CN118139696A - Device and method for processing biological samples - Google Patents

Abstract

Cartridge modules, devices, and methods for improved pollution control during biological analysis or nucleic acid analysis protocols are provided. The cartridge module (10) includes a hollow tube (36) and a container (18). The cover layer (23) isolates the container (18) from the surrounding environment, while the container (18) allows the tube (36) to dispense reagent liquid (12) or biological sample (21) to the container (18) through the cover layer (23) only from the container top opening (22) when attached to the syringe (32). The cover layer (23) substantially blocks aerosol exchange between the environment and the container (18) throughout the analysis. Contamination of the environment due to accidental spillage of the container (18) is also prevented. The air connection space (104) reduces air pressure variations within the container (18) during aspiration or dispensing of liquid.

Description

Device and method for processing biological samples

Field of the invention

The present invention relates to devices and methods for processing biological samples, and in particular, devices and methods for performing nucleic acid extraction, amplification reactions, and detection.

Background

Extraction and amplification techniques of nucleic acids are increasingly important for application in molecular biology, food safety and environmental monitoring. Many biological researchers have used the Polymerase Chain Reaction (PCR) in the work of nucleic acid analysis because of its high sensitivity and high specificity. Generally, PCR is performed by a thermal cycling process that effects denaturation, annealing, and extension of DNA by heating and cooling a container containing a reaction material. In real-time PCR detection, the instrument detects positive reactions by accumulation of fluorescent signals. The instrument typically performs fluorescence imaging multiple times or after each thermal cycle in order to record the progressive changes in the biological sample and to detect the amount of target nucleic acid in the sample or biological sample. The PCR amplification of the trace DNA can be used for scientific research, forensic analysis, wild animal research, ultrasensitive diagnosis and the like. Isothermal amplification techniques have also been used. In a general procedure, sample preparation steps such as nucleic acid extraction and purification are required prior to PCR, wherein multiple liquid handling steps including aspiration of liquid, pushing of liquid, and mixing of liquid are required. The operations in both the sample preparation step and the nucleic acid amplification step may lead to contamination, mainly due to aerosol generation and/or evaporation of the liquid. A typical method of preventing contamination of laboratory space by nucleic acid analyzers or sample processing instruments is to seal the PCR container during amplification and place the sample preparation instrument in a biosafety cabinet or fume hood with negative pressure, where vapors generated during aerosol and/or liquid processing are removed from the biosafety cabinet. In a nucleic acid detection laboratory, in order to prevent the occurrence of the contamination, a sample preparation step for nucleic acid extraction and a PCR step are performed in different spaces, the air pressure and the air flow direction of which are strictly controlled. Outside of PCR labs, such as schools, pharmacies, airports, or commercial buildings, distributed Care Testing Sites (POCTs), the use of such biosafety cabinets or safety covers, and controlled testing environments are technically unsuitable or not cost effective.

Nested PCR with a pre-amplification step is a technique that reduces non-specific amplification of DNA templates, which typically uses two primer sets or reagents, and two consecutive PCR reactions. The first set of primers was used for the initial PCR reaction. The amplicon resulting from the first PCR reaction will be used as a template for the second set of primers and the second amplification step. However, as the practitioner is required to open the container used for the first round of amplification to perform additional liquid transfer and processing of the amplicon product from the first round of amplification in order to prepare for the second round of amplification. The possibility of contamination of the laboratory environment by the above process is also generally increased. In order to minimize contamination residues between the first and second rounds of amplification, the different steps of the process need to be physically separated from each other and preferably performed in a completely independent experimental space. The detection of amplicons in nested PCR detection is the same as in PCR. Thus, when all steps are performed in one instrument box, it is difficult to automate the nested PCR steps in the same instrument because of the high possibility of contamination.

Prior to the nucleic acid amplification step, the nucleic acid extraction and amplification process typically results from sample contamination of laboratory surfaces, reagents, used pipettes and pipette tips, laboratory coats, glove boxes, and waste baskets. Aerosols and volatiles from the sample and reagent liquids can contaminate the surrounding environment. The higher the sensitivity of the detection, the more susceptible it is to contamination. Successful aerosol pollution control, whether genotyping, creating NGS libraries, studying single cells, or testing sensitive clinical specimens, is a reliable mainstay of numerous experimental data. In the laboratory, the extraction step is very convenient using unsealed well plates and provides flexibility for many testing protocols. It is also low cost and easy to put in and out the liquid handling device. In use, however, several separate rooms are required to perform the steps of reagent preparation, sample preparation, PCR, etc., to control aerosols and/or vapors and other contaminants.

Such facilities exist only in laboratories with trained personnel, air management and regulations. Generally, such laboratories have also had corresponding decontamination protocols when contamination occurs. Conditions separating rooms, anti-pollution facilities, and trained personnel are not available in POCT environments.

Even minor contamination of biological samples can have serious adverse effects, particularly when such contamination is amplified during amplification and subsequently leads to false detection and diagnosis. Thus, both contamination of the biological sample to the environment and contamination of the sample by the environment need to be prevented.

Microfluidic cartridge modules were developed to provide a sealed environment that contained internal microchannels, valves, and pumps or pump interfaces, all of which required complex machine interfaces to operate the cartridge module. BioFire FilmArray provides a new standard for molecular diagnosis of infectious diseases in syndromes, which integrates the functions of sample preparation, amplification, detection and analysis, patent publication No.: US2018/0214864A1. In addition, the product GeneXpert also describes the use of a reaction cartridge module. The disposable cartridge module consists of a micro valve and a micro pump for transferring liquid between chambers, and the thermal cycle for amplification and the real-time optical detection are performed in the chambers. However, such cartridge modules are expensive because of the complex design and the complex production operations. High complexity may also lead to low performance reliability and require fabrication in good production systems. Microfluidic cartridge modules cannot be flexibly used for different protocols. The number of samples that can be tested is also limited by the design of the cartridge module. In addition, the cartridge module design does not control sample preparation and cross-contamination between samples and the environment during PCR.

Thus, the market is greatly demanding the development of simple and low cost liquid handling devices for performing complex aerosol and/or vapor pollution free nucleic acid extraction and sample preparation steps for PCR. Also, biological analysis of infectious or malodorous samples such as stool is a challenge in pollution control, particularly in POCT settings.

The present invention provides several improvements to the apparatus, instruments and methods for sample preparation, amplification, detection and analysis. The invention makes the disposable more affordable and reliable by using simple designs and functions, thereby providing a great positive impact on biological analysis. The invention makes it possible to speed up the amplification process, provide significant pollution control, especially for POCT settings, and also solves the problems faced when bioanalytically analyzing infectious or malodorous samples, such as stool.

Disclosure of Invention

Unless specifically stated otherwise, the terms "comprising" and "including" and grammatical variants thereof are intended to mean "open" or "inclusive" language such that they include the recited elements, but also allow for the inclusion of additional non-recited elements. The word "substantially" does not exclude "complete". The term biological sample has been used for biological samples containing nucleic acids, either before or after mixing of the reagent liquids. The biological sample may be derived from any biological form, such as a swab, saliva, blood, etc. "reagent solution" may refer to any kind used in the art, such as PCR reagents for treating biological samples, extraction solutions, purification solutions, deamination solutions, desulfonation solutions, and the like for treating biological samples. In the present disclosure, the container may be in an integrated form like an orifice plate, or in an exploded form.

According to a first aspect, the present invention provides a method for improving pollution control during a biological analysis or nucleic acid analysis procedure comprising nucleic acid extraction, nucleic acid amplification and detection. The cartridge module includes: at least one hollow tube; a plurality of containers; at least one cover layer to isolate the containers from the environment outside the cover layer, wherein each container is capable of allowing a tube connected to a syringe or pipette to pass through the cover layer and to push and aspirate reagent fluid in use from the container through only an opening in the top of the container, the cover layer being capable of substantially blocking aerosol exchange between the environment and the container throughout a biological analysis or nucleic acid analysis process even if the tube pierces the cover layer and is retracted, the cover layer being further capable of preventing contamination of the environment due to spillage of reagent fluid or biological sample from the container or cartridge module, the cover layer being connected to: 1) At least one container top opening, or 2) a cartridge module or container, and maintaining an environmentally isolated connection air space between the cover and the container top opening that reduces the variation in air pressure in the isolated container when the tube aspirates or pushes the biological sample and reagent fluid. This coating advantageously scrapes the majority of the reagent liquid or biological sample adhered under the tube under the coating as the tube is retracted from the container. The preferred tube is a beveled tip. The outer diameter of the tube is between 0.1mm and 5 mm. The thickness of the cover layer is between 0.1mm and 50 mm.

According to one embodiment, at least a portion of the cover layer material is porous when the cover layer is attached to the container top opening. The porous cover layer helps to regulate the gas pressure during liquid displacement and aspiration. The rise or fall in the internal pressure of the container can also affect the volume of reagent fluid or biological sample that is originally programmed to be pushed toward or drawn from the tube. The term effect may only cause a small volume effect, but when the container volume is also small, the effect becomes significant. Fig. 7A and 7B illustrate further problems. The porous coating should have porosity, tortuosity, and internal pore structure so as to substantially block aerosols generated in nucleic acid analysis in typical experimental practices.

According to one embodiment, at least a portion of the cover material is elastomeric. The elastomeric coating helps substantially close the coating gap created by the penetration of the tube.

The material of the cover layer is any one or combination of the following materials: a) a porous elastomer, b) a porous non-elastomer, c) a non-porous elastomer, and d) a non-porous non-elastomer. The material of the cover layer may be selected from the four types described above to optimize between pressure regulation, aerosol blocking and spill prevention.

According to one embodiment, a layer of coating liquid is pre-pushed or provided to be pushed onto the coating layer, wherein at least the portion pierced by the tube is pushed to form a stack or to soak the coating layer with coating liquid or to sandwich the coating liquid in the coating layer, the coating liquid not reacting with the reagent liquid and the biological sample. The covering liquid may also be on the underside of the covering layer. The covering liquid is advantageously adapted to enhance the insulating effect of the covering layer by blocking the gap formed in the covering layer where the tube is pierced. If the cover layer is non-porous, the insulation may be airtight. The coating liquid also improves the barrier to aerosols and spills, especially for porous coatings. With a coating liquid, in particular with a non-elastomeric coating, the container can be kept airtight from the environment both before and after the tube has pierced the coating, for example when performing steps that do not involve high temperatures. The cover liquid closes the cover apertures of the inelastic cover. As the tube passes through the covering liquid, trace amounts of reagent liquid or biological samples remaining on the tube are covered by the covering liquid. This prevents reagent liquids or biological samples adhering to the tube from being exposed to the environment and contaminating it. The covering liquid also covers a portion of the tube inserted into the container, so that any contamination from the environment into the container can be reduced. When the covering liquid is located below the covering layer, the covering liquid is also better protected from physical and handling damage. The preferred cover fluid is immiscible with the reagent fluid and biological sample and is capable of wetting the tube and cover layer. The covering liquid may be a viscous liquid polymer or monomer or oil or liquid wax or any other suitable material in the art.

