MAHMOUD, Ra’ad Munir David (GB)
TAGGART, Robert Anthony (GB)
| CLAIMS 1. A reactor apparatus for controlling the temperature of a reaction volume whilst monitoring optical properties of a reaction therein comprising: a housing formation defining within it at least one reaction volume; a heat source disposed in association with and selectively operable to input heat to the housing formation, and thereby to affect a temperature of the reaction volume; a photon source; a photon detector; a photon transmissive region provided in the housing formation and configured to define an optical path between the photon source and the reaction volume; a photon transmissive region provided in the housing formation and configured to provide an optical path between the reaction volume and the photon detector. 2. A reactor apparatus in accordance with claim 1 , wherein an optically transmissive region is configured to provide both an optical path between the photon source and the reaction volume and an optical path between the reaction volume and the photon detector. 3. A reactor apparatus in accordance with claim 1 or claim 2, wherein a first optically transmissive region is configured to provide an optical path between the photon source and the reaction volume, and a second separate optically transmissive region is configured to provide an optical path between the reaction volume and the photon detector. 4. A reactor apparatus in accordance with any preceding claim, comprising a plurality of optically transmissive regions each configured to provide an optical path between a photon source and a given reaction volume, and a plurality of optically transmissive regions each configured to provide an optical path between the reaction volume and the photon detector. 5. A reactor apparatus in accordance with any preceding claim, defining a plurality of reaction volumes each associated with at least one optically transmissive region configured to provide an optical path between a photon source and a given reaction volume, and at least one optically transmissive region configured to provide an optical path between the reaction volume and the photon detector. 6. A reactor apparatus in accordance with any preceding claim, wherein at least one optically transmissive region is defined as an apertured portion in the housing formation, configured to define the desired optical path. 7. A reactor apparatus in accordance with any preceding claim, wherein at least one optically transmissive region is formed of optically transmissive material. 8. A reactor apparatus in accordance with any preceding claim, wherein the housing formation defines at least one reaction volume directly, the internal wall surfaces of the housing formation themselves constituting walls of the reaction volume. 9. A reactor apparatus in accordance with any preceding claim, wherein the housing formation defines at least one volume adapted to contain and retain a reactor vessel, the apparatus of the invention further comprising such a reactor vessel; wherein the reactor vessel is provided with optically transmissive vessel walls, at least in the region of any wall of the reactor vessel which coincides with and forms part of an optical path into the reaction volume in use. 10. A reactor apparatus in accordance with claim 9, wherein the housing formation defines an externally accessible apertured portion to provide access to the reaction volume into which the reactor vessel may be inserted. 11. A reactor apparatus in accordance with any preceding claim, wherein the heat source comprises a thermal heater, for example including one or more resistance heating elements, provided in direct contact with the housing 19 formation; and the housing formation is fabricated from a thermally conductive metallic material. 12. A reactor apparatus in accordance with any preceding claim, further comprising a temperature control system including at least a thermocouple and PID controller. 13. A reactor apparatus in accordance with any preceding claim, wherein the housing formation defines a reactor volume by means of a recessed portion within the body of the housing formation which leads to an aperture at the top, the top aperture being open to serves as a means to add reagents to the reaction volume and/or as a means to provide at least one of the optical paths. 14. A reactor apparatus in accordance with any preceding claim, wherein the photon source is a light source comprising at least one LED source to emit visible and/ or ultraviolet light. 15. A reactor apparatus in accordance with any preceding claim, wherein the photon detector is a camera. 16. A reactor apparatus in accordance with any preceding claim, wherein the photon detector is a colorimeter. 17. A reactor apparatus in accordance with any preceding claim, comprising a housing formation configured to provide a plurality of reaction volumes. 