Thermal isolation of reaction sites on a substrate

A thermal block assembly is provided. The assembly can comprise a substrate comprising a first surface configured with a plurality of reaction sites each reaction site configured to contain a biological sample and a sample block comprising a plurality of pedestals configured to thermally modulate the plurality of biological samples wherein each pedestal is thermally coupled to one of the reaction sites. The assembly can further comprise cooling blocks, slots and insulating rings associated with reaction sites each capable of minimizing heat flow between reaction sites. A method for thermally isolating reaction sites is also provided. The method can comprise providing a substrate including a plurality of reaction sites, each reaction site configured to contain a biological sample, providing a sample block comprising pedestals, each pedestal having a dimension substantially equal to a dimension of the reaction site and thermally coupled to the reaction site, thermally isolating the reaction sites with a thermal isolating feature, modulating the temperature of the pedestals through a sequence of temperature and hold times and cooling the reaction sites with cooling blocks.

BACKGROUND

Polymerase Chain Reaction (PCR) is a frequently used tool in genetic analysis to amplify samples of DNA. The process can involve placing biological samples of DNA into or onto a sample holder suitable to isolate each sample from other samples at a reaction site. Such sample holders are well known in the art and can take many forms such as, but not limited to, microtiter plates, microcards, individual tubes, strips of connected tubes, capillaries, micro arrays and slides. Additionally, the number of samples contained in a sample holder can vary depending, for example, on the type of analysis required, and can be from 1 sample to thousands of samples in or on a single sample holder.

One of the challenges encountered with sample holders designed for multiple functions such as, for example, mixing, fluid transfer, heating and cooling is that it can be difficult to thermally manipulate one region of the holder without thermally affecting adjacent regions. This can also be challenging in the case of processing small, closely spaced samples in, for example, polymerase chain reactions (PCR). Amplifying DNA (Deoxyribose Nucleic Acid) using the PCR process, involves cycling a specially constituted liquid reaction mixture through several different temperature incubation periods in a thermal cycler. The reaction mixture is comprised of various components including the DNA to be amplified and at least two primers sufficiently complementary to the sample DNA to be able to create extension products of the DNA being amplified. A key to PCR is the concept of thermal cycling in a thermal cycling instrument. The thermal cycler can be designed to alternating steps of denaturing DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of double-stranded DNA. In thermal cycling the PCR reaction mixture is repeatedly cycled from high temperatures of around 95° C. for denaturing the DNA, to lower temperatures of approximately 50° C. to 70° C. for primer annealing and extension.

In some previous PCR instruments, sample tubes are inserted into sample wells on a metal block. To perform the PCR process, the temperature of the metal block is cycled according to prescribed temperatures and times specified by the user in a PCR protocol. The cycling is controlled by a computer and associated electronics. As the metal block changes temperature, the samples in the various tubes experience similar temperature changes. Such a heated metal block can be used with any of the sample holders mentioned above. However, when performing PCR on very small samples, for example, the device can be subjected to very demanding thermal protocols not typical in some previous PCR instruments. One such protocol can require one or several samples to be heated to, for example, 95° C. while an adjacent sample or samples needs to be maintained at a temperature substantially different from 95° C. In the case of samples requiring different temperatures the block can heat up not only the desired reaction sites but can also transfer heat to the surrounding area and other samples. This thermal transfer can adversely influence the other samples and negatively affect the amplification or incubation of the samples. It would therefore be desirable to have sample holders and that can be used to conduct, for example, thermal cycling reactions for PCR without thermally affecting adjacent reactions.

SUMMARY

In one embodiment of the present invention, a thermal block assembly is provided. The assembly comprises a substrate comprising a first surface configured with a plurality of reaction sites each reaction site configured to contain a biological sample wherein the substrate is configured with a feature to improve thermal isolation of the reaction sites and a sample block comprising a plurality of pedestals configured to thermally modulate the plurality of biological samples wherein each pedestal is thermally coupled to one of the reaction sites.

