Patent Application
20240279727
The present invention relates to the device and method for processing biological samples, and particularly for performing extraction, amplification reaction and detection of nucleic acid.
Extraction and amplification of nucleic acids is increasingly important to molecular biology, food safety and environmental monitoring. A large number of biological researchers use polymerase chain reaction (PCR) in their work on nucleic acid analyses, due to its high sensitivity and specificity. The PCR is typically conducted by thermal cycling process that is adapted to heat and cool receptacles containing the reaction material to different temperatures for DNA denaturation, annealing and extension. In a real time PCR assay, a positive reaction is detected by accumulation of a fluorescent signal. Fluorescence imaging is typically conducted multiple times or after every thermal cycle to record the progressive change in the biological sample, and the amount of the target nucleic acid in the sample or biological sample is detected. PCR amplification of minute quantities of DNA is conducted for applications like research, forensic analyses, wildlife studies, ultrasensitive diagnostics and the kind. Isothermal amplification is also used. Typically, the PCR is preceded by steps of sample preparation such as nucleic acid extraction and purification, in which multiple steps of liquid handling are performed, including liquid aspiration, dispensing, and mixing. Both sample preparation steps and the nucleic acid amplification steps may cause contamination, mainly due to aerosol and/or vapor generation. Typical approach to prevent the nucleic acid analyzer or sample processing instrument from contaminating a lab space is to seal the PCR receptacles during an amplification process and put the sample preparation instrument in a biological safety cabinet or a hood having negative pressure where the aerosol and/or vapor generated from liquid handling is removed from the biological safety cabinet. Typically, to prevent said contamination in a nucleic testing lab, sample preparation for nucleic acid extraction and PCR steps are performed in different rooms with controlled air pressure and air flow direction. Use of such a biological safety cabinet or a hood and a controlled testing environment is not technologically suitable or cost effective for nucleic acid testing to be performed outside a PCR lab, such as in distributed point of care testing (POCT) sites such as school, pharmacy, airport, or a commercial building.
Nested PCR with a pre-amplification step is a technique that reduces nonspecific amplification of the DNA template, typically with the use of two primer sets or reagents and two successive PCR reactions. The first set of primers are used in an initial PCR reaction. Amplicons resulting from the first PCR reaction are used as template for a second set of primers and a second amplification step. However, the potential for contamination of a lab environment is typically also increased due to opening of the cap of the PCR receptacles used in the first round of amplification to carry out additional liquid transfer and processing of the amplicon products from the first round of amplification in order to prepare for the second round of amplification. To minimize such contamination carryover between the first and the second rounds of amplification, different parts of the process need to be physically separated from one another and conducted preferably in entirely separate rooms. Amplicons from nested PCR assays are detected in the same manner as in PCR. As such, the nested PCR process is hard to be automated in one instrument, due to a large chance of contamination when all steps are performed within an instrument box.
Prior to the nucleic acid amplification step, the extraction and amplification processes for the nucleic acid are often hampered by contamination of the sample from the laboratory surface, reagents, carry over pipettes and pipette tips, lab coats, glove boxes, and waste baskets. The aerosol and/or vapors generated from the samples and reagent liquids contaminate the surrounding ambience. Higher the sensitivity of the assays, more prone are they to the effects of contamination. Successful aerosol contamination control is the backbone of numerous experiments—whether for genotyping, creating NGS libraries, studying single cells, or testing sensitive clinical samples. In the lab, using unsealed wellplates for extraction steps is convenient and provides flexibility for many test protocols. It also has low cost and is easy to have a liquid handling access to the liquids. But, in use, a few separated rooms are needed for the steps of reagent preparation, sample preparation, PCR and the like to control aerosol and/or vapor along with other contaminations. Such facilities are provided in laboratories having trained personnel with air controlling facilities and protocol. Such laboratories generally also have decontamination protocol when contamination occurs. In POCT settings, there are no separated rooms, contamination facilities or trained personnel provided.
Even trace amounts of contamination by the biological samples can have serious negative impact particularly when these get amplified during the amplification process and subsequently cause erroneous detection and diagnosis. Thus both, contamination of the ambience by the biological sample and contamination of the sample by the ambience need to be arrested.
Microfluidic cartridge modules are developed to provide sealed environments, containing internal microchannels, valves, and pumps or pump interfaces requiring complex machine interface to operate the cartridge module. The BioFire FilmArray System provides a new standard for syndromic infectious disease molecular diagnostics, with integrated sample preparation, amplification, detection, and analysis. Patent Publication No: US2018/0214864 A1 and the product GeneXpert describes use of reaction cartridge modules. The disposable cartridge modules constitute micro-valves and micro-pumps for fluid transfer from chamber to chamber. The thermal cycling for the amplification and the real time optical detection are conducted within the chamber itself. However, such cartridge modules are complex to manufacture due to the complex design, hence are expensive. The high complexity also may potentially lead to low performance reliability and be demanding to manufacture with a good production yield. The microfluid cartridge modules are not flexible to be used for different protocols. Number of samples to be evaluated are limited by the design of the cartridge module. Besides, the cartridge module design does not control cross-contamination between the sample and the ambience during the sample preparation and the PCR.
