Patent ID: 12220706

DETAILED DESCRIPTION OF THE INVENTION

Exemplary embodiments of the present disclosure include apparatus and methods for polymerase chain reaction (PCR) thermal cycling. Particular embodiments are discussed below with reference to the drawings included in the figures.

Referring initially toFIGS.1-3, an apparatus100for thermal cycling is shown comprising a housing160with a retaining member150coupled to housing160. In the embodiment shown, retaining member150is configured to retain a sample module180. In particular embodiments, sample module180may comprise one or more sample chambers suitable for use in a polymerase chain reaction (PCR) process. In certain embodiments, sample module180is a separate component from apparatus100that can be inserted and removed from retaining member150, and sample module180is shown in the figures for illustration of operational aspects of apparatus100. For purposes of clarity, not all components illustrated in the figures are labeled with reference numbers. It is also understood that the components shown inFIGS.2and3that are not labeled are equivalent to those labeled inFIG.1. Certain components included in the view ofFIG.1are also not shown inFIGS.2and3for purposes of clarity. It is understood that the embodiment shown inFIGS.2and3includes each of the components shown inFIG.1.

In the illustrated embodiment, apparatus100further comprises a first pivot arm110configured to pivot around a first pivot axis101and a second pivot arm120configured to pivot around a second pivot axis102. In the embodiment shown, apparatus100also comprises a first thermal mass111and a second thermal mass112each coupled to first pivot arm110, as well as a third thermal mass123and a fourth thermal mass124each coupled to second pivot arm120. In particular embodiments, thermal masses111,112,123and124may comprise material with a high coefficient of thermal conductivity. In specific embodiments, thermal masses111,112,123and124may comprise copper or aluminum blocks of material.

First arm110and second arm120are shown in a first position inFIG.2and in a second position inFIG.3. InFIG.1, first arm110and second arm120are shown in an intermediate position between the first and second positions. First thermal mass111and third thermal mass123are proximal to the retaining member when first pivot arm110and the second pivot arm120are in the first position shown inFIG.2. In addition, second thermal mass112and the fourth thermal mass124are proximal to retaining member150when first pivot arm110and second pivot arm120are in the second position shown inFIG.3.

As shown inFIG.1, sample module180comprises a first side181(e.g. the top side in the orientation shown) and a second side182(e.g. the bottom side in the orientation shown). When sample module180is retained in retaining member150, first and second pivot arms110and120can pivot between the first and second position such that certain thermal masses111,112,123and124alternately contact the first side181and second side182of sample module180. In the illustrated embodiment shown, first thermal mass111is in contact with first side181and third thermal mass123is in contact with second side182when first pivot arm110and second pivot arm120are in the first position shown inFIG.2. In addition, second thermal mass112is in contact with second side182and the fourth thermal mass124is in contact with the first side181when first pivot arm110and second pivot arm120are in the second position shown inFIG.3.

In the embodiment shown, first thermal mass111comprises a heating element117and third thermal mass123comprises a heating element127. In certain embodiments, heating elements117and127are electrically coupled to a power source135via wires136,137,138and139that have sufficient length for to allow first and second pivot arms110,120to pivot from the first position to the second position while remaining electrically coupled to power source135. During operation of apparatus100, heating elements117and127can increase the temperature of first thermal mass111and third thermal mass123. In the embodiment shown, second thermal mass112and fourth thermal mass124do not include heating elements. Accordingly, heating elements117and127can be activated to increase the temperature range of first thermal mass111and third thermal mass123to a temperature range that is higher than the temperature range of second thermal mass112and fourth thermal mass124.

In specific embodiments, first thermal mass111and third thermal mass123are maintained at a temperature range at or above the denaturing temperature of the target oligomer(s), and second thermal mass112and fourth thermal mass124are maintained at or below the annealing temperature of the target oligomer(s). The first thermal mass111and the third thermal mass123could therefore be referred to as heating masses, and the second thermal mass112and the fourth thermal mass124could be referred to as cooling masses. During operation of apparatus100, first and second arms110and120can pivot from the first position shown inFIG.2to the second position shown inFIG.3to cycle the temperature of the contents of sample module180from a higher temperature to a lower temperature as used in PCR processes.