In another embodiment, a thermal block assembly is provided. The assembly comprises a substrate comprising a first surface configured with a plurality of reaction sites each reaction site configured to contain a biological sample, a sample block comprising a plurality of pedestals configured in one or more rows, wherein each pedestal is thermally coupled to one of the reaction sites and configured with a feature to improve thermal isolation of the reaction sites and a plurality of cooling blocks, each cooling block associated with one reaction site and capable of minimizing heat flow between reaction sites.

In another embodiment a method for thermally isolating reaction sites is provided. The method comprises the steps of providing a substrate including a plurality of reaction sites, each reaction site configured to contain a biological sample, providing a sample block comprising pedestals, each pedestal having a dimension substantially equal to a dimension of the reaction site and thermally coupled to the reaction site, modulating the temperature of the pedestals through a sequence of temperature and hold times with thermoelectric devices, thermally isolating the reaction sites from each other and cooling the reaction sites with cooling blocks.

Additional aspects, features, and advantages of the present invention are set forth in the following description and claims, particularly when considered in conjunction with the accompanying drawings in which like parts bear like reference numbers.

DESCRIPTION OF VARIOUS EMBODIMENTS

Embodiments of apparatuses, systems and methods for providing thermal isolation between elements of a substrate are described in this specification. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.

It will be appreciated that there is an implied “about” prior to the temperatures, distances, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. In this application the term “substrate” is used to refer to all sample holder formats known in the art and is not intended to be limiting to any specific format. Further, it is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

The present teachings disclose various embodiments of a substrate and sample block having improved thermal isolation throughout the devices. As will be discussed in more detail subsequently, various embodiments of substrates and sample blocks having an improved thermal isolation provide for desired performance of bio-analysis instrumentation utilizing such devices.

In the case of PCR, for example, it can be desirable to change the sample temperature between the required temperatures in the cycle as quickly as possible for several reasons. First the chemical reaction has an optimum temperature for each of its stages and as such less time spent at non-optimum temperatures can mean a better chemical result is achieved. Secondly a minimum time is usually required at any given set point which sets minimum cycle time for each protocol and any time spent in transition between set points adds to this minimum time. Since the number of cycles is usually quite large, this transition time can significantly add to the total time needed to complete the amplification.

The absolute temperature that each reaction site attains during each step of the protocol is critical to the yield of product. It is therefore advantageous to thermally isolate regions of the substrate in order to minimize the influence of one region on another. The geometries that can be found in substrates can frequently be small resulting in hot or warm regions being located very close to cooler regions of the device. This close proximity can result in cooler regions being warmer than intended due to heat flow through the device from the hot or warm region to the cool region thereby compromising the performance of the cooler regions. Additionally the small geometries can make it difficult to thermally isolate the hot and cool regions.

FIG. 1depicts an embodiment not uncommon in the art. In this embodiment substrate140is shown upon which are two rows110and120of eight reaction sites. Coupled to the underside of substrate140is heated block130in the shape of a bar under row110and opposite the substrate from row110. Such a relationship between a substrate, reaction sites and heated block is also not uncommon in the art. Heated blocks are also well known in the art and are frequently fabricated of materials with high thermal conductivities. Some examples of suitable materials for heated blocks include, but are not limited to, silver, gold, aluminum, magnesium and copper. In some embodiments a heated block can be fabricated in a rectangular geometry, but can also be circular, square or any other shape suitable for the application. Frequently a surface of the block has an array of depressions capable of receiving a sample containment device sometimes referred to as a plate or tube. Array sizes vary and can be, for example, 8, 16, 24, 48, 96, 384, 1536 or more. In some applications the metal block can include a flat surface without depressions to accommodate an array of reaction sites that may have a flat surface such as, for example, a glass slide.

