 16. The heating of the assay card 10 in this manner may facilitate the deformation of the channel 16.
c) illustrates the assay card 10 being deformed by the tool 43 applying pressure to the assay card 10. In the embodiment shown, the tool 43 is positioned to the side of the channel 16, and the shape of the tool 43 in addition to the motion of the tool 43 (shown as arrow A) are such that some portion of the heated substrate is caused to be displaced, i.e., is pushed into, the channel 16. It should be apparent that the position of contact of the tool 43 relative to the channel 16, the direction of motion of the tool and the amount of pressure applied may be varied to achieve suitable deformation of the assay card in the region of channel 16. In the embodiment shown in
Additionally or alternatively, heat may be applied to at least a portion of the assay card 10 on a second surface of the substrate, i.e., a surface to which the channel 16 is directly or most nearly adjacent. Such an arrangement is illustrated in, e.g.,
As set forth above, there are various ways that the heated assay card 10 may be deformed such that plastic of the substrate 41 at least partially obstructs the channel 16. Various ones of the figures described hereinabove, e.g.,
It should also be apparent that varying amounts of contact and/or pressure (including no contact and/or pressure) may be employed depending on various factors, e.g., the type or grade of plastic used, degree of softening of the plastic, the size and shape of the channel 16, the size and shape of the tool 43, in addition to other factors. It should also be apparent that the relative positions of the tool 43 and the assay card 10 may also be varied, depending on such factors. Moreover, the heated assay card 10, or at least a portion of it may be deformed by the application of contact and/or pressure to the surface of the substrate to which the channel 16 is directly or most nearly adjacent, or, additionally or alternatively, to the opposite surface of the substrate.
Still further, the heated assay card 10, or at least a portion of the assay card 10, may be deformed by methods that do not require application of contact and/or pressure to a surface of the substrate. For example, the assay card 10, or at least a portion of the assay card 10, may be deformed by the application of a surface tension on the softened region of the substrate. Additionally or alternatively, the assay card 10, or at least a portion of the assay card 10, may be deformed by permitting gravity to deform the softened region of the assay card 10. Also, the assay card 10, or at least a portion of the assay card 10, may be deformed by the application of a pneumatic pressure or vacuum to the substrate. Furthermore, the assay card 10, or at least a portion of the assay card 10, may be deformed by moving the assay card so as to cause an inertial stress on the substrate, thereby deforming the softened plastic.
The invention has been described hereinabove as having various arrangements in which a tool 43, is employed to heat and deform a region of the assay card 10 to isolate a single reaction chamber 18. Alternatively, the invention may employ an arrangement in which each one of one or more tools may be employed to heat and deform a region of the substrate that includes two or more channels. In this manner, a single tool may be employed to isolate more than one reaction chamber.
Each one of these depressions 191 may be configured to receive a tool, such as shown in
While various embodiments set forth hereinabove describe the substrate 41 being formed of a single type of plastic, the invention includes embodiments in which the substrate 41 is formed of more than one type of plastic. For example,
The assay card of the invention, in accordance with any of the various embodiments described hereinabove, may be configured for use in a system. For example,
Isolating the reaction chambers 18 may accomplish a number of desirable objectives, including prevention of cross-contamination of the reactions in the respective reaction chambers by preventing diffusion of reactants from one chamber to another through the channels, and preclusion of air bubbles from entering the reaction chambers. In the assay card and the method of the invention, chamber isolation can be accomplished safely, accurately and reliably. For example, as set forth above, the assay card 10 may be heated by a device, e.g., a heated tool 43 or an ultrasonic horn 51, that is positioned within, or in close proximity to, depressions 191 that are defined in the assay card 10. Such depressions 191 enable the region of the assay card 10 that includes the channel 16 to be heated by a tool that is located on a side of the assay card 10 that is opposite the side on which the channel 16 is located. Such an arrangement may reduce the amount of heat that is required in order to heat the region of the assay card 10 that includes the channel 16, and may reduce the likelihood of unintentionally heating a sample in the reaction chamber or inadvertently softening or deforming any portions of the substrate 41 that are desired to remain intact.