18. A method of controlling the temperature of a reaction volume whilst monitoring optical properties of a reaction therein comprising: providing a photon source and a photon detector; providing a housing formation defining within it: at least one reaction volume; a photon transmissive region provided configured to define an optical path between the photon source and the reaction volume; 20 a photon transmissive region configured to provide an optical path between the reaction volume and the photon detector; associating a heat source with the housing formation; adding reagents to the reaction volume to initiate a chemical reaction; monitoring the progress of the reaction therein by detecting changes by: causing photons from the photon source to be incident into the volume; detecting photons emergent from the volume at the photon detector; controlling the temperature of the reaction volume by operating the heat source. The method of claim 18, wherein internal wall surfaces of the housing formation are configured to define a reaction volume directly, and the step of adding reagents to the reaction volume to initiate a chemical reaction comprises adding reagents to this volume directly. The method of claim 18, wherein the internal wall surfaces of the housing formation are configured to define a volume adapted to contain and retain a reactor vessel, and the method comprises: introducing a reactor vessel into the volume so adapted; adding reagents to the reactor vessel to initiate a chemical reaction. The method of claim 20, wherein the housing formation defines an externally accessible apertured portion to provide access to the reaction volume, and the method comprises first introducing a reactor vessel through the externally accessible apertured portion and then adding reagents to the reactor vessel to initiate a chemical reaction through the externally accessible apertured portion. The method of one of claims 18 to 21 , comprising the step of monitoring the progress of the reaction by detecting a colour change in the photons emergent from the reaction volume. The method of claim 22, comprising use of a colorimeter to detect the colour change. 21 The method of claim 23, comprising use of a camera wherein an image is obtained and processed to determine a colour change. The method of one of claims 22 to 24 comprising the step of monitoring the progress of the reaction by detecting a colour change in a fluorescent reporter material, the method comprising the steps of: adding a fluorescent reporter material to the reagents in the reaction volume; monitoring the progress of the reaction therein by detecting changes in the photons emergent from the fluorescent reporter material. |
Field of Invention
The invention relates to the determination of optically-manifest changes in temperature controlled processes, and to a reactor apparatus and reaction method to facilitate the same.
The invention is a method and apparatus to control the temperature of reaction whilst monitoring the change in the optical properties of the reaction to determine its state. In embodiments, the invention comprises a component to control the temperature of a reagents held within a container, apparatus for generating photons to impinge on the reagents, apparatus for detecting the emergent photons, and a method to determine from the detected photons that a reaction has taken place.
Background of Invention
A significant number of chemical and biological processes can be monitored through changes in optical properties of an indicator chemical reaction. These include indicators that can lead to a change in the colour of the chemical indicator (chromogens) or in the emission of photons (luciferin, fluorescent molecules, etc.).
For a proportion of these processes it is desirable to control the temperature of the reaction. For some processes an isothermal process may be desirable, for others it may be beneficial to be able to change the temperature in a controlled manner, add or remove heat. For example, biological enzymatic reactions occur at their maximum rate at a specific temperature, therefore temperature control of the reaction is essential.
There may be circumstances where it is desirable to control the temperature in a reaction volume of a reactor or during a reaction process by maintaining isothermal conditions. There may be circumstances where it is desirable to effect such control by varying the temperature as the reaction progresses. In both these cases, an ability to monitor the progress of the reaction, and to feed back information regarding the progress of the reaction as a determining parameter for effecting that temperature control is likely to be beneficial.
However, reaction progress monitoring techniques need to be developed which do not tend to interfere with the progress of the reaction, or do not tend to interfere with the process of controlling temperature. In particular, it may be desirable to develop a method of monitoring of the reaction process which is non-invasive, in that it allows the progress to be monitored externally of a reactor vessel, without requiring any interference with the reagents therein, or with the vessel itself.
The invention is directed at the development of such a non-invasive reaction progress monitoring system and method in association with an effective means of reaction volume temperature control, for example to maintain isothermal conditions and/or to vary those conditions in a controlled manner as the reaction progresses and responsive to the progress of the reaction.
Summary of Invention
In accordance with the invention in a first aspect, a reactor apparatus for controlling the temperature of a reaction volume whilst monitoring optical properties of a reaction therein comprises: a housing formation defining within it at least one reaction volume; a heat source disposed in association with and selectively operable to input heat to the housing formation, and thereby to affect a temperature of the reaction volume; a photon source; a photon detector; a photon transmissive region provided in the housing formation and configured to define an optical path between the photon source and the reaction volume; a photon transmissive region provided in the housing formation and configured to provide an optical path between the reaction volume and the photon detector.
