WO2004055499A1

WO2004055499A1 - Assay apparatus

Info

Publication number: WO2004055499A1
Application number: PCT/GB2003/005488
Authority: WO
Inventors: Tony Canas
Original assignee: Molecular Sensing Plc
Priority date: 2002-12-17
Filing date: 2003-12-17
Publication date: 2004-07-01
Also published as: GB0229409D0; AU2003288563A1

Abstract

A light transmitting module for use in an analytical apparatus comprising: - a light receiving portion for receiving light from a sample vessel, - a light emitting portion, - a locating means for positioning a sample vessel in optical communication with the light receiving portion, - a light transmitting portion located between the light receiving portion and the light emitting portion, the light transmitting portion comprising an internally reflective cavity with an internal cross sectional width in a direction perpendicular to a line joining the light receiving and emitting portions, which increases from the end proximate to the light receiving portion to it’s the proximate to the light transmitting portion, the light receiving portion being formed by an opening at the light receiving end of the cavity.

Description

Assay apparatus

This invention relates to spectrophotometric detection apparatus for diagnostic, experimental and other laboratory procedures and methods associated therewith.

Many diagnostic procedures include steps in which spectrophotometric properties of a sample are investigated. Conventional spectroscopy methods and apparatuses are well known in the art (as described, for example, in "Instrumental Methods of Analysis", H.H. Willard et al., Wadsworth Publishing Company, 1988). In a general spectrophotometry experiment, light (of a single or of a mixture of different wavelengths) is shone onto or through a sample and the amount of light transmitted, reflected or emitted from the sample (of the same or a different wavelength from the source light) is analysed.

The sensitivity of any spectrophotometric experiment depends on the intensity of the light source, the efficiency of light transmission to and from the sample and the sensitivity of the light detector. High intensity light sources may be large and/or expensive and they are prone to generating a large amount of heat. High sensitivity light detectors are also generally expensive and may take up a large amount of space. The degree to which the sensitivity of an experiment can be improved by use of an improved light source or detector is thus somewhat limited. In most devices, sensitivity therefore depends on the efficiency of light transmission to and from the sample.

In many diagnostic and research applications, the sample volume is small. Furthermore, the vessel in which the sample is held may be subject to various design constraints, for example by the need to control the temperature of the sample accurately or to keep the sample sterile. In the diagnostic and research fields, it has become common for analyses to be carried out on arrays of samples (for example in 96-, 384- or 1536-well plates). In such multiple sample arrays, each sample is small and the array layout places constraints on the accessibility of the samples.

High sensitivity spectrophotometric analysis of samples is desirable in a large number of diagnostic, research and experimental procedures. Many spectrometers depend on efficient light transmission. Such spectrometers include UV-visible, infrared and fluorescence spectrometers. High sensitivity spectrophotometric analysis of samples is desirable in various apparatus for molecular biological applications, for example in apparatus for restriction digest experiments, isothermal or variable temperature amplification experiments, nuclease or protease digests, or protein expression experiments.

One application in which high sensitivity spectrophotometric analysis of small samples is important is in the analysis of nucleic acids, in particular the analysis of nucleic acid amplification reactions. A particularly important area is the analysis of polymerase chain reactions (PCRs), for example in real time. The principle of the PCR nucleic acid amplification technique is described in US Patent US 4,683,195 (Cetus Corporation/Roche). Apparatus for carrying out the PCR reaction have been described in, for example, European Patent application EP 0236 069 (Cetus Corporation/ Roche/PE). Such apparatus are commonly referred to as "thermocyclers".

Briefly, in a PCR reaction, a sample is subjected to a cycling between three phases: 1. Denaturation, during which a mixture of the target DNA, individual nucleotide bases

(usually A,T,C and G), primers and a suitable DNA polymerase are heated to a relatively high temperature (typically over 80 °C) so that the two strands of the target DNA separate; 2. Annealing, during which the primers are allowed to anneal to the target DNA at a relatively low temperature (typically around 50 °C to 60 °C); and 3. Extension, during which the DNA polymerase synthesises strands of oligonucleotides complimentary to the target strands at an intermediate temperature (typically around 70 °C). In theory the quantity of target DNA present is doubled in each cycle. The cycle is repeated as many times as necessary to obtain a desired quantity of product, typically around 30 times.

It is useful to the user to know how a PCR is progressing during the course of the reaction.

