Patent Application
20240017256
The present disclosure generally relates to an apparatus and method for processing and analysing one or more samples. The samples may be biological samples. The present disclosure stems in some embodiments from work based on the disclosure of an earlier application WO2016/144192 filed on 4 Mar. 2016, the entire contents of which are incorporated herein by reference.
WO2016/144192 discloses a method and device for preparing, extracting, separating and/or purifying biological samples, for example a biomolecule such as nucleic acid from a biological sample. An apparatus is disclosed comprising a sample tube configured to hold a biological sample. The biological sample in the tube may be heated for a predetermined time to form a processed sample. The processed sample in the tube can then be further heated at a temperature sufficient to deform the tube to reduce the volume of the tube. This deformation of the tube forces the processed sample from the tube, from where the expelled processed sample can be analysed, for example using known laboratory techniques.
WO2016/144192 discloses an apparatus configured for extracting or purifying nucleic acids, such as deoxyribose nucleic acid (DNA) or ribonucleic acid (RNA) for a variety of molecular biology applications. For example, the method and apparatus of the invention may be used to produce a composition comprising nucleic acid extracted from a sample that is suitable for immediate use for a polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR (qPCR or RT-qPCR), isothermal amplification (LAMP, RPA or other methods), forensic DNA fingerprinting, fluorescence-based detection, chip-based hybridisation detection, evaporation enrichment, DNA sequencing, RNA sequencing, molecular beacons, electrophoresis, direct electronic detection or nanopore analysis.
The sample tubes of WO0016/144192 is an example of how a biological sample can be processed into a form suitable for subsequent analysis. However, that analysis would typically be achieved using traditional laboratory conditions and equipment, and typically bench based, non-portable equipment.
US20160016171 discloses a portable system for extracting, optionally amplifying, and detecting nucleic acids or proteins using a compact integrated chip in combination with a mobile device system for analysing detected signals and comparing and distributing the results via a wireless network. Disclosed is portable DNA extraction and analysis device, with a removable chip for holding a biological sample, a heater/cooler, and with a simple laser/LED and processor-based analysis system. The analysis system analyses the basic fluorescence produced by illuminating the sample.
It is an object of the present disclosure to provide an apparatus for processing and analysing a biological sample, and/or that will at least provide the public or a medical profession with a useful choice.
According to an aspect of this disclosure, there is provided an apparatus for processing and analysing a biological sample; the apparatus comprising:
The sample extraction system may be configured to receive or hold a body at least partially formed of a heat-deformable material, the body defining
The sample extraction system may comprise a sample holder comprising at least one cavity in which a sample is held. The sample holder may comprise a plurality of cavities and may thus be configured to simultaneously hold a plurality of samples.
The sample holder may comprise a sample holding block, the sample holding block being a thermal block configured to be temperature regulated by the thermal system.
The sample holding block preferably has a relatively low thermal mass.
The thermal system may be controlled by the or a controller according to:
The thermal system preferably comprises:
The Peltier assembly preferably comprises:
The cooling system may comprise a heat transfer system, in thermal contact with the Peltier device and configured to transfer thermal energy from the Peltier device to the cooling system.
The heat transfer system may comprise a vapour chamber in thermal communication with the Peltier device, the cooling system comprising a cooler in thermal contact with the vapour chamber.
The heat transfer system may comprise one or more heat transfer elements in thermal contact with the vapour chamber and the Peltier device.
The cooler may comprise one or more heat sink(s) and/or fan(s), downstream of the vapour chamber.
The apparatus may comprise an air inlet fan, and an air outlet fan, the air inlet fan being configured to draw in cooling air to the cooling system, the air outlet fan being configured to expel heated air from the cooling system.
The thermal system may comprise a plurality of Peltier assemblies, a first Peltier assembly being configured to thermally regulate the sample when in the sample extraction system, and a further Peltier assembly being configured to regulate the sample when in the microfluidic chip.
The further Peltier assembly may comprise a Peltier device positioned adjacent the microfluidic chip, and preferably below the microfluidic chip.
The further Peltier assembly comprises a thermal plate adjacent the further Peltier device, the thermal plate comprising a contact surface being in contact with the microfluidic chip when the sample is being analysed, and a contact surface in contact with the further Peltier device.
One or each contact surface may be planar.
The thermal plate may comprise:
The thermal plate may comprise synthetic diamond or sapphire plate embedded in its contact surface.
The Peltier assemblies may share a common cooling system.
Each Peltier assembly may comprise a respective heat sink.
The optical system may be configured to generate spectrally discrete excitation and fluorophore emission wavelengths bands such that an optical signal received at the optical detector can be attributed to one excitation wavelength and matching target fluorophore pair.
The optical system may comprise a plurality of light sources each configured to emit light that is incident on the sample in the microfluidic chip. The plurality of light sources may be provided by a single optical unit (for example a single optical unit configured to selectively, sequentially, or simultaneously emit light from different excitation emission wavelength bands), or from discrete optical units.
The light sources may be configured to generate three excitation emission wavelength bands.
The light sources may be configured to generate blue, red and green excitation emission wavelength bands.
The optical system may comprise an optical filter configured to allow only a peak wavelength band from each light source to pass through the filter. The peak wavelength band may be less than 100 nm, preferably less than 70 nm, more preferably less than 50 nm and in some examples 40 nm.
Each light source may be consecutively and sequentially excited.
At least one light source may emit light along an excitation path that is not aligned with an excitation path of light from at least one other light source.