Those skilled in the art will appreciate that the containers to be amplified by heat treatment need to be provided with a separate cover layer to be able to heat the containers and transfer them to the amplification module outside the cartridge.

The invention has several advantages as follows: 1) By isolation, once the reagent fluid and biological sample are pushed out into the container, these fluids will never be exposed to the environment, from bioanalytical or nucleic acid extraction processes, amplification, imaging to disposal. Thus, in the present invention, the contamination from the environment to the biological sample and the contamination from the biological sample to the environment are significantly reduced compared to the most advanced kit. The isolation significantly prevents aerosols and/or vapors from escaping or entering the container during analysis. 2) The mechanism of the container with precautions and only liquid communication from the opening above the container allows the cartridge module to operate in a relatively low complexity environment. Thus, the cartridge module reduces costs and improves reliability of detection and analysis without any valves, thermal cycling modules, or optical detection modules. Moreover, the present invention reduces cross-contamination between biological samples in the containers because there is no fluid communication between the containers. This feature also allows the containers to be used on custom computer programming, as opposed to microchannels. 3) In addition, the low cost allows the cartridge module to be discarded after the reagent fluid is consumed, thereby further reducing the likelihood of contamination. 4) The cartridge provides the user with greater flexibility in selecting a bath for the device for amplification, such as: the user may achieve faster thermal cycling by using several baths maintained at respective target temperatures without limiting the thermal cycling rate to single bath or heating block applications. 5) The simple nature of the cartridge module makes it easy to carry. 6) The cartridge module allows for the use of disposables commonly used in the industry, such as separate containers and well plates. 7) The cover layer isolation also solves the problems faced when performing biological analysis on infectious or malodorous samples such as feces in a POCT environment.

According to one embodiment, the cartridge further comprises a scrubbing layer secured to 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 the scrubbing layer being adapted to substantially scrub the cover liquid adhering to the tube when the tube is retracted from the container. Without the scrubbing layer, the cover fluid may be depleted faster when the number is smaller or the tube is longer.

According to one embodiment, the cartridge further comprises at least one syringe and provides at least one feature from the group of: a) An O-ring is arranged between the plunger of the syringe and the barrel and a cover liquid is provided on the O-ring, which cover liquid is remote from the push or suction tube tip of the barrel, and b) at least two O-rings are arranged between the plunger of the syringe and the barrel and a cover liquid is provided between the two O-rings, which cover liquid is immiscible and non-reactive with the reagent liquid and the biological sample. The O-ring with the covering liquid tends to provide the syringe with better air tightness, thereby further reducing any contamination that may enter the container from the environment, and also from the container. In addition, when the plunger is pushed down into the syringe barrel, a minute amount of reagent liquid or biological sample adhering to the inner wall of the plunger and the outer wall of the syringe barrel may be covered with the covering liquid. The cover helps to avoid exposure of the reagent fluid or biological sample to the environment and thus to avoid contamination of the environment.

According to one embodiment, the cartridge further comprises a rigid retaining layer positioned above or below the cover layer, the rigid retaining layer having a retaining layer aperture aligned above the top opening of the container to allow the tube to pierce the cover layer. The rigid retention layer advantageously prevents the cover layer from disengaging or bending as the tube pierces or retracts from the container.

According to one embodiment, the cartridge further comprises at least one container holder for receiving at least one container, the container holder being detachable from the cartridge. The container holder is useful when placing or removing containers into or from a cartridge module, such as in a module on an apparatus for biological analysis or nucleic acid analysis protocols including nucleic acid extraction, nucleic acid amplification and detection.

According to one embodiment, the cartridge further comprises a liquid sealant that seals the opening in the top of the container, both before and after curing, when pushed over the opening in the top of the at least one container, the liquid sealant being immiscible and non-reactive with the reagent liquid and the biological sample. The liquid sealant can be pushed through the tube through the cover layer or laminate and out, creating an air gap between the biological sample or reagent liquid in the container. The liquid sealant may be viscous, which may provide a significant resistance to leakage of pressurized air from the container. The sealing layer helps prevent aerosols or vapors from the biological sample within the container from escaping through the cover layer wells or the stack wells when the container is heated during the amplification step. The sealing layer also controls any such contamination that may reach the container from the environment. Thanks to this air gap, the fluorescence from the liquid sealant is not captured together with the fluorescence from the biological sample during imaging. The liquid sealant may be any tacky material, such as wax or glue. Waxes that cure at room temperature make transport of the test kit easier. It can be locally heated to a liquid form as it is sucked and pushed out by the tube.

According to one embodiment, the cover layer is provided with grooves to contain the cover liquid, and the grooves are interconnected. The tip of the hollow tube can then be moved between the dimples while remaining immersed in the covering liquid and not exposed to the environment. If the tip of the tube is exposed to the environment after aspiration or ejection of the biological sample containing nucleic acids, any dripping of the biological sample can contaminate the environment. There are several possible factors that cause unnecessary sample dripping, such as software errors or instrument errors. This embodiment avoids the possibility of such contamination, as any such biological sample droplets will be captured in the covering liquid.

According to one embodiment, the cover layer over at least one of the container top openings is flexible and has at least one slit or slit that can be partially therethrough in order to allow a blunt tube, such as a pipette, to be pierced more easily and with less effort. A blunt tube is preferred when the container is made of a deformable material. Sharp tubes have the potential to pierce deformable materials.

According to one embodiment, the cover layer has a recessed top for guiding the tube tip of the tube before piercing the cover layer when the tube tip passes through a base region of the recessed top, the recessed top having an inclined side to provide a taper towards the base region, the bottom region being smaller than the container top opening. When the tube is in the form of a sharp needle, the tapered surface is preferably a hard surface to prevent the needle from penetrating the tapered surface. This provides better tolerance for misalignment between the tip of the syringe or pipette and the top opening of the cuvette. This feature is particularly helpful when the container is in the form of a very narrow capillary tube of the type employed to enhance heat transfer efficiency for nucleic acid amplification in biological samples. The inclined sides are made of a rigid material to prevent penetration by the tip of the tube.

The cartridge module may comprise at least one container containing magnetic beads for binding nucleic acids from a biological sample. At least one of the containers may contain oil or water or DNA removing agent as a cleaning fluid for cleaning the tubing and syringe at any stage during nucleic acid analysis, including the interior and exterior surfaces of the needle cannula, and FIG. 24 shows the fluid retention space, i.e., the area where the syringe engages the tip. These areas leave a surface shape left by the previous processing steps that is undesirable to researchers. This cleaning effect also removes biological samples from previous treatments when one syringe is used to treat several samples.

According to one embodiment of the cartridge module, at least a portion of the container is made of a deformable material such that, in use, (i) the container in its initial state or shape can be inflated to contain a reagent fluid or biological sample being expelled by the instrument. (ii) The container in its initial state or shape may collapse as reagent fluid or biological sample pushed by the instrument is aspirated out of the container so that the buffer tube changes with respect to the air pressure in the isolated container as the biological sample and reagent fluid are aspirated or pushed out. This embodiment is particularly useful for non-porous coverings. The rise or fall in the internal pressure of the container can also affect the volume of reagent fluid or biological sample that is originally programmed to be pushed toward or drawn from the tube. This effect may only have a small effect on the volume, but when the volume of the container is also small, the effect becomes significant.

According to one embodiment, the cartridge module having the air connection space further comprises: a sealing layer which seals the top opening of the container or containers so as to prevent any reagent fluid and biological sample within the container from escaping when the cartridge module is not in the upright position, the sealing layer being pierceable by the tube. During use, the device needs to create a vent hole in the sealing layer through a tube attached to the syringe or pipette mechanism so that the container is in air communication with the air connection space during subsequent aspiration or pushing from the container into the container.

According to one embodiment, when the cover layer is attached to the container top opening, a plurality of isolation containers are placed vertically in a stack such that the tips of the hollow tubes are able to pierce the cover layer over each container in the stack to reach from the uppermost container in the stack to the lowermost container in the stack. This embodiment helps to reduce the footprint of the cartridge module, which is desirable for portable devices.

According to one embodiment, the cartridge module further comprises: a fixed pattern that can be detected by the device when the device automatically positions the tube over the top opening of the container.

According to one embodiment, the cartridge module further houses at least one sample container having a cap, at least a portion of which comprises a cover layer that is pierceable by the tube, so as to allow the tube to aspirate and push biological sample from the sample container into the container. This improves contamination control because the biological sample is transferred by the tube into the container while the sample container is within the cartridge module. The cover and the cover layer in the cover prevent any possible spillage of the biological sample throughout the analysis process until the cartridge module is discarded.

According to a second aspect, the present invention provides an apparatus for improving pollution control during a biological analysis or nucleic acid analysis procedure comprising nucleic acid extraction, nucleic acid amplification and detection. The apparatus includes: a) A receiving module for receiving the cartridge module according to the foregoing; b) A mechanically controlled syringe or pipette mechanism for operating the syringe or pipette connected to the syringe or pipette to aspirate or push a biological sample or reagent fluid from or into the container through the top opening of the container and the cover layer or laminate or sandwich; c) An amplification module for providing isothermal heating or cyclic heating, the amplification being performed while the container containing the biological sample is inside or outside the cartridge module; d) An optical imaging mechanism for imaging the biological sample while or after the container containing the biological sample is inside or outside the cartridge module; and e) a robotic transfer device that may be configured to transfer containers held by the container holding rack from within the cartridge module or within the apparatus to outside the cartridge module when the amplification is performed outside the cartridge module.

According to one embodiment, the device is computer programmable to operate the syringe or pipette device such that it presses a tube attached to the syringe against a fixed surface in the cartridge module or device and bends the tube, then embeds the syringe and bent tube in the cartridge module. The advantages of which have been described in the first aspect. Bending the tubing attached to the syringe or pipette after it has been used facilitates easy storage in the cartridge prior to disposal.

The apparatus also includes an Ultraviolet (UV) curing module for curing a UV resin deposited at the top opening of the container. This in situ curing prior to amplification helps to prevent vapors from the biological sample in the container from contaminating the environment through the cap layer pores formed in the cap layer due to the puncturing of the tube.

According to one embodiment, the apparatus is further programmable by the computer to position the container holding rack such that the length of tubing in contact with the biological sample within the container does not move out of the air connection space as the syringe or pipettor aspirates and pushes reagent fluid or biological sample into and out of the plurality of containers. This significantly reduces contamination.

According to one embodiment, the device is computer programmable so as to perform at least two amplification steps for a biological sample treated with a reagent solution between any two consecutive amplification steps by a cover layer with or without a cover solution, during which the container containing the biological sample is kept inside or outside the cartridge module but inside the device. The invention enables nucleic acids in biological samples to be amplified automatically in nested duplex in the same container with a coating or stack. The PCR amplicon does not need to be removed from the container after one amplification in order to mix it with the reagent solution before another amplification. Thus, the method is suitable for a rapid point of care testing (POCT) PCR process. The product can automatically perform highly complex and time-consuming manual procedures while minimizing the possibility of contamination. In order to increase the detection sensitivity of the DNA and RNA studied by the user, it may be necessary to perform amplification more than two times.

