TECHNICAL FIELD
The present invention relates to a method and an apparatus for performing amplification reaction of nucleic acids in a sample.
BACKGROUND
Polymerase chain reaction (PCR) is increasingly important to molecular biology, food safety and environmental monitoring. A large number of biological researchers use PCR in their work on nucleic acid analyses, due to its high sensitivity and specificity. The time cycle of a PCR is typically in the order of an hour, primarily due to a time-consuming PCR thermal cycling process that is adapted to heat and cool reactors containing the sample to different temperatures for DNA denaturation, annealing and extension. Typically, the thermal cycling apparatus and method employs moving the reactors between two heating baths whose temperatures are set at the target temperatures as required for nucleic acid amplification reactions. Researchers have been constantly striving to increase the speed of thermal cycling.
Thermoelectric cooler (TEC) or Peltier cooler is also used as the heating/cooling element. However, it provides a typical ramping rate of 1-5 degree C./sec which is rather slow in changing the temperature of the reactor and disadvantageously increases the time of the thermal cycling.
As an attempt to increase the PCR speed by reducing thermal mass, microfabricated PCR reactor with embedded thin film heater and sensor was developed to achieve faster thermal cycling at a cooling rate of 74 degree Celsius/s and a heating rate of around 60-90 degree Celsius/s. However, such a wafer fabrication process for making the PCR device is extremely expensive and thus is impractical in meeting the requirement of low cost disposable applications in biological testing.
Hot and cold air alternately flushing the reactors in a closed chamber to achieve higher temperature ramping than the TEC-based thermal cycler has been described. However, from the heat transfer point of view, air has much lower thermal conductivity and heat capacity than liquid, hence the temperature ramping of the air cycler is slower than that with a liquid. The TEC needs a significant amount of time to heat and cool itself and the heat block above the TEC. Further there is also need to overcome the contact thermal resistance between the heat block and the reactors.
Alternating water flushing cyders were also developed in which water of two different temperatures alternately flush the reactors to achieve PCR speed. However, such devices contain many pumps, valves and tubing connectors which increase the complexity of maintenance and lower the reliability while dealing with high temperature and high pressure. With circulating liquid bath medium, the liquid commonly spills out from the baths.
Traditional water bath PCR cyclers utilize the high thermal conductivity and heat capacity of water to achieve efficient temperature heating and cooling. But, such cyders have large heating baths containing a large volume of water which is hard to manage in loading and disposal, and also makes the heating time to target temperatures too long before thermal cycling can start. Such cyclers also have large device weight and high power consumption. The water tends to vaporize with usage and needs to be topped up. Besides, during the thermal cycling every time the reactor is alternately inserted into the baths, a layer of water remains adhered on the reactor body when taken out of each bath, thereby causing the change in temperature inside the reactor to get slower undesirably.
Researchers also tested moving heated rollers of different temperatures to alternately contact the reactors. However, use of long tubing reactors make it not only cumbersome to install and operate a large array of reactors, but also expensive. When the reactors are in a large array or a panel, it may be challenging to achieve heating uniformity among all the reactors.
The present invention provides an improved method and apparatus for enabling thermal cycling nucleic acid at an ultra-fast speed at affordable cost without using complex and expensive components or consumables. The apparatus is robust, light weight, easy to use, needs a small amount of bath medium in the baths and can handle disposable reactors for the reaction material to avoid cross contamination from one reactor to the next. This invention provides a great positive impact on biological analysis.
SUMMARY
Unless specified otherwise, the term “comprising” and “comprise” and grammatical variants thereof, are intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, unrecited elements. The word “substantially” does not exclude “completely”. The terminologies ‘first bath’, ‘second bath’ . . . ‘sixth bath’ do not constitute the corresponding number of baths in a sequence but merely are names for ease of identification with respect to the purpose they serve. These baths may not represent separate physical entities as some of them may be sharable.
According to a first aspect, apparatus for thermal cycling nucleic acid in a thermal profile is provided. The apparatus employs a reactor holder for holding reactor(s) each accommodating reaction material containing the nucleic acid and the reactor(s) being in any form such as tube(s) or wellplate(s) or chip(s) or cartridge(s), the apparatus comprising: a first bath; and a second bath, bath mediums in the baths being respectively maintainable at two different temperatures; and a transfer means for allowing the reactor(s) to be in the two baths in a plurality of thermal cycles to alternately attain: a predetermined high target temperature THT, and a predetermined low target temperature TLT; and reciprocating means to enable relative reciprocating motion between the holder and at least one bath while the reactor(s) is/are placed in the at least one bath, the relative reciprocating motion being executable either by shaking the at least one bath or shaking the holder or shaking the at least one bath and the holder. The reciprocating motion increases the convectional rate of heat transfer between the bath mediums to the reactor(s) and homogenizes the temperature field inside the bath thereby improving temperature uniformity among the reactors. The reciprocating motion thus increases the temperature ramp-up and ramp-down rates in the reactor(s) thereby increasing the speed of thermal cycling. Similar effect can otherwise be achieved by letting the reactor travel in one direction in the bath thereby requiring a much larger bath size as against the small bath size required with reciprocating motion. Such larger bath sizes not only requires a long time and large power to heat up to the target temperatures for the nucleic acid analysis, but also are too heavy to be portable for many field applications. The small baths in this invention due to the use of reciprocating motion greatly reduces the volume of bath medium required, hence also reducing the times for pre-heating and cooling, thereby reducing overall time for a genetic test. The advantageous impact of the reactor shaking feature has been demonstrated by experimental graphs at FIGS. 10(c) and (d) where the time of thermal cycling with forty cycles has been shown to reduce to less than half.
According to an embodiment, a third bath is provided where the transfer means and the reciprocating means allow the reactor(s) to be in the third bath with the reciprocating motion to attain a predetermined medium target temperature TMT.
The reciprocating motion is substantially in horizontal direction. This is advantageous over vertical direction where the amplitude is limited by the small vertical length of the reactor(s) besides undesirably having inconsistent heat transfer along the vertical length of the reactor(s). Sufficiently submerging the reactor(s) would mitigate this issue but in such a case the upper part of the reactor body above the lower part containing the reaction material to enter the bath medium adds to the thermal mass thereby lowering the speed of the thermal cycling. Reciprocating motion in the horizontal direction overcomes both of these issues. The reciprocating motion is at a frequency above 0.2. Hz, with more than 1 mm amplitude. Such a frequency range substantially improves on the heat transfer between the bath medium and the reactor(s) and hence speeds up the thermal cycling as against merely agitating the bath medium at a much slower speed as in the art for maintaining a more uniform bath temperature.