In particular embodiments, the photon source is provided externally of the housing formation and the photon transmissive region is provided through the housing formation and configured to define an optical path between the photon source external to the housing and the reaction volume within the housing formation.
In particular embodiments, the photon detector is provided externally of the housing formation and the photon transmissive region is provided through the housing formation and configured to define an optical path between the reaction volume within the housing formation and the photon detector is provided external to the housing formation.
Thus, the reactor apparatus of the first aspect of the invention combines in a simple but effective manner a means to control the temperature of a reaction within the reaction volume and a means to monitor the progress of a reaction within the reaction volume in cases where that progress can be so monitored by a determination of optical properties of the reaction in the broadest sense that photons incident on the reaction volume undertake or cause a detectable change in the photons that emerge from the reaction volume, which detectable change varies over the course of the reaction.
Many reactions exhibit such a detectable change For example a detectable change may be manifest in the energy profile of transmitted photons, for example being a colour change, or may be manifest in a process causing the emission of photons, such as fluorescence.
For such reactions where an optically-manifest change occurs over the course of the reaction process, the apparatus of the invention may therefore provide a technical solution for the non-invasive reaction progress monitoring of such reactions in association with an effective means of reaction volume temperature control, for example to maintain isothermal conditions and/or to vary those conditions in a controlled manner as the reaction progresses and responsive to the progress of the reaction. The apparatus provides this for such reactions in a simple manner which does not require invasive probes or other invasive monitoring of the reaction volume but can be carried out externally via the photon source and photon detector.
In accordance with the principle of the invention, the housing formation is provided with at least one optically transmissive region to provide an optical path between the photon source and the reaction volume, and at least one optically transmissive region to provide optical path between the reaction volume and the photon detector.
The principles of the invention encompass both embodiments where a particular optically transmissive region is configured to provide both an optical path between the photon source and the reaction volume and an optical path between the reaction volume and the photon detector, and embodiments where a first optically transmissive region provides an optical path between the photon source and the reaction volume, and a second separate optically transmissive region that provides an optical path between the reaction volume and the photon detector.
Embodiments of the former might be applicable for example where an optically- manifest change is intended to be observed using reflective light for light emitted through an interactive process such as fluorescence. Examples of the latter may be more suitable for the detection of transmitted photons or photons emitted from interactive processes such as scattering.
A single reactor apparatus in accordance with the first aspect of the invention may embody either or both principles.
A single reactor apparatus in accordance with the first aspect of the invention may have a plurality of optically transmissive regions each configured to provide an optical path between a photon source and a given reaction volume, and/ or a plurality of optically transmissive regions each configured to provide an optical path between the reaction volume and the photon detector.
A single reactor apparatus in accordance with the first aspect of the invention may define a plurality of reaction volumes each associated with at least one optically transmissive region each configured to provide an optical path between a photon source and a given reaction volume, and at least one optically transmissive regions to provide an optical path between the reaction volume and the photon detector.
In some embodiments an optically transmissive region as above described may be defined as an apertured portion in the housing formation, configured to define the desired optical path. Additionally or alternatively an optically transmissive region may comprise a portion formed of optically transmissive material. For example a portion formed of optically transmissive material may comprise a window in that portion of the housing formation. Alternatively, an optically transmissive region may comprise an open portion formed in the body of the housing formation.
Embodiments of the invention may be contemplated in which the housing formation defines at least one reaction volume in one of two ways. In a first alternative, internal wall surfaces of the housing formation may define at least one such volume directly, the internal wall surfaces of the housing formation themselves constituting walls of the reaction volume in which reaction reagents may be held in use. In a second alternative, the internal wall surfaces of the housing formation may define a volume adapted to contain and retain a reactor vessel, the apparatus of the invention optionally therefore further comprising such a reactor vessel in which reaction reagents may be directly held in use.