According to one embodiment, the apparatus further comprises: a detection module for detecting relative misalignment between the tip of the tube and the top opening of the container due to the puncturing of the cover layer; and an automatic calibration module for allowing the syringe or pipette device to compensate for misalignment. The degree of misalignment of the tube tips depends on the material, thickness, etc. of the cover layer. The thinner the tube, the more easily it will bend when it pierces the cover layer and is stressed during use when manufactured, assembled onto the cartridge module. Thus, the tube may not be able to enter the container through the container top opening, for example for use in PCR or glass capillaries. The tube may also not enter the desired reach within a container during magnetic extraction and impact with the magnetic bead mass may not be avoided. One method of locating the tip is to bring the tip closer to a fixed pattern on the cartridge module before or during operation of the cartridge module, and to use a camera or sensor for recording images or signals of the tip and pattern, and then to calculate the coordinates of the position of the tip relative to the position of the pattern. The coordinates calculated by the system are sent to a controller in the device to generate movement instructions for moving the tip to a target position as required by the test protocol. The sensor may be optical, electrical or magnetic.

According to one embodiment, a detection module on the apparatus optically detects misalignment of the tip of the tube relative to a fixed pattern on the cartridge module.

According to one embodiment, the apparatus further comprises: a syringe pick-up module for picking up at least one syringe from the cartridge module.

According to one embodiment, the apparatus further comprises: a tube pick-up module for mounting the tube provided in the cartridge module to the syringe.

According to one embodiment, the apparatus further comprises: a positioning module for detecting the fixing pattern on the cartridge module and for positioning the tube over the top opening of the container when the tube is attached to the syringe or pipette device. Before and during operation of the cartridge module, the tip is brought close to a fixed pattern on the cartridge module, and a camera or sensor records images or signals of the tip and the pattern, and then calculates the relative position between the tip and the pattern. The coordinates calculated by the system are sent to a controller in the device to generate movement instructions for moving the tip to a target position as required by the test protocol. The sensor may be optical, electrical or magnetic.

According to one embodiment, the device further accommodates at least one sample container having a cap for holding a biological sample, at least a portion of the cap comprising a cover layer, the syringe or pipette device being computer programmable to facilitate penetration of the cover layer and aspiration of the biological sample from the sample container, and pushing of the biological sample into the container. This improves contamination control because the biological sample is transferred by the tube into the container while the sample container is within the cartridge module. The cover and the cover layer in the cover prevent any possible spillage of the biological sample throughout the analysis process until the cartridge module is discarded.

According to a third aspect, the present invention provides a method for improving pollution control during biological analysis and nucleic acid analysis protocols, including nucleic acid extraction, nucleic acid amplification and nucleic acid detection, employing any of the cartridge modules and devices described above; loading a cartridge module containing containers containing a reagent solution and a biological sample into a receiving module; aspiration or pushing of the reagent fluid and biological sample from the container into the container by puncturing and collapsing the cap layer with the tube, performing biological or nucleic acid analysis within the device without exposing the reagent fluid and biological sample to the environment; the cartridge module containing the container as well as the cover layer, reagent fluid, biological sample and tubing is then discarded. The advantages of which have been described in the second aspect.

According to one embodiment, the method comprises: operating the syringe or pipette operating mechanism according to one of the following group: a) after pushing the reagent liquid or biological sample into the container, when the tip of the tube has reached the top of the reagent liquid or biological sample in the container, letting the syringe or pipette perform a pumping mode for a predetermined period of time in order to release at least a part of the excess air pressure in the hermetically isolated container caused by the pushing, b) before the tube is inserted into the container, and before pumping, when the tip of the tube is above the reagent liquid or biological sample in the container, letting the syringe or pipette perform a spraying mode for a predetermined period of time in order to raise the air pressure in the hermetically isolated container in order to at least partially compensate for the vacuum due to pumping the reagent or biological sample, and c) when the tube is taken out of the container, keeping the position of the tube for a predetermined period of time in order to release a part of the reagent liquid or biological sample adhered to the tube in the covering liquid. This mechanism reduces the variation in air pressure during liquid handling as liquid is drawn from and pushed to the container. The air pressure in the container increases due to the pushing of the tube. Subsequently, when the tube is retracted from the hermetically-sealed cover layer, the reagent liquid or biological sample is dropped under atmospheric pressure, thereby exposing the biological sample or reagent liquid to the environment. Similarly, the air pressure within the container may decrease due to the suction of the tube. Subsequently, when the tube is retracted from the hermetically sealed cover layer, the air in the environment is forced into the tube, creating an opportunity for contamination from the environment. The rise or fall in the internal pressure of the container can also affect the volume of reagent fluid or biological sample that is originally programmed to be pushed toward or drawn from the tube. This effect may only have a small effect on the volume, but when the volume of the container is also small, the effect becomes significant.

According to one embodiment, the method further comprises: when the containers are in the cartridge module, the covering liquid provided in at least one container is pushed out over the specified several areas on the covering layer. The ejector may be in any form known in the art. Pushing the covering liquid onto the covering layer outside or inside the device just before use helps to prevent the covering liquid from being displaced or rubbed off during transport or handling.

According to one embodiment of the method, the invention employs a tube attached to a pipette, the pipette comprising a filter and a liquid layer separated by a first gap, the liquid layer being relatively close to the tip of the tube; and operating the syringe or pipette operating mechanism to maintain a second gap between the liquid layer and the reagent liquid or aspirated biological sample. The filter and liquid layer block vapors or aerosols from the biological sample and allow them to enter the pipette to only a limited extent, thereby reducing contamination of the device.

According to one embodiment, the method comprises the step of taking any of the group consisting of: a) Injecting a liquid sealant through the cover layer to seal the top opening of the container prior to amplifying the biological sample in the container, and b) disposing the liquid sealant onto the cover layer or stack over the top opening of the container prior to amplifying the biological sample in the container. The liquid sealant may be sufficiently viscous to provide a substantial resistance to the pressurized aerosol and/or vapor escaping through the cover layer aperture or the stack aperture when the container is heated during amplification. The liquid sealant may be cured by any means known in the art. The open area of the top of the container is here sufficiently narrow to provide sufficient surface tension in retaining the liquid sealant. The liquid sealant forms an air gap over the biological sample such that during imaging, fluorescence from the liquid sealant is not unnecessarily captured along with fluorescence from the biological sample. The liquid sealant may be wax or glue. The wax, which cures at room temperature, facilitates transportation of the cartridge module. During the suction and expulsion by the tube, it can be locally heated to a liquid form.

According to one embodiment, the method includes programming the syringe or pipette mechanism with a computer such that the tube pierces only a few selected locations in the cover layer over the top opening of the container, so as to minimize the size of the pierced hole in the cover layer over multiple pierces.

According to one embodiment, during the analysis, the method further comprises at least one time: the tube and the syringe are cleaned by sucking and pushing out the cleaning liquid using the tube.

According to one embodiment, the method comprises: adopting a cartridge module with a sealing layer; and creates a vent hole in the sealing layer over the top opening of the container through a tube connected to the syringe or pipette mechanism so that the air of the container and the air connection space are in communication with each other during subsequent aspiration or pushing from the container into the container.

According to one embodiment, the method comprises: using the apparatus to detect misalignment of the tube relative to the top opening of the container due to puncturing of the cover layer; and calibrating the syringe or pipette mechanism to compensate for the misalignment.

According to one embodiment, the method comprises: at least once, aspirating the filling liquid in the syringe through the tube; and pushing the filling liquid such that the filling liquid: a) Fill any existing liquid retention space in the cartridge that might otherwise retain reagent liquid and biological sample during subsequent pushing after aspiration, or b) replace reagent liquid and biological sample from previous treatments that previously occupied the liquid retention space, the fill liquid being immiscible with, heavier than, and not reactive with the reagent liquid and biological sample. The dilution of the reagent solution and the biological sample is maintained due to the use of the filling solution. Moreover, there is no loss in volume of the reagent fluid and biological sample during aspiration and ejection.

According to a fourth aspect, the present invention provides a sample container having a cap which, in use, contains a biological sample, the cap comprising at least in part a cover layer as described in the first aspect.

Brief description of the drawings

In the following figures, like reference numerals refer to like elements throughout.

The drawings are not drawn to scale and emphasis instead is placed upon illustrating concepts.

Fig. 1A is a front view and a cross-sectional view of a cartridge module of an embodiment of the present invention, wherein a cover layer is attached to a container top opening.

Fig. 1B is a front view and a cross-sectional view of a cartridge module of an embodiment of the invention, wherein a cover layer is attached to the cartridge module.

Fig. 1C is a front view and a cross-sectional view of a cartridge module of an embodiment of the invention, wherein a cover layer is attached to a container.

Fig. 2A and 2B show front, front and cross-sectional views, respectively, and a folded side view of an embodiment of the invention, wherein the container is made of a deformable material. Fig. 2C shows the pattern of the article of fig. 2B in an expanded state.

Fig. 3A shows a plan view of an embodiment of the cartridge module without a cover layer. Fig. 3B is a perspective view of the cartridge module under the cover of fig. 3A.

Fig. 5 is an elevational cross-sectional view of an embodiment in which a region of the cover overlaps the cover.

Fig. 6A and 6B are elevation cross-sectional views of an embodiment in which the cover layer separates individual containers from multiple containers in the cartridge module, respectively.

Fig. 7A and 7B show partial sectional views in elevation for describing the problem of using an airtight cover layer.

Fig. 8A shows an elevational cross-section of an embodiment of a test kit including a cartridge module, a syringe, and a pipette. Fig. 8B shows fig. 8A in operation.

Figure 9A illustrates an elevation cross-sectional view of a tube coated with a coating liquid within a container in one embodiment. Fig. 9B shows the situation when the tube in fig. 9A is retracted from the container and a covering liquid is coated on the biological sample.

Fig. 10 illustrates an elevational cross-sectional view of a cartridge module with a scrubbing layer in accordance with an embodiment of the present invention.

Fig. 11A and 11B show an elevational cross-section of one embodiment in which the syringe has a covering liquid, and one and two O-rings.

Fig. 12 shows an embodiment in which the cover layer is tightly clamped by a rigid holding layer having a rigid layer aperture for insertion of the tube.

Fig. 15A and 15B show an elevational cross-sectional view of an embodiment of a biological sample prior to amplification, wherein the liquid sealant is pushed out over or under the stack, respectively.

Fig. 15C and 15D illustrate embodiments in which more containers create an air gap between the liquid sealant and the biological sample.

Fig. 16A shows a plan view of an embodiment of a cover layer in which grooves containing a cover liquid are interconnected. Fig. 16B shows a front view and a cross-sectional view of the cover layer at the cutting line a-B in fig. 16A.

Fig. 17A and 17B are plan views of embodiments having a via or a portion of a via on a cover layer. 17C and 17D show the flexible nature of the cover layer at the aperture.