Additional advantages are obtained through use of a tool to heat and deform a region of the substrate that includes two or more channels. Specifically, and as illustrated for example in
Further advantages are obtained where the tool device include a plurality of tools, each one of which may be employed to heat and deform a region of the substrate that includes a channel. Specifically, and as illustrated for example in
Still further, the assay card of the invention, may provide advantages by virtue of the substrate 41 being formed of more than one type of material, e.g., by co-molding. Specifically, and as illustrated for example in
The method of the invention, in accordance with an embodiment thereof, was tested with an assay card 100 such as that shown in
In addition, the above-described test was also performed using various conditions, e.g., temperatures ranging from 180° C. to 250° C. and pressures ranging from about 50 to 150 lbs. In all of these tests, results similar to the result of the above-described test were obtained.
In order to determine whether, and thereby minimize the likelihood that, the high temperature of the tool 43 will be undesirably conducted to an analyte sample located in a reaction chamber 18, further testing was carried out to measure the temperature in a reaction chamber 18 during the application of the tool. A thermocouple (Omega Engineering, model 5SRTC-TT-T-40-36) was embedded in a reaction chamber 18 with a thermally conductive epoxy (Omega Engineering, model OB 200). A soldering iron having a surface temperature of about 300° C. was manually pushed against the channel region of the COP substrate 141 for 3 seconds and the temperature reading from the thermocouple was monitored. The measured temperature was determined to be dependent upon the location of the tool 43 relative to the reaction chamber 18, and ranged from 24° C. to 38° C. The temperature measurement results indicate that the method of the invention would not adversely affect an analyte sample and the polymerase chain reaction (PCR) mix or the reverse-transciptase polymerase chain reaction mixture (RT-PCR) in the reaction chamber 18.
Numerical calculation data was collected in order to describe temperatures in the reaction chambers 18 when tools 43 having a temperature of 200° C. were contacted for 2.5 seconds, the tools 43 being contacted with an assay card 100 that included depressions 191 having a depth of 500 μm. It was determined that the maximum temperature in the reaction chamber 18 was about 34° C. The numerical calculation confirmed again that the hot temperature on the tool 43 would not adversely affect an analyte sample and the PCR mix in the reaction chamber 18.
After the above-described test was performed, DNA amplification by real-time PCR was performed in an assay card 100. A sample of Streptococcus pneumoniae (concentration of 10 k copies/uL) was provided in all 24 chambers of an assay card 100. Then, the channels 16 and the vent channels 20 were occluded by applying a soldering iron having a surface temperature of about 300° C. For comparison purpose, as tool at room temperature was applied to another assay card prepared with the same target sample. DNA amplification by real-time PCR was performed in an Applied Biosystems 7900HT Real-Time PCR System for the two assay cards.
DNA amplification by real-time PCR including reverse-transcriptase (RT) at 48° C. was also performed in an assay card 100. A sample of Flu A was provided in all 24 wells of an assay card 100. Then, the channels 16 and the vent channels 20 were occluded by applying a thermal isolation with a thermal isolation teeth temperature of about 240° C. For comparison purpose, a tool at room temperature was applied to another assay card prepared with the same target sample. DNA amplification by real-time PCR was performed in an Applied Biosystems 7900HT Real-Time PCR System. 5 different concentrations of Flu A samples were amplified in 5 different assay cards.
The invention, according to various embodiments thereof also relates to a method of manufacturing or fabricating a tool device.
At step 1402 of the flowchart illustrated in
At step 1403 of the flowchart illustrated in
At step 1405 of the flowchart illustrated in
Referring now to
The method of manufacturing or fabricating the tool device as set forth above, and the use of same in a method for isolating chambers of an assay card, may provide advantages as compared to conventional manufacturing and fabrication methods. Again, by introducing heat into the staking process, excessive downward forces on the assay card may be greatly reduced or avoided. Furthermore, the amount of heat that is employed to melt and/or deform the substrate material is very localized. Still further, the invention may, in accordance with various embodiments described herein, reduce the thermal mass of any heated surface thus reducing the potential to introduce heat to sensitive areas of the assay card.
Thus, the several aforementioned objects and advantages of the invention are most effectively attained. Those skilled in the art will appreciate that numerous modifications of the exemplary embodiment described hereinabove may be made without departing from the spirit and scope of the invention. For example, the invention may be employed in other biochemical assays, such as isothermal amplification, clinical chemistry assays, and others. Although various exemplary embodiments of the invention have been described and disclosed in detail herein, it should be understood that this invention is in no sense limited thereby.
The present invention may have been made with support from the U.S. Government under United States Air Force Contract No. FA7014-06-C-0017. The U.S. Government may have certain rights in the inventions recited herein.