The optical detector may be spaced vertically from the sample in the microfluidic chip, a straight vertical fluorescence path being defined between the optical detector and the microfluidic chip, light from at least one light source being emitted along an excitation path that is perpendicular to the vertical fluorescence path.
Each light source is configured to emit light in a different direction.
At least one light source may be configured to emit light substantially at 90° to another light source.
The blue and red light sources may be configured to emit blue and red light in the same direction, and wherein the green light source is configured to emit light in an orthogonal direction.
Each light source may be associated with a respective filter, each filter allowing only a predetermined wavelength or range of wavelengths of light to pass through the filter.
Each filter may only allow a peak wavelength of the respective light colour through the filter.
The light sources may be controlled to consecutively excite the sample with light from the light sources sequentially.
The sample may be analysed using PCR or qPCR in which the sample is subject to multiple temperature cycles, wherein the sample is excited from light from each light source once per temperature cycle.
The sample may be analysed using a single, isothermal temperature cycle, for example isothermal amplification (LAMP, RPA or other methods).
The optical system may comprise a dichroic filter positioned between the sample in the microfluidic chip and the optical detector, and configured to reflect excitation light wavelengths to the sample, and to allow fluorescent wavelengths to pass through the dichroic filter from the sample to the optical detector.
The optical system may comprise an emission filter, between the dichroic filter and the optical detector, and configured to pass only fluorescent wavelengths from the sample.
The optical path from each light source may change direction through at least 90° between the light source and the optical detector.
The optical path from at least one light source may change direction through substantially 180° between the light source and the optical detector.
The light from each light source may change direction at least three times, and preferably at least four times, between the light source and the photodetector.
The optical detector may be positioned vertically above the sample in the microfluidic chip.
The light sources may be spaced from the microfluidic chip so as to not be in the optical path between the microfluidic chip and the optical detector.
Each light source may comprise at least one photo-diode array.
The optical detector may comprise, for example, a photo-diode detection array, and/or a CCD/CMOS camera detector.
The components of the optical system may be contained in an optical housing, that is mounted or configured to be mounted centrally in the apparatus housing.
The thermal system may be substantially located around the periphery of the optical housing.
The thermal system may be configured substantially in a ‘U’ shape when the apparatus is viewed from above, the optical housing being located between the arms of the ‘U’.
The optical system may comprise a plurality of optical components, the optical components having no moving parts.
The apparatus may comprise any one or more of:
The sample microfluidic chip may be configured to receive the processed sample from the sample extraction system, and locate the processed sample in a desired position in the apparatus.
The microfluidic chip may be substantially oblong, and may be inserted into or ejected from a chip slot in the apparatus.
The apparatus may comprise a chip cassette configured to removably receive the chip. Alternatively, the chip may be configured to be directly received in the chip slot of the apparatus, without a chip cassette.
The chip cassette may comprise a closure configured to positively locate and engage the chip in the cassette.
The chip cassette may comprise an engagement feature, the engagement feature configured to engage the apparatus when the cassette is received in the slot, the engagement retaining the closure in a closed position.
The chip may comprise a plurality of sample inlets.
The number of inlets may correspond to the maximum number of samples held by the sample extraction system.
Each inlet may lead to a respective microfluidic passageway, each microfluidic passageway leading to a respective sample well.
The sample wells may be arranged in an array below an exposure window provided in an upper surface of the chip, the window being in optical communication with optical system.
The chip may comprise a thermal surface, adjacent with and in contact with the or each sample well, the thermal surface configured to be thermally regulated by the thermal system.
According to another aspect of this disclosure there is provided an apparatus for processing and analysing a biological sample; the apparatus comprising:
According to another aspect of this disclosure there is provided an apparatus for processing and analysing a biological sample; the apparatus comprising:
According to a further aspect of this disclosure there is provided an apparatus for processing and analysing a biological sample; the apparatus comprising:
According to a further aspect of this disclosure there is provided an apparatus for processing and analysing a biological sample; the apparatus comprising:
An apparatus according to any one of the preceding statements may be configured to analyse the sample, when in the microfluidic chip, using PCR, such as qPCR for example.
The housing is preferably hand portable.
According to another aspect of this disclosure there is provided a microfluidic chip configured for use with an apparatus according to any one of the above statements.
According to a further aspect of this disclosure there is provided a microfluidic chip assembly comprising an elongate, planar microfluidic chip, the microfluidic chip comprising:
According to a further aspect of this disclosure there is provided a microfluidic chip comprising an elongate, planar body, the microfluidic body comprising:
The microfluidic chip may comprise a bore, configured to be in selective fluid communication with the sample inlet, the bore being configured to receive a sample tube.
The bore may be coaxial with the longitudinal axis.
The body may be transparent, or comprises one or more transparent regions.
The well array may comprise a pair of sub-arrays, each sub-array comprising at least one wells.
Each sub-array may comprise a plurality of wells.
The sub-arrays may opposed across the body, the longitudinal axis of the body being in between the sub-arrays.
The wells in each sub-array may be arranged in a straight line.
A distal end of the microfluidic chip may comprise an arcuate profile, to facilitate insertion of the microfluidic chip into an apparatus for processing and analysing a biological sample.
The microfluidic chip may be substantially self-supporting. In other embodiments the microfluidic chip may comprise one or more reinforcing elements. The one or more reinforcing elements may comprise a peripheral frame. The microfluidic chip is therefore preferably a single component.
According to an aspect of this disclosure, there is provided an apparatus for processing and analysing a biological sample; the apparatus comprising:
Further aspects of the disclosure, which should be considered in all its novel aspects, will become apparent from the following description.