18A, 18B and 18C show elevation cross-sectional views to depict operation of an embodiment in which the top of the cover layer has a recess.

Fig. 19A-19D are elevation cross-sectional views of an embodiment in which the device has been folded over the tube attached to the syringe for receipt into a cartridge prior to disposal.

Fig. 20A illustrates an embodiment in which the tube sprays air into the sealed isolation container prior to aspiration, as shown in fig. 20B.

Fig. 21A shows an elevational cross-section of an embodiment in which a tube pushes liquid into a hermetically isolated container, while fig. 21B shows the tube drawing air from the container after pushing.

FIG. 22A is an elevational cross-sectional view showing an embodiment in which a tube pushes liquid into a hermetically sealed container. Fig. 22B shows the tube retracted from the container after pushing. Fig. 22C shows the tube being held briefly in the covering liquid.

Fig. 23A-23C are elevation cross-sectional views, drawn according to one embodiment, to use a filter in a pipette.

Fig. 24A shows an embodiment when the tip is attached to the tube. Fig. 24B illustrates one embodiment of a method of filling a liquid retention space with a filling liquid.

FIG. 25 is a flow chart of an embodiment of a nested PCR method.

FIG. 26 shows a plan view of an embodiment of a cartridge module, and a description of the sequences used for extraction and sample loading for PCR.

Detailed Description

The following description presents several preferred embodiments of the invention in sufficient detail to enable those skilled in the art to make and use the invention.

Fig. 1A illustrates an embodiment of a disposable cartridge module 10 for improved pollution control during a nucleic acid analysis procedure including nucleic acid extraction and/or nucleic acid amplification. A cover layer 23 is attached over the container top opening 22. The containers 18 do not have means for liquid communication between the containers 18 while only allowing the tube to push liquid from the container top opening 22 to each container 18 and to draw liquid from each container 18. The tube 36 passes through the covering liquid 25 contained by the covering layer 23. The assay solution 12 in the container 18 is used for nucleic acid extraction and/or nucleic acid amplification. The cover layer 23 provides a cover liquid 25 to form a stack 55 provided by the container 18, the cover liquid 25 being immiscible and non-reactive with the reagent liquid 12 and the nucleic acid containing biological sample 21. The coating liquid 25 is used to fill the coating hole 60 formed by the tube 36 when the coating 23 is pierced, which tube 36 is used to push liquid into the container 18 and/or to suck liquid from the container 18. The tube 36, the coating 25 and the coating 23 are compatible with one another to form an isolation layer to control aerosol contamination into and out of the container 18. The preferred cover liquid 23 is immiscible with the reagent liquid 12 and biological sample 21 and is capable of wetting the tube 36 and the cover layer 23 so as to completely cover the reagent liquid 12 or sample 21 on the tube 36 when the tube 36 is removed from the container 18. The material of the covering liquid 25 may be selected from any suitable materials known in the art, including liquid silicone, oil, and liquid wax. The reagent solution 12 may be various, such as a lysis buffer, a magnetic bead buffer, a washing buffer, an elution buffer, a PCR reagent, etc. Fig. 21 already describes the function of the magnet 49. The cover layer 23 may include a piece of sponge material in the stack 55 and over the opening 22 at the top of the container for soaking the cover liquid 25 to better retain the cover liquid 25 within the cover layer 23. Some of the containers 18 may be loaded with a reagent solution 12 for nucleic acid extraction and amplification. Cartridge module 10 may provide at least one void 48 to accommodate at least one or several containers 18 in use. The vertical and horizontal movement of the tube 36 needs to be provided by the device. The cover layer 23 may comprise an elastomeric material or a non-elastomeric material or a combination of both. The elastomeric material may be a non-porous material such as natural or synthetic rubber or silicone or any other suitable material in the art. The elastomeric material may be a porous material, such as a sponge. The non-elastic material may be a non-porous material such as a plastic film or a metal film or a laminate thereof, or a porous material such as a fibrous mat. The cover layer 23 may be made of more than one of the above materials. Tube 36 may be metal or plastic or any other suitable material. Cartridge module 10 may include at least one container 18 without a cover layer 23 to allow a user to isolate container 18 from cover layer 23 in use. The cover liquid 25 may be immersed in the cover layer 23 or sandwiched between cover layer materials or form a stack 55 with the cover layer 23. Multiple layers of the material of the cover layer 23 may be helpful, particularly when the cover layer 23 comprises a non-elastic material. Cartridge module 10 may or may not include a syringe 32 or pipette 89 or reagent solution 12. Tubing 36, syringe 32 or pipette 89 is placed in cartridge module 10 for disposal. This embodiment provides an air connection space above the containers 18 and below the cover layer 23 to allow for air communication between several containers 18. The empty container 18 shown in the figures helps to add bulk to the air connection space 104. The cover layer 23 is tightly clamped by a rigid holding layer 46, which rigid holding layer 46 has a hole 45 for inserting the tube 36. The tube 36 passes through the covering liquid 25 held by the covering layer 23. Fig. 1C shows an embodiment in which a cover layer 23 is attached to the container 18, while the air connection space 104 is above the container 18 and below the cover layer 23, in order to allow several containers 18 to communicate air. The cartridge module 10 may accommodate more than one syringe 32 or more than one tube 36 of different types or sizes, depending on the process requirements. The device 100 is provided with a replacement mechanism for the syringe 32 and tube 36. Cartridge module 10 has a receiving, releasing and receiving: a) a syringe 32, b) a tube 36, and c) a mechanism of the syringe 32 with the tube 36 mounted. Cartridge module 10 contains a UV curable resin and a cleaning fluid for cleaning tubes 36. Cartridge module 10 contains magnetic beads for nucleic acid binding or porous filter-like elements for binding nucleic acids under vacuum, pressure and centrifugal force. Cartridge module 10 has a pattern for identifying the position of tip 37.

Fig. 2A illustrates an elevational cross-sectional view of a front view of a container 18 according to one embodiment, wherein the container 18 is made of a deformable material. Fig. 2B is a side view of the container 18 of fig. 2A in a contracted state. The container 18 indicated by the black arrow in fig. 2C shows how the deformable container 18 can increase the internal volume of the container 18 from the contracted state 6 by expanding in order to contain the reagent liquid 12 or the biological sample 21. The increase in internal volume may be accomplished without the need to increase the air pressure in the container 18. The deformable material may be appropriately selected from the prior art and needs to be non-reactive with the reagent liquid 12 and the biological sample 21.

Fig. 3A shows a plan view of an embodiment of a cartridge module 10. Fig. 3B is a perspective view of the cartridge module 10 with the cover layer 23 and the window 41 for receiving ultraviolet light. The cartridge module 10 may be loaded to and/or unloaded from an apparatus (not shown) for nucleic acid extraction and polymerase chain reaction PCR for amplifying nucleic acids. Although some of the containers 18 have been previously filled with the reagent solution 12 and the cover solution 25, the remaining containers are shown as empty, allowing the user to use these empty containers 18 for the intended purpose of mixing the biological sample 21 with the reagent solution 12. Biological sample 21 as defined herein may or may not include one or more reagent solutions 12. Before amplification, PCR reagents as reagent solutions 12 are added to the amplification modules within the apparatus. The covering liquid 25 may be more than one liquid each loaded in a different container 18. The apparatus may have one or several container holders 33 for holding one or several tubular containers 18. The container 18 may also be in the form of an orifice plate having a plurality of tubular containers 18. Window 41 is shown transparent to ultraviolet light so that when ultraviolet light is allowed to cure, the sealant (not shown) contained in top opening 22 of container 18. The uv source may be adjustable to project uv light at an upward angle to protect the biological sample 21 loaded at the bottom of the container 18 from exposure to uv light provided in the apparatus. In particular, the sealing and curing steps are performed before the amplification step. The container holder 33 can be detached from the cartridge module 10. The container 18 may be fixedly attached to the container holder 33. All components in contact with the biological sample 21 and the reagent liquid 12 are disposable and disposable consumables after use. The cartridge module 10 includes a liquid bath medium 75 in at least one container 18 for being heated during nucleic acid amplification in a biological sample. The apparatus may then be calibrated in advance for heating the bath medium 75, and the user may only need to fine tune the calibration in the field. The liquid bath medium 75 also allows for easy transfer of the liquid bath medium 75 between baths in the apparatus and between containers 18 in the cartridge module 18. During transport of the apparatus, the liquid bath medium 75 may be transferred back to the container 18 in the cartridge module 10. Storing the liquid bath medium 75 in the container 18 also helps to reduce the effects of evaporation when the device is not in use.

Fig. 4A shows a plurality of containers 18 made of deformable material and stacked so that they are in a collapsed condition when not loaded with any objects. Fig. 4B shows an elevation cross-sectional view of fig. 4B. Wherein the container 18 is in an expanded state when containing the reagent solution 12 and the biological sample 21.

Fig. 5 shows an embodiment of the invention in which the cover layer 23 is embedded in the lid 35 of the container 18 and extends through the entire thickness of the lid 35, while the cover region 39 covers both above and below the lid 35. A portion of the cover layer 23 on the cover 35 is pierceable by the tube 36, the cover layer 23 being embedded in the cover 23 and extending through the entire thickness of the cover 23, and having first and second cover areas 39 above and below the cover 35, the cover areas being resistant to any relative movement between the cover layer 23 and the cover 35 during piercing and retraction of the tube 36.

Fig. 6A shows that the cover layer 23 individually surrounds each container top opening 22 with an attachment 43 to individually cover each container 18 in the cartridge module 10. Fig. 6B shows that the cover layer 23 covers several containers 18 in the cartridge module 10 by surrounding the several containers 18 by the attachment 43.

Fig. 7A and 7B illustrate some practical problems of the airtight isolation cover layer 23. As shown in fig. 7A, the air pressure inside the container 18 increases due to the pushing of the tube 36. When the tube 36 is thereafter retracted from the hermetically-sealed cover layer 23, the biological sample 21 is dropped from the tube tip 37 under atmospheric pressure, thereby exposing the biological sample 21 to the environment and contaminating the environment. Similarly, the air pressure inside the container 18 is reduced by the suction of the tube 36. Thereafter, when the tube 36 is retracted from the hermetically sealed cover layer 23, air in the environment may be forced into the tube 36, thereby giving the environment a chance to contaminate the interior of the tube 36.