Therefore, there is a large demand to develop simple and low-cost liquid handling device and method to run complicated sample preparation steps for nucleic acid extraction and for PCR without aerosol and/or vapor contamination. Likewise, biological analyses with infectious or foul-smelling samples like faeces is also a challenge with respect to contamination control, particularly in POCT setting
The present invention provides several improvements towards device, apparatus and method for sample preparation, amplification, detection, and analysis. This invention provides a great positive impact on biological analysis by making the disposables much more affordable and reliable due to their simplistic design and functionality. The invention potentially makes the process of amplification faster, provides a notable contamination control particularly for POCT settings and also addresses the issues with biological analyses for infectious or foul-smelling samples like faeces.
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 term biological sample has been used for the biological sample containing nucleic acid, whether before or after mixing a reagent liquid. The biological sample may originate in any biological form such as swab, saliva, blood and the kind. The reagent liquid may refer to any kind used in the art, such as PCR reagent, extraction liquid, purification liquid, deamination liquid, desulphonation liquid and the kind, which are used for processing of the biological sample. In this disclosure, the receptacles may be in the integrated form as wellplates, or as in disintegrated form.
According to a first aspect, a cartridge module is provided for improving contamination control during biological analyses or nucleic acid analyses protocol including nucleic acid extraction, nucleic acid amplification and detection. The cartridge module comprises: at least one hollow tube; and a plurality of receptacles; at least one cover layer to isolate the receptacles from the ambience outside the cover layer, wherein each of the receptacles allow the tube when attached to a syringe or a pipette in use to dispense and aspirate an in use reagent liquid or in use biological sample to and from the receptacle through the cover layer only from a receptacle top opening, the cover layer is to substantially block aerosol exchange between the ambience and the receptacle during the whole process of biological analyses or nucleic acid analyses even after the tube pierces the cover layer and retracts, the cover layer also prevents contamination of the ambience due to any spillage of the reagent liquid or the biological sample from the receptacles or the cartridge module, the cover layer being attached to: 1) at least one of the receptacle top openings, or 2) the cartridge module or the receptacles while maintaining a connecting air space being isolated from the ambience of the cartridge by the cover layer between the cover layer and the receptacle top openings, the connecting air space facilitating a reduction in change of air pressure inside the isolated receptacles during aspiration or dispensation of the biological sample and the reagent liquid by the tube. The cover layer advantageously scrubs off a significant portion of the reagent liquid or the biological sample adhering on the tube underneath the cover layer while the tube is retracted out of the receptacle. The tube preferably has a bevelled tip. The outer diameter of the tube is between 0.1 mm and 5 mm. The cover layer has a thickness range from 0.1 mm to 50 mm.
According to an embodiment, at least a portion of the cover layer material is porous when the cover layer is attached to the receptacle top openings. The porous cover layer helps in regulating pressure during liquid dispensation and aspiration. The increased or decreased air pressures inside the receptacle also affect the volumes of the reagent liquid or the biological sample that are programmed to be dispensed or aspirated from and into the tube. These effects may impact in small liquid volumes but are significant when the receptacle volumes are also small. More issues are described under
According to an embodiment, at least a portion of the cover layer material is elastomeric. The elastomeric cover layer helps in substantially closing the cover layer aperture created in the cover layer due to the piercing of the tube.
The material of the cover layer is any one or a combination of the following: a) porous elastomeric, b) porous non-elastomeric, c) non-porous elastomeric, and d) non-porous non-elastomeric. The material of the cover layer can be selected from the four types as described above to optimize between the pressure regulation, blocking of aerosols and preventing the spillage.
According to an embodiment, a cover liquid is pre-dispensed or provided to be dispensed on the cover layer at least where the tube pierces, to form a stack or be soaked by the cover layer or be sandwiched within the cover layer, the cover liquid being non-reacting with the reagent liquids and the biological sample. The cover liquid may also be underside of the cover layer. Advantageously, the cover liquid is suitable for enhancing the isolation of the cover layer by blocking a cover layer aperture caused by the piercing of the cover layer by the tube, particularly when the cover layer is non-elastomeric. The isolation can be hermetic if the cover layer is non-porous. The cover liquid also improves the aerosol blockage as well as the spillage, particularly for porous cover layers. With the cover liquid, particularly for the non-elastomeric cover layer, the receptacle could be maintained hermetically isolated from the ambience before and after piercing of the cover layer by the tube, such as for the steps involving no high temperature. The cover liquid closes the cover layer aperture of non-elastomeric cover layer. Trace amounts of the reagent liquid or the biological sample remaining on the tube get covered by the cover liquid as the tube passes through the cover liquid. This prevents the reagent liquid or the biological sample adhering to the tube from being exposed to the ambience and contaminating the same. The cover liquid also covers a portion of the tube that is inserted within the receptacle, hence entry of any contamination from the ambient into the receptacle is also reduced. When the cover liquid is under the cover layer the cover liquid is also better protected from physical and handling damages. The cover liquid is preferably immiscible with the reagent liquids and the biological sample and is wetting over the tube and the cover layer. The cover liquid may be a viscous liquid polymer or monomer or an oil or liquid wax or any other suitable material in the art.