The heating masses and cooling masses can be used in at least two major modes of operation—a steady-state mode or a transient mode. In a steady-state mode, the heating masses are set to the denaturing temperature, and the cooling masses are set to the annealing temperature. Alternatively, the heating masses can be set to a temperature with a range centered around the denaturing temperature, and the cooling masses can be set to begin at a temperature within a range centered around the annealing temperature. During the denaturing phase, the heating masses remain clamped around the sample module180until the temperature of the fluid in the sample chambers18reaches or substantially reaches the temperature of the heating masses. During the annealing phase, the cooling masses remain clamped around the sample module180until the temperature of the fluid in the sample chambers18reaches or substantially reaches the temperature of the cooling masses. In steady-state mode, the heating masses could be maintained at a constant temperature within a temperature range such as 85-98 C, or 90-98 C, or even 94-98 C. The cooling masses could be maintained within the range of 50-70 C, or 55-65 C, or 58-62 C. These ranges are merely illustrative, as the desired temperatures may vary depending on the assay.

In a transient mode of operation (or alternatively called overshoot mode), the heating and cooling masses are set to temperatures that overshoot the desired denature and anneal temperatures, and they contact the sample module180for only enough time for the fluid in the sample chambers18to reach the desired denaturing or annealing temperatures. For example, the heating masses could be set to maintain a steady temperature of 120 Celsius, and the cooling masses could begin at room temperature, around 24 Celsius. The sample module180and fluid within the sample chambers183-190might also begin at or near room temperature. Even though the heating masses are at 120 Celsius, they contact the sample module180only for sufficient time for the fluid within the sample chambers183-190to reach the desired denaturing temperature, e.g. 95 Celsius. Similarly, the cooling masses contact the sample module180only for enough time for the fluid within the sample chambers183-190to reach the desired annealing temperature, e.g. 60 C. In one embodiment, the time required for fluid within the sample chambers183-190to increase in temperature from 60 to 95 Celsius ranges from a couple to several seconds, and the time required for the fluid within the sample chambers183-190to decrease from 95 to 60 Celsius ranges from one to several seconds. The heating masses can be maintained at a steady temperature within the range of 98-130 C, 110-130 C, or 115-125 C. The cooling masses can begin thermal-cycling at a temperature within the range of 15-40 C, 20-30 C, or 20-25 C.

Note, however, that the denaturing and annealing temperatures can be dictated by the chemistry and assay requirements, and the contacting times for heating and cooling may be adjusted accordingly. For example, instead of a 35-Celsius cycling delta (difference between 95 Celsius and 60 Celsius), a smaller cycling delta might be preferred. A 20-Celsius delta might be seen by cycling between denaturing and annealing temperatures of 85 Celsius and 65 Celsius. An even smaller cycling delta might be seen by cycling, for example, from around 70 Celsius to mid-80's Celsius.

In the embodiment shown, apparatus100comprises a controller130configured to control apparatus100including the movement of first pivot arm110and second pivot arm120between the first position and second positions. For example, controller130can actuate a first actuator119coupled to first pivot arm110and actuate a second actuator coupled129to second pivot arm120. In other embodiments, controller130may actuate a single actuator that is coupled to both first pivot arm110and second pivot arm120via a belt, gear, or other suitable configuration.

In certain embodiments, controller130is configured to maintain first pivot arm110and second pivot arm120in the first position for a specific period of time and maintain first pivot arm110and second pivot arm120in the second position for a different period of time. In some embodiments, controller130is configured to maintain first pivot arm110and second pivot arm120in the first position for an initial time period, and then alternate first pivot arm110and second pivot arm120between the first and second position for different time periods. For example, controller130can maintain first pivot arm110and second pivot arm120in the first position for an initial period of several minutes and then alternate first pivot arm110and second pivot arm120between the first and second positions for periods of time at each position ranging from about a second to several seconds.