In this embodiment block130was fabricated as a metallic bar with a flat upper surface and dimensioned to thermally couple to the underside of substrate140opposite the reaction sites of row110. Row120was kept at room temperature or approximately 27° C. Block130was heated to 95° C. and the temperature of rows110and120were determined.FIG. 2Adepicts the thermal results. It is clear from the thermal map that both rows110and120are heated to approximate the same temperature and the individual reaction sites are not distinguishable.FIG. 2Bshows a more detailed interaction between heated row110and unheated row120. Heated row110is shown to be a uniform high temperature as indicated by the uniform shading. This temperature provides a high enough temperature difference between rows110and120that heat flows across and through substrate140from row110to row120. The temperature scale included inFIG. 2Bindicates the temperature of the region of unheated row120closest to heated row110is approximately 50° C. Additionally it can also be seen that there is a significant temperature gradient across unheated row120of approximately 20° C. These results demonstrate the magnitude of interaction between closely spaced adjacent rows having significantly different temperature requirements. When performing PCR, it can be common to heat one row to 95° C. for as long as 10 minutes, for example, while requiring an adjacent row to be maintained at less than 37° C. To ensure no adverse thermal interaction by a heated row on an unheated adjacent row it would be advantageous to minimize the thermal effect on the adjacent row. The data presented inFIGS. 2A and 2Bconfirms the thermal interaction between adjacent rows and demonstrates the need for an embodiment that would minimize this effect.

There are many factors that can contribute to thermal interaction between reaction sites. Some of these factors can include, but not be limited to, spacing, reaction site size which can be related to spacing, the temperature difference between the reaction sites and substrate materials. One skilled in the art would understand that one or a combination of factors may be required to achieve the degree of thermal isolation desired. With this understanding, solutions for thermal isolation are presented as follows.

Substrate Materials

Materials used for fabricating substrates can be, for example, elastomers such as, for example, polymers that display rubber-like elasticity. One skilled in the art will further know that there are many types of elastomers, such, for example as saturated rubbers, unsaturated rubbers, thermoplastic elastomers and polysulfide rubber. These materials all exhibit relatively poor thermal conductivity, and as such provide a level of thermal isolation between adjacent rows of reaction sites. As an example, thermally conductive elastomers from CoolPoly® exhibit thermal conductivities of up to 5 W/mK. As a comparison, metals such as Aluminum, Copper, Gold and Silver exhibit thermal conductivities from 205 W/mK to 406 W/mK. However as discussed above, even though they are poor thermal conductors they still conduct heat sufficiently to affect the temperature of reactions in adjacent wells of a substrates.

Substrates can also be fabricated from polymers including, for example, polypropylene, although other polymers could also be used. By way of example, polypropylene has a thermal conductivity on the order of 0.1 W/mK to 0.22 W/mK. This degree of conductivity can still result in reaction sites affecting other reaction sites if the temperature difference between the sites is sufficiently large as presented above.

Thermal Block Geometry

It is evident from the thermal map ofFIG. 2Bthat heating sample block130to 95° C. provided enough heat to row110and substrate140to heat at least a portion of row120to approximately 50° C. which as presented above would be sufficient to adversely affect the reactions in row120.FIG. 3Adepicts a thermal map which is similar to the thermal map depicted inFIG. 2A. However, in the embodiment represented byFIG. 3A, block130ofFIG. 2Bhas been replaced with block150. Block150differs from block130in that the flat upper surface of block130has been replaced with a series of pedestals. The top surface of each pedestal can have a shape that is substantially circular (as illustrated inFIG. 3A), square, oval, rectangular or any other shape suitable for the application. Additionally, the top surface450(as illustrated inFIG. 4) of each pedestal can, for example, have an area that can be substantially equal to the area of the contact surface of a corresponding reaction site, smaller than the area of the contact surface of a corresponding reaction site or larger than the area of the contact surface of a corresponding reaction site. Regardless of shape and size block150can be located under substrate140with each pedestal surface450(as illustrated inFIG. 4) aligning with a region of a substrate surface620(as illustrated inFIG. 6B) opposite the reaction site.