Fluorescence-based approaches to real-time measurement of PCR amplification products have been proposed and are in common usage. Some such approaches have employed double-stranded DNA binding dyes (for example major or minor groove binding intercalating dyes, for example SYBR Green I (RTM) or ethidium bromide) to indicate the amount of double stranded DNA present. Other approaches have employed probes containing fluorescer-quencher pairs (for example the "TaqMan" (RTM) approach) that are cleaved during amplification to release a fluorescent product the concentration of which is indicative of the amount of double stranded DNA present. Such fluorescer-quencher pairs methods typically make use of fluorescence resonance energy transfer (FRET), for example in a dual probes arrangement (for example in a "HYB-Probes" approach). Adaptations of those approaches are known (as described in, for example, WO 95/30139), in which two or more dyes are used.

Fluorescence-based approaches have become the standard methods for monitoring PCR reactions in real time. Accordingly, there is demand for apparatus in which a small sample can be subjected to heating and cooling whilst simultaneously being spectrophotometrically analysed.

The efficiency of a PCR amplification procedure is heavily dependent on the rates at which the sample is cycled between the various temperatures and the accuracy of the temperature control and accordingly it is desirable for accurate, yet rapid, heating and cooling to be used.

Accordingly, the spectrophotometric analysis of PCR reactions is heavily constrained by the need for the sample to be positioned in a heater apparatus and the need for the sample container to be shaped so as to allow efficient heating of the sample. The constraints on the spectrophotometric analysis are exacerbated in the case of an apparatus for simultaneous analysis of many samples in an array.

One widely used PCR apparatus is the LightCycler® device, available from Roche Diagnostics (Roche Diagnostics Ltd., Bell Lane, Lewes, East Sussex, BN7 1LG, U.K.). A device with many of the features of the Lightcycler device is described in WO 97/46707 and WO 97/46712. In the Lightcycler device, a carousel having a plurality of sample tube receiving slots is located in an enclosed housing. The housing is in communication with a fan and a heater. In use, sample capillary tubes containing the samples of interest are inserted into the carousel. During a heating phase, the fan pushes hot air into the housing, causing the samples to be heated. During a cooling phase, the apparatus is vented and the fan pushes cold air into the housing. An optical detection unit comprising a light source and a fluorescence detector is arranged to interrogate the contents of one capillary tube at a time along the length of the tube. The carousel rotates such that each sample tube may be aligned with the optical detection unit in turn.

The Lightcyler device enables the progress of several PCR reactions to be monitored simultaneously in "real time", i.e. whilst the reaction is still progressing.

In WO01/35079 there is described a combined fluorometer and thermal cycler in which several samples may be analysed for fluorescence characteristics simultaneously. The fluorometer comprises a plurality of low heat-generating hght sources, means for positioning a plurality of containers for containing potentially fluorescing sample into optical communication with said light sources, wherein each light source corresponds with one of said containers when in position, a first optical path means for guiding light from said light source to said corresponding container, an optical signal sensing means in optical communication with the sample in said positioned containers, and a second optical path means for guiding emitted light from the sample to said optical signal sensing means.

One low heat-generating light source is provided for each of the containers in one-to-one correspondence. The low heat-generating light sources are defined as light sources operated at a level below the level at which active cooling of the light source, such as via a fan, is required.

The low heat-generating light sources provide adequate power to the samples because light is not wasted on the spaces between the positioned containers. The thermal cycler portion of the device of WO 01/35079 comprises a thermally controlled base having a plurality of wells for receiving sample containers, the base being fabricated on a thermoelectric heater/cooler element, and a thermally controlled cover having a plurality of apertures. The thermally controlled cover may be an electrically heated plate.

In the quest for ever cheaper or useful spectrophotometric detection devices, there remains a need for devices with more efficient and sensitive detection capabilities. In addition, many devices of the prior art require very accurate locating of the sample in order for detection to be efficient. Those low tolerances require the devices to be accurately manufactured and to be carefully operated. It has now, surprisingly, been found that improved sensitivity and tolerance can be obtained in a detection device by use of a light transmitting module of the invention.