It will be appreciated by those skilled in the art that the receptacles to undergo amplifications by heat treatment need to be provided with a separate cover layer, to enable heating and transferring the receptacles to an amplification module outside the cartridge.
The claimed features have several advantages as described hereafter: 1) With the isolation, once the reagent liquid and the biological sample are dispensed in the receptacles, these liquids are never exposed to the ambience, right through the processes of biological analyses or nucleic acid extraction, amplification, imaging and disposal. Hence, the contamination of the biological sample from the ambience and vice versa is significantly reduced in comparison to the state-of-the-art cartridges. The isolation significantly prevents aerosols and/or vapors from escaping or entering the receptacle during the analyses. 2) The feature of having the receptacles with provision for liquid communication only from the receptacle top openings, enables the cartridge module to operate with lesser complexity. Thus the cartridge module, in the absence of any valve, thermal cycling module or optical detection module, lowers the cost and enhances the reliability of the detection and analysis. Also, cross-contamination between the biological samples in the receptacles is reduced as there is no provision for liquid communication between the receptacles. This feature also allows customized computer programming of using the receptacles as against the microchannels. 3) In addition, the low cost allows the cartridge module to be disposed once the reagent liquids are consumed, thereby further reducing the contamination possibilities. 4) The cartridge module allows greater flexibility for the user on the choice of baths in an apparatus for amplification, like for a faster thermal cycling the user may use separate baths maintained at the target temperatures in the apparatus instead of cycling the temperature in a single bath or heat block. 5) The simplistic feature of the cartridge module allows it to be easily portable. 6) The cartridge module allows using commonly used disposables in the industry like the individual receptacles and the wellplates. 7) The cover layer isolation also addresses the issues with biological analyses in POCT settings with infectious or foul-smelling samples like faeces.
According to an embodiment, the cartridge further comprises a scrubbing layer that is fixed over the stack such that the cover layer, the cover liquid and the scrubbing layer form a composite layer, the scrubbing layer being pierceable by the tube and substantially scrubs off the excess cover liquid sticking to the tube when being retracted out of the receptacle. Without the scrubbing layer, the cover liquid may get depleted faster when in small quantity or when the tube is long.
According to an embodiment, the cartridge further comprises at least one syringe, providing at least one feature from the group consisting: a) one o-ring between a plunger and a barrel of the syringe, a cover liquid being provided above the o-ring and away from a dispensing or aspirating nozzle of the barrel, and b) at least two o-rings between a plunger and a barrel of the syringe, a cover liquid being provided in between the two o-rings, the cover liquid being immiscible and non-reacting with the reagent liquids and the biological sample. The o-ring with the cover liquid tends to make the syringe better airtight, so that any potential contamination within the receptacle from the ambience and vice versa is further reduced. Besides, the trace amounts of the reagent liquid or the biological sample in the syringe sticking along the inner wall of the plunger and the outer wall of the barrel get covered with the cover liquid when the plunger is pushed downwards into the barrel. This covering helps the reagent liquid or the biological sample from getting exposed to the ambience and contaminating the latter.
According to an embodiment, the cartridge further comprises a rigid holding layer as positioned over or under the cover layer, the rigid holding layer having holding layer apertures aligned over the receptacle top openings for the tube to pierce the cover layer. Advantageously, the rigid holding layer prevents the cover layer from being undesirably detached or flexed when the tube is pierced into or retracted from the receptacle.
According to an embodiment, the cartridge further comprises at least one receptacle holder for holding at least one receptacle, the receptacle holder being detachable from the cartridge. The receptacle holder is useful for carrying the receptacles in and out of the cartridge module, as within the modules of an apparatus for biological analyses or nucleic acid analyses protocol including nucleic acid extraction, nucleic acid amplification and detection.
According to an embodiment, the cartridge further comprises a liquid sealant which when dispensed over at least one of the receptacle top openings can seal the receptacle top openings whether before or after curing, the liquid sealant being immiscible and non-reacting with the reagent liquids and the biological sample. The liquid sealant is dispensable by a tube passed through the cover layer or the stack thereby creating an air gap in the receptacle between the biological sample or the reagent liquid. The liquid sealant may be a viscous one which provides a large resistance to the pressurized air in the receptacle to leak out of the liquid sealant. The sealing helps to prevent aerosols and/or vapors from the biological sample inside the receptacle from emerging out through the cover layer aperture or the stack during the step of amplification where the receptacle is subjected to heat. Any such contamination from the ambience reaching the receptacles is also controlled. Due to the air gap, during imaging fluorescence from the liquid sealant is not unwantedly captured along with that from the biological sample. The liquid sealant may be any viscous material such as wax or glue. Wax that solidifies at room temperatures are easier for shipment of the kit. During its aspiration and dispensation by the tube, it can be locally heated to a liquid form.