If such a reactor vessel is used, it will be necessary that it should be provided with optically transmissive vessel walls, at least in the region of any wall of the reactor vessel which coincides with and forms part of an optical path into the reaction volume as herein described. Most conveniently, such a secondary reactor vessel is preferably fabricated entirely of optically transmissive material, and is for example optically transparent.
In a convenient embodiment, the housing formation defines an externally accessible apertured portion to provide access to the reaction volume into which a reactor vessel may be inserted. Conveniently, this apertured portion may also serve as an optically transmissive portion defining at least one of the light paths hereinabove described.
Thus, in a particular convenient embodiment, a transparent reactor vessel such as a test tube or similar tube-like container defines a reaction volume in which the reagents for the reaction are directly held in use. A least one such vessel is placed in a housing formation as above described which has an apertured portion manufactured to accept the reactor vessel within the body of the material of the housing formation. The heat source provides a means to control the temperature of the housing formation to allow control of the temperature of the reagents within the reactor vessel, for example to maintain isothermal conditions or to vary the temperature responsive to measured progress of the reaction.
In embodiments, a heat source such as a thermal heater, for example including one or more resistance heating elements, is provided in thermal association and for example direct contact with the housing formation (either internally or externally).
To facilitate heating of the reaction volume via operation of such a heat source, the housing formation is preferably fabricated from a thermally conductive material, and for example the housing formation is fabricated from a metallic material, for example being a monolithic block. Preferably, the housing formation comprises aluminium or alloys based on aluminium.
In operation an appropriate temperature control protocol used and the apparatus preferably further comprises a temperature control system. A thermocouple and PID controller may be used for this purpose.
In typical operation, an apparatus in accordance with the first aspect of the invention will be operated in an environment defining an up direction, and convenient embodiments of the apparatus can be discussed with reference to this in use direction.
In such cases, the housing formation preferably defines a reactor volume by means of a recessed portion within the body of the housing formation which leads to an aperture at the top. Conveniently, the top aperture is open and serves as a means to add reagents to the reaction volume and/or as a means to provide at least one of the optical paths. Alternatively, the top aperture may be provided with a transparent window.
In embodiments, the top aperture may in use serve to define both an incoming and an outgoing optical path. In alternative embodiments, an incoming or outgoing optical path may be provided by a second apertured portion, and for example a portion having a window of transparent material, defined on a side of the housing. Thus, in a possible mode of operation, incident photons from the source are directed into the reaction volume by means of a hole through the body of the housing formation that also serves to allow passage of reagents into the reaction volume, for example being the top aperture above described. In an alternative embodiment, incident photons from the source are directed through the housing formation and into the reaction volume via an alternatively defined pathway.
To allow the photons emerging from the reaction volume to be detected at the photon detector it is necessary to allow a minimal absorption path out of the housing formation. In one embodiment this can be through the reaction volume and for example in the case where a reaction vessel is provided through the reagent and vessel and out through the aperture designed to hold the vessel in place. In an alternative embodiment, a separate output window may be created to allow passage out of the housing formation.
References herein to photons will be understood to be references to photons with an energy or energy range that is suitable to monitor an optically-manifest process occurring during the progress of a particular reaction. The skilled person will have no difficulty in selecting a suitable energy or energy range for incident photons from the photon source, and a consequent suitable energy or energy range for the detector, depending on the optically-manifest process which is being monitored for a given reaction to monitor the progress of the reaction.
Accordingly, the photon detector is configured to detect photons within a target range constituting at least a part, and for example a substantial part, of an expected range of energies of photons emitted from the reaction volume having regard to the particular emission process and reaction taking place, and having regard to the energies of the photons from the photon source.
In preferred embodiments photons may be in the visible range, in the ultraviolet range etc.
In preferred embodiments, the photon source is for example a light source, including, but not limited to, a visible light source, an ultraviolet light source etc. Suitable photon detectors might include a camera, a colorimeter or the like.
In one embodiment, where a change is manifested by a colour change the photons are optical photons, for example from an LED source. In an alternative embodiment, the photons are at the appropriate wavelength for the excitation of the fluorescent reporter dyes, such as in the ultra-violet range of the electromagnetic spectrum.