A number of embodiments of the disclosure will now be described by way of example with reference to the drawings in which:
Despite advances in diagnostic technologies, the ability to rapidly and accurately diagnose infectious disease lags behind what the world requires. Diagnostic tools able to deliver immediate actionable information (i.e. at the point-of-care or in-field) would help to address the health and environmental challenges that are being faced globally. Some examples include water contamination, HPV testing to provide cervical screening in hard to reach populations, containment of new and emerging diseases like Ebola, SARS and Covid, and prevention of antibiotic resistance. An apparatus in accordance with this disclosure provides a relatively simple, accessible, accurate and hand portable device, configured to be used in the field, and which can yield relatively rapid results. An apparatus in accordance with this disclosure is capable of processing a biological sample, and analysing the processed sample, in the field, without recourse to a laboratory or other permanent or non-portable device.
In accordance with this disclosure we therefore provide a ‘sample-to-result’ molecular diagnostics platform designed for simplicity that allows non-experts to deliver results rapidly at the point-of-care, enabling frontline professionals to take immediate action. In accordance with this disclosure we provide a single diagnostic apparatus. This apparatus will purify samples, detect the diagnostic marker and report the result to the operator relatively quickly, for example in under 15 minutes. The apparatus can comprise a user interface which will guide the operator through apparatus set up and sampling, whereas intelligent algorithms will automate data interpretation and results will be presented unambiguously.
With reference to
The housing is preferably hand portable in that the housing can be picked up and carried with a user in the field, for example by being held in a user's hand, carried in or by one hand, or carried in a bag or backpack or the like.
The apparatus 1 may further comprise one or more data transceivers, such as wireless, Bluetooth or NF transceivers configured to send and/or receive data to a client device 13. The client device 13 may for example comprise a portable electronic data processor such as a laptop, smartphone or tablet.
The apparatus and method of this disclosure are suitable for preparing, extracting, purifying or separating biomolecules from a wide range of samples, and subsequently analysing the processed sample, using a single apparatus, in the field, that is, without having to send the sample to a traditional laboratory, for processing with traditional non-portable laboratory equipment, and/or without requiring specialist, highly trained users.
The prior art discussed above discloses, at a relatively high level, a portable apparatus for processing and analysing a sample in the field. The present disclosure relates to various significant improvements over the prior art, necessary for such a portable apparatus to perform correctly in the real-world.
Referring to
The housing H in this example is oblong and comprises a front panel FP having various controls C, and a slot S to receive the microfluidic chip 5. A front closure FC is provided to close the slot S, when the chip 5 is fully received in the slot. The housing H further comprises a top panel TP having a closure TC, in the form of a hinged flap in this example, that can open and close a sample receiving sample block 21. The top of the sample block 21 can be seen in
With reference to
Although not shown in the Figures, the housing H may further comprise any one or more of:
With reference to
With additional reference to
The thermal system 7 comprises two aspects: 1) a thermal system for thermally controlling the samples in sample block 21 during extraction of the sample from the sample tubes into the microfluidic chip 5, such a system being shown for example in
For completeness,
With reference to
The thermal system 7 is configured to thermally regulate the temperature of the sample block 21. The sample block 21 is in thermal communication with one or more Peltier devices 19, for example being two dual stage thermoelectric heaters/coolers which operate in electrical and thermal parallel. The Peltier devices 19 are thermally coupled to the sample block 21 on the cold side, while the hot side of the Peltier devices 19 is thermally coupled to a heat spreading vapour chamber 23 via one or more copper transfer elements 20. The chamber 23 is in turn thermally coupled to a cooling assembly 15 comprising one or more heat sinks 29 and one or more cooling fans 31, to dissipate heat from the thermal system 7 as required.
The thermal system 7 of
Referring to
The thermally controlled sample in the tubes in sample block 21 is delivered to the microfluidic chip 5, for example by heating the sample tubes such the tubes contract sufficiently to force the sample from one or more of the tubes into the microfluidic chip 5. Once delivered to the chip 5, the temperature of the chip 5, and of the samples in the chip 5, is also controlled by the thermal system 7 as described above. Consequently for any given set of samples, the thermal system 7 is operative according to two thermal stages each having independent thermal control: 1) a sample processing stage with the samples in the tubes, and 2) an analysis stage in which the samples in the chip 5 are thermally regulated before and during analysis, for example using qPCR techniques.
With reference to
Referring now to
Each LED array 43, 45, 47 is associated with a respective planar lens array 49, 51, each oriented parallel to, and in the light path of, the respective LED arrays 43, 45, 47. Lens array 49 is adjacent blue and red LED arrays 43, 45, whilst lens array 51 is adjacent LED array 47. Each LED array is also associated with a respective planar optical filter 53, 55. Filter 53 is a blue and red filter, and is positioned downstream of the lens array 49. Filter 55 is a green filter and is positioned downstream of the lens array 51.
Downstream of the filters 53, 55 is a combination diagonal 56 which serves to direct the blue, red and green light substantially horizontally through a common optical aperture 59, aperture 59 being planar and vertically oriented, and then through a downstream, vertical excitation filter 60.
Downstream of the excitation filter 60 is an inclined dichroic filter 61 which serves to redirect the blue, red and green light vertically downwardly onto the horizontal lens array 49 and subsequently onto the samples in the wells 35 that are optically exposed through window 39 in chip 5.