According to an embodiment, the reciprocating means reduces speed of the reciprocating motion as the reactor(s) approach the target temperatures. This is useful to slow down the change of temperature at the target temperatures before the reactor(s) is/are lifted out of the corresponding bath so that the reactor(s) are subjected to a better temperature control and do not substantially cross the target temperatures.
According to an embodiment, the reciprocating means stops the reciprocating motion during fluorescent imaging of the reactor(s). This is helpful when the imaging system is outside the bath and the reactor(s) need to be stationary for the illumination beam to reliably reach the reaction material and the emitted rays are reliably captured for analyses. According to an alternate embodiment the reciprocating means continues the reciprocating motion during fluorescent imaging of the reactor(s) when optical means for illuminating the reaction material and collecting the emitted light from the reaction material is moving with the reactor(s). This provides faster thermal cycling as the imaging can be conducted without having to stop the reciprocating motion and only for imaging purpose.
According to an embodiment, a fourth bath is provided to allow an additional process for the reactor(s) before the thermal cycling, the additional process being one from the group consisting: reverse transcription-polymerase chain reaction (RT-PCR), hot start process and isothermal amplification reaction, where the transfer means and the reciprocating means allow the reactor(s) to be in the fourth bath with the reciprocating motion to attain an additional process target temperature TAPT. The third and fourth baths advantageously allow flexibility to attain various thermal profiles, depending on the type of the reaction material and the process of analysis. The reciprocating motion increases the temperature ramp-up and ramp-down rates in the reactor(s) thereby increasing the speed of the thermal processing.
According to an embodiment, the apparatus comprises a reactor guard comprising reactor confining means to partially confine the reactor(s) to prevent the reactor(s) from getting deformed under resistive forces of the bath medium and the THIGH when the reactor(s) is/are received in the bath medium comprising high thermal conductivity powder and during the reciprocating motion. The reactor guard may preferably be made up of materials comprising metal or glass or high temperature plastics or ceramics to withstand the resistive forces and high temperature conditions in the bath. The reactor guard is preferably an extension of the reactor holder to minimize the complexity of the structure. According to an embodiment, the reactor guard allows a portion of the reactor(s) to remain exposed for facilitating fluorescent imaging from a direction below the tip(s). The reactor confining means preferably facing the direction of the reciprocating motion to take the impact of the resistive forces.
According to an embodiment, the apparatus further comprises bottom support means in the bath bottom for supporting the bottom tip(s) of the reactor(s) during the reciprocating motion when the bath medium is powder, for reducing the bending moment on the reactor(s) wherein the reciprocating motion to the reactor(s) is provided by any one of the methods: moving the reactor holder, moving the bath bottom, and moving the bath bottom and the reactor holder in opposite directions depending on which mode reduces the bending moment to the maximum extent.
The THT can be set in the region 85-99 degree Celsius for pre-denaturation and denaturation of the nucleic acid, the THT can be set in the region 45-75 degree Celsius for annealing of primers or probes onto nucleic acid or for primer extension, the first and the second baths being for thermal cycling the reactor(s) to attain polymerase chain reaction (PCR) amplification or primer extension. These two temperatures are typically useful for amplification of nucleic acid.
The apparatus may further comprise a fifth bath for a temperature stabilization step in the thermal profile as desirable for certain kinds of analysis of the nucleic acid. The temperature stabilization step may be at one of the target temperatures when advantageously no separate bath is required for stabilization.
The apparatus may further comprise: fluorescence imaging means or electrochemical detection means for analyses of the nucleic acid when the reactor(s) is/are in any of the baths or in air outside the baths.
The reciprocating means may provide a three-stage shaking of the reactor(s) in the second bath such that a higher speed shaking is followed by a lower speed shaking as the target temperatures approach followed by no shaking for taking fluorescence images.
The apparatus may further comprise: a sixth bath to contain a liquid or hot air maintainable at 40-80 degree C., wherein at least a portion of the bath wall is transparent to allow transmission of illumination light from a light source and transmission of emitted light from the reactor(s).
The bath medium in any of the baths may be in any phase including air, liquid, solid, powder and a mixture of any of these to suit the application.
The apparatus may further comprise a reactor temperature sensor that is capable of moving with the reactor holder during thermal cycling, to monitor the real-time temperature of the reactor(s) more accurately. The apparatus may further comprise a vessel containing a substance to encapsulate the reactor temperature sensor, the vessel and the substance having similar construction or heat transfer characteristics to that of the reactor(s) and the reaction material for sensing the reactor temperature more accurately.
The apparatus may further comprise a seventh bath that can receive the reactor(s) and be progressively heated while conducting melt curve analysis after the thermal cycling. This helps in conducting further analyses after the thermal cycling with an integrated process flow and is particularly advantageous when any of the baths for thermal cycling or additional processing can be shared with progressive heating feature for melt curve analysis.
The apparatus may further comprise altering means for altering temperature in any bath during thermal cycling. This feature provides more flexibility for designing the thermal profiles during thermal cycling.
The baths preferably have a high-aspect-ratio shape, the length to width ratio being 2-10:1 for accommodating the reactor holder with a plurality of the reactors with the reciprocating motion accommodated along the length. This feature advantageously reduces the required quantity of the bath medium and also saves energy for their heating. The splashing of the liquid bath medium is reduced as well during the insertion of the reactor(s). The volume of the baths thus being minimal, the bath heating times are reduced and the apparatus preparation time before the start of the thermal cycling is shortened. The baths may preferably be disposed with heating means along larger surface(s) lengthwise for a more uniform and efficient heating of the bath mediums and heat transfer with the reactor(s) due to short heat transfer characteristic length for fast heating of the baths.
According to a second aspect, methods corresponding to the first aspect are provided.
The present invention also enables the entire process of thermal processing of nucleic acid to be completed in a very short time duration of a few minutes, from bath heating preparation, to reactor thermal cycling and fluorescence signal acquisition.
BRIEF DESCRIPTION OF THE DRAWINGS
In the following drawings, same reference numbers generally refer to the same parts throughout. The drawings are not to scale, instead the emphasis is on describing the concept.