According to an embodiment, the cover layer is provided with pits to contain the cover liquid, and the pits are interconnected. A tube tip of the hollow tube can then move between the pits while remaining dipped in the cover liquid and without being exposed to the ambience. If the tube tip is exposed to the ambience after aspiration or dispensation of the biological sample containing the nucleic acid, any dripping of the biological sample contaminates the ambience. The unwanted dripping may occur due to various factors like software error or instrumental error. Such a possibility of contamination is avoided in this embodiment as any such dripped biological sample remains trapped within the cover liquid.
According to an embodiment, the cover layer over at least one of the receptacle top openings is flexible and is provided with at least one through aperture or a partially-through aperture to make the piercing by blunt tubes like with the pipettes easier and with lesser force applied. Blunt tubes are preferred particularly when the receptacles are made with deformable material. Sharp tubes have the potential to pierce through the deformable material.
According to an embodiment the cover layer has a recessed top for guiding a tube tip of a tube before the tube tip pierces the cover layer through a base region of the recessed top, the recessed top having slanted sides to provide a tapering towards the base region, the base region being smaller than the receptacle top opening. The tapering surface is preferred to be a hard surface when the tube is in the form of a sharp needle to prevent the needle from penetrating the tapering surface. This provides better tolerance for misalignment of the tube tip of the syringe or the pipette with reference to the small receptacle top opening. This feature is helpful particularly when the receptacles are in the form of very narrow capillaries to increase the efficiency of heat transfer during the amplification of the nucleic acid in the biological sample. The slanted sides are made of a rigid material to prevent piercing by the tube tip.
The cartridge module may include at least one receptacle containing magnetic beads for binding nucleic acids from the biological sample. At least one receptacle may contain oil or water or a DNA removing agent as a cleaning liquid for cleaning the tube and the syringe at any stage during the nucleic acid analyses, including inner and outer surfaces of the needle tube, the liquid retaining space as shown in
According to an embodiment of the cartridge module, at least a portion of the receptacles is made of a deformable material, so that in use, (i) the receptacles in an initial condition or shape can bloat to accommodate the dispensed reagent liquid or the biological sample, and (ii) the receptacles in an initial condition or shape can collapse when the dispensed reagent liquid or the biological sample is aspirated out, thereby facilitating a reduction in change of air pressure inside the isolated receptacles during aspiration or dispensation of the biological sample and the reagent liquid by the tube. This embodiment is useful particularly for the non-porous cover layer. The increased or decreased air pressures inside the receptacle also affect the volumes of the reagent liquid or the biological sample that are programmed to be dispensed or aspirated from and into the tube. These effects may impact in small liquid volumes but are significant when the receptacle volumes are also small.
According to an embodiment, the cartridge module with the connecting air space further comprises: a sealing layer sealing the receptacle top openings individually or in plurality to prevent any spillage of the reagent liquid and the biological sample from within the receptacles when the cartridge module is not in upright position, the sealing layer being pierceable by the tube. During use vent apertures needs to be generated in the sealing layer by a tube attached to the syringe or pipette mechanism, so that the receptacles gets in air communication with the connecting air space during subsequent aspiration or dispensation from or into the receptacles.
According to an embodiment, when the cover layer is attached to the receptacle top openings, a plurality of the isolated receptacles are vertically positioned in a pile, such that a tube tip of the hollow tube can pierce through the cover layers of each receptacle in the pile to reach a lower most receptacle in the pile from a topmost receptacle in the pile. This embodiment helps to reduce the foot-print of the cartridge module which is desirable for a portable apparatus.
According to an embodiment, the cartridge module further comprises: a fixed pattern that can be detected by an apparatus during auto-positioning of the tube over the receptacle top openings.
According to an embodiment the cartridge module further accommodates at least one sample container with a cap that in use contains the biological sample, the cap at least in part includes the cover layer that can be pierced by the tube to aspirate the biological sample from the sample container and dispense into the receptacle. This improves the contamination control as the biological sample is transferred into the receptacle by the tube while the sample container is in the cartridge module. The cap and the cover layer in the cap prevent any possible spillage of the biological sample during the whole analysis until disposal of the cartridge module.
According to a second aspect, apparatus is provided for improving contamination control during biological analyses or nucleic acid analyses protocol including nucleic acid extraction, nucleic acid amplification and detection. The apparatus comprises: a) receiving module for accommodating the cartridge module according to any one preceding claim; b) robotically controlled syringe or pipette mechanism for operating the hollow tube attached to a syringe or a pipette to aspirate and dispense the biological sample or reagent liquid from and into the receptacles via the receptacle top openings and through the cover layers or the stacks or the sandwich; c) an amplification module to provide isothermal heating or cyclic heating for amplification of the nucleic acid in the receptacle, the amplification being conductible while the receptacle containing the biological sample is within the cartridge module or outside; d) an optical imaging mechanism for imaging of the biological sample during or after the amplification, while the receptacle containing the biological sample is within the cartridge module or outside; and e) a robotically controlled transfer mechanism that is configurable to position the receptacle as held by a receptacle holder as provided in the cartridge module or in the apparatus, to outside the cartridge module, when the amplification is conducted outside the cartridge module.