In certain embodiments, apparatus100comprises an illumination module145configured to illuminate contents of sample module180retained by the retaining member150. Apparatus150may further comprise a detection module147configured to detect contents of sample module180illuminated by illumination module145. In certain embodiments, detection module147is configured to detect contents of sample module180that fluoresce in response to excitation energy provided by illumination module145. In the illustrated embodiment, retaining member150comprises illumination module145and detection module147. The location of illumination module145and detection module147shown is for illustrative purposes only, and in other embodiments retaining member150may comprise different locations for illumination module145and detection module147(e.g. above and below sample module180, or both proximal to one end of sample module180, etc.). In other embodiments, other components of apparatus100may comprise an illumination module and a detection module. For example, first thermal mass111, second thermal mass112, third thermal mass123and/or fourth thermal mass124may comprise an illumination module and/or a detection module. In certain embodiments, illumination module145may comprise light-emitting diodes (LEDs) or lasers emitting light at different frequencies. In particular embodiments, detection module147may comprise photodetectors or other light-sensing elements. In specific embodiments, apparatus100may comprise fiber-optic elements in communication with detection module147.

During operation, apparatus100provides efficient thermal cycling of sample module180. For example, the ability to provide heat transfer simultaneously to both first side181and second side182can increase the heat transfer rate to the contents of sample module180as compared to systems that provide heat transfer to only one side of a sample module180. Contacting each side of sample module180with first and third thermal masses111,123, for example, transfers heat through more of the available surface area of the sample module180compared to single-sided heating systems. This can reduce the amount of time needed to bring the contents of sample module180to the desired temperature during a cycle. Certain sample processing techniques require a significant number of cycles (e.g. 50-100), so reducing the time for a single cycle can have a substantial reduction in the overall processing time.

Similarly, engaging both sides of sample module180with second and fourth thermal masses112,124can reduce the amount of time needed to lower the temperature of sample module180to the desired temperature range. The increased surface area contacted by second and fourth thermal masses112,124(as compared to single sided contact embodiments) can also reduce the amount of time required for each thermal cycle.

In the embodiment shown, first pivot arm110is shown comprising a first bracket arm113and a second bracket arm115with a spacer bar116extending between them. Similarly, second pivot arm120is shown comprising a first bracket arm121and a second bracket arm125with a spacer bar126extending between them. For discussion purposes, first pivot arm110includes first bracket arm113, second bracket arm115and spacer bar116, while second pivot arm120includes first bracket arm121, second bracket arm125and spacer bar126. It is understood that other embodiments may comprise a different configuration for first and second pivot arms110,120, including for example, single arms for each pivot arm.

Referring now toFIG.4, an embodiment of apparatus100is shown that is equivalent to the embodiment shown inFIGS.1-3, but also includes cooling elements161and162. In certain embodiments, cooling elements161and162may be configured as electric fans configured to direct air from outside housing160into housing160. For purposes of clarity, not all components illustrated inFIG.4are labeled with reference numbers. It is understood that the components shown inFIG.4operate in a manner equivalent to those previously discussed with respect toFIGS.1-3.

As previously discussed, during operation of apparatus100, heating elements117and127heat first and third thermal masses111,123which transfer the heat to sample module180. As a result of the heat generated by heating elements117and127and transferred to other components of apparatus100, the temperature within housing160can become elevated. As a result, the temperature of second and fourth thermal masses112,124can also increase over time. Cooling elements161and162are positioned within housing160such that they direct air from outside housing160toward second thermal mass112and fourth thermal mass124when first and second pivot arms110,120are in the first position (i.e. second thermal mass112and fourth thermal mass124are not in contact with sample module180). The air from outside housing160will typically be a lower temperature than the air within housing160during operation of apparatus100. The air from outside housing160will also typically be a lower temperature than the temperature of second thermal mass112and fourth thermal mass124. Accordingly, cooling elements161and162can direct air to second thermal mass112and fourth thermal mass124that reduces the temperature of second thermal mass112and fourth thermal mass124, for example, by convective heat transfer. In certain embodiments, apparatus100may comprise one or more vents163to increase air flow within housing160and reduce the temperature increase within housing160during operation of apparatus100. If desired, the vents163can be located on the wall opposite that with the cooling elements161and162such that cooler external air tends to interact with and flow past the cooling second and fourth thermal masses112and124more than the heating first and third thermal masses111and123. In certain embodiments, vent163may comprise a fan configured to evacuate air from within housing160.