In one embodiment where a colour change is to be determined, a colorimeter may be used to detect the emerging photons and such a colour change. Alternatively, a camera may be used, together with an appropriate means to characterise the colour. A particular embodiment may determine the hue across several pixels determined to cover the reagent region, and an appropriate algorithm used to assign a value to the reagent, and another appropriate algorithm to determine a change in this value is associated with a change in colour.
In a possible embodiment, the progress of a reaction may be manifested in a change in the reagents as the reaction progresses.
In an alternative embodiment, the progress of a reaction may be manifested in a change in the properties of a fluorescent material such as a fluorescent reporter. In one particular embodiment, this fluorescence can be instigated by the presence of a UV light source.
The invention is not restricted to the processing of one reaction volume. The invention is amenable to the processing and monitoring of multiple reaction volumes together.
In embodiments of the invention to facilitate this, a reactor apparatus may be provided comprising a housing formation including a plurality of reaction volumes as herein described.
More completely, a reactor apparatus for controlling the temperature of a reaction volume whilst monitoring optical properties of a reaction therein comprises: a housing formation defining within it at a plurality of reaction volumes as hereinabove described; a heat source disposed in association with and selectively operable to input heat to the housing formation, and thereby to affect a temperature of the reaction volumes; and associated with each reaction volume: a photon source; a photon detector; a photon transmissive region provided in the housing formation and configured to define an optical path between the photon source and the reaction volume; a photon transmissive region provided in the housing formation and configured to provide an optical path between the reaction volume and the photon detector.
Such an apparatus may have at least one photon source and/or photon detector that provides photons to or detects photons from each such reaction volume separately, or may be provided with a common source or a common detector providing photons to or detecting photons from more than one reaction volume simultaneously as the case may be.
In a further aspect of the invention, a method of controlling the temperature of a reaction volume whilst monitoring optical properties of a reaction therein comprises: providing a photon source and a photon detector; providing a housing formation defining within it: at least one reaction volume; a photon transmissive region provided configured to define an optical path between the photon source and the reaction volume; a photon transmissive region configured to provide an optical path between the reaction volume and the photon detector; associating a heat source with the housing formation; adding reagents to the reaction volume to initiate a chemical reaction; monitoring the progress of the reaction therein by detecting changes by: causing photons from the photon source to be incident into the volume; detecting photons emergent from the volume at the photon detector; controlling the temperature of the reaction volume by operating the heat source. The method is thus a method to control the temperature of a reaction within the reaction volume while also monitoring the progress of a reaction within the reaction volume in cases where that progress can be so monitored by a determination of optical properties of the reaction in the broadest sense that photons incident on the reaction volume undertake or cause a detectable change in the photons that emerge from the reaction volume, which detectable change varies over the course of the reaction.
Many reactions exhibit such a detectable optically-manifest change for example being a colour change of the reagents, or in a process causing the emission of photons, such as fluorescence. For such reactions where an optically-manifest change occurs over the course of the reaction process, the method of the invention may therefore provide a technical solution for the non-invasive reaction progress monitoring of such reactions in association with an effective reaction volume temperature control for example to maintain isothermal conditions and/or to vary those conditions in a controlled manner as the reaction progresses and responsive to the progress of the reaction.
In particular, the step of temperature control may be responsive to the step of monitoring the progress of the reaction therein by detecting such optically-manifest changes. For example the step of temperature control may be responsive to maintain isothermal conditions until an optically-manifest change suggests a reaction or a stage thereof is complete. For example the step of temperature control may be responsive to change a temperature of the reaction volume in response to an optically-manifest change that suggests a reaction or a stage thereof is complete.
The method is most preferably a method that makes use of an apparatus of the first aspects of the invention, and preferred features of each will be understood by analogy with the description of the other. In particular, preferred modes of operation of the apparatus will by analogy be understood to correspond to preferred steps of the method where appropriate.
In embodiments, internal wall surfaces of the housing formation may define a reaction volume directly, and the step of adding reagents to the reaction volume to initiate a chemical reaction comprises adding reagents to this volume directly. In other embodiments, the internal wall surfaces of the housing formation may define a volume adapted to contain and retain a reactor vessel, and the method comprises: introducing a reactor vessel into the volume so adapted; adding reagents to the reactor vessel to initiate a chemical reaction.