The invention provides a light transmitting module for use in an analytical apparatus comprising:

- a light receiving portion for receiving light from a sample vessel,

- a light emitting portion,

- a locating means for positioning a sample vessel in optical communication with the light receiving portion,

- a light transmitting portion located between the light receiving portion and the light emitting portion, the light transmitting portion comprising an internally reflective cavity with an internal cross sectional width in a direction perpendicular to a line joining the light receiving and emitting portions, which increases from the end proximate to the light receiving portion to its end proximate to the light transmitting portion, the light receiving portion being formed by an opening at the light receiving end of the cavity.

In many devices of the prior art, there are inefficiencies in the transmission of light from the sample to the detector. Those may arise from refraction or reflection at interfaces or from light emitted in certain directions not reaching the detector. The light transmission device of the invention ameliorates those problems and enables up to around 10⁴ times more light to reach the detector than in an equivalent device in which light is transmitted from the sample to the detector via a junction with an optical fibre. In use, light from the sample tube enters the light transmission device at a wide variety of angles. The shape of the light transmitting portion causes the light to be emitted at the light emitting end at a narrower set of angles thus enabling a large portion of the light to reach a detector.

Preferably, the internal cross sectional width in a direction perpendicular to a line joining the light receiving and emitting portions, increases monotonically from the end proximate to the light receiving portion to its end proximate to the light transmitting portion.

Preferably, the light transmitting portion comprises an internally reflective cavity with an internal cross section, in a plane containing the light receiving and emitting portions, which is curved for at least a part of its length. A curved shape further aids the gathering of light from the sample vessel.

Preferably, the light transmitting portion comprises an internally reflective cavity with an internal cross sectional width in a direction perpendicular to a line joining the light receiving and emitting portions, which increases from its end proximate to the light receiving portion to its end proximate to the light transmitting portion with a decreasing rate with respect to the displacement along a line joining the light receiving and emitting portions for at least a part of its length. For example, the internally reflective cavity may have an internal cross section, in a plane containing the light receiving and emitting portions, which has the shape of a truncated parabola for at least a part of its length, the truncation being at the turning point of the parabola at the light receiving end of the light transmitting portion.

Preferably, the internal shape of the cavity of the light transmitting portion is symmetrical about an axis in the direction of a line joining the light receiving and emitting portions. Preferably, the light transmitting portion comprises an internally reflective cavity with the internal shape of a truncated paraboloid of revolution for at least a part of its length.

In certain embodiments of the invention, the light transmitting portion comprises an internally reflective cavity with an internal cross sectional width in a plane containing the light receiving and emitting portions, which is elliptical, hyperbolic, arcuate or conical for at least a part of its length.

The internal cross sectional width of the internally reflective cavity may comprise two or more sections of different cross sectional width shapes. Preferably, the two or more sections are joined together with a continuous slope.

In a particularly preferred embodiment of the invention, the light transmitting portion comprises an internally reflective cavity with a section with an internal cross sectional width in the direction perpendicular to a line joining the light receiving and emitting portions which has a truncated conical shape at the end proximate to the light receiving portion and a section with an internal cross sectional width in the direction perpendicular to a line joining the light receiving and emitting portions which is parabolic at the end proximate to the light emitting portion. The parabolic and conical sections are preferably joined together with continuous slope. Such a modified compound parabolic concentrator is also known as an angle transforming concentrator.

It has been found that improved optical transfer from the light receiving portion to the light emitting portion is achieved if no images are formed.

The reflective internal surface of the cavity may be reflective by virtue of the material from which the module, or at least the relevant part of the module, is made being a reflective material. For example, the module or relevant part of the module may be made of a metal, for example aluminium or silver. Alternatively, the cavity may be mirrored, that is to say that a reflective material may have been applied to the internal surface of the cavity. For example, silver or any other reflective material may have been applied to the internal surface of the cavity. The reflective properties of the reflective surface may be enhanced by polishing the surface. The light receiving portion is formed by an opening at the light receiving end of the cavity of the light transmitting portion. The opening is preferably of a suitable size to receive a sample vessel, i.e. the opening is slightly wider than the external cross section of the vessel. Preferably, the majority of the sample vessel is located outside the opening and only the tip of the vessel protrudes into the cavity of the light transmitting module.

In a preferred embodiment, th sample vessel is a tube, optionally with a rounded tip. In a light transmitting module for use with such a tube, the opening may be wide enough for the tube to protrude into the cavity and the locating means may be arranged to hold the sample tube in a position whereby the rounded end of the tube is within the cavity. The light transmitting module may be constructed to be suitable for use with a tube of any desired width, for example up to 25mm in width. Preferably, the light transmitting module is suitable for use with a tube of up to 15mm width.