Fig. 8A shows an embodiment of a kit 110 comprising a cartridge module 10, a syringe 32 and a cover pipette 89. In one of the containers 18, a cover liquid 25 is provided, and a pipette 89 is used to push the cover liquid 25 to a selected area, such as a recess 27 in the cover layer 23, when the container 18 is in the cartridge module 10. Fig. 8B shows an embodiment in which a pipette 89 pushes the cover liquid 25 from the container 18 onto the cover layer 23, as indicated by the dashed arrow. The solid arrows represent the process of aspirating reagent fluid 12 from container 18 through syringe 32 and pushing it out into container 18 containing biological sample 21. The pipette 89 may be of any form known in the art. Pushing the covering liquid 25 onto the covering layer 23 inside the device just before use helps to protect the covering liquid 25 from being displaced or rubbed off during transport or handling. In this embodiment, the cartridge module 10 also houses a sample container 115 with a lid 116, the sample container 115 housing a biological sample 21 for analysis. The cap 116 includes a cover layer 23 that is pierced by the tube 36 to aspirate the biological sample 21 and pushed into the container 18. As depicted in fig. 25, when cartridge module 10 is loaded into apparatus 100, the syringe or pipette device 84 is computer programmable to aspirate biological sample 21 from sample container 115 and expel into container 18 with tube 36. This improves contamination control as the tube 36 transfers the biological sample 21 into the container 18 during the time that the sample container 115 is in the cartridge module 10. The cover 116 and the cover layer 23 in the cover 116 prevent any possible spillage and aerosol leakage of the biological sample 21 during the entire analysis process until the cartridge module 10 is discarded. This allows the user to avoid the complex manual transfer operations that require training in using the syringe 32 or pipette 89 in order to push the biological sample 21 into the container 18 without contaminating the surrounding environment. This method is particularly important for the analysis of infectious biological samples.

Fig. 9A shows tube 36 of syringe 32 inserted into container 15 through stack 55 and pushing biological sample 21 into container 15. As the tube 36 passes through the covering liquid 25, a quantity of the covering liquid 25 covers the tube 36 that enters the container 18. The cover liquid 25 does not cause contamination problems, since the cover liquid 25 is not miscible with the reagent liquid 12 and the biological sample 21 in use and does not react. Fig. 9B shows the situation when the tube 36 is retracted from the container 18 through the stack 55. Consistent with the expectation, by default, the tube 36 is covered by a layer of biological sample 21, and as the tube 36 passes through the covering liquid 25, a layer of covering liquid 25 also covers the layer of biological sample 21, as contemplated by the present invention. Thus, the present invention prevents trace layers of biological sample 21 from being exposed to the environment, thereby reducing contamination. The coating fluid 25 also helps to seal the coating aperture 60 when the tube 36 pierces the coating. The coating liquid 25, coating 23 and tube 36 need to be compatible with each other to ensure sealing of the coating aperture 60. The figure shows only a part of the cover layer 23.

Fig. 10 illustrates an embodiment in which the scrubbing layer 28 is secured to the laminate 55 such that the cover layer 23, the cover liquid 25, and the scrubbing layer 28 form a composite layer 30 for penetration by the tube 36 and into the container 18. As shown, the scrubbing layer 28 helps to scrub off excess coating liquid 25 adhering to the upper portion of the tube 36, which excess coating liquid 25 may deplete the coating liquid 25 when the amount of coating liquid 25 is smaller or the tube 36 is longer.

Fig. 11A shows an embodiment in which a syringe 32 containing a biological sample 21 has two O-rings 68 between the plunger 66 and barrel 67 of the syringe 32. The covering liquid 25 is disposed between the two O-rings 68 and away from the tube head 62 which engages the barrel 67. Fig. 11B shows the situation when plunger 66 is pushed further into barrel 67 to push away biological sample 21. The covering liquid 25 between the two O-rings 68 substantially covers the trace biological sample 21 adhering to the inner wall of the cylinder 67 and the outer wall of the plunger 66, so that the biological sample 21 exposed to the environment is substantially maintained at a negligible level.

Fig. 12 shows an embodiment in which the cover layer 23 is tightly sandwiched by a rigid retainer layer 46 below the cover layer 23, the rigid retainer layer 46 having a rigid layer aperture 45 aligned with the container top opening 22 for insertion of the tube 36 to penetrate the cover layer 23.

Fig. 13 shows cartridge module 10 with air connection space 104, with sealing layer 120 on container top opening 22. The apparatus 100 is computer programmable to allow the tube 36 to pierce the laminate 55 and the sealing layer 120 to create a vent 61 in the sealing layer 120 above the container top opening 22 to allow air communication between the container 18 and the air communication space 104 during aspiration and expulsion of the tube 36.

Fig. 14A to 14D show an embodiment of the operation of the apparatus 100. Here, the cartridge module 10 is provided with a coupling mechanism 99, which coupling mechanism 99 is sealingly fitted on the cartridge module wall 98 and connected to the container holder 33, allowing the device 100 to operate the coupling mechanism 99 from outside the cartridge module 10 in order to move the container holder 33 in the cartridge module 10 back and forth in the horizontal direction for positioning the container top opening 22 below the tip 37 during the container 18 is inside the cartridge module 10. The syringe or pipette device 84 is computer programmable to allow the tube 36 to be moved in a vertical direction so that the tube 36 does not need to be moved out of the air communication space 104 during aspiration from the container 18 and pushing of the reagent liquid 12 and biological sample 21 into the container 18. Fig. 14A shows a state when the tube 36 is inserted into the left outermost container 18. Fig. 14B shows a state when the tube 36 is lifted out of the left outermost container 18 without the tube tip 37 moving out of the air communication space 104. Fig. 14C shows a state when the tube 36 is reinserted into the right outermost container 18. Fig. 14D shows a state when the tube 36 is lifted out of the right outermost container 18 without the tube tip 37 moving out of the air communication space 104. According to an alternative embodiment (not shown), vertical motion is provided to cartridge module 10 instead of tube 36. The airtight coupling mechanism 99 helps to isolate the container from the environment. Alternatively, the device 100 may operate the coupling mechanism 99 from outside the cartridge module to move the cartridge module in a horizontal direction, such that the coupling mechanism 99 does not need to be sealingly attached to the cartridge module wall 98.

Fig. 15A shows that the liquid sealant 73 is arranged onto the stack 55 before the biological sample 21 in the container 18 is amplified, and then the liquid sealant 73 is cured by the heat source or light source or chemical in the device 100 during the time that the container is within the cartridge module 10. Fig. 15B represents an alternative embodiment in which there is an orifice layer 74 below the cover layer 23, the orifice layer 74 having a sealing orifice 75, the sealing orifice 75 being sealable by injecting a liquid sealant 73 into the sealing orifice 75 prior to heat treatment for amplification of the biological sample 21 in the container 18. Tube 36 may inject the liquid sealant 73 through the cap layer hole 60 into the seal hole 75. The thermal or optical or chemical treatment cures the liquid sealant 73 when the container 18 is positioned in the cartridge module 10. This helps prevent any aerosols and/or vapors generated during the heat treatment from escaping from the overlay hole 60. Any such contaminants entering the container 18 from the environment are also controlled. In the case of nested PCR, the liquid sealant 73 is used just prior to the second amplification. If the sealant is used and cured prior to the first amplification, it is not possible for the device to add reagent liquid 12 through the layer of already hardened sealant liquid 73 prior to the second amplification. In the case of nested PCR, after the device completes the first amplification, the sealant liquid 73 used in the first amplification may be in a liquid state or may be converted back to a liquid state (e.g., by heating the liquid sealant 73 in the form of wax) to allow the tube 36 to aspirate or push liquid from or to the container 18 in preparation for PCR. If the liquid sealant 73 is cured before the first amplification, it is impossible to add the reagent liquid 12 through the liquid sealant 73 that has been hardened before the second amplification. Alternatively, without the use of sealant liquid 73, cover layer 23 may be pierced directly by tube 36 after the first amplification is complete, so that tube 36 can aspirate and/or push liquid from container 18 to container 18 prior to the first amplification. In alternative embodiments, the sealant liquid 73 may have a high viscosity and cannot be hardened or cured prior to the first amplification. In yet another embodiment, the sealant liquid 73 may be a wax-like material, may be hardened or cured prior to the first amplification, and may be softened by heating after the first amplification to allow the tube 36 to aspirate or push liquid from or toward the container 18 prior to the second amplification. The liquid sealant may be cured by ultraviolet light or heat or chemicals, and may be any suitable material in the art, such as oils, polymers, monomers, and waxes. 15C and 15D illustrate a container 18 with an air gap 16 created and maintained between the liquid sealant 73 and the biological sample 21. Here, this embodiment selects a combination of liquid sealant 73 and container 18 such that the container can retain liquid sealant 73 at container top opening 22 as liquid sealant 73 is pushed in. A syringe or pipette device 84 is used to aspirate the liquid sealant 73 and push it out near the container top opening 22. Biological sample 21 is then pushed through liquid sealant 73 into container 18. Here, the covering liquid 25 is located below the covering layer 23. Prior to amplification, the liquid sealant 73 may be cured so that it becomes hard. The liquid sealant 73 may be in the form of wax or glue. Curing is completed by exposing the liquid sealant 73 to ultraviolet rays. The ultraviolet rays may be irradiated to the liquid sealant 73 at an upward angle so that the biological sample 21 is not exposed. In-line curing helps to further integrate and automate the process prior to heat treatment to avoid evaporation problems of biological samples 21 that lead to contamination inside the device. Any other method used in the art for curing may also be employed, for example by chemical or thermal treatment. In the case of nested PCR, the liquid sealant 73 is used just prior to the second amplification. If the sealant is used and cured prior to the first amplification, it is not possible for the device to add reagent liquid 12 through the layer of already hardened sealant liquid 73 prior to the second amplification. The liquid sealant 73 may be non-reactive and non-miscible with the reagent liquid 12 and the biological sample 21. The air gap 16 is used to further isolate the biological sample 21 or the reagent liquid 12 from the environment.

Fig. 16A shows a plan view of an embodiment of the cover layer 23, in which two grooves 27 filled with the cover liquid 25 are in communication with each other. Fig. 16B shows an elevation cross-sectional view of the cover layer 23 at the a-B cut line in fig. 14A, wherein the tube tip 37 of the hollow tube 36 moves between the interconnected grooves 27 while remaining immersed in the cover liquid 25 and not exposed to the environment. Tube 36 aspirates biological sample 21 from container 18. After the biological sample 21 is aspirated through the tube 36, it is retracted from the container 18 and moved over the container 18, as indicated by the block arrow. Thereafter, tube 36 enters container 18 by piercing cover layer 23. Tube 36 then pushes biological sample 21 onto reagent fluid 12 in container 18. Tube 36 may be aspirated and expelled multiple times to mix the two liquids. After pushing, the tube 36 will be retracted from the container 18, a step not shown here. Thus, during this operation, the tube tip 37 is held within the container 18 or within the covering liquid 25. When the tip 37 leaves one container 18 and moves to another, it is not exposed to the environment. This prevents biological sample 21 from dripping from tip 37 and being exposed to the environment.

The plan view of fig. 17A shows the unidirectional through holes 96 on the cover layer 23. The plan view of fig. 17B shows a cross-shaped through hole 96 in the cover layer 23, particularly for a blunt tube 36 such as used with a pipette 89. Any other shape may be used. The elevation cross-section of fig. 17C shows the blanket bending downward as the tube pierces the hole 96. The elevational cross-sectional view of fig. 17D shows that the cover layer 23 is substantially bent back to its original planar shape as the tube 36 is retracted from the container 18 through the aperture 96. Since the aperture 96 is substantially curved back to its original shape, cross-contamination between the interior of the container 18 and the surrounding environment is controlled. The cover liquid 25 may also be pushed over the holes 96 to enhance isolation. The aperture 96 needs to be sufficiently narrow to prevent the covering liquid 25 from leaking into the container 18.