Referring now toFIG.5, an embodiment of sample module180is shown in an exploded view. In certain embodiments, sample module180may be formed by heat-sealing biaxially oriented polypropylene film (BOPP) to a polypropylene base material. In the embodiment shown, sample module180comprises a core layer170that is generally planar and comprises a first major face173, a second major face174and an outer edge178. Sample module180further comprises a first film171bonded to first major face173and a second film172bonded to second major face174of a core layer170. In the embodiment shown, sample module180comprises first side181on first film171and second side182on second film172.

In addition, sample module180comprises an inlet176, channel175and multiple sample chambers183-190in fluid communication with corresponding air spring chambers191-198. In the embodiment shown, inlet176extends through the thickness of first film171, while channel175is formed in the first major face173(but does not extend through to the second major face174), and sample chambers183-190and air spring chambers191-198are formed by cutting out portions of core layer170. Core layer170comprises an inner edge177that extends along sample chambers183-190and air spring chambers191-198. Inner edge177, first film171and second film172define a volume179for sample chambers183-190and air spring chambers191-198. First film171and core layer170define channel175.

Air spring chambers191-198can be configured such that fluid can flow from sample chambers183-190to the corresponding air spring chamber191-198. In certain embodiments, air spring chambers191-198can be sized and configured such that the pressure within sample chambers183-190and air spring chambers191-198is approximately 20 pounds-per-square inch gauge (psig) as fluid begins to flow from a sample chamber to an air spring chamber—at which point all of the air within the previously empty chip has been compressed into the air spring chambers191-198. In the embodiment shown, sample chambers183-190are configured such that the length L of each sample chamber183-190is greater than the width W of each sample chamber183-190.

FIGS.11and12further illustrate aspects of air spring chambers and sample chambers. In the embodiment shown, sample module180comprises inlet176, channel175, sample chambers187-190and air spring chambers195-198.FIG.11illustrates sample module180before fluid enters, whileFIG.12illustrates sample module180after sample fluid199enters sample chambers187-190via inlet176and channel175.

As shown inFIG.12, when sample fluid199is loaded into sample module180, sample fluid199fills sample chambers187-190. Air displaced from sample chambers187-190by sample fluid199is compressed in air spring chambers195-198. As previously discussed, the U-shaped configuration of sample chambers187-190and air spring chambers195-198permit optical excitation and detection of labeled analytes through end portions201-204that contain sample fluid199. In particular embodiments, at least half of the total empty air volume in sample chambers187-190is compressed into air spring chambers195-198once sample fluid199is loaded onto sample module180.

In the embodiment shown inFIG.12, sample fluid199can be introduced into sample module180via inlet176and channel175. Sample fluid199can then be transferred to sample chambers187-190by applying pressure via inlet176. The air displaced from channel175and sample chambers187-190can then be compressed in air spring chambers195-198. In certain embodiments, air is compressed in air spring chambers195-198from 5 to 50 pounds-per-square inch gauge (psig), 10 to 30 pounds-per-square inch gauge (psig), or 15 to 25 pounds-per-square inch gauge (psig).

In the embodiment ofFIG.5, illumination module145comprises a plurality of illumination elements145A-145H configured to illuminate sample chambers183-190, respectively. In certain embodiments, illumination elements145A-145H may be individual light emitting diodes (LEDs) or lasers. In addition, detection module147comprises detection elements147A-147H configured to detect a response signal (e.g. a fluorescent signal resulting from illumination by illumination elements145A-145H) from sample chambers183-190, respectively. In the embodiment shown, illumination elements145A-145H are configured to illuminate sample chambers183-190along the length L of sample chambers183-190. Similarly, detection elements147A-147H configured to detect a response along the length L of sample chambers183-190. As illustrated inFIG.5, illumination element145A is shown illuminating sample chamber183with an excitation signal148and detection element147A is shown detecting a response signal149from sample chamber183. As shown, excitation signal148and response signal149are emitted and detected in a direction parallel to the length (e.g. the largest dimension of sample chamber183). Such a configuration can be beneficial in detecting a response signal from a small volume contained within the sample chamber. It is understood that illumination elements145B-145H and detection elements147B-147H are configured equivalent to illumination element145A and detection element147A, respectively. Accordingly, illumination module145and detection module147can provide excitation signals and detect response signals to a plurality of sample chambers on a single sample module180. In certain embodiments, illumination elements145A-145H can emit excitation signals with different wavelengths and/or detection elements147A-147H can detect signals of different wavelengths. In particular embodiments, sample chambers183-190may comprise different reagents that react with different target analytes to provide different response signals. Accordingly, a single sample can be loaded into sample module180and simultaneously analyzed for multiple target analytes.