The blue, red and green light passes into, and is reflected and scattered from, the sample in the wells 35 and travels vertically upwardly, through dichroic filter 61, through an emission filter 63, through a further lens array 65, through a further optical aperture 67, and onto an optical detector which in this example is a photodiode detection array 69. Emission filter 63, lens array 65 and optical aperture 67 are all planar and are arranged in a parallel and adjacent configuration. The path of light reflected from sample wells 35 is indicated by arrow L in
Photodiode detection array 69 is configured to generate an electrical output signal indicative of the light L reflected from the chip 5. That output signal is processed, either by a controller in housing H, or by a remote processor for example on a user's smartphone, to indicate one or more properties of the sample. In other examples photodiode detection array 69 may instead comprise a different type of optical detector, such as a CCD/CMOS camera detector.
The photodiode detection array 69 is a printed circuit board with photodiodes for fluorescence detection. The emission filter 63 passes fluorescent wavelengths (indicated by arrow L) of interest while the dichroic filter 61 reflects excitation wavelengths to the sample chip (red green and blue) and passes fluorescent signals (indicated by arrow L) to the emission filter 63 for further filtering.
The combination diagonal 56 combines the red blue and green excitation colours into one source. The green filter 55 selects the green excitation band from the green LED spectrum. The blue/red led filter 53 does the same task for the blue and red led array 51. Apertures 59, 67 are to mask off stray light and otherwise define beam diameters as required.
A method of processing and analysing a sample, for example a biological sample, is now described.
The sample chip 5 is prepared and loaded into the chip cassette 5A and then inserted into the apparatus 1. The tabs 5C in the cassette closure 5B engage the sample slot ceiling in the slot S of the apparatus 1 in which the chip 5 is received, and press the sample chip 5 firmly against an internal qPCR thermocycling surface 50, as can be seen in
Next, the sample tubes are loaded into the sample block 21 and the top closure TC closed. Magnetic inserts can provide pressure to the tops of the tubes T via the closure TC to prevent the tubes from coming loose under pressure during the sample processing cycle. This cycle is described in WO2016/144192. The apparatus 1 is then controlled to first perform a sample processing protocol and subsequently the qPCR sample analysis protocol. The apparatus software user interface then reports the results of the sample analysis having run an analysis algorithm against the signal generated from the sample, and stored, predetermined qPCR data. Throughout both protocols, which may be controlled by the same or respective algorithms, the apparatus 1 is thermally regulated by thermal system 7. Thermal system 7 thermally regulates the chip 5 and sample(s), and also the sample block 21 and tubes T.
As described above, the apparatus 1 in one embodiment is configured to be used in conjunction with sample processing tube system as described in WO2016/144192. That tube system, in accordance with this disclosure, uses a Peltier driven thermally regulated sample block 21 which houses the tubes T and produces the required temperature profile for the tube/reagent. The sample block 21 is thermally coupled to one or more Peltier device(s) 19 on one side and one or more heatsink(s) 29 on the other side, as described above. The vapour chamber 23 helps transport heat to a finned heatsink 29 and variable speed fan assembly 31 to cool the apparatus 1 as required. This process produces a processed sample that is then analysed to determine one or more properties of the sample. This thermal control process, of the sample block 21, is outlined in
The analysis of the processed sample is controlled by an analysis algorithm run via a controller 11 of the apparatus 1, the controller 11 including one or more data processors and associated circuitry which control the thermal system 7 (if this does not have its own discrete controller 17) and the optical system 9. The apparatus 1 uses a PCR analysis system. The PCR analysis system comprises a PCR thermal surface 50 in contact with the sample chip 5, and the optical system 9.
The thermal surface 50 in this example is a low mass planar section of gold-plated copper which has embedded in its upper interface a section of synthetic diamond plate measuring, again in this example, 20×20×0.5 mm. The gold plated copper section interfaces with the underside of the sample chip 5. The copper section also contains the temperature sensor. This configuration assists in heat transport and ensure thermal uniformity under the sample chip well array 37. A temperature sensor (for example a platinum RTD) embedded in the PCR thermal surface 50 provides feedback to the control circuitry and controller 11 in order to thermocycle and maintain the required temperature of the processed sample(s) in the chip 5. The copper/diamond PCR surface 50 is thermally coupled to a Peltier device 59 of the thermal system 7 on one side, and cooling system 15 comprising the finned fan 31 and heatsink 29 array of the thermal system 7 on the other side, which vents to the exterior of the apparatus 1 via vents V. Vapour chamber 23 is coupled directly to and forms part of the heatsink array 29 in order to aid in rapid heat transport to and from the PCR surface 50 as required. A single stage Peltier device 53 is used in order to maximize heat transport to and from the PCR thermal surface 50. In order to achieve the maximum heat flow, the heatsink 29 is maintained near 40° C. via a feedback cooling system.
The optical system 9 is relatively compact with no moving parts. The optical components are arranged in varying relative orientations within the optical housing 9H, to both fit in all of the required optical components, and provide the required optical path. Traditionally used componentry such as rotating filter wheels are not required in system 9. In order to achieve this, spectrally discrete (LED) excitation and fluorophore emission wavelengths bands are required such that an optical signal received at the photodiode detection array 69 can be attributed to one excitation wavelength and matching target fluorophore pair. As more than one fluorophore may be present in the sample, this approach requires that, for example, a fluorophore intended to be excited by the blue 470 nm led source 43 is not excited to any significant degree by one of the other possible system excitation wavelengths (green/red, 546/635 nm) from LED sources 45, 47 respectively.
If interference between fluorophores occurs, then an intensity subtractive method is required to determine the intensity contribution from various dyes as present in the sample.