The housing formation preferably defines an externally accessible apertured portion to provide access to the reaction volume and the step of adding reagents to the reaction volume comprises introducing reagents through the externally accessible apertured portion.
For example, the method comprises first introducing a reactor vessel through the externally accessible apertured portion and then adding reagents to the reactor vessel to initiate a chemical reaction through the externally accessible apertured portion.
The heat source is preferably a thermal heater, for example including one or more resistance heating elements, and is provided in thermal association and for example direct contact with the housing formation (either internally or externally) and operated to control the temperature of the housing formation to allow control of the temperature of the reagents within the reactor vessel, for example to maintain isothermal conditions or to vary the temperature responsive to measured progress of the reaction.
To facilitate heating of the reaction volume via operation of such a heat source, the housing formation is preferably fabricated from a thermally conductive material as above described.
In operation an appropriate temperature control protocol used and the apparatus preferably further comprises a temperature control system. A thermocouple and PID controller may be used for this purpose. The method comprises use of the same to control operation of the heat source.
In a convenient embodiment of the method, the housing formation defines a reactor volume by means of a recessed portion within the body of the housing formation which leads to an aperture at the top and the step of adding reagents to the reactor vessel to initiate a chemical reaction comprises introducing them through the aperture at the top.
Further in a possible mode of operation, incident photons from the source are directed into the reaction volume by means of a hole through the body of the housing formation that also serves to allow passage of reagents into the reaction volume, for example being the top aperture above described. In an alternative embodiment of the method, incident photons from the source are directed through the housing formation and into the reaction volume via an alternatively defined pathway.
In preferred embodiments photons may be in the visible range, in the ultraviolet range etc.
In one embodiment of the method a colour of the reagents in the reaction volume and for example a colour change is determined. A method of the invention may comprise the step of monitoring the progress of the reaction by detecting colour changes in the photons emergent from the reaction volume.
A colorimeter may be used to detect the emerging photons. In an alternative embodiment an image is obtained and processed to determine a colour of the reagents in the reaction volume. A particular embodiment may determine the hue across several pixels determined to cover the reagent region, and an appropriate algorithm may be used to assign a value to the reagent, and another appropriate algorithm to determine a change in this value is associated with a change in colour.
In an alternative embodiment, the progress of a reaction may be manifested in a creation of or change in the properties of a fluorescent material and for example a change in a fluorescent reporter material. In one particular embodiment. A method of the invention may comprise the steps of: adding a fluorescent reporter material to the reagents in the reaction volume; monitoring the progress of the reaction therein by detecting changes in the photons emergent from the fluorescent reporter material. This fluorescence can be instigated by the presence of a UV source, the method comprising causing photons from the photon source in the ultraviolet range to be incident into the volume.
By further analogy, a further aspect of the invention comprises the use of the apparatus of the first aspect to perform the method of the second aspect.
Brief Description of Drawings
The invention will now be described by way of example only with reference to figures 1 to 2 of the accompanying drawings, in which:
Figure 1 is a lateral schematic of an example embodiment;
Figure 2 shows a top-down view of a multi-vessel system;
Detailed Description of Preferred Embodiments
The examples are discussed with reference to Loop Mediated Isothermal Amplification (LAMP). LAMP is a technique for the amplification of DNA and has been used to detect specific genetic sequences, among them from certain viruses. The target sequence is amplified at a constant temperature of 60-65°C using either two or three sets of primers and a polymerase. The amplification product can be detected by measuring the turbidity caused by the magnesium pyrophosphate precipitate in solution as a by-product of amplification. Reporter dyes can be used to create a visible colour change. In this example, if a target virus is present, its amplification causes a colour change from red to orange. The dye is a pH indicator, in this instance Phenol Red that turns yellow at low pH, driven by the accumulation of acidic DNA amplified molecules in the solution when the reaction occurs.
The general principle of the invention as it might be applied to this or other techniques is illustrated in the figure 1 schematic.