FIG. 1 is a schematic view of a set up for thermal cycling a reaction material containing nucleic acid according to an embodiment of the invention.
FIG. 2 is an isometric view of an embodiment of the apparatus for the process of thermal cycling as at FIG. 1.
FIG. 3 is an isometric view of an embodiment of the reactor array baths and optics modules of the apparatus in FIG. 2.
FIG. 4 is an isometric view of an embodiment of the reactor array transfer mechanism module of the apparatus in FIG. 2, along with a reciprocating motion generator.
FIG. 5(a) is an isometric view of an embodiment of the reciprocating mechanism module of the reciprocating motion generator of FIG. 4.
FIG. 5(b) is an isometric view of FIG. 5(a) with the reactors attached.
FIG. 6 is a set of views showing the working principle of a low inertia Scotch-Yoke mechanism for the reciprocating motion.
FIG. 7(a) is a schematic diagram showing the reciprocating motion of a high-aspect-ratio reactor array in a matching high-aspect-ratio reactor array bath, according to an embodiment of the invention.
FIG. 7(b) is a schematic view of an exemplary 2×16 high-aspect-ratio reactor array according to an embodiment of the invention.
FIGS. 8 is a graphical representation of an exemplary reactor temperature variations with time during thermal cycling using the apparatus previously described.
FIG. 9 is a perspective view of an embodiment of the illumination and fluorescence emission detection module with a low temperature reactor array bath having a transparent glass window and reactors having transparent sealing caps.
FIG. 10(a) is a graphical representation of experimental reactor temperature variations with time during forty thermal cycles using the apparatus previously described and without shaking the reactors.
FIG. 10(b) is a graphical representation of experimental reactor temperature variations with time during forty thermal cycles using the apparatus previously described and with shaking the reactors.
FIG. 11 is an elevation cross sectional view of the reactors illustrating the reciprocating motion under an extra support provided by the bath bottom.
FIG. 12(a) is a cross-sectional diagram of an array of tubular reactors partially confined within reactor guards.
FIG. 12(b) is a top view of one reactor of FIG. 6, along with the reactor guard and reactor holder.
FIG. 13(a) and (b) are perspective views of the reactors being accommodated in a card with specially designed edges for executing the reciprocating motion in the bath medium.
FIG. 13(c) is a cross-sectional view of a metallic reactor using optical fibers for imaging.
DETAILED DESCRIPTION
The following description presents several preferred embodiments of the present invention in sufficient detail such that those skilled in the art can make and use the invention.
FIG. 1 shows a schematic view of an embodiment of a portion of the thermal cycling apparatus for thermal cycling of nucleic acid such as for PCR, primer extension or other enzymatic reactions. The apparatus has two baths 50 and 51 each containing the bath medium 75, a bath heater 17 and a bath temperature sensor 39 mounted along the bath surface to enable control of the temperature of the bath medium 75. The bath temperature sensors 39 may be positioned inside the baths 50, 51. In this embodiment, the bath 50 is suitable for the step of denaturation and the bath 51 is suitable for the step of annealing and/or extension. The cooler 16 is useful when the bath 51 needs to be actively cooled to below room temperature. Active cooling device such as a thermoelectrical cooler or a fan can also be installed for above cooling purpose. The bath medium 75 shown here is liquid, however any other type of fluid or powder or solid bath medium 75 may also be used. For some embodiments, the bath heater 17 on the low temperature bath 51 is optional, if bath 51 does not have to be heated. For the thermal cycling, the reactor 15 is alternately transferred between the baths 50, 51 multiple times. To enable fast plunging of the reactor 15 into the bath medium 75, slim reactor 15 is preferable such as glass capillaries. The reactor 15 is sealed with a sealant or a cap 77 and a portion of the reactor 15 herein is transparent to allow light to pass through for dye or probe excitation and fluorescence imaging. A temperature monitoring unit 34 is installed on the reactor holder 33 and moves along with the reactor 15 between the baths 50, 51. The temperature monitoring unit 34 contains a fast response temperature sensor 38 inside. The temperature monitoring unit 34 has a shape similar to that of the reactor 15 and is constructed to have a similar or the same steady state and transient thermal characteristics as those of the reactor 15, for the temperature reading and thermal response to be similar or same as those of the reactor 15 unless another reactor 15 itself is used for the purpose. For example, the temperature monitoring unit 34 may have the fast response temperature sensor 38 inserted into water or oil or a layer of oil over water 22 and sealed. Although only one reactor 15 is shown, according to other embodiments the reactor holder 33 may accommodate a plurality of reactors 15. The reactor 15 may be in the form of tube(s) as shown or as wellplate(s) or chip(s) or cartridge(s). The reactor transfer mechanism 85 transfers the reactor 15 and the temperature monitoring unit 34 at high speed among the baths 50 and 51 to expose them alternately to the different temperatures in the baths 50 and 51 as required for the thermal cycling. There are many possible designs of the reactor transfer mechanism 85. One such mechanism is comprised of an X stage 86 moving along an X axis linear guide 87 for the reactor 15 and the temperature monitoring unit 34 to reach to a region above the baths 50 and 51, and a Z stage 88 moving along a Z axis linear guide 89 for the reactors 15 to move them down to enter the bath medium 75 or to be withdrawn from the bath medium 75. Such a transfer mechanism 85 can also consist of a rotary arm (not shown) that moves the reactor 15 and the temperature monitoring unit 34 in an angular direction along with the Z stage 88 moving along the Z axis linear guide 89. The reactors 15 have an opening for loading and the reaction material 21 and the openings are sealable. The sealant 77 may be made of a silicone rubber or UV cured polymer, hot melt and/or wax and/or gel which is in solid phase during thermal cycling. The sealing can also be achieved using liquid such as oil, viscous polymer, and gel. The highly viscous liquid can be applied to the opening and/or top section of the reactors 15 to block the vapor generated from the reaction material 21 from leaking out. The arrangement for the fluorescent imaging may be in any form as in the art. Herein, the bath 51 has a transparent window 25 so that illumination from the illumination source 44 reaches the reactor 15 inside and the emitted beam is received by the receiver 43. Within the baths 50, 51 the reactors 15 undergo reciprocating motion or shaking as shown by the block arrow, to create a higher and forced convection heat transfer scenario on the surface of the reactor 15. In case of smaller sized baths, the reactor reciprocating motion can be in a vertical or the depth direction (not shown) of the baths 50/51.