Fig. 18A, 18B and 18C illustrate one embodiment of a container 15 for a nucleic acid analysis protocol including nucleic acid extraction or nucleic acid amplification. As shown, the container 15 has a cover layer 23 that covers the container top opening 14 and isolates the container 15. The cover layer 23 has a recessed top 76 for guiding the tip 37 of the tube 36 in use before the tip 37 pierces the cover layer 23 through the base 72 of the recessed top 76. The recessed top 76 has sloped sides 71 that act as guide surfaces to provide a taper to the base 72 for the tube 36 to reach the base 72, the base 72 being smaller than or equal to the container top opening 14. The sloped side 71 is stiffer than the cover layer 23 such that the tip 37 slides along the sloped side 71 and reaches the base region 72 without piercing the surface 71, as shown in fig. 10B, to pierce the cover layer 23, as shown in fig. 18C. This provides for better tolerance for misalignment between the tip 37 of the syringe 32 relative to the capsule top-opening 14. The inclined surface 71 may be made of a metal or a hard plastic material.

Fig. 19A shows an embodiment in which the tube 36 attached to the syringe 32 is bent so as to allow the cartridge module 10 to house the tube 36 after use and before disposal. Fig. 19B, 19C and 19D show with block arrows how the syringe or pipette device 84 in the apparatus 100 is operated stepwise to press the tube 36 sideways and downwards against one of the fixed surfaces 42 in the cartridge module 10 or in the apparatus 100 in order to bend the tube 36. Thereafter, the syringe 32 with the tube 36 having been bent is mounted on the cartridge module 10 as shown in fig. 19A. The hollow tube 36 is flexible and may be made of any suitable material, such as metal.

FIG. 20A shows that when the device inserts the tube 36 into the container 18 and prior to aspiration, the syringe operating means 84 is computer programmable such that when the tip 37 of the tube 36 reaches the assay solution 12 or biological sample 21 in the container 18, the syringe 32 performs the spray mode for a predetermined period of time to create an excess air pressure within the hermetically isolated container 18 that aids in releasing the partial vacuum created after aspiration of the assay solution 12 or biological sample 21 by the tube 36 as shown in FIG. 20B.

Fig. 21A shows an embodiment of the device. When the tube 36 is removed from the container 18 after ejection, a syringe handling device (not shown) may be computer programmed to operate according to the following steps: as shown in FIG. 21B, when tip 37 of tube 36 has reached the top of assay solution 12 or biological sample 21 in container 18, syringe 32 performs a pumping mode for a predetermined period of time to release at least a portion of the excess air pressure in hermetically isolated container 18. The curved arrow shows the suction for excess air pressure. Excess air pressure is created as a result of the assay solution 12 or biological sample 21 being pushed into the hermetically isolated container 18. Reducing the excess air pressure within the container 18 helps to expel less of the analysis liquid 12 or biological sample 21 from the container 18 as the tube tip 37 exits the cover layer 23.

Fig. 22A shows an embodiment of an apparatus. When tube 36 is removed from container 18, the syringe operating mechanism is computer programmable to operate according to the following steps: as shown in FIG. 22B, when the tip 37 of the tube 36 has reached between the cover layer 23 and the cover liquid 25, the tube 36 is maintained in this position for a predetermined period of time to release the assay liquid 12 or biological sample 21 attached to the tube 36 therein. Fig. 22C is an enlarged view of the dotted circle area of fig. 22B.

Fig. 23A illustrates a method in which the pipette device 84 aspirates or pushes the biological sample 21 through one of the filters 17. Fig. 23B shows another embodiment in which the pipette device 84 aspirates and aspirates biological sample through a liquid layer 94 and filter 17 and a first gap 93 is maintained between the liquid layer 94 and filter 17. Fig. 23C shows the biological sample 21 in fig. 23B being aspirated such that the biological sample 21, the liquid layer 94, and the filter 17 remain separated from one another. A second gap 95 is maintained between the biological sample 21 and the liquid layer 94. This significantly reduces nucleic acid aerosol and/or vapor contamination during the drawing and pushing of the container 18 into the container 18.

Fig. 24A depicts an embodiment where one tube 36 is attached to tube head 62 with glue 109. The tube tip 112 of the tube 36 protrudes above the bottom 110 of the tube head 62 to prevent the glue 109 from blocking the tube tip 112. This forms a liquid retention space 108. Fig. 24B shows that after the filling liquid 107 is sucked into the syringe 32 by the tube 36 for the apparatus and the filling liquid 107 is pushed out again, the liquid-retaining space 108 filled with the filling liquid 107 is filled with any liquid-retaining space 108 present in the tube head 62 by the filling liquid 107, which liquid-retaining space 108 would otherwise retain the reagent liquid 12 and the biological sample 21 during pushing out after suction. The filling liquid also replaces any reagent liquid 12 or biological sample 21 from previous processing steps that remains in the liquid retention space 108. This two-step aspiration and push process may be performed several times before one or more of the following steps: 1) The purified biological sample 21 is aspirated from the elution buffer containing the purified nucleic acids. In a direct PCR or other direct amplification procedure that does not require a nucleic acid purification step, 2) aspirating PCR reagent solution 12 to prepare a final reaction mixture containing purified nucleic acid, 3) aspirating the final reaction mixture for loading into a PCR container 18, and 4) any step in a nested PCR procedure. The nucleic acid extraction step or the PCR step is optional. Other sample processing may also be performed on cartridge module 10 with tube 36, including direct PCR with release of nucleic acids from the sample and no purification steps, isothermal PCR with or without nucleic acid purification steps.

Fig. 25 is a flow chart in an apparatus 100 for nested PCR according to one embodiment of the present invention. The apparatus 100 is used to improve pollution control during nucleic acid analysis protocols including nucleic acid extraction, nucleic acid amplification and detection. In this embodiment, the cartridge module 10 and the biological sample 21 are loaded into the device 100 by the receiving module 102. The syringe or pipette device 84 transfers the bath medium 75 from the container 18 in the cartridge module 10 to a bath (not shown) in the amplification module 101. Syringe or pipette device 84 is used to manipulate hollow tube 36 along syringe 36 or pipette 89 to aspirate and expel fluid from container top opening 22 through cover layer 23 at a predetermined location. The syringe or pipette device 84 mixes the biological sample 21 with the reagent fluid 12 in the container 18 with the cover layer 23 for extraction and PCR. The transfer device 85 then transfers the containers 18 from the cartridge module 10 to the amplification module 101. In the amplification module 101, the transfer device 85 performs a first thermal cycle for the biological sample 21 in the container 18 having the covering liquid 23 and held by the container holding frame 33. Or the amplification module 101 may provide isothermal heating rather than cyclic heating as shown for amplification of nucleic acids in the vessel 18. The transfer device 85 then transfers the containers 18 held by the container holder 33 back to the cartridge module 10. With the container 18 in the cartridge module 10, the syringe or pipette device 84 further mixes the reagent liquid 12 with the biological sample 21 through the cover layer 23. The transfer device 85 then performs a second thermal cycle for the biological sample 21 in the container with the cover layer 23 in the amplification module 101. During and after amplification, the imaging mechanism 19 performs fluorescence imaging. Since in this embodiment the amplification is performed outside the cartridge module 10, the robotic controlled transfer device 85 is configurable to position the containers 18 held by the container holding rack 33 outside the cartridge module 10. Or the container 18 may be retained in the cartridge module 10 to undergo amplification by the device 100. Thereafter, the transfer device 85 transfers the containers 18 held by the container holding frame 33 back to the cartridge module 10. The syringe or pipette device 84 then transfers the bath medium 75 from the bath to the container 18 in the cartridge module 10. Thereafter, the cartridge module 10 is unloaded from the device 100. As soon as the reagent solution 12 is consumed, the cartridge module 10 is discarded. The apparatus 100 is computer programmable to process the biological sample 21 with the reagent fluid 12 and at least one of nucleic acid extraction, nucleic acid amplification, and detection of the biological sample 21 during the positioning of the container 18 in the cartridge module 10. Such an embodiment of nested PCR has wide application in point of care testing (POCT). Imaging module 19 may optically image with container 18 either inside or outside of cartridge module 10. The use of the tube 36 and cover layer 23, as well as the cover layer and cover liquid 25 concepts described above, allows extraction and nested PCR with dual amplification to be performed within the apparatus 100 without the need to remove the cartridge module 10 in the process. In one nested PCR, the containers 18 used for the first amplification, when mixed with the reagents 12 used for the second PCR, and the second amplification, should all be covered by one or more cover layers 23, either on a single container 18 or on all containers 18.

FIG. 26 is a plan view of a cartridge module, which partially depicts a flow chart of an extraction sequence and PCR, and FIG. 1 may also be referenced.

P26 extraction sequence

1. Mixing biological samples in container 18 with tube 36

2. Biological sample 21 is aspirated into lysis vessel 18 and mixed using tube 36

3. Binding buffer (magnetic beads) is drawn into lysis vessel 18 and mixed using tube 36

4. Moving magnet 49 upward from position B to position A and holding in position A for a predetermined period of time

5. During the raising of the magnet 49, the solution is mixed in the cleavage vessel 18

6. The supernatant is discarded into a waste container 18

7. Moving magnet 49 downwardly from position a to position B

8. The wash 1 buffer is aspirated and pushed into the lysis vessel 18 and mixed using tube 36

9. Moving magnet 49 upward from position B to position a and holding in position a for a predetermined period of time

10. During the raising of the magnet 49, the solution is mixed in the cleavage vessel 18

11. The supernatant is discarded into a waste container 18

12. Moving magnet 49 downwardly from position a to position B

13. Wash 2 buffer is aspirated and pushed into lysis vessel 18 and mixed using tube 36

14. Moving magnet 49 upward from position B to position a and holding in position a for a predetermined period of time

15. During the raising of the magnet 49, the solution is mixed in the cleavage vessel 18

16. The supernatant is discarded into a waste container 18

17. Moving magnet 49 downwardly from position a to position B

18. Starting heating the pyrolysis container to a predetermined temperature

19. Cleaning tube 36 at clean 1 vessel 18, clean 2 vessel 18, and clean 3 vessel 18

20. Stopping heating the pyrolysis container 18

21. 100Ul of elution buffer is drawn into the lysis vessel 18 and mixed using tube 36