According to an embodiment, the apparatus is computer programmable to operate the syringe or pipette mechanism to press the tube as attached to the syringe against a fixed surface in the cartridge module or in the apparatus and bend the tube, and thereafter mount the syringe with the bent tube in the cartridge module. The advantage has been described under the first aspect. Advantageously, bending the tube as attached to the syringe or pipette after use enables easier for accommodation in the cartridge before disposal.
The apparatus further comprises an ultraviolet (UV) curing module to cure a UV resin deposited at the receptacle top opening. Such in-situ curing before amplification is helpful to block the vapors from the biological sample in the receptacle to contaminate the ambience through the cover layer aperture formed in the cover layer due to piercing of the tube.
According to an embodiment, the apparatus is further computer programmable to position the receptacle holder or the cartridge module such that during aspiration and dispensation of the reagent liquid and the biological sample by the syringe or pipette operating mechanism from and into the plurality of receptacles, the portion of the tube that contacted the biological sample in the receptacle does not move out of the connecting air space. This reduces the contamination significantly.
According to an embodiment, the apparatus is computer programmable to conduct at least two steps of amplifications with treatment of the biological sample by the reagent liquid between any two consecutive steps of amplifications and through the cover layer with or without the cover liquid, while the receptacle containing the biological sample remains within the cartridge module or outside but remains within the apparatus. The invention enables nested dual amplification of the nucleic acid in the biological sample to be carried out automatically within the same receptacle with the cover layer or the stack. The PCR amplicons need not be taken out of the receptacle after one amplification, for mixing with the reagent liquid before another amplification. The method is thus suitable for a fast point of care testing (POCT) PCR process.
This product automates the highly complex and time-consuming manual procedures, with minimal chances for contamination. More than two amplifications may be desirable to improve the sensitivity of detection of the DNA and RNA under investigation.
According to an embodiment, the apparatus further comprises: a detection module to detect misalignment of a tube tip with respect to the receptacle top openings due to piercing through the cover layer; and an auto-calibrating module for the syringe or pipette mechanism to compensate the misalignment. The extent of the misalignment suffered by the tube tip depends on the material of the cover layer, its thickness and the kind. Thinner the tube, more susceptible it is to bending during making, assembly onto the cartridge module and during use where it subjected to forces during piercing the cover layer. Therefore, the tube may be unable to enter the receptacle through the receptacle top opening such as for PCR or in a glass capillary. The tube may also be unable to enter a desirable area of a receptable during magnetic extraction and avoid hitting the magnetic beads clump. One way to find the tube tip position before and during the operation of the cartridge module is that the tube tip approaches a fixed pattern on the cartridge module, and a camera or a sensor records image or signals of the tube tip and the pattern and calculates the coordinates of the tube tip in reference to a position of the pattern. The calculated coordinates are sent to an instrument controller in the apparatus to generate a motion command to move the tube tip to a target position required in a test protocol. The sensor may be optical or electrical or magnetic.
According to an embodiment, the apparatus the detection module optically detects misalignment of the tube tip with respect to a fixed pattern on the cartridge module.
According to an embodiment, the apparatus further comprises: a syringe pickup module to pick up at least one syringe from the cartridge module.
According to an embodiment, the apparatus further comprises: a tube pick up module for mounting the tube as provided in the cartridge module onto a syringe.
According to an embodiment, the apparatus further comprising: a positioning module to detect a fixed pattern on the cartridge module and position the tube over the receptacle top openings when the tube is attached to the syringe or pipette mechanism. Before and during the operation of the cartridge module the tube tip approaches a fixed pattern on the cartridge module, and a camera or a sensor records image or signals of the tube tip and the pattern and calculates the coordinates of the tube tip in reference to a position of the pattern. The calculated coordinates are sent to an instrument controller in the apparatus to generate a motion command to move the tube tip to a target position required in a test protocol. The sensor may be optical or electrical or magnetic.
According to an embodiment, the apparatus further accommodates at least one sample container with a cap that in use contains the biological sample, the cap at least in part includes the cover layer, the syringe or pipette mechanism being computer programmable to aspirate the biological sample from the sample container by piercing the tube through the cover layer and dispense into the receptacle. This improves the contamination control as the biological sample is transferred into the receptacle by the tube while the sample container is in the cartridge module. The cap and the cover layer in the cap prevent any possible spillage of the biological sample during the whole analysis until disposal of the cartridge module.
According to a third aspect, a method is provided for improving contamination control during biological analyses and nucleic acid analyses protocol including nucleic acid extraction, nucleic acid amplification and nucleic acid detection, the method employing any of the cartridge modules and the apparatus as described above; loading the cartridge module with the reagent liquids and biological sample in the receptacles into the receiving module; conducting biological analyses or nucleic acid analysis within the apparatus without exposing the reagent liquid and biological sample to the ambience, by aspirating and dispensing the reagent liquid and the biological sample from and into the receptacles by piercing and retracting the tube through the cover layer; and disposing the cartridge module with the receptacles with the cover layer, reagent liquids, the biological sample and the tube. The advantages have been described under the second aspect.