In an alternative embodiment, the sample vessel may be a cuvette. Such a cuvette may have any conventional size and shape, for example it maybe square. Preferably a cuvette has a width of from 2mm to 20mm, more preferably from 5mm to 10mm.

As mentioned above, the tip of the vessel may protrude into the cavity of the light transmitting module. For example, the sample vessel may protrude into the cavity to a distance of less than 10mm from the light receiving portion opening. Preferably, the sample vessel may protrude into the cavity to a distance of less than 5mm from the light receiving portion opening. For example, the sample vessel may protrude into the cavity to a distance of 2mm from the light receiving portion opening. Preferably, the sample tube has a hemispherical tip and the sample vessel protrudes into the cavity to a distance such that the centre of hemisphere is located at the centre of the light-receiving aperture.

Alternatively, to maintain cleanliness in the light transmitting portion cavity, the light receiving portion may comprise a transparent cover over the opening at the light receiving end of the light transmitting portion. In such an embodiment, the sample vessel is held in position in close proximity to the light receiving portion. It may be in physical contact with the cover, but that is not essential. Close proximity to the light receiving portion increases the efficiency of transmission of light into the device. Typically, the sample vessel may be located not more than 10 mm away from the light receiving portion. Preferably, the sample vessel is located not more than 6 mm, more preferably not more than 3mm away from the light receiving portion.

The width of the internally reflective cavity at the light-emitting end determines the angles at which light is emitted from the light-emitting portion; a wider opening and cavity results in light being emitted at a narrower spread of angles. Preferably the width of the cavity at the light- emitting end is several times wider than at the light-receiving end, typically 2 to 8 times wider, for example 3 to 5 times wider.

In the case of a sample tube with external cross section of approximately 1 to 2mm, an opening at the light receiving end of around 1.5 to 3.5mm cross section and an opening at the light emitting end of around 5 to 14mm cross section is typically suitable. In the case of a light transmitting module for use with a larger vessel, a larger opening is appropriate. For example for use with a cuvette of 10mm width, an opening of from slightly more than 10 to 15mm is suitable.

The length of the light transmitting portion is not critical to the operation of the device. The length is generally greater than the width of the light transmitting portion at the light emitting end. Typically, for a device for use with a sample tube of 1 to 2 mm external cross sectional width, a length of from 10mm to 80mm is suitable. For a device for use with a larger sample vessel, a longer device is suitable.

Generally, the sample vessel is removable from the apparatus. The locating means may optionally comprise a sample vessel locating cup to guide or hold the sample vessel in a favourable position near or in the light receiving portion.

In a PCR apparatus it is necessary to heat and cool a sample as rapidly as possible, as described above. The vessels are therefore generally arranged to have a large surface area to volume ratio. Long, thin tubes are therefore frequently used. Such tubes may be of circular cross section (despite the low surface area to volume ratio that that entails in comparison to, for example, an oblong or square cross-section) or may have any other suitable shape. If a tube is used, it is preferable for the locating means to position the sample tube such that its end is in optical communication with the light receiving portion. In that way, the light receiving portion is aligned with as much of the sample in the tube as possible. The reflective light transmitting portion cavity serves to concentrate the light from a sample in the direction of a detector. It can thus be referred to as a light concentrator. An example of such an element is known as a compound parabolic concentrator (CPC).

Preferably, the light transmission device is arranged to transmit light to a detector. The detector may be of a type known in the art. Suitable detectors include charge-coupled devices (CCDs), photomultiplier tubes (PMTs), photodiodes, avalanche photodiodes and photohybrids. If more than one wavelength of light is used or is to be detected, it may be desirable for the detector means to include a demultiplexer to separate different wavelengths of light for detection.

The term "light" is understood herein to include light outside the visible part of the electromagnetic spectrum. It therefore includes, in particular, light in the far and near infrared and in the ultraviolet and vacuum ultraviolet as well as light in the visible region. The light transmission device of the invention is especially suitable for use with light in the near infrared, the visible or the u.v. regions of the electromagnetic spectrum.