FIG. 2 shows an isometric view of the apparatus with the reactor transfer mechanism 85, wherein an optics module 206 carries out fluorescent detection of nucleic acid inside the reactors 15, a temperature controller module 205 controls the bath temperatures, a motion controller 207 controls all motions and a system controller 208 controls the system, with data communication and processing, image processing and data analysis. A reactor shaker 100 shakes the reactors 15 and the reactor temperature monitoring unit 34 when in a bath in the heating bath module 204. The bath module 204 has five baths 50, 51, 52, 53, 54 placed next to each other and each maintainable at a predetermined temperature. In this embodiment, in the optics module 206 the reactors 15 are situated in the low temperature bath 51 having air or transparent liquid as the bath medium 75. The additional processes like reverse transcription-polymerase chain reaction (RT-PCR), hot start process, and isothermal amplification reaction can also be carried out in any of the baths for thermal cycling which can be set at temperature TAP before the thermal cycling and reset to the temp for thermal cycling after the completion of the above additional processes. The reactor shaker 100 providing the reciprocating motion is explained under FIG. 4.
FIG. 3 shows a blown up illustration of the bath module 204 and the optical module 206. The array of reactors 15 with the temperature monitoring unit 34 are transferred among the baths 50 to 54 of a high-aspect-ratio (HAR) shape and placed next to each other along the narrow sides of the baths 50 to 54. The orientation of the typical HAR reactor array 15 matches with that of the HAR baths 50 to 54. In this embodiment, the bath 51 has air or transparent liquid as the bath medium 75 along with a transparent window 25 at the bottom to pass the illumination light and emission light to and from the reactor 15 as shown with the arrows for the ray paths. The bath heaters 17 or cooler 16 are connected to the sidewalls of the baths. The apparatus may further comprise a hot air zone (not shown) for placing the reactors 15 particularly during imaging. This simplifies the apparatus. There may be an electrical heater or an infrared heater to form the hot air zone above a bath. The heater may preferably be installed with the reactor holder 33 so that only the air in the vicinity of the reactors 15 is heated while the reactor(s) 15 is/are moving between the baths. This feature saves energy and saves the other parts of the apparatus from getting undesirably heated. The multiple baths 50 to 54 may be used as required under the thermal profile for the thermal cycling and for steps before and after the thermal cycling. In a three-step thermal profile for thermal cycling, the reactor 15 is inserted into three baths within each thermal cycle. In between the baths 50 and 51, the reactor 15 with the monitoring unit 34 may be inserted into a 3rd bath at a medium temperature or positioned in hot air for a period of time required for annealing and or extension. Before the thermal cycling, the reactor 15 with the temperature monitoring unit 34 may be inserted into a fourth bath, that is maintained at a predetermined temperature for an additional processing before the thermal cycling for nucleic acid amplification. Melt curve analysis may be conducted using a bath after the thermal cycling.
One embodiment of the low inertia motion mechanism or a reactor transfer module 85 is shown in FIG. 4, in which a low inertia X stage 86 and Z stage 88 are designed with a stationary motor for driving Z stage 88 and a driving mechanism using cam arm 112 to drive Z stage 88 without a need to use a linear guide and driving screw. A blown up view of the reactor shaker 100 is shown in FIG. 4. A motorized shaker stage 101 is connected to a shaker axis linear guide 102. The shaker stage 101 is connected to the reactor holder 33 that further holds the reactors 15 and the reactor temperature monitoring unit 34. The reactor shaker 100 is attached to the Z axis to enable the reactors 15 to be transferred among the baths 50 and/or 51 while moving back and forth at high speed in a reciprocating manner in the baths 50 and/or 51. Unlike the water bath in the conventional thermal cyders, the reactor shaker 100 can move the reactors 15 at a high speed in a reciprocating manner, in a small and confined bath domain to achieve strong forced convective heat transfer on the reactors 15 in the bath medium 75 thereby shortening the time to reach the target temperatures in the baths 50 and/or 51. The reactor transfer module 85 is capable of rapidly moving the X stage 86 and Z stage 88 which do not carry a Z stage motor to move along. To move the low inertia X stage 86 and Z stage 88, a stationary motor turns an X axis driving belt 105 that is connected to the X stage 86, and another stationary motor turns the Z ball spline shaft 106 that rotates the Z timing pulley (driving) 107 and subsequently the Z timing pulley (driven) 108 through the Z timing belt 109. Rotation of the Z timing pulley (driven) 108 causes the connected Cam arm 112 to rotate, which turns the link joint 110 on the Cam arm 112 and moves the Cam linkage lever 111 and the Z stage joint 113 up and down, achieving Z movement of the Z stage 88 connected to the Z stage joint 113 along the Z axis linear guide 89. The shaker axis liner guide 102 attached to the Z stage 88, the shaker motor 103 and the shaker stage 101 that is linked to the reactor holder 33 generates the high speed reciprocating motion for reactors 15. An exemplary reciprocating motion generator module 99 is shown in FIG. 5(a). A Scotch Yoke 115 with a Yoke pin 116 is mounted to the shaft of the shaker motor 103. The shaker motor 103 is mounted on the Z stage 88, but the Scotch Yoke 115 is not connected to the Z stage 88. When the shaker motor 103 shaft rotates, the Scotch Yoke 115 rotates with it, causing the Yoke pin 116 to move left and right. As shown in FIG. 5(b), the shaker stage 101 is mounted on the shaker axis linear guide 102 mounted on the Z stage 88 with the Yoke pin 116 inserted into the Cam slot 114 which is a part of the shaker stage 101. When the Scotch Yoke rotates 115, the Yoke pin 116 in the Cam slot 114 pushes the shaker stage 101 to move left and right in a reciprocating manner which is further shown in FIG. 6,