According to an embodiment, the method comprises: operating the syringe or pipette operating mechanism according to one step from the group consisting: a) after dispensing the reagent liquid or the biological sample in the receptacle, when a tube tip of the tube has reached above the reagent liquid or the biological sample in the receptacle, conducting the syringe or the pipette in a suction mode for a predetermined time to release at least a part of an excess air pressure in the hermetically isolated receptacle that is created due to the dispensing, b) while the tube is inserted into the receptacle and before aspirating, with a tube tip of the tube being above the reagent liquid or the biological sample in the receptacle, conducting the syringe or the pipette in an ejection mode for a predetermined time to increase an air pressure in the hermetically isolated receptacle for at least partially compensating a vacuum to be created by the aspiration of the reagent liquid or the biological sample, and c) while the tube is taken out of the receptacle when a tube tip of the tube has reached the cover liquid in the stack, holding the tube for a predetermined time to release within the cover liquid a portion of the reagent liquid or the biological sample adhering to the tube. This feature reduces air pressure variation during liquid handling from and to the receptacles during liquid aspiration and dispensing. The air pressure inside the receptacle is increased due to dispensation by the tube. When the tube is thereafter retracted out of the hermetically isolating cover layer, the reagent liquid or the biological sample drips under the atmospheric pressure, thereby exposing the biological sample or the reagent liquid to the ambience. Similarly, the air pressure inside the receptacle is decreased due to aspiration by the tube. When the tube is thereafter retracted out of the hermetically isolating cover layer, the ambient air forces into the tube thereby causing chances for contamination from the ambience. The increased or decreased air pressures inside the receptacle also affect the volumes of the reagent liquid or the biological sample that are programmed to be dispensed or aspirated from and into the tube. These effects may impact in small liquid volumes but are significant when the receptacle volumes are also small.
According to an embodiment the method further comprises: dispensing the cover liquid as provided in at least one of the receptacles, on selected regions of the cover layer while the receptacle is in the cartridge module. The dispenser may be in any form in the art. Dispensing the cover liquid on the cover layer outside or inside the apparatus just before use is helpful in protecting the cover liquid from getting displaced or wiped off during transport or handling.
According to an embodiment of the method, employing the tube being attached to a pipette containing a filter and a liquid layer in the pipette, the filter and the liquid layer being separated by a first gap in between and the liquid layer being closer to a tube tip; and operating the syringe or pipette operating mechanism to maintain a second gap between the liquid layer and the reagent liquid or the biological sample as aspirated. The filter and the liquid layer block the vapors or aerosols from the biological sample to enter the pipette only to a limited extent thereby reducing contamination of the apparatus.
According to an embodiment the method comprises adapting to either step according to the group consisting: a) injecting a liquid sealant through the cover layer to seal the receptacle top opening before amplification of the biological sample in the receptacle, and b) disposing a liquid sealant on the cover layer over the receptacle top opening or on the stack, before amplification of the biological sample inside the receptacle. The liquid sealant may be viscous enough to provide a large resistance to the pressurized aerosols and/or vapors in the receptacle to leak out of the cover layer aperture or the stack during the step of amplification where the receptacle is subjected to heat. The liquid sealant may be cured by any means in the art. Herein, the receptacle top opening region is narrow enough to provide enough surface tension for holding the liquid sealant. The liquid sealant forms an air gap over the biological sample so that during imaging, fluorescence from the liquid sealant is not unwantedly captured along with that from the biological sample. The liquid sealant may be wax or glue. Wax that solidifies at room temperatures are easier for shipment of the cartridge module. During its aspiration and dispensation by the tube, it can be locally heated to a liquid form.
According to an embodiment the method comprises computer programming the syringe or pipette mechanism for the tube to pierce the cover layer or the stack at selected locations of the cover layer over the receptacle top openings, such that a size of a cover layer aperture created on the cover layer is minimized during multiple piercings.
According to an embodiment, at least once during analyses, the method further comprises: cleaning the tube and the syringe by aspirating and dispensing a cleaning liquid by the tube.
According to an embodiment, the method comprises: employing the cartridge module with the sealing layer; and generating a vent aperture in the sealing layer over the receptacle top opening by the tube attached to the syringe or pipette mechanism, so that the receptacle is in air communication with the connecting air space during subsequent aspiration or dispensation from or into the receptacle.
According to an embodiment, the method comprises: employing the apparatus to detect misalignment of the tube with respect to the receptacle top openings due to piercing through the cover layer; and calibrating the syringe or pipette mechanism to compensate the misalignment.
According to an embodiment, the method comprises: at least once, aspirating a filler liquid in the syringe by the tube; and dispensing the filler liquid, such that the filler liquid: a) fills any existing liquid retaining space in the tube head that could otherwise retain the reagent liquid and the biological sample during the subsequent dispensation after aspiration, or b) replaces the reagent liquid and the biological sample pre-occupying the liquid retaining space from previous processing, the filler liquid being immiscible with, heavier than and non-reacting with the reagent liquid and the biological sample. With the filler liquid, the dilutions of the reagent liquid and the biological sample is maintained. Also, there is no loss of volume for the reagent liquid and the biological sample during aspiration and dispensation.