In order to achieve a well-defined excitation and matching fluorophore pair the LED outputs from the LED arrays are initially filtered using a filter which passes only the peak LED wavelength with a spectral width of similar to 40 nm. The blue and red LEDS are placed on the same array so that little to none of the blue LED emission spectra (470 nm) is allowed through the spectral window reserved to the red LED array (635 nm). The green LED array has a single spectral window and so is not subject to the same filter constraints.
Once this optical configuration is realized the method is to consecutively excite the sample(s) with each excitation wavelength in turn and record the sample response which is produced from the target dye in the sample. This occurs once per qPCR temperature cycle as the target DNA is amplified. The data is then output for analysis.
The firmware of optical system 9, and an outline of the optical control protocol can be seen with reference to
With reference to
Apparatus 101, in accordance with this disclosure comprises, in a single housing:
Apparatus 101 comprises a hand portable housing H in which all of the components necessary to receive and analyse a given sample are contained. The housing H is of a size that renders the apparatus 1 hand portable.
The housing H in this example is oblong and comprises a front panel FP having various controls C, and a slot S to receive a microfluidic chip 105. A front closure FC is provided to close the slot S, when the chip 105 is fully received in the slot. The housing H further comprises a top panel TP having a closure TC, in the form of a hinged flap in this example, that can open and close a sample receiving sample block 21. The top of the sample block 21 can be seen in
With reference to
The chip 105 comprises a single sample inlet 131. The inlet 131 leads to a microfluidic passageway 133, the microfluidic passageway 133 then leading to a plurality of sample wells 135. The sample wells 135 are arranged in an array within an exposure aperture or window 139 of the chip 105. The array 13 comprises two sub-arrays of four sample wells 135 arranged in adjacent parallel straight lines along the chip 105, each sub-array being a respective side of the longitudinal axis of the chip 105.
In this example a single sample inlet 131 is provided on the chip 105, and is positioned so as to be aligned with the rightmost tube T in the sample block 121.
In a modified embodiment of chip 105, one or more additional sample inlets 131 can be provided, each inlet 131 being positioned on the chip 105 to be aligned with a respective sample tube T. For example one to four sample inlets 131 can be provided, in the embodiment shown in
The window 139 is in optical communication with optical system 109 as will be described further below. The sample block 121 and the chip 105 are sealingly connected, when the chip 105 is fully received in the slot of the apparatus 101. Thus, the samples in the tubes T in the sample block 121 are isolated from the ambient environment as the samples are ejected from the tubes T into the inlet 131 in the chip 105. The isolated samples pass along the passageways 133 until they reach the wells 135. The samples when in the wells 135 remain isolated from the ambient environment and thus are isolated from external contaminants.
With reference to
In apparatus 101, optical system 109 comprises a plurality of optical/processing components in a self contained optical unit that is positioned adjacent the sample tubes T, with a lower part of the optical system 109 being located directly above the chip 105.
In this embodiment, and with particular reference to
Common thermal system 107 is located at the base of the apparatus 101, below the optical system 109, with part of the thermal system 107 being located at one end of the apparatus 101, adjacent the tubes T. Heat from the sample extraction process is transferred to a common heat removal system at the base of the apparatus 101, this heat removal system also being used during sample analysis using the optical system 109. This configuration is relatively compact, and minimises the space required to thermally control the tubes T and chip 105.
Thermal system 107 is configured by way of componentry and control, to allow precise and relatively quick heating and or cooling of the sample, both in the sample extraction system 3, and in the microfluidic chip 105 during analysis of the sample, for example using qPCR techniques, using optical system 109. As with apparatus 1, the sample block 21 is configured to have relatively low thermal mass, such that the temperature of block 21 can be varied relatively easily and quickly.
With additional reference to
In this example a pair of heat transfer pipes 107A is provided at each side of the sample block 121.
The heat transfer pipes 107A transfer heat from the vapour chamber to a common cooling assembly, which may comprise one or more heat sink(s) 29 and fan(s), as described in respect of apparatus 1, but located in the base of the apparatus 101, and also used during sample analysis.
Thermal controller 17 may be a common controller that controls the thermal system 107 during both the sample extraction and sample analysis steps.
As can be best seen in
Sample block 121 comprises an external structure and/or profile configured to minimize the thermal mass of sample block 121 to aid in rapid temperature change. The sample block 121 comprises a plurality of elongate recesses 121A and cutouts 121B configured reduce the thermal mass of the sample block 121.
As with apparatus 1, the sample tubes T used with apparatus 101 are inserted substantially vertically down into the top panel TP of the apparatus 101. The chip 105 is inserted substantially horizontally into the front panel FP of the apparatus 101, perpendicularly to and beneath the sample block 121. The chip 105, when fully inserted into the housing H, is substantially below the optical system 109 which is located centrally and towards the front of the housing H. The thermal system 107 is substantially underneath, and adjacent a front end of, the optical system 9, and is thermally coupled to both the sample block 121, and the chip 105.
This configuration of the sample tubes T, sample block 121, and thermal system 107 can best be seen in
Referring now to
The chip 105 is located below sapphire window 150 which receives light from a plurality of LED arrays 43, 45, 47, one for each of blue, red and green light wavelengths. The blue, red and green LED arrays 43, 45, 47 are located together, in a horizontal orientation, at the rear of the optical system housing 109H. The LED arrays may comprise sub-arrays of a single LED unit which is RGB capable.