In one of our experiments, we used a reactor 15 made of glass capillary of 1.1 min ID and 1.5 mm OD that was filled with 10 μl PCR reagent and submerged in a bath of the dimensions of 110 mm in length, 12 mm in width, and 25 mm in depth filled with a mixture of glycerol and water at a mixing ratio of 75:25. The temperature inside the reactor 15 was measured by inserting a miniature thermocouple into the bottom section of the glass capillary that moves together with the reactors 15 with the reactor transfer mechanism 85. When the above reactor 15 moves in a reciprocating manner at 600 cycles per minute inside the mixture heating liquid in the baths 50 and 51 with a stoke of 6 mm along the long side of a narrow bath (or in the direction of length of the bath) under a Scotch-Yoke shaking mechanism as described in FIGS. 5(a, b) and FIGS. 6(a, b), it is equivalent to the reactor 15 moving at a linear speed of 2×6×600/60=120 mm per second. We found that the reactor temperature can ramp up from 58° C. of annealing temperature to 95° C. of denaturation temperature in 0.8-1.5 second after the reactors 15 entering the high temperature bath set at 125° C. In our experiment to cool the same reactor 15 under the same reciprocating motion, the reactor temperature can decrease from 95° C. to 58° C. in 0.8-1.5 second after the reactors 15 entering a low temperature bath 51 set at 25-40° C. In the above experiment, the reactors 15 travelled a distance of 120 mm per second during reciprocating movement when entering the bath. If such motion is in a one-directional manner (Reference 1: Jared S. Farrar and Carl T. Wittwer, Extreme PCR: Efficient and Specific DNA Amplification in 15-60 Seconds. Clinical Chemistry 61:1 p145-153 (2015)), rather than in a reciprocating manner, a long bath of minimum of 340 mm in length (240 mm reactor travel when move for 2 second in the bath+100 min reactor array length) needs to be used if a reactor array 30 of 100 mm length is used. This would be a large bath that is impractical for lab use and field use in comparison with a much smaller bath of 1.06 mm long (6 mm travel+1.00 mm reactor array length) of this invention, if the reciprocating motion methodology is adopted. Without reciprocating motion, the longer the time for the reactors 15 to stay in a bath as often required for annealing step of PCR, the larger is the bath size required to provide an enough space inside the bath for the reactors 15 to travel in one-direction. Such a large bath is not favorable for practical use, since it not only requires a long time and consumes large power to heat up to the target temperatures for nucleic acid analysis, but also too heavy to be portable for many field applications. Moreover, the related equipment also becomes bulky and hard to manage the large amount of heating liquid. The small baths in this invention due to the use of reciprocating motion greatly reduces the liquid volume to be heated and cool, thus requiring a much shorter time to pre-heat the bath to prepare for PCR, reducing overall time for a genetic test. The above small liquid volume also can be managed by simple pipetting for loading before the test and removal after the test, and the associated lighter equipment weight and smaller equipment size make the field applications feasible. The path of the reciprocating motion is preferred to be of a high-aspect-ratio shape with its orientation matching the orientation of the high-aspect-ratio shaped baths. Furthermore, the high-aspect-ratio shaped reactor array 30 also matches the orientation of the high-aspect-ratio shaped baths 50, 51 after the reactor 15 entering the baths 50, 51.
In another embodiment (not shown), the Z stage 88, or X stage 86, or both can be optionally used as the reciprocating motion generator to move the reactors 15 to generate strong forced convention.
Serving as a reference, the following section further describes and quantifies heat transfer enhancement of the reactors 15. One type of our reactor in operation comprises a reactor tube containing reaction liquid. The tube material and the liquid have different thermal and other material properties. To illustrate the heat transfer characteristics of the reaction liquid loaded reactor submerged in heat medium in heat bath during thermal cycling, we approximate the submerged reactor tube loaded with the reaction liquid as a cylinder made of a homogeneous material having the outer surface area As, the radius R, the length L, the volume of the submerged cylinder V, the density, the heat capacity cp. In order to estimate the time of heating of the cylinder submerged in the high temperature bath from the low target temperature TLT when the cylinder enters the high temperature bath to the high target temperature THT during thermal cycling, we determine the average rate of heat transfer {dot over (Q)}avg from Newton's Law of Cooling by using the average surface temperature Ts, avg of the cylinder [Reference 1: Y. A. Cengel and A. J. Ghajar, Heat And Mass Transfer: Fundamentals And Applications, Fifth Edition In SI Units by McGraw-Hill Education, 2015]. That is,
{dot over (Q)}
avg
=−hA
s(Ts, avg−TH), Eq. (1)
where As is the outer surface area of the cylinder, TH is the temperature of bath medium in the high temperature bath, Ts, avg=(TLT+THT)/2. Generally, under a practical application or instrument condition, TH≠THT. Note: an example of the low target temperature TLT is the annealing temperature in PCR, and an example of the high target temperature THT is the denaturation temperature in PCR.
- Next, we determine the total heat transferred from the cylinder [Reference 1], which is simply the change in energy of the cylinder as it heats from TLT to THT:
Q
total
=Vc
p(THT−TLT), Eq. (2)
In this calculation, we assumed that the entire cylinder is at uniform temperature over the domain of the cylinder. With this assumption, the time of heating the cylinder from TLT to THT, Δt, is determined to be
Because of the assumption made above, Eq (3) does not yield accurate temperature value, but it reveals the factors of influencing the time of heating up the cylinder.
The heat transfer coefficient h in Eq(3) is related to the reactor shaking or moving speed, which can be obtained from the following analysis of convective heat transfer in an external flow across a cylinder [Reference 1]:
The average Nusselt number for flow across the cylinder can be expressed compactly as
Nu=CRemPrn, Eq(7-37) of Reference 1
where the constant C, m, and n are related to the Reynolds number Re which is defined as
where D is the diameter of the cylinder, ρ is the density of the liquid in bath, μ is the dynamic viscosity of the liquid in bath, and ν is the moving speed of the cylinder in the bath.
For example, when the reactor in this invention is moved reciprocatingly at 600 cycles per minute by a motorized shaker with a stoke of 6 mm, it is equivalent to the reactor moving at a linear speed of 2×6×600/60=120 mm per second, Re is calculated to be
where the values of D, ρ, μ, and ν shown in the above expression of the Reynolds number were estimated at a film temperature 90° C.
- if Re is in the range of 40-4,000, Eq(7-37) can be rewritten as
Nu=0.683Re0.466Pr1/3 Eq (4),
which is as shown in Table 7-1 in said book by Cengel and Ghajar.
Since
where h is the heat transfer coefficient on the cylinder surface, D is the diameter of the cylinder, and k is the thermal conductivity of liquid in the bath, Eq(4) can be rewritten as
Inserting Eq (5) into Eq (3), we obtain
Eq(6) shows that the higher the shaking speed v, the shorter the time Δt of heating the cylinder from TLT to THT.