22. Moving magnet 49 upward from position B to position a and holding in position a for a predetermined period of time

23. During the raising of the magnet 49, the solution is mixed in the cleavage vessel 18

24. Moving magnet 49 downwardly from position a to position B

25. The eluate is extracted for PCR

All mixing steps are here performed by several rounds of aspiration and expulsion through the tube 36. Here, in step 1, the biological sample 21 is mixed in the container 18. In step 2, a volume of sample 21 is aspirated and pushed into extraction vessel 18 containing the lysate and mixed. In step 3, another volume of binding buffer with magnetic beads is added to the lysis vessel 18 via tube 36. Then, in step 4, the magnet 49 is then moved upward from position B to position a near the cleavage vessel 18 for a predetermined period of time. In step 5, the solution in the lysis vessel 18 is mixed while the magnetic beads with DNA and RNA attached are located on the wall of the lysis vessel 18 and facing the magnet 49, while the magnet 49 is in position A. In step 6, tube 36 transfers supernatant/waste liquid from the lysis vessel to the binding buffer vessel 18. Then, in step 7, the magnet 49 is moved downward from the position a to the position B. In step 8, a volume of wash 1 buffer is aspirated from the wash 1 buffer container 18 and pushed into the lysis container 18 and then mixed. In step 9, magnet 49 is again moved upward from position B to position a and held in position a for a predetermined period of time. In step 10, the solution in cleavage vessel 18 is mixed during the raising of magnet 49. Then, in step 11, the tube 36 transfers the supernatant/waste liquid from the lysis vessel 18 into the wash 1 buffer vessel 18. Then, in step 12, the magnet 49 is moved downward from position a to position B. In step 13, a volume of wash 2 buffer is aspirated from the wash 1 buffer container 18 and pushed into the lysis container 18, and subsequently mixed. In step 14, magnet 49 is again moved upward from position B to position a and held at position a for a predetermined period of time. In step 15, the solution in cleavage vessel 18 is mixed during the raising of magnet 49. Then, in step 16, the tube 36 transfers the supernatant/waste liquid from the lysis vessel 18 into the wash 2 buffer vessel 18. Then, in step 17, the magnet 49 is moved downward from the position a to the position B. Subsequently, in step 18, the lysis vessel 18 is heated to a predetermined temperature for a predetermined period of time until the washing in the wash 3 vessel is completed. In step 19, the apparatus cleans tube 36 by several rounds of aspiration and pushing in wash 1 vessel 18, then cleans tube 36 in the same manner in wash 2 vessel 18, and then cleans tube 36 in the same manner in wash 3 vessel 18. Then, in step 20, the heating of the cleavage vessel 18 is stopped. In step 21, tube 36 aspirates a volume of elution buffer from elution buffer container 18 and is then pushed into lysis container 18 and mixed. In step 22, magnet 49 is again moved upward from position B to position a and held in position a for a predetermined period of time. In step 23, the solution in cleavage vessel 18 is mixed during the raising of magnet 49. Then, in step 24, the magnet 49 is moved downward from position a to position B. Then, in step 25, the tube 36 aspirates the eluate for PCR. FIG. 24 also illustrates the sample loading sequence for PCR after the previous extraction sequence. Here, in step 26, PCR buffer is mixed in PCR buffer container 18. In step 40, a predetermined volume of PCR buffer is aspirated from the PCR buffer container 18 and pushed into the PCR enzyme container 18 containing the PCR buffer enzyme and then mixed to form a PCR premix. In step 28, a predetermined volume of PCR premix is aspirated from the PCR enzyme container and pushed into the negative control container 18, and then mixed. In step 29, a mixed predetermined volume of negative control PCR premix is aspirated and pushed into the glass capillary vessel 1 (not shown). In step 30, the iron 49 is again moved upward from position B to position a and held in position a for a predetermined period of time. In step 31, a predetermined volume of liquid is aspirated from the extraction tube and pushed into the PCR enzyme container and then mixed. In step 32, a predetermined volume of biological sample 21 from the PCR enzyme container 18 is aspirated and pushed into the glass capillary 2 (not shown). In step 33, a predetermined volume of liquid is aspirated from the PCR buffer container 18 and pushed out to the PCR enzyme container 18, and then mixed. In step 33, a predetermined volume of the solution is aspirated from the PCR enzyme container and pushed out into the glass capillary container 3 as a positive control-PC. In step 34, a predetermined volume of liquid is aspirated from the UV buffer reservoir 18 and pushed into the glass capillary reservoir 18. Subsequently, in step 35, the glass capillary vessel 18 is sealed with UV light. PCR was then performed.

A peel-off layer (not shown) may be disposed over stack 55 to prevent displacement of the covering liquid 25 when the container 18 is not in the upright position, the peel-off layer 31 being peelable prior to puncturing. Or the peel-off layer may be pierced by a suitable tube 36 for pushing liquid through the laminate 32 toward the container 18 and/or for drawing liquid from the container 18.

The bath structure and construction disclosed herein is not limiting as to the scope of achieving any type of thermal profile. By properly placing the containers 18 in a bath maintained at a predetermined temperature in a specified order for a specified period of time, any user-specified thermal profile can be achieved. The container 18 can quickly cross additional baths in the direction of the bath (if any) that does not contribute to the thermal profile.

The tube 37 may be wedge-shaped in order to more easily pierce the cover layer 23. Typically, the outer diameter of the tube 36 or the tube 36 with beveled or wedge-shaped tips is between 0.1mm and 5mm, and preferably less than 1mm. The thicker the cover layer 23, the greater the chance that the cover layer aperture 60 will be able to close completely when the tube 36 is removed. The preferred thickness of the cover layer 23 is greater than 1mm. When the cover liquid 25 is stored at 10 degrees celsius to 50 degrees celsius, it remains in liquid form for at least one month.

A vibration device (not shown) may be used to vibrate the container 18 at a predetermined frequency, amplitude and time to disperse the magnetic beads within the biological sample 21 in use. This is another method of mixing suction and push. Alternatively, the user may alternatively press and release the container 18 in the cartridge module 10 with a press release device (not shown) for mixing the biological sample 21 in the container 18, the container 18 being elastic or deformable when pressed. The apparatus 100 may also include a motorized platform (not shown) to position the container top opening 22 below the tube tip 37 during retention of the container 18 in the cartridge module 10.

The container 18 may be of any shape known in the art and may be made of any material known in the art. The container 18 may be made in the form of a small diameter glass capillary, for example a glass tube having an outer diameter of between 0.1mm and 4mm and an inner diameter of between 0.02mm and 3 mm. If the bath temperatures are appropriate, the user can advantageously perform simultaneous PCR for the different biological samples 21.

From the foregoing description, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design, construction and operation may be made without departing from the present invention as set forth in the following claims.

Claims (43)

1. A cartridge module (10) for improved pollution control during biological analysis or a nucleic acid analysis protocol comprising nucleic acid extraction, nucleic acid amplification and detection, the cartridge module (10) comprising:

At least one hollow tube (36);

A plurality of containers (18); and

At least one cover layer (23) to isolate the containers (18) from the environment outside the cover layer (23), wherein each container (18) allows a tube (36) attached to the in-use syringe (32) or pipette (89) to push in-use reagent liquid (12) or in-use biological sample (21) into the container (18) or to aspirate from the container (18) with only through the container top opening (22),

Even after the tube (36) pierces the cover (23) and retracts, the cover (23) substantially blocks aerosol exchange between the environment and the container (18) throughout the biological or nucleic acid analysis process,

The cover layer (23) also prevents any contamination of the environment due to spillage of the reagent liquid (12) or biological sample (21) from the container (18) or cartridge module (10),

The cover layer (23) is attached to:

1) At least one container top opening (22), or

2) The cartridge module (10) or container (18), during which the air connection space (104) between the cover layer (23) and the container top opening (104) is maintained, the cover layer (23) isolates the air connection space (104) from the cartridge's environment, reducing the variation in air pressure within the container (18) that has been isolated by the tube (36) during aspiration or dispensing of the biological sample (21) and reagent fluid (12).

2. The cartridge module (10) of claim 1, wherein at least a portion of the material of the cover layer (23) is porous when the cover layer (23) is attached to the container top opening (22).

3. Cartridge module (10) according to claim 1, wherein at least a part of the material of the cover layer (23) is an elastomer.

4. The cartridge module (10) of claim 1, the cartridge module (10) further comprising:

A covering liquid (25) which has been pushed or provided in advance and is ready to be pushed at least where the tube (36) on the covering layer (23) pierces, in order to create a laminate (55) in the covering layer (23) or to let the covering layer (23) to sandwich the covering liquid (25), which covering liquid (25) is non-reactive with the reagent liquid (12) and the biological sample (21).

5. The cartridge module (10) of claim 4, wherein the covering liquid (25) satisfies at least one of the following conditions:

a) The covering liquid (25) is immiscible with the reagent liquid (12) and the biological sample (21), and

B) The covering liquid (25) wets the surfaces of the tube (36) and the covering layer (23).

6. Cartridge module (10) according to claim 1, wherein the outer diameter of the tube (36) is between 0.1mm and 5 mm.

7. The cartridge module (10) of claim 1, the cartridge module (10) further comprising:

A scrubbing layer (28) secured to the stack (55) such that the cover layer (23), the cover liquid (25) and the scrubbing layer (28) form a composite layer (30), the scrubbing layer (28) being pierceable by the tube (36) and the scrubbing layer (28) substantially scrubbing excess cover liquid (25) adhering to the tube (36) when the tube (36) is retracted from the container (18).

8. The cartridge module (10) of claim 1, the cartridge module (10) further comprising at least one syringe (32) and providing at least one feature from the group of:

a) An O-ring (68) is provided between the plunger (66) and the barrel (67) of the syringe (32), a covering liquid (25) is provided above the O-ring (68), the covering liquid (25) is remote from a tube head (62) attached to the barrel (67), and

B) At least two O-rings (68) are arranged between the plunger (66) and the barrel (67) of the syringe (32), and a covering liquid (25) is arranged between the two O-rings (68),

The cover liquid (25) is immiscible and non-reactive with the reagent liquid (12) and the biological sample (21).

9. The cartridge module (10) of claim 1, the cartridge module (10) further comprising:

a rigid retaining layer (46) above or below the cover layer (23), the rigid retaining layer (46) having a plurality of retaining layer apertures (45) aligned with the container top opening (22) for allowing the tube (36) to pierce the cover layer (23).

10. The cartridge module (10) of claim 1, the cartridge module (10) further comprising:

At least one container holder (33) for holding at least one container (18), the container holder (33) being detachable from the cartridge module (10).

11. The cartridge module (10) of claim 1, the cartridge module (10) further comprising:

A liquid sealant (73), when pushed over or under a cover layer (23) over at least one of the container top openings (22), the liquid sealant (73), with or without curing, being able to seal a cover layer aperture (60) made in the cover layer (23) by puncturing the cover layer (23) by a tube (36), the liquid sealant (73) being immiscible and non-reactive with the reagent liquid (12) and the biological sample (21).

12. Cartridge module (10) according to claim 4, wherein the cover layer (23) has a number of grooves (27) for receiving a cover liquid (25), and the grooves (27) are in communication with each other.

13. Cartridge module (10) according to claim 1, wherein the cover layer on at least one of the container top openings is flexible and is provided with at least one through hole (96) or part of the through hole (96).