According to a fourth aspect, a sample container with a cap is provided, that in use contains a biological sample, the cap at least in part includes the cover layer as described in aspect one.
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.
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.
There may be a peeling layer (not shown) over the stack 55 to prevent the cover liquid 25 from displacement when the receptacle 18 is not in upright position, the peeling layer 31 being peelable before the piercing. Alternately the peeling layer is pierceable by suitable tube 36 for dispensing and/or aspirating liquids to and from the receptacle 18 through the stack 32.
The structure and configuration of the baths disclosed under this invention do not limit the scope of achieving any kind of thermal profile. Any user specified thermal profile may be attained by suitably placing the receptacles 18 in a specified sequence and for specified time periods in the baths that are maintained at pre-determined temperatures. The receptacle 18 can rapidly cross-over the extra baths (if any) along the line of the baths that do not contribute to the thermal profile.
The tube tip 37 may be wedge-shaped for easier piercing of the cover layer 23. Typically, the outer diameter of a tube 36 or a tube 36 with beveled or wedge-shaped tip is between 0.1 mm and 5 mm, and is preferred to be less than 1 mm. The thicker the cover layer 23, the higher is the chance that the cover layer aperture 60 can be fully closed upon removal of the tube 36. The preferred thickness of the cover layer 23 is larger than 1 mm. The cover liquid 25 remains in liquid form for at least one month, when stored at 10 degrees Celsius to 50 degrees Celsius.
A vibration mechanism (not shown) may be used for vibrating the receptacles 18 at predetermined frequency, amplitude and time for dispersing in use magnetic beads within the biological sample 21. This is an alternate method to mixing by aspirating and dispensing. A press-release mechanism (not shown) for alternately pressing and releasing the receptacles 18 in the cartridge module 10 may also be used instead, for mixing the biological sample 21 in the receptacle 18 that are flexible or elastically deformable under the pressing. The apparatus 100 may further comprise a motorised stage (not shown) to position the receptacle top openings 22 under the tube tip 37, while the receptacle 18 remains in the cartridge module 10.
The receptacles 18 may be of any shape and be made of any material in the art. The receptacle 18 may be made in the form of a glass capillary of small diameters such as 0.1 mm-4 mm OD and 0.02 mm-3 mm ID. Simultaneous PCR can be advantageously conducted for non-identical biological samples 21 if the bath temperatures are suitable.
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.
The terminology ‘substantially’ in the claims is to be interpreted as: intended to be completely or 100%, with small deviations or offsets for practical reasons.
It may be appreciated that when the apparatus 100 is computer programmable to position the cartridge module 10 such that during aspiration and dispensation of the reagent liquid 12 and the biological sample 21 by the syringe or pipette operating mechanism 84 from and into the plurality of receptacles 18, the portion of the tube 36 that contacted the biological sample 21 in the receptacle 18 does not move out of the connecting air space 104, the syringe or pipette operating mechanism 84 also needs to move together with the cartridge module 10.
The cover layer 23 when elastomeric at least where the tube 36 pierces the cover layer 23 and retracts helps in the blockage of aerosol exchange and the spillage of the reagent liquid 12 or the biological sample 21 from the receptacles 18 or the cartridge module 10 into the ambience. Elastomers are rubber-like solids with elastic properties. Aerosols are typically defined as solid/aqueous particles suspended in the air and are typically of size 1−9m-10−4m, as generated by natural and anthropogenic sources. A typical diameter of air molecule is reported to be about 4×10−10 m.
The syringe pick-up module (not shown) in the apparatus 100 picks up at least one syringe 32 or at least one pipette 89 from the cartridge module 10 and releases back into the cartridge module 10 after one or more operations such as the aspiration and the dispensation. The tube pick-up module (not shown) in the apparatus 100 is for mounting the tube 36 as provided in the cartridge module 10 onto the syringe 32 or the pipette 89.
The transfer mechanism 85 picks up the reactor holder 33 for thermal cycling and returns to the cartridge module 10 after the cycling. The syringe pick-up module and the tube pick up module pick up the syringe 32 and the tube 36 by the syringe 32 from the cartridge module 10 and put back both into the cartridge module 10 after the test, after bending the tube 36. When returning the tube 36 to the cartridge module 10, the tube 36 as attached to the syringe 32 or the pipette 89 may be dipped in a protective liquid (not shown) to be covered with the protective liquid to cover the tube tip 37 to prevent contamination during the tube 36 or the syringe 32 returning process. The protective liquid may be a viscous UV glue.