Thermal block150was set to 95° C. and the results are depicted inFIG. 3A.FIG. 3Aclearly shows the individual reaction sites that were not visible inFIG. 2A. The addition of pedestals to thermal block150demonstrates more localized heating of the reaction sites of row110without dramatically affecting row120.FIG. 3Bprovides more thermal detail of rows110and120. Once again, row110is shown with uniform shading which indicates a uniform temperature. The temperature scale provided inFIG. 3Bnow indicates that the warmest region of row120is now approximately 46° C., and the gradient across the reaction sites is approximately 16° C. Comparing these results to the results shown inFIG. 2Aindicates the addition of the pedestals to thermal block150reduces the absolute temperature of row120and the size of the thermal gradient across the reaction sites by approximately 4° C. or approximately an 8% improvement. One skilled in the art will recognize that the dimensions and geometries of the pedestals and reaction site dimensions can be modified for other applications as presented above.

Another embodiment to improve thermal isolation between adjacent reaction sites as compared toFIG. 2Ais depicted inFIG. 4. Thermal block440is shown with pedestals430and pedestal surfaces450. In this example the shape of the pedestals is substantially circular. One skilled in the art however, would recognize that the pedestals can be any shape or size as presented above. In this case the pedestals have been modified to include a radius around an upper circumference to accommodate rings410and420. Rings410are shown to have a smaller thickness than rings420and are substantially circular to match the shape of the pedestals. One skilled in the art would know that the rings can be any shape or size to match the shape of the pedestals, and therefore may not be described as rings in some embodiments. Rings410and420can also comprise a thermal insulating material to further prevent heat leak from thermal block440to an adjacent row. The results of heating thermal block440to 95° C. for 10 minutes are shown inFIG. 5. According to the scale inFIG. 5, row120was heated by row110to a maximum temperature of approximately 43° C.FIG. 5also illustrates that the pedestals with rings410are warmer at the adjacent reaction sites than the pedestals with rings420, thus establishing that modifying the geometry of the pedestals and including thermal resistive rings reduces the thermal interactions between the rows. In this embodiment, unheated row120reached a maximum temperature of approximately 43° C. with a thermal gradient of 14° C. which translates to an overall improvement in the temperature of row120of 14% as compared to the data inFIG. 2A.

Slots Between Reaction Sites

FIG. 6Adepicts another embodiment in which slots610were located between rows110and120. Slots610can introduce a thermal resistance between the rows and thereby decrease the flow of heat from a heated row to an unheated row. Factors that can affect the flow of heat in the device can include, but not be limited to, length of the gap, width of the gap and the location of the gap.FIG. 6Ashows a perspective view of a portion of a substrate that includes heated block150located under substrate140and aligned opposite the reaction sites of row110. Block150has the same configuration as block150ofFIG. 4.FIG. 6Afurther shows slots located between each reaction site of row110and row120. The distance between row110and row120is 10 mm and the dimensions of the slots is 1 mm×4 mm One skilled in the art would know that the dimensions of the slots and distance between rows, are arbitrary and selected to provide the performance required by the embodiment. In this example, the distance between rows and slot dimensions are based on the dimensions of the substrate and corresponding reaction sites as well as the density of the reaction sites.

To determine the effectiveness of this embodiment, block150was heated to 95° C. and held at that temperature for 10 minutes. As presented above this time/temperature combination is not uncommon in a PCR protocol and can represent a worst case scenario for heat leak to adjacent reaction sites.FIG. 6Bshows thermal results and is a view of the underside of substrate140. Block150is oriented such that the upper four pedestals are fitted with 0.5 mm thick rings and the lower four pedestals are fitted with 1.0 mm thick rings. Slots610retarded the flow of heat from row110to row120such that the maximum temperature of the upper4reaction sites of row120ranged from 36° C.-38° C., and maximum temperature of the lower4reaction sites ranged from 35° C.-37° C.