Similarly, the time of cooling the cylinder from THT to TLT, Δt, is determined to be
Where TL is the lower the under-heated bath temperature. Eq(7) shows that the higher the shaking speed v, the shorter the time Δt of cooling the cylinder from THT to TLT.
The term ‘liquid’ used in the above description and the entire description is a general term for ‘heating medium’, including heating medium of different forms, such as liquid, water, water mixed with solvent or other chemical fluid or other solid particles, solid particles, metal particles, copper particles and powders
As shown in FIGS. 7(a) and (b), the reactors 15 are arranged in a high-aspect-ratio reactor array 30, in which the number of columns is much larger than the number of rows of the reactors 15 in the array 30. This enables use of a matching high-aspect-ratio bath geometry (shown in FIG. 3) to reduce thermal mass of the bath medium 75 in the baths 50 or 51. The high-aspect-ratio bath geometry refers to a narrow shaped geometry whose opening surface of the top of the bath 50 or 51 for reactors 15 to enter is narrow. An example of a high-aspect-ratio reactor array 30 is a 1 by 8 or a 2 by 8 array of reactors 15 in the form of glass capillaries, and each glass capillary is typically vertically placed and has an internal diameter of 0.9 mm-1.1 mm and an outer diameter of 1.2 mm with the bottom ends of the capillaries sealed and its top end open for sample loading and then sealed for thermal cycling. The high-aspect-ratio reactor shape of the reactor array 30 matches the shape of the narrow bath 50 and 51 whose top opening is a narrow side of a rectangular bath, and the depth of the bath 50 and 51 can be deep to match the large length of the reactors 15.
An embodiment shown in FIG. 8 describes a method of conducting thermal cycling. After entering the low temperature bath 51, the reactors 15 move rapidly under a high speed reciprocating motion to speed up the cooling. Once the reactors 15 reach the TLT or a vicinity of TLT, the reactors 15 stop or take a lower speed of the reciprocating motion to reduce the forced convection heat transfer with the surrounding heat medium, which enables the reactors 15 to stay in the vicinity of the TLT annealing and extension for a longer period of time t. After that, the reactors 15 are moved to the high temperature bath 50 to continue thermal cycling. This method can also be applied to the high temperature bath 50 case.
The bath heaters 17 and or coolers 16 may preferably be over the larger surfaces 50b of the high-aspect-ratio bath 50 instead of the smaller surface 50a, as shown in FIG. 9. Due to the resulting small characteristic length 305 of heat transfer, rapid heat transfer to the entire bath medium 5 in the bath 51 can be achieved and therefore stable target temperatures can be reached rapidly during thermal cycling. For the high temperature bath 50, transfer of the cold reactors 15 from the low temperature bath 51 that tends to reduce the bath medium temperature in the high temperature bath 50 is thus rapidly compensated by the bath heaters 17 along the larger surfaces 50b. For the low temperature bath 51, transfer of the hot reactors 15 from the high temperature bath 50 tends to increase the bath medium temperature in the low temperature bath 51. The bath coolers 16 along the larger surfaces 50b thus help to rapidly remove the extra thermal energy brought in by the hot reactors 15, rather than relying on natural heat dissipation to remove the extra thermal energy. In another embodiment, the reactors array 30 is not arranged in high-aspect ratio layout. Similarly, the bath does not have a high-aspect-ratio geometry. An apparatus and a method for imaging the reactors 15 is also described. The apparatus is consisting of an illuminator 44a and a fluorescence detector 43. The illuminator 44a may comprise light emitting device like LED with optical filters 81 to emit light of specific wavelengths to excite dyes and probes in the reactors 15. The fluorescence detector 43 may comprise a light detector such as a camera or a photodiode or a photomultiplier tube with the optical filters 81 to detect light of specific wavelengths from the reactors 15. The filters 81, illuminator 44a and detector 43 can be mounted on a motorized wheel to carry out multiplex fluorescence detection. In a preferred embodiment, during fluorescence detection of amplicons inside the reactors 15, the reactors 15 are located inside the bath medium 75 in the low temperature bath 51, while the shaking under the reciprocating motion of the reactor 15 has stopped. When the detector 43 capable of taking images at a high speed is used, fluorescence imaging of the reactors 15 can be carried out while the reactor 15 is shaking under reciprocating motion. Fluorescence imaging of the reactors 15 while the reactors 15 are inside the bath 51 is important since the reactors 15 may have to be maintained at the annealing or extension temperature for a prolonged period of time when multiple images of different wavelengths are taken for multiplex detection or to acquire fluorescence images of control genes. In another embodiment, during fluorescence detection of amplicons inside the reactors 15, the reactors 15 are located outside the bath medium 75 in the low temperature bath 51. To avoid dropping of reactor temperature significantly below the annealing or extension temperature, heated air can be applied to the reactors 15 when moving out of the low temperature bath 51. The illumination light 46 passes through a band-pass filter 81 reflects downward from a dichroic mirror 82 and reaches the reaction material 21 inside the reactors 15 from the transparent cap 32 on top of the reactors 15, and emission light 47 from the reaction material 21 passes through the dichroic mirror 82 and a band-pass filter 81 and reaches at least one detector 43, or PMT or a camera. In another embodiment, to enable illumination and imaging of reactor 15, a portion of the low temperature bath 51 has a transparent window 25 and the illuminator 44b with the illumination light 46 are shown by broken lines. In a preferred embodiment, a three-stage shaking of the reactors 15 in the low temperature bath 51 is adopted; that is, Stage 1: a high speed shaking is used to rapidly cool the reactors 15 when the reactors 15 from the high temperature bath 50 enters the low temperature bath 51, and Stage 2: reactor shaking speed is reduced when the reactor temperature approaches the low target temperature TLT or annealing temperature, and finally Stage 3: the reactors 15 stop moving for taking fluorescence images before being transferred back to the high temperature bath 50. In said three-stage shaking, the speed reduction in Stage 2 enables more precise and repeatable positioning of the reactors 15 from cycle to cycle when the reactors 15 stop moving for taking fluorescence images.