FIG.6shows another embodiment in which the apparatus is enclosed within a housing260, and a sample module280is loaded vertically rather than horizontally. The housing260has at least one vent263for enabling a cooling element261and a cooling element262(on the opposite side, as shown inFIG.7) to exchange hot air within the device with cooler ambient air. Views A-D show the progression of loading a sample module280through a sample module port264into the apparatus and closing a lid265.

FIG.7is a perspective view of the embodiment inFIG.6, where the housing260is transparent so that the internal elements are visible. Except where otherwise stated, the major internal elements ofFIGS.7-10operate in a fashion similar to the elements of the embodiment shown inFIGS.1-4. For example, the first pivot arm210, second pivot arm220, first through fourth thermal masses211,212,223,224, first actuator219, second actuator229, controller230, and cooling elements261,262ofFIG.7operate similarly to the first pivot arm110, second pivot arm120, first through fourth thermal masses111,112,123,124, first actuator119, second actuator129, controller130, and cooling elements161,162ofFIGS.1-4. A relay unit231contains one or more relays for switching and/or modulating current to heating elements, as dictated by the controller230.

FIG.8is a perspective view of the embodiment ofFIG.6with portions of the housing either removed or made transparent so elements within are visible, and where all thermal masses are unclamped from the sample module. In this embodiment, first thermal mass211and second thermal mass212are each coupled to first pivot arm210, while third thermal mass223and fourth thermal mass224each coupled to second pivot arm220. In the illustrated embodiment, first pivot arm210comprises a first bracket arm213and a second bracket arm215. Similarly, second pivot arm220comprises a first bracket arm221and a second bracket arm225. In this embodiment, the illumination module elements245A-H are contained within one of the cooling thermal masses, such as the third thermal mass223. Small ports through the sample-module-facing side of the thermal mass223allow light from the illumination module elements245to pass through and excite fluorescent material within the sample module280when the cooling thermal masses are clamped around the sample module280. A detection module247is embedded within one of the retaining members250. The detection module247contains one or more detection module elements247A-H, which measure the emitted fluorescence from within the sample chambers of the sample module280. The heating thermal masses such as the first thermal mass211and the third thermal mass223have ports218for heating elements, as well as one or more ports for214for temperature measurement probes. The temperature measurement probes can comprise but are not limited to thermocouples, resistance temperature detectors (RTDs), and thermistors. The heating elements can be controlled by the controller230and switched on/off using one or more relays in a relay unit231(shown inFIG.9). Control of the heating elements can be based on a feedback loop with measurements from temperature measurement probes.

FIG.9is a perspective view of the embodiment ofFIG.8where the second and fourth thermal masses are clamped around the sample module. The controller230receives power through a power input232. The controller can optionally comprise a computer connector233for sending and/or receiving data and/or commands to or from a computer. However, other common means of connectivity can be included, such as Bluetooth and/or WiFi. Regarding cooling of the sample module280, the cooling second and fourth thermal masses212,224can optionally comprise cooling fins or other features that increase surface area for enhanced cooling.

FIG.10is a perspective view of the embodiment ofFIG.8where the first and third thermal masses are clamped around the sample module. Ports in the fourth thermal mass224allow light from the illumination module245to pass through and excite fluorescent material within the sample module280when the fourth thermal mass224is placed in optical communication with the sample module280.

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

V. REFERENCES

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