The results illustrated inFIG. 6Billustrate that the use of slots between reaction sites can also be an effective way of breaking the heat flow to considerably lower the maximum temperature in the adjacent row as compared toFIG. 2A. One skilled in the art can see that further optimization of the slot is possible. One such embodiment is shown inFIG. 7Athat shows slots slightly offset between the reaction sites.FIG. 7Billustrates the thermal results of heating the thermal block to 95° C. and maintaining 95° C. for 10 minutes. As indicated by the temperature scale the maximum temperature of the adjacent reaction sites has dropped to approximately 34° C. This result demonstrates the effectiveness of slots to improve thermal isolation between reaction sites as compared toFIG. 2A

Row Spacing Vs. Ambient

According to another embodiment thermal isolation can be improved overFIG. 1by varying the distance between the rows of reaction sites.

FIG. 8shows the spacing increased from the spacing presented above inFIG. 1. After heated block150is heated to 95° C. and held at 95° C. for 10 minutes at an ambient temperature of 25° C.,FIG. 9Ashows a thermal map representing the results after the 10 minute hold time. Referring to the scale inFIG. 9Athe temperature of adjacent row120achieves a maximum temperature of approximately 26° C. This maximum temperature is a significant improvement in isolation when compared to the spacing shown inFIG. 1. InFIG. 9Bthe effect of an increase in ambient to 30° C. is shown. Referring to the temperature scale the increase in ambient of 5° C. results in approximately 4° C. increase in row120.FIG. 10Ashows similar results for a row spacing of 15 mm at 25° C. ambient conditions, andFIG. 10Bshows the results for the same 15 mm simulation at 30° C. ambient. This data demonstrates that increasing the spacing between the reaction sites and reducing the ambient temperature can result in improved thermal isolation between reaction sites as compared toFIG. 2A. One skilled in the art will realize that the chosen row spacing can be dependent on the geometry of the substrate as well as the reaction site density.

Cooling Blocks

In yet another embodiment the adjacent row presented above can be cooled.FIG. 11depicts an embodiment of the underside of substrate140. Heated block150is also shown located opposite the reaction sites of row110, as presented previously, and cooling blocks1110are shown opposite the reaction sites of row120with one cooling block aligned with each reaction site.

In one embodiment, cooling blocks1110can be used to provide greater thermal mass to adjacent row120in much the same way that a heat sink can be used to remove heat from a hot object to ambient. As such, it would be advantageous for the cooling blocks to comprise a thermally conductive material such as, for example aluminum and copper. One skilled in the art can appreciate that the effectiveness of the cooling blocks in this embodiment can also be dependent, for example, on the size of cooling blocks1110relative to the size of the reaction sites.

An alternative embodiment can provide active cooling to cooling blocks1110. Active cooling can be provided by any number of implementations known in the art. For example various implementations of active cooling can include, but not be limited to, thermoelectric cooling, chilled fluid pumped through the cooling blocks and heat pipes. The thermal results of an active cooling solution are shown inFIG. 12. Heated block150was heated to 95° C. and held at 95° C. for 10 minutes and cooling blocks1110were cooled and held at 25° C. after which the thermal map ofFIG. 12was captured. As can be seen on the temperature scale the warmest edges of row120are maintained at approximately 28° C. which is approximately one half of the maximum temperature of row120inFIG. 2A. It can also be seen inFIG. 12that the thermal gradient across adjacent row120has been significantly reduced as compared to the thermal map ofFIG. 2Afurther demonstrating the effectiveness of cooling blocks610in thermally isolating the reaction sites.

While the principles of this invention have been described in connection with various embodiments of a thermal block and substrate, it should be understood clearly that these descriptions are made only by way of example and are not intended to limit the scope of the invention. What has been disclosed herein has been provided for the purposes of illustration and description. It is not intended to be exhaustive or to limit what is disclosed to the precise forms described. Many modifications and variations will be apparent to the practitioner skilled in the art. What is disclosed was chosen and described in order to best explain the principles and practical application of the disclosed embodiments of the art described, thereby enabling others skilled in the art to understand the various embodiments and various modifications that are suited to the particular use contemplated. It is intended that the scope of what is disclosed be defined by the following claims and their equivalence.