14. Cartridge module (10) according to claim 1, wherein above the container top opening (22) the cover layer (23) has a recessed top (76) with an inclined side (71) to provide a conical zone to a base area (72) smaller than the container top opening (22), the inclined side (71) being used to guide the tip (37) of a hollow tube (36) in use into the base area (72) before the tip (37) pierces the cover layer (23) through the base area (72).

15. The cartridge module (10) according to claim 1, wherein at least one container (18) comprises magnetic beads for binding nucleic acids from the biological sample (21).

16. The cartridge module (10) of claim 1, wherein at least one container (18) contains a cleaning liquid (53) for cleaning the tube (36) and the syringe (32) at any stage during nucleic acid analysis.

17. Cartridge module (10) according to claim 1, wherein at least a portion of the container (18) is made of a deformable material, such that in use,

(I) The container (18) in its initial state or shape is expandable to contain the reagent liquid (12) or biological sample (21) expelled by the device, and

(Ii) When the reagent liquid (12) or the biological sample (21) pushed out by the device is sucked out of the container (18), the container (18) in the initial state or shape can be contracted,

Thereby facilitating a reduction in the variation of the air pressure in the isolated container (18) during aspiration or expulsion of the biological sample (21) and the reagent liquid (12) by the tube (36).

18. The cartridge module (10) of claim 1, wherein the cartridge module (10) having the air communication space (104) further comprises: a sealing layer (120) sealing the top opening (22) of the container or containers to prevent any spillage of the reagent fluid (12) and biological sample (21) from the container (18) when the cartridge module (10) is not in the upright position, the sealing layer (120) being pierceable by the tube (36).

19. Cartridge module (10) according to claim 1, wherein a number of isolated containers (18) are placed vertically in a stack (47) with the cover layer (23) attached to the container top opening (22) such that the tips (37) of the hollow tubes (36) can pierce the cover layer (23) of each container (18) in the stack (47) in order to reach from the uppermost container (18) in the stack (47) to the lowermost container (18) in the stack (47).

20. The cartridge module (10) of claim 1, the cartridge module (10) further comprising:

a fixed pattern that can be detected by the apparatus (100) when automatically positioning the tube (36) over the container top opening (22).

21. The cartridge module (10) of claim 1, said cartridge module (10) further containing at least one sample container (115) having a cap (116), the sample container (115) containing the biological sample (21) during use, and at least a portion of the cap (116) containing a cover (23) pierceable by the tube (36) for drawing the biological sample (21) out of the sample container (115) and into the container (18).

22. An apparatus (100) for improving pollution control during biological analysis or a nucleic acid analysis protocol comprising nucleic acid extraction, nucleic acid amplification and detection, the apparatus (100) comprising:

a) A receiving module (102) for receiving a cartridge module (10) according to any one of the preceding claims;

b) Controlled by a robot, for operating a syringe or pipette device (84) attached to a hollow tube (36) of a syringe (36) or a pipette (89) to aspirate or push a biological sample (21) or reagent liquid (12) from or into a container (18) through a container top opening (22) and through a cover layer (23) or laminate (55) or sandwich;

c) An amplification module (101) for providing isothermal or cyclic heating for amplification of nucleic acids within a biological sample (21) in a container (18), the amplification being performed while the container (18) is inside or outside the cartridge module (10);

d) An optical imaging module (19) for imaging the biological sample (21) during or after amplification when the container (18) containing the biological sample (21) is inside or outside the cartridge module (10); and

E) When the amplification is performed outside the cartridge module (10), a robotically controlled transfer device (85) may be configured to position the container (18) held by the holding frame (33) provided in the cartridge module (10) or the apparatus (100) to the outside of the cartridge module (10).

23. The apparatus (100) of claim 22, the apparatus (100) being computer programmable to operate a syringe or pipette device (84) to:

The tube (36) attached to the syringe (32) is pressed against a fixed surface in the cartridge module (10) or the device (100) and the tube (36) is bent, and then the syringe (32) is mounted into the cartridge module (10) together with the tube (36).

24. The apparatus (100) of claim 22, said apparatus (100) further comprising a UV curing module (106) for curing UV resin deposited at said container top opening (22).

25. The apparatus (100) of claim 22, the apparatus (100) being further programmable by the computer to position the container holder (33) or the cartridge module (10) such that the portion of the tube (36) in contact with the biological sample (21) in the container (18) does not move out of the air connection space (104) during aspiration and dispensing of the reagent liquid (12) and the biological sample (21) from the plurality of containers (18).

26. The device (100) according to claim 22, the device (100) being computer programmable so that at least two amplification steps are performed while the container (18) containing the biological sample (21) is kept outside or inside the cartridge module (10) but inside the device (100), and the biological sample (21) is processed by the reagent liquid (12) between any two consecutive amplification steps and by the cover layer (23) with or without the cover liquid (25).

27. The apparatus (100) of claim 22, said apparatus (100) further comprising:

a detection module for detecting misalignment of the tip (37) of the tube (36) relative to the top opening (22) of the container due to the penetration of the cover layer (23); and

An automatic calibration module for compensating for the misalignment of the syringe or pipette device (84).

28. The apparatus (100) of claim 27, wherein said detection module optically detects misalignment between the tip (37) with respect to a fixed pattern on the cartridge module 10.

29. The apparatus (100) of claim 22, said apparatus (100) further comprising:

a syringe pick-up module for picking up at least one syringe 32 from the cartridge module 10.

30. The apparatus (100) of claim 22, said apparatus (100) further comprising:

a tube pick-up module for mounting the tube 36 provided in the cartridge module 10 to the syringe 32.

31. The apparatus (100) of claim 22, said apparatus (100) further comprising:

a positioning module for detecting a fixed pattern on the cartridge module (10) and a puncture location of the cover layer 23 over the container top opening 22 to position the tube (36) over the container top opening (22) when the tube (36) is attached to the syringe or pipette device (84).

32. The device (100) according to claim 22, the device (100) further accommodating at least one sample container (115) with a lid (116) for holding the biological sample (21) in use, the lid (116) at least partially comprising the cover layer (23), and the syringe or pipette device (84) being computer programmable for aspirating and dispensing the biological sample (21) from the sample container (115) into the container (18) by puncturing the cover layer (23) with the tube (36).

33. A method for improving pollution control during biological analysis or a nucleic acid analysis protocol comprising nucleic acid extraction, nucleic acid amplification and detection, the method comprising:

use of a cartridge module (10) and an apparatus (100) according to any of the preceding claims;

Loading a cartridge module (10) with a container (18) containing a reagent liquid (12) and a biological sample (21) into a receiving module (102);

Biological analysis or nucleic acid analysis is performed within the device (100) by piercing the tubing (36) through the cover (23) and retracting from the cover (23) without exposing the reagent liquid (12) and the biological sample (21) to the environment, by aspirating the reagent liquid (12) and the biological sample (21) from the container (18) and pushing them out of the container (18); and

The cartridge module (10) is discarded, the cartridge module (10) comprising a container (18) with a cover layer (23), a reagent liquid (12), a biological sample (21) and a tube (36).

34. The method of claim 33, the method further comprising:

Operating the syringe or pipette operating mechanism (84) according to any one of the following group:

a) After the reagent solution (12) or biological sample (21) is dispensed into the container (18), when the tip (37) of the tube (36) reaches above the reagent solution (12) or biological sample (21) in the container (18),

The syringe (32) or pipette (89) is manipulated to perform the aspiration mode for a predetermined period of time to at least partially relieve excess air pressure created in the hermetically isolated container (18) due to the dispensing.

B) Before the tube (36) is inserted into the container (18) and aspirated, when the tip (37) of the tube (36) is above the reagent liquid (12) or biological sample (21) in the container (18), the syringe (32) or pipette (89) is manipulated to perform a spray pattern for up to a predetermined length of time in order to raise the air pressure in the hermetically isolated container (18) for at least partially compensating for the vacuum created by aspiration of the reagent liquid (12) or biological sample (21), and

C) When the tip (37) of the tube (36) reaches over the covering liquid (25) in the stack (55) while the tube (36) is removed from the container (18), the position of the tube (36) is maintained for a predetermined period of time to release a portion of the reagent liquid (12) or biological sample (21) adhering to the tube (36) in the covering liquid (25).

35. The method of claim 33, the method further comprising:

During the time that the containers (18) are in the cartridge module (10), the covering liquid (25) provided in at least one of the containers (18) is dispensed over selected areas of the covering layer (23).

36. The method of claim 33, the method comprising:

Attaching the tube (36) to a pipette (89) containing a filter (17) and a liquid layer (94), the filter (17) and liquid layer (94) being separated therefrom by a first gap (93), and the liquid layer (94) being relatively close to the tip (37); and

The syringe or pipette handling device (84) is operated to maintain a second gap (95) between the liquid layer (94) and the aspirated reagent liquid (12) or biological sample (21).

37. The method of claim 33, the method comprising:

Any steps consisting of the following group are taken:

a) Injecting a liquid sealant (73) through the cover layer (23) to seal the container top opening (22) before amplifying the biological sample (21) in the container (18), and

B) Before amplification of the biological sample (21) in the container (18), a liquid sealant (73) is pushed onto the cover layer (23) or onto the stack (55) over the top opening (22) of the container.

38. The method of claim 33, the method comprising:

The syringe or pipette device (84) is computer programmed such that the tube (36) pierces a particular number of locations on the cover layer (23) or stack (55) over the container top opening (22) to minimize the size of the cover layer aperture (60) created during the number of pierces.

39. The method of claim 33, the method further comprising:

during analysis, the tube (36) is cleaned at least once by sucking and dispensing a cleaning liquid using the tube (36).

40. The method of claim 33, the method comprising:

Cartridge module (10) with a sealing layer (120) according to claim 18; and

In the sealing layer (120) above the container top opening (22), a vent hole (61) is made with a tube (36) attached to a syringe or pipette device (84) so that the container (18) is in air communication with the air communication space (104) during the subsequent act of sucking from or dispensing to the container (18).

41. The method of claim 33, the method further comprising:

Detecting misalignment between the tube (36) and the container top opening (22) due to puncturing of the cover layer (23) with the apparatus (100); and

The syringe or pipette device (84) is calibrated to compensate for the misalignment.

42. The method of claim 33, the method further comprising:

At least once, sucking the filling liquid (107) into the syringe (32) with the tube (36); and dispensing the filling liquid (107) such that the filling liquid (107):

a) Filling the tube head (62) with any existing liquid retention space (108) which might otherwise retain the reagent liquid (12) and the biological sample (21) during dispensing after subsequent aspiration, or

B) The previously occupied liquid retention space (108), reagent liquid (12) from a previous treatment and biological sample (21) are replaced,

The filling liquid (107) is immiscible and non-reactive with the reagent liquid (12) and the biological sample (21), and the filling liquid (107) is also heavier than the reagent liquid (12) and the biological sample (21).

43. A sample container (115) with a lid (116) for containing a biological sample during use, said lid (116) at least partially comprising the cover layer (23) according to claim 1.