Optionally, the light path from the transmission device to the detector includes one or more filters and/or one or more lenses. Several filters may be present in a filter-wheel. A stack of filters may be used. Examples of filters that may be present include high-pass, low-pass, band-pass, band- reject and interference filters. A filter serves to reduce the quantity of light of wavelengths that are not of interest that reaches the detector. The filter or filters can be chosen in dependence on the wavelength of light to be observed. For example, if a SYBR green dye is being observed, the filter or filters is/are chosen so as to favour transmission of light with a wavelength in the region of 520nm. One or more lenses serve to focus light onto the detector.

In a preferred embodiment of the light transmitting module of the invention, the module is further arranged to transmit illuminating light from a light source towards the location of the sample vessel. The light source may be of a type known in the art. Suitable light sources include Light Emitting Diodes (LEDs), lasers and conventional bulbs, including halogen bulbs. The light source may produce light of a single wavelength, of a number of single wavelengths or of a mixture of wavelengths.

Preferably, the light source is an LED. In view of the fact that a strong output from the LED is generally required, the LED is generally operated at a high power level and it may become hot. Locally, the LED may reach temperatures of over 100°C. A raised temperature of the LED interferes with its performance causing it to emit light inconsistently and to fail sooner than would be the case under operation at a lower temperature. To control those problems and to enhance the performance of the LED, it is preferred for the LED to be cooled. Preferably, the LED is cooled all the time the device is switched on.

The LED may be cooled, for example, by its being attached with good thermal contact onto a plate, for example a metal plate or a printed circuit board incorporating metal. In turn, the plate may be cooled by air being moved by a fan, a Peltier device or a heat conducting strip, for example a copper strip, in contact with a cold body. If the device is one in which the sample itself is, at times, cooled the apparatus part for cooling the LED may be the same apparatus part as cools the sample. For example, the same fan may be arranged to cool the sample and the LED.

In addition to increasing the efficiency of light transmission from the sample to the detector, the light fransmitting module also increases the efficiency of light transmission from the light source to the sample. The device may be bi-directional. That is to say, it can accept a wide-angle beam at the narrow light emitting portion and concentrate it into a narrow angle-beam at the wide light receiving portion and it can accept a narrow-angle beam at the wide light emitting portion and concentrate it into a wide angle-beam at the narrow light receiving portion. Accordingly, the device enables efficient transmission of excitation light to the sample and also enables efficient transmission of emitted light from the sample. The system gain is thus multiplicative and sensitive analysis is made possible.

Preferably, the light transmission device allows highly sensitive photometric analysis of samples. For example, the detection of a fluorochrome, for example fluorescein, is preferably possible using a device in accordance with the invention when the fluorochrome is present at a concentration of lOnM in a 20μl sample, preferably at a concentration of 3nM in a 20μl sample, more preferably at a concentration of 0.3nM in a 20 μ\ of sample, more preferably at a concentration of 0.03nM in a 20 μl of sample, even more preferably at a concentration of 3pM in a 20μl sample. Optimisation of parameters associated with the light source and light detector (for example their exact locations, the power level of the light source or the gain of the light detector) may allow yet more sensitive detection. The light transmitting module may comprise a filter positioned so as to direct light from the light source towards the light transmitting portion and the location for the sample vessel. The filter may be dichroic. Light from the sample passes through the light transmitting portion and, rather than being reflected at the dichroic filter, passes through the dichroic filter towards the detector. An alternative arrangement whereby a different type of dichroic filter is positioned so as to direct light from the sample towards the detector and light from the light source passes through the dichroic filter towards the light transmitting portion and the location for the sample vessel, is also possible.

Preferably, the dichroic filter is located proximate to the light emitting portion of the light transmitting device of the invention. The dichroic filter may be positioned at 45 degrees to the axis of the light transmitting portion. In that arrangement, the path of the light reflected at the dichroic filter changes direction by 90 degrees.

An excitation filter may be provided between the light source and the light transmission module. For example, if a dichroic filter is present, the excitation filter may be located between the light source and the dichroic filter.

The invention further provides a light transmitting block for use simultaneously with a plurality of samples. A hght transmitting block of the invention comprises a plurality of light transmitting modules of the invention. That is to say, it may comprise a plurality of light transmitting cavities. In the device of the invention there may be a single optical detection means or there may be a plurality of optical detection means, for example one for each sample vessel. The emitted light from each of the plurality of samples is focused onto its respective detection means or onto a common detection means.