In the illustrated embodiment, a housing formation is a solid block 1 of appropriate material (such as a material with high thermal conductivity, for example aluminium). The block 1 includes a heating element 3 to control the temperature. As the block is conductive, the temperature of the reaction volume can be effectively maintained or varied as required.
In the illustrated embodiment, the reagents 5 are held within the lower part of a tapered transparent vessel 7, such as a test tube or similar tube-like container. The vessel is placed in a hole manufactured to accept the vessel within the body of the material of the block 1. The element 3 acts as a means to control the temperature of the block 1 to allow control of the temperature of the reagents 5. A thermal heater is brought into contact with the block (either internally or externally, in the example illustrated, internally) and an appropriate temperature control protocol used. A thermocouple and PID controller (not shown) may be used for this purpose.
The invention requires that there are photon sources and detectors and that there is a photon transmissive region provided in the housing formation and configured to define an optical path between the photon source and the reaction volume; and a photon transmissive region provided in the housing formation and configured to provide an optical path between the reaction volume and the photon detector
In the illustrated embodiment, a light source 11 is disposed above the block such that the incident photons are directed to the reagent 5 by means of top aperture which also serves to hold the vessel and allow passage to the reagents. The photons are directed through the vessel and reagent from above the block. In alternative embodiments other optical paths may be provided through the block.
In one embodiment, where a change is manifested by a colour change the photons are optical photons, for example from an LED source. In an alternative embodiment, the photons are at the appropriate wavelength for the excitation of the fluorescent reporter dyes, such as in the ultra-violet range of the electromagnetic spectrum.
To allow the photons emerging from the reagent to be detected it is necessary to allow a minimal absorption path out of the block region. This can be through the reagent and vessel and out through the hole designed to hold the vessel in place. In the illustrated embodiment, a separate hole (output window 13) may be created to allow passage out of the block and an output optical pathway created to the detector 15. In one embodiment where a colour change is to be determined, a colorimeter may be used to detect the emerging photons. In the illustrated embodiment a camera 15 is used, together with an appropriate means to characterise the colour of the reagent. A particular embodiment may determine the hue across several pixels determined to cover the reagent region, and an appropriate algorithm used to assign a value to the reagent, and another appropriate algorithm to determine a change in this value is associated with a change in colour.
In an alternative embodiment, the progress of a reaction may be manifested in a change in the properties of a fluorescent reporter. In one particular embodiment, this fluorescence can be instigated by the presence of a UV light source.
Figure 1 illustrates a simple example with a single vessel. The invention is not restricted to the processing of one reagent vessel. The invention is amenable to the processing and monitoring of multiple reagent-containing reagents through multiple holes in the block for vessel access. In one embodiment a single photon source may be used to illuminate all vessels. In an alternative embodiment multiple light sources may be used.
In the figure 2 embodiment, to allow the testing of multiple samples, an aluminum block 21 is created with five holes to hold five vessels 27 containing the sample and reagents. An LED light source (not shown), appropriately positioned, directs light into each of the vessels from above. Horizontal holes in the block allow the light to emerge from the reagent through the output windows 33, with the holes angled so that all of the vessels can be imaged by one optical camera 35. The camera 35 outputs the images at a fixed interval, and a connected computer (not shown) monitors for changes in the hue of each vessel. Once the hue has changed by a certain amount or according to a certain pattern, a change is judged to have occurred, and the virus detected. As the LAMP method amplifies the target exponentially, by determining the time taken for the change to occur, a measure of the concentration of the virus within the sample can be obtained.
A single camera, colorimeter or other device may image all vessels. A suitable image processing method may be used to determine the reagent regions from each vessel. In an alternative embodiment, multiple cameras, colorimeters or other devices may be used.
In an alternative example of a LAMP process, in-vessel detection may be achieved using similar apparatus with the visible light source replaced by a UV source.
Methods exist, such as using manganese loaded calcein, which starts fluorescing upon complexation of manganese by pyrophosphate during in vitro DNA synthesis. An appropriate camera system to detect the emergent UV light can allow the state of the reaction to be monitored and the concentration of the target DNA in the original sample determined.
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