FIG. 10(a) is a graphical representation of experimental reactor temperature variations with time during forty cycles of thermal cycling using the apparatus previously described and without shaking the reactors. The time taken is 1450 seconds as seen from the time-axis. FIG. 10(b) is a graphical representation of experimental reactor temperature variations with time during forty cycles of thermal cycling using the apparatus previously described and with shaking the reactors, where the time taken is only 680 seconds as seen from the time-axis. Thus, the reactor shaking feature allows to speed up the thermal cycling significantly by reducing the time to less than half. In this experiment, the amplitude of shaking has been maintained to 3 mm, at the frequency 6 Hz using copper powder of size 60 mesh as the bath medium 75. Enhancing the speed of thermal cycling may also be explored with other magnitudes for the amplitude and frequency along with other types of the bath medium 75 or other mesh sizes for the copper powder.
FIG. 11 shows another embodiment of reciprocating motion of reactor 15 by introducing a bottom support region 73 at a bath bottom 69 to reduce the bending moment on the reactor 15 with the possibility of breaking the reactor 15 during the reciprocating motion in a powder bath medium 75. In this embodiment, the reactor 15 is made to be in contact with the bath bottom 69 at a bottom support region 73 when the reactor holder 33 provides the reciprocating motion to the reactor 15 as shown by the block arrow. Similarly, the bath bottom 69 instead of the reactor holder 33 can also provide the reciprocating motion to the reactor 15 as shown by the line arrow, to reduce bending moment on the reactor 15 while executing the reciprocating motion. In another embodiment, the bath bottom 69 and the reactor holder 33 may execute the reciprocating motion in opposite directions. The support regions 73, 74 provide sufficient flexibility for the reactor 15 to undergo the reciprocating motion. In other previous described embodiments, during the reciprocating motion provided by the reactor holder 33, the reactor 15 is not in contact with the bath bottom 69 and there is only one top support region 74 on the reactor 15, which may generate a large enough bending moment to break the reactor 15 at the top support region 74.
FIG. 12(a) is a cross-sectional diagram of an array of tubular reactors 15 partially confined within a reactor guard 7 that may be optionally used. The reactor guard 7 provides physical support to prevent the plastic reactors 15 from getting deformed under the resistive forces and the THIGH or the glass reactors 15 from breaking under the resistive forces when the reactors 15 are inserted into the powder bath medium 75 and thereafter on the body of the reactor 15 during the reciprocating motion in the powder bath medium 75. Typically, such force is maximum on the reactor tip 81 when the reactor 15 is inserted into the powder bath medium 75. The reactor guard 7 may be made up of materials comprising metal or glass or high temperature plastics or ceramics and faces the direction of reciprocating motion. FIG. 12(b) shows a top view of one reactor 15 with the reactor guard 7 and the reactor holder 33. As shown, the reactor guard 7 covers only a small portion of the reactors 15 hence does not obstruct fluorescent imaging.
FIG. 13(a) is a biocchip 31 consisting of reactors 15 in the form of wells. The reaction material 21 is dispensed from the opening of the reactors 15 and sealed by a cover or sealing fluid 30. Then, the biochip is mounted onto the reactor holder 33. FIG. 13(b) is a perspective view of the reactors 15 being accommodated in a card 31 for use with the baths. Herein, a plurality of the reactors 15 are arranged in the card 31. The edges of the card 31 may be disposed with a material (not shown) that retains rigidity under the reciprocating motion at high THIGH and when used with powder bath medium 75. There is at least one inlet 313 which is in fluid communication with the reactors 15 via a network of channels 315. The reaction material 21 to be tested can be loaded into the inlet 315 that subsequently flow into the reactors 15. For moving inside the powders bath mediums 75. FIG. 13(c) shows an embodiment where the reactor 15 is made of a metal tubing which is suitable for metal powder as the bath mediums 75. For the fluorescent imaging, an optical fiber 309 transmits light from an illumination light source such as an LED (not shown) into the reaction material 21 inside the reactor 15. Optical fiber 310 is for light transmission from the reaction material 21 to a photodetector (not shown). The sealant or cap 77 holds the optical fibers 309, 310. This facilitates the optical detection for the non-transparent reactor 15. FIG. 13(c) In another embodiment, a set of optical fibers can guide the illumination light 46 into each of the reactors 15, and a second set of the optical fibers can guide the emission light 47 out from each of the reactors 15 to reach at least one light detectors 43.
Various materials can be used as the bath medium 75, including liquid and/or solid powder and/or a mixture of liquid and solid powder or beads. Single silicon powder and copper powder can be in the heating bath, but the hardness of the silicon powder and the copper powder could not cause the reactor to rupture. The bath medium 75 can also be air. The bath medium 75 also comprises one or more selected from a group consisting of water, oil, glycerin, chemical liquid, liquid metal, gas, air, metal powder and silicon carbide powder and/or beads and their mixture. The materials used to construct the reactors 15 may be plastics, elastomer, glass, metal, ceramic and their combinations, in which the plastics include polypropylene and polycarbonate, the glass reactor 15 can be made in a form of a glass capillary of small diameters such as 0.1 mm-3 mm OD and 0.02 mm-2 mm ID, and the metal can be aluminum in form of thin film, thin cavity, and capillary. Reactor materials can be made from non-biological active substances with chemical or biological stability. At least a portion of the reactor 15 is preferred to be transparent. The volume of the at least one reactor may be in the range 1 μL to 500 μL. Smaller the volume, faster is the reciprocating motion possible, higher is the speed of PCR, smaller are the required bath sizes and more compact is the apparatus. The reaction material in all the reactors 15 in the reactor holder 33 may not be identical. Simultaneous PCR can be advantageously conducted for different materials if the bath temperatures are suitable.