The cover layer 23 may comprise of multiple layers and be in multiple phases such as the cover liquid 25, an air gap, a pierceable metal foil such as for sealing the receptacle 18, sponge and a hermetic material like rubber or plastic or silicone. The cover layer 23 may include paper, plastic or aluminum. The cover layer 23 may be a special paper that satisfies the qualities as defined under the invention. The cover liquid 25 may be a gel, particularly when soaked in the cover layer 23 or sandwiched within the cover layer 23. The tube 36 preferably has a sharp tip 37 for piercing the cover layer 23 with minimum damage in the cover layer 23. The tube 36 may be a hypodermic needle. The tube 36 may preferably be disposed after use. If the tube 36 is reused, any trace of DNA left would be amplified and cause contamination. In order to assist the pressure regulation inside the receptacle 18, the cover liquid 25 does not fully block the cover layer 23 over receptacle top opening 22, so that air can still pass through the porous layer. Apart from closing the cover layer aperture 60, the cover liquid 25 is also capable of closing the cracks generated if any in the cover layer 23 during the piercing of the tube 36. The sponge in the cover layer 23 may be soaked in cover liquid 25. The cover layer 23 may include paper, plastic or aluminum. The cover layer 23 may be a special paper that satisfies the qualities as defined under the invention. The PCR tube can be sealed by a UV curable resin, oil, gel, polymer or monomer liquid, etc. The cover liquid 25 can be stored in a receptable 18 such that the tube 36 aspirates it and dispenses to the penetration area before penetration. This is to prevent the cover liquid 25 from flowing out of the penetration area.
According to an embodiment, the apparatus 100 is programmable for enabling the syringe or pipette mechanism 84 to robotically: i) engage with a syringe 32 or a pipette 89 in the cartridge 10, and ii) disengage from the syringe 36 or the pipette 89 after placing in the cartridge 10. The apparatus 100 may comprise at least one heater module (not shown) for extraction of nucleic acid from the biological sample. The receiving module 102 is such that at least one receptacle 18 engages with the heater module for the extraction process. The syringe or pipette mechanism 84 engages with the syringe 36 or the pipette 89 in the cartridge module 10 to transfer out of the cartridge module 10 and disengages the syringe 36 or the pipette 89 after returning back to the cartridge module 10.
The sample container 115 may also have the cover layer 23 and the cover liquid 25 and may be transferrable to the cartridge module 10 for analyses. The syringe and tube pick up modules may return it back to the cartridge module 10 after the analyses.
The tube 36 preferably has a sharp tip for piercing the cover layer 23 with minimum damage in the cover layer 23. During the processes of aspiration and dispensation of the reagents 12 or the biological sample 21, at least one receptacle 18 within the cover layer 23 is maintained empty to act as an air reservoir for assisting in the air pressure regulation in other receptacles 18. An air filter 13 in cover layer 23 may be provided that blocks aerosol and spillage but allows air flow.
After the analyses, the cartridge module (10) with the receptacles (18) with the cover layer (23), reagent liquids (12), the biological sample (21), the tube (36), and the syringe (36) or the pipette (89) are disposed. Otherwise, any trace DNA left would be amplified and cause contamination.
According to an embodiment, the tube 36 used for the steps of nucleic acid extraction is wider than the tube 36 for the steps of PCR preparation before amplification. The wider tube 36 can accommodate magnetic beads in use and the protein molecules in the biological samples 21 without clogging and causing high resistance to their flow within the tube 36. The narrower tube 36 is used for the steps for PCR preparation for handling the reagents 12 and the biological sample 21 and loading into the receptacle 18 for amplification. The narrower tube 36 provides enough capillary force to avoid liquid dripping in air from the tube tip 37. The cartridge module 10 is provided with such tubes 36 and the tube pick-up module (not shown) in the apparatus 100 is capable of selecting the tube 36 that is appropriate for the step under consideration. The wider tube 36 may have an internal diameter of 0.5 mm to 3 mm and the narrower tube 36 may have an internal diameter of 0.1 mm to 0.45 mm. Other dimensions may also be used as required.
While replacing the tube 36, in order to minimize the aerosol contamination in the syringe 32 and the ambience, the method comprises the following steps: a) pushing down the plunger 66 to discharge aerosol contaminated air into the cartridge module 10, b) moving the syringe 32 with the tube 36 to a cleaner area; c) allowing the syringe 32 to aspirate air in the cleaner area; d) replacing the tube 32 by the apparatus 100 after following the steps at a) to c) in that order at least once, the tube (36) being attached to the syringe 32 or being a part of the set 145 comprising the tube 36, a tube holder 135 and a tube filter 140.
According to an embodiment, the cartridge module 10 may be provided with all the features as recited in the last claim.
The cover layer 23 may comprise an air passage filter 13 to facilitate the reduction in change of the air pressure, while substantially preventing the contamination due to spillage and blocking the aerosol exchange.
The method may further comprise: exempting at least one of the receptacles 18 from the aspiration and the dispensation, for further facilitating the reduction in change of air pressure by increasing the volume of the connecting air space 104.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202111690U | Oct 2021 | SG | national |
| 10202205151V | May 2022 | SG | national |
This application is a continuation-in-part application of International Application No. PCT/SG2022/050748, filed on Oct. 20, 2022, which is based upon and claims priority to Singapore Patent Applications No. 10202111690U, filed on Oct. 21, 2021, and No. 10202205151V, filed on May 18, 2022, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/SG2022/050748 | Oct 2022 | WO |
| Child | 18641454 | US |