The LED array 43, 45, 47 is associated with a respective planar lens array 149 in the light path of the LED arrays 43, 45, 47. The LED arrays 43, 45, 47 are also associated with a respective planar optical excitation filter 153, and a diffuser 154.
Downstream of the excitation filter 153 and diffuser 154 is an inclined dichroic filter 161 which serves to redirect the blue, red and green light vertically downwardly onto the sapphire window 150 and subsequently onto the samples in the wells 135 that are optically exposed through window 139 in chip 105.
The blue, red and green light passes into, and is reflected and scattered from, the sample in the wells 135 and travels vertically upwardly, through dichroic filter 161, and through a second dichroic filter 162, also inclined and parallel to the first dichroic filter 161. Second dichroic filter 162 redirects the light horizontally through an emission filter 163, through a camera lens 165 of camera 171. Camera 171 is positioned towards the top and rear of the optical system housing 109H, above the LED arrays 43, 45, 47. The blue, red and green light is incident on the sample in the wells 135 and fluoresces the sample, The fluorescent light reflected and scattered from the sample travels vertically upwardly and is ultimately incident on the camera lens 165. The light may be scattered in many directions. The light which is finally scattered and or reflected towards the detectors is that which is detected. The remainder may be lost.
Each dichroic filter 161, 162 comprises a dichroic material which causes the light from the sample to be divided into separate beams of different wavelengths. The purpose of the dichroic filter 161 is to reflect LED excitation to the sample and pass fluorescent light to the detector. Dichroic filter 162 reflects sample fluorescence to the camera 171 while double filtering out any remaining excitation light passing the first dichroic 161. A satisfactory result may still be achieved by using a standard broadband mirror in place of dichroic filter 162, as the emission filter 163 is the primary means of removing residual excitation light.
Camera 171 is controlled via a micro-controller 172, such as a Raspberry Pi™ microcomputer, according to one or more algorithms which analyses the image generated by the camera 171 indicative of the light L reflected from the chip 5, to indicate one or more properties of the sample. The algorithms may compare the image with other images in one or more databases stored on, or accessible by, the microcontroller.
The camera 171 and micro-controller 172 looks at how the intensity of the sample images vary with cycle number in each of the RGB channels. The camera 171 can also be used in conjunction with intelligent software to recognise and detect defects within the wells 135, such as bubbles, and omit these from the average well intensity (typically found by averaging all of the RGB pixels over the sample well area for any given cycle reading). The RGB data can be combined or treated separately depending on the number of wavelengths being detected and the principal wavelengths. Data analysis algorithms can then be used to determined curve fit and cycle threshold (CT) values for the data.
Referring now to
Optical system 209 also uses a camera 171 and associated controller as described above with reference to apparatus 101.
For clarity,
As can best be seen in
Chip 205 comprises a single component, formed from a transparent material.
Chip 205 may be configured such that there is a single or parallel fluid path to each sample well 235.
Chip 205 may comprise a valving system comprising one or more valves configured to prevent fluid pumping between sample wells 235. For example, the inlet and/or outlet to each sample well 235 may comprise a valve.
Referring to
Alternatively, optical system 209 may use one or more photodiodes, as described above with reference to the apparatus 1 of
It is also envisaged that the sample chip 205 may be heated from both sides, with excitation and detection taking place from the short edge faces 241 only. In other words, detection occurs in a direction perpendicular to the longitudinal axis of the chip 205, and in the direction of one or both sides of the chip 205, rather than in the direction of the front or rear of the chip 205. In this case there are no apertures 213A in the Peltier device 213 and the optically clear sides of the chip 205 are the windows through which excitation light is passed and fluorescent light is detected. An array of ×4 LEDS/photodiode pairs is then required on each side of the chip 205, or strip type CMOS camera detectors.
With reference to
Chip 205 differs from chip 105 in that chip 205 is self-supporting and not mounted in a separate cassette. Chip 205 is planar and oblong. Chip 205 is a single component that is inserted into the apparatus 101, with no moving or separate parts.
As chip 205 is inserted into the slot S in the front of the apparatus 101, engaging elements inside the slot S engage the chip 205 and retain and stabilise the chip 205 in the correct position in the slot S. The engaging elements may comprise movable elements, such as one or more rollers for example.
We refer now to
We have shown that an apparatus 1, 101, 201 in accordance with this disclosure generates sufficient force to load a Microfluidic chip 5, 105, 205. In the example given below four tubes T were loaded with water coloured with red food dye. The tube outlet nozzles were aligned, through an incubation apparatus, with four receiving ports on a microfluidic chip 5. The tubes T traversed the heat block of the incubation device. The microfluidic chip 5 had a foam port interface into which the tubes T were pressed by downward pressure from the apparatus housing. This prevented leaking at the point where the microfluidic chip land tube T met.
The tubes T were heated to 95° C. This caused the inner tube lining to shrink and the fluid retention valve to burst. This pushed the liquid out of the tubes T and into the chip 5. The images of
AIM: Test thermal cycling and optical detection with GFP assay with prototype chip.
Experimental Design (Methods):
Results:
As can be seen in
As can be seen in
Referring to
ImageJ (https://imagej.net/software/fiji/) was used to determine fluorescence from screenshots taken during thermal cycling.