There are many other advantages of adopting reactor motion in a reciprocating manner:
- a) For example, in high-aspect-ratio baths 30 shown in FIG. 1.4(a) in which a 1×4 reactor array and FIG. 1.4(b) a 2×16 reactor array both of length M and width N are placed, respectively, use of the small bath width, H, one can achieve both small volume and a high aspect ratio L/H of a bath 30 when reciprocating motion is used. For example, reciprocating motion of the reactors 15 along the length direction of the high-aspect-ratio reactor array yields small shaking amplitude l which can reduce bath size significantly since L=M+l+2 regardless of the motion speed. This significantly reduces thermal mass of bath medium in the bath 30, which is a key to achieve rapid bath pre-heating to target temperatures before thermal cycling starts, or fast bath preparation, and reduce the overall time for a genetic test and equipment size for improved portability. Such fast bath preparation before thermal cycling cannot be achieved for conventional water baths which have large thermal mass associated with large water bath dimensions. For example, instead of using a reciprocating motion, Reference 2 [Jared S. Farrar and Carl T. Wittwer, Extreme PCR: Efficient and Specific DNA Amplification in 15-60 Seconds. Clinical Chemistry 61:1 p145-153 (2015)] describe a high speed one-directional linear motion or a “sweeping-through motion” to enhance heat transfer around the reactors 15. For a reactor to stay in a bath for a long enough time especially for annealing and extension, their one-directional linear “sweeping-through motion” requires a very large l which can be calculated to be l=vt, in which v is the velocity of the reactor motion in the bath and t is the time the reactor stays in the bath during “sweeping-through motion”. Therefore, to have a high velocity v to achieve strong forced convection heat transfer with a sufficiently long time t the reactor 15 needs to stay in the bath 30 that is typically required for the annealing step of PCR, l becomes large and a very large length of the bath 30 (L=M+l+2) has to be built, which causes many problems in usability such as management of a large amount of water, long pre-heating time before PCR, large size and weight of equipment. With a small and thin-shaped bath, said low thermal mass in this invention may also facilitate responsive temperature control during thermal cycling.
- b) Another important advantage of a small and high-aspect-ratio bath shape is that a user can manage the bath medium much more easily. For example, since the amount of the bath medium in the baths is small, a user can easily dispense and remove the entire content of the bath medium in and out of all baths for each thermal cycling operation, without worrying about disposal of a large amount of water in a conventional water bath cycler and adding in a large amount of water into conventional bath cycler for a new test and wait for a long time for the large baths to be heated to the target temperatures to start a new thermal cycling process.
When using the above described methods and devices for nucleic acid analysis and processing, the reaction system comprises reaction constituents including at least one enzyme, nucleic acid and/or particle containing at least one nucleic acid, primers for PCR, primers for isothermal amplifications, primers for other nucleic acid amplifications and processing, dNTP, Mg2+, fluorescent dyes and probes, control DNA, control RNA, control cells, control micro-organisms, and other reagents required for nucleic acid amplification, processing, and analysis. The particle containing nucleic acid mentioned above comprises at least one cell virus, white blood cell and stromal cell, circulating tumor cell, embryo cell. One application may be to use the methods and devices to test different kind of reaction systems against the same set of primer and probes, such as test more than one sample. For such application, different kinds of reaction material 21 containing no target primers and/or probes are each loaded into one reactor 15 in a reactor array 30, with all the reactors 15 being pre-loaded with the same set or the same sets of PCR primers and/or probes. For the same application, different kinds of reaction materials pre-mixed with respective PCR target primers and/or probes are each loaded into one reactor 15 in a reactor array 30, with all the reactors 15 being not pre-loaded with the same set of PCR primers and or probes. The reaction materials 21 can include control genes and/or cells and corresponding fluorescent dyes or probes. In the above situations, the different probes emit light of different wavelengths. Another application of the methods and devices are used to test the same reaction system against different sets of primer and probes. One example of such an application is to test one type of sample for more than one purpose. For this application, a single reaction material 21 is added into the reactors 15 each loaded with at least one different set PCR primers and or probes. For example, a first reactor 15 can be loaded with primer and probe set 1, and a second reactor 15 can be loaded with primer and probe set 2, Another example under this application is that the first reactor 15 is loaded with primer and probe set 1 and 2, and the second reactor 15 can be loaded with primer and probe set 3, 4 and 5. The reaction material 21 can include control genes and/or cells and corresponding fluorescent dyes or probes. In the above situations, the different probes emit light of different wavelengths. The above reaction material 21 is used in polymerase chain reaction, reverse transcription-PCR, end-point PCR, ligase chain reaction, pre-amplification or target enrichment of nucleic acid sequencing or variations of polymerase chain reaction (PCR), isothermal amplification, linear amplification, library preparations for sequencing, bridge amplification used in sequencing. The variation of the polymerase chain reaction mentioned above comprises reverse transcription-PCR, real-time fluorescent quantitative polymerase chain amplification reaction and real-time fluorescent quantitative reverse transcription polymerase chain amplification reaction, inverse polymerase chain amplification reaction, anchored polymerase chain amplification reaction, asymmetric polymerase chain amplification reaction, multiplex PCR, colour complementation polymerase chain amplification reaction, immune polymerase chain amplification reaction, nested polymerase chain amplification reaction, the target enrichment of pre-amplification or nucleic acid sequencing, ELISA-PCR.
Use of the reactors in the form of capillaries is advantageous for cycling between the baths at high speed and also during shaking. Due to smaller surface area, capillaries offer lower resistance of movement within the bath medium, particularly when the bath medium is in powder form. Additionally, the splashing of the bath medium when in the liquid form is reduced.
The reciprocating means include a reactor shaker for shaking the reactor holder. Herein, the shaking is independent of any movement of the holder as provided by the transfer means. This feature is advantageous when the shaker in the reciprocating means has an inertia of motion that is significantly lower than that of the transfer means. In such a situation, the energy consumption and the noise level may be reduced. Additionally, high frequency shaking may be easier to attain with the shaker.
According to an alternate embodiment the transfer means include the reciprocating means and in operation the shaking of the holder is executed by the transfer means at a specified frequency and amplitude. This feature reduces the complexity of the apparatus. In this embodiment, the reactor shaker is not used and may or may not be provided with the apparatus.
It is implied that the reciprocating means include the software program to provide the shaking at selected frequency and amplitude.
The choice and optimization of the frequency and amplitude may be made based on factors like the nature of the bath medium being used, the mechanical loading on the transfer means, the nature of the reactors and the kind. The objective is to reduce the cycling time as described under FIG. 10(a) and FIG. 10(b). The ranges of the choices may be : frequency above 1 Hz with above 0.5 mm amplitude, frequency above 0.2 Hz with above 5 mm amplitude, frequency above 1 Hz with above 5 mm amplitude, and frequency above 3 Hz with above 0.5 mm amplitude, and frequency above 3 Hz with above 5 mm amplitude.
The reactors may be in any form, such as tubes or wellplates or chips or cartridges. The tubes include capillaries.
From the foregoing description it will be understood by those skilled in the art that many variations or modifications in details of design, construction and operation may be made without departing from the present invention as defined in the claims.
Patent Application
20190105656