If a single detection means is present, the light sources are preferably each arranged to illuminate the samples in turn for a relatively short period of time. For example, each sample may be illuminated for one second or less, preferably for 0.5s or less, more preferably for 0.1s or less, for example for from 20ms to 80ms, for example for 60ms. Typically illumination for at least 1ms is required. The light detected at the detector is generally light derived from the illumination of a particular single sample and, as the control means has information regarding which sample is illuminated at a given time, the signal can be allocated to the correct sample. The device of the invention may be arranged to receive any convenient number of sample vessels. Preferably, the device is arranged to receive from 2 to 384 sample vessels, more preferably from 3 to 96 sample vessels, still more preferably from 5 to 20 sample vessels, for example 12 sample vessels.

The light transmitting device of the invention or a light transmitting block of the invention may be incorporated in a spectrophotometric device. Such a device may comprise a means for controlling the temperature of a sample. The light transmittmg device of the invention or a light transmitting block of the invention may be incorporated in a thermal cycler apparatus.

The device is preferably operated in an automated fashion. Accordingly, the device preferably further comprises a control means for controlling each heater means. The control of the heater means preferably enables the operator to control the temperature of each sample independently.

Suitably the control means comprises a computer. The control means is preferably arranged to receive instructions from an operator regarding the required temperature of a given sample. For many molecular biology applications, including, for example, PCR, the temperature of a sample is varied with time. In that case, the control means is arranged to receive instructions from the operator regarding the required temperature profile over time for a particular sample. Preferably, the control means is arranged to control the temperature of each sample independently.

Preferably, the device of the invention further comprises a temperature sensor for determining the temperature of a sample vessel or the temperature of a sample in a sample vessel. The temperature sensor may be present in the sample vessel and be in direct physical contact with the sample. In that arrangement, the close physical contact enables a very accurate assessment of the temperature of the sample to be made. Alternatively, the temperature sensor may be present in the proximity of the sample vessel. In that arrangement, the temperature sensor is of a more simple construction and thus cheaper and simpler to manufacture and maintain. The temperature experienced by a temperature sensor in the proximity of the sample vessel may not be the same as the actual temperature of the sample. However, with information from calibration experiments, the temperature of a temperature sensor in the proximity of the sample vessel provides a measurement from which the temperature of the sample may be inferred. The temperature sensor may, for example be a platinum resistive thermometer, a thermistor, a thermocouple, for example a small semi-conductor sensor. Preferably, the device comprises an individual temperature sensor for each sample vessel. Preferably, the temperature sensor provides to the control means an input signal representing the temperature of a sample or sample vessel and the control means provides an output to the heater means dependent on the input signal and a preset required sample temperature. The temperature sensor may be arranged to act as a safety feature. For example, it may be arranged to ensure that the temperature of given sample does not exceed a pre-set temperature limit, above which the sample or a part of the apparatus may be damaged.

In one embodiment of the device of the invention, the device is arranged to receive a sample holder unit, the sample holder unit comprising a plurality of sample vessels or sample vessel receiving spaces each for receiving a sample vessel.

For user convenience an apparatus preferably comprises more than one device of the invention, each device of the invention optionally being independently operable within the apparatus. For example, an apparatus of the invention may comprise two devices of the invention. The commercial apparatus product may thus comprise two independent devices with two fans, two optical detection units, and two arrays of sample receiving spaces.

Sample vessels for use in the device of the invention are preferably tubular in shape with a closed end and an open end. The internal diameter of the tubes is preferably in the range of from 0.2 mm to 20mm. More preferably from 0.5 mm to 10mm, still more preferably from 1mm to 3mm, for example 1.5mm.

The sample vessels for use with a device of the invention are preferably cylindrical. Alternatively, they may be frustoconical, that is to say that they may have a taper, preferably becoming narrower towards the closed end.

Preferably, the tip of the sample vessel is in optical communication with the light transmitting module of the invention. The tip is preferably transparent. The tip is preferably curved or spherical in shape for a portion of its surface, for example it may be hemi-spherical. A curved tip acts as a lens and improves transmission of light from the vessel to the light transmitting module of the invention. Certain embodiments of the invention will now be described in more detail with reference to the accompanying figures in which:

Figure 1 shows a cross sectional view of a light transmitting module in accordance with the invention.

Figure 2 shows a cross sectional view of a light transmitting module of Figure 1 with a sample tube in place.

Figure 3 shows a cross sectional view of a spectrophotometric analysis unit incorporating a light transmitting module of