The LEDs in the array were not calibrated in this example. Therefore, it is more correct to treat each position as an independent measurement. An average is not likely to be valid. Subtraction of the initial fluorescence reading from the final fluorescence reading indicates a general increase in fluorescence over the course of the amplification reaction as would be expected for a successful qPCR amplification, as shown in Table 3 below:
Conclusion:
AIM: Performance comparison between prototype QX microfluidic chip and 3M adhesive microfluidic chip with COVID-19 WarmStart LAMP assay incubated on a QX prototype device (an apparatus using photodiode detection in accordance with the apparatus of
Experimental Design (Methods):
This experiment compared incubation and fluorescence sampling using the Warm Start® LAMP assay system from NEB, Ltd (https://international.neb.com/products/e1700-warmstart-lamp-kit-dna-rna#Product%20Information) to which SYTO9 fluorescent dye was added (https://www.thermofisher.com/order/catalog/product/S34854#/S34854). The master mix was loaded into the standard microfluidic chip ‘standard’ in accordance with
The LAMP master mix contained COVID-19 N-gene primers (Zhang et al, 2020: https://doi.org/10.1101/2020.02.26.20028373) and a dilution of Twist 2 control RNA (https://www.twistbioscience.com/resources/product-sheet/twist-synthetic-sars-cov-2-rna-controls). The sample wells of all microfluidic chips had valves 1 and 4 (top and bottom-most) lightly sanded as part of the assembly process. After the reagent was loading, Platsil Gel-10 silicone (https://www.barnesnz.co.nz/addition-cured/platsil-gel-10-silicone-rubber-1605) was injected into the valves to prevent liquid movement during incubation. Exhaust ports were sealed with Tegaderm (https://www.3mnz.co.nz/3M/e_NZ/p/d/v000089540/) after pipetting master mix and before incubation.
Microfluidic chip preparation was as follows:
Microfluidic chips were assembled from component parts. The first and fourth sample valves of all chip bodies were sanded with fine, 600 grit sandpaper for this experiment. Prototype ‘Standard’ chip assembly requires the chip body, front and back plastic sheets and the adhesive layers that adhere the plastic sheets to the chip body. The ‘3M’ chip assembly requires a front and back piece adhered to the same chip body. Both chips had a hydrophobic membrane placed around the exhaust ports. This is where the Tegaderm was placed to seal the sample wells. The chip template used in this experiment is shown in
A Warmstart lamp master mix was used as per Table 4 below:
To better visualise any unintentional liquid movement in the microfluidic chip during incubation, coloured liquids were used to fill the unused sample wells adjacent to and between the sample wells. Filling the wells also helped to prevent a vacuum from forming during heating that would pull samples from the test wells. A Coomassie stain dilution was used in these wells and labelled ‘purple’ in the chip diagram of
Prototype ‘Standard’ chips generally accepted 18-20 μl of master mix when loaded into the chip via the exhaust port, without filling the channels leading to the centre chamber.
All chips were allowed to sit for an extended amount of time (>10 min) after injecting the silicone into the centre wells, prior to use. 800 μl Part A, 800 μl Part B of Platsil were mixed thoroughly and loaded into a 1 ml syringe. A 27 G needle was attached to inject silicone into the centre wells of each tested chip.
Experiment Order was as follows:
There were concerns regarding temperature differences when using the test bench TEC between experiments. The “standard” chip was started from room temp. In order to lower the temperature of the TEC after this incubation a dry bath incubator aluminium block was place on the TEC to act as a heat sink. This brought the temperature much closer to room temp for the next incubation using the 3M chip.
QX prototype Settings were as follows, and used for all chips:
To best understand sensor positioning and labelling in relation to chip well orientation please see
Test Bench Settings were as follows, and used for all chips:
Data Analysis was as follows:
Photos and screenshots were taken of the experiments for use as visual references or data points to manually log into excel for analysis.
Test bench sample analysis included uploading the photos taken throughout the incubation into FIJI (https://imagej.net/software/fiji/). A set area within the same well was sampled for fluorescent intensity throughout the incubation and a graph formed using the collected data imported into excel.
QX prototype sample analysis came from the data files generated by the purpose written software interface throughout the incubation. The LAMP 2 program sampled fluorescence in the wells every 60 seconds. Those measurements were extracted from the file and imported into excel for analysis.
In most cases, the data shown in the graphs of
Results were as follows:
The results of the control LAMP Assay can be seen in
Prototype “Standard” Chip Results:
A direct comparison of averaged fluorescence against time between test bench (top pair of lines) and QX (bottom pair of lines) incubations with standard chips is shown in
Prototype “3M” Chip Results:
A direct comparison of averaged fluorescence against time between test bench (top pair of lines) and QX (bottom pair of lines) incubations with 3M chips is shown in
A comparison of averaged fluorescence against time between the standard and 3M microfluidic chips from the Test Bench run is shown in
A comparison of averaged fluorescence against time between the standard and 3M microfluidic chips from the QX Prototype run is shown in
Conclusion:
We describe below a set of Standard Operating Procedures for producing a sample that can be used in an apparatus in accordance with this disclosure, and for use in the above experiments.
Introduction:
Assay Details:
The following is a list of consumables required:
To make up 1× TE:
To make up the DNA template:
To make up forward and reverse primers:
To make up probe:
A thermal cycle program for GFP assay can be used in accordance with Table 6 below (noting that the step time may need optimising for the qPCR device):
For all assays:
For SYBR Green Assays:
For probe assays:
References herein to a controller or microcontroller are intended to indicate that one or more components of the apparatus 1, 101, 201 are controlled by such. The controller or microcontroller may be associated with only one or more components, or may comprise a controller of all of the components of the apparatus. These terms are intended to cover the apparatus comprising a single controller, a master controller and one or more slave controllers, or a plurality of controllers.
The sample may be a biological sample. The sample may be derived from a human or a non-human animal, plant or environmental subject. The sample may be obtained from a microorganism. The sample may comprise bacteria, yeast, fungi, endophytes or spores. The sample may be derived from plant tissue such as from leaves, stems, roots, flowers, seeds, sap, bark, pollen or nectar of a plant. The sample may be a filtrate. The sample may be a crude or unprocessed sample. For example, the sample may be a crude sample obtained from a subject or source and applied directly to the apparatus 1 without any processing or purification steps undertaken.
The sample may comprise a partially purified preparation comprising a biomolecule. For example, in various embodiments the sample comprises a cell lysate, partially degraded tissue, or a sample that has undergone one or more partial purification steps.
The term “sample” as used in this specification refers to any material from which a biomolecule is to be prepared, extracted, purified or separated. The sample may comprise a natural or biological sample, for example, a sample of urine, whole blood, blood cells, serum, plasma, urine, faecal matter, cells, tissue, saliva, sputum, cultured cells, vaginal fluid, a swab, plant tissue, fungus, or a microorganism. The sample may comprise a natural or biological sample such as those listed above that is bound to a sample-holding matrix, for example, a swab or storage card. In some cases the sample-holding matrix will have been used to obtain the sample from a source (for example, a buccal swab) and is able to be added directly to the device of the disclosure for extraction, purification, separation or preparation of the biomolecule from the sample. The sample can also be soil, water and water filtrate taken from the environment.
The term “nucleic acid” as used in this specification refers to a single- or double-stranded polymer of deoxyribonucleotides (DNA), ribonucleotide bases (RNA) or known analogues of natural nucleotides, or mixtures thereof. The term includes reference to synthetic, modified or tagged nucleotides.
The method may comprise extracting nucleic acid from a sample comprising cells, and subsequently analysing that sample, in the field, that is, not in a traditional laboratory. In this embodiment the method comprises the steps of maintaining the sampling block 21 at a temperature of from about 70° C. to about 75° C., for a duration of less than about 1 minute to about 10 minutes, and maintaining the apparatus 1 at a temperature of from about 90° C. to about 95° C., for a duration of about 1 minute to about 5 minutes.
It will be appreciated by those skilled in the art that the apparatus and methods of the disclosure are suitable for the preparation, extraction, separation, or purification of various types of biomolecules from a range of sample types for many medical, laboratory, horticultural, veterinary, agricultural, environmental, forensic or diagnostic applications.
The method and apparatus of the disclosure are useful for applications where the sample comprises minute quantities of the biomolecule, where the biomolecule is of relatively poor quality, or where it is critical that the composition comprising the biomolecule comprises low or no contaminants.
The method and apparatus of the disclosure are particularly useful for extracting or purifying nucleic acids, such as deoxyribose nucleic acid (DNA) or ribonucleic acid (RNA) for a variety of molecular biology applications. For example, the method and apparatus of the disclosure may be used to produce a composition comprising nucleic acid extracted from a sample that is suitable for immediate use for a polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR (qPCR or qRT-PCR), isothermal amplification, forensic DNA fingerprinting, fluorescence-based detection, chip-based hybridisation detection, evaporation enrichment, DNA sequencing, RNA sequencing, molecular beacons, electrophoresis, direct electronic detection or nanopore analysis.
The method and apparatus of the disclosure are suitable for the preparation of nucleic acids for applications where the concentration of nucleic acid in the sample may be very low and where contamination may lead to an incorrect analysis of the nucleic acid.
An advantage of the disclosure is that the apparatus 1 is hand portable and that therefore both the processing of the sample, and the analysis of the processed sample, can take place in the field, and provide an output indicative of one or more properties of the processed sample in real time, that is within less than 30 minutes, and preferably less than 15 minutes.
Unless the context clearly requires otherwise, throughout the description, the words “comprise”, “comprising”, and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of “including, but not limited to”.
Where reference is used herein to directional terms such as ‘up’, ‘down’, ‘forward’, ‘rearward’, ‘horizontal’, ‘vertical’ etc., those terms refer to when the apparatus is in a typical in-use position, and are used to show and/or describe relative directions or orientations.
Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of “including, but not limited to”.
The terms “approximately,” “about,” and “substantially” as used herein represent an amount close to the stated amount that still performs a desired function or achieves a desired result. For example, in some embodiments, as the context may permit, the terms “approximately”, “about”, and “substantially” may refer to an amount that is within less than or equal to 10% of, within less than or equal to 5% of, and within less than or equal to 1% of the stated amount.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world.
The disclosed apparatus and systems may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.
Where, in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.
Depending on the embodiment, certain acts, events, or functions of any of the algorithms, methods, or processes described herein can be performed in a different sequence, can be added, merged, or left out altogether (for example, not all described acts or events are necessary for the practice of the algorithms). Moreover, in certain embodiments, acts or events can be performed concurrently, for example, through multi-threaded processing, interrupt processing, or multiple processors or processor cores or on other parallel architectures, rather than sequentially.
It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the disclosed apparatus and systems and without diminishing its attendant advantages. For instance, various components may be repositioned as desired. It is therefore intended that such changes and modifications be included within the scope of the disclosed apparatus and systems. Moreover, not all of the features, aspects and advantages are necessarily required to practice the disclosed apparatus and systems. Accordingly, the scope of the disclosed apparatus and systems is intended to be defined only by the claims that follow.
| Number | Date | Country | Kind |
|---|---|---|---|
| 766359 | Jul 2020 | NZ | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/NZ2021/050107 | 7/16/2021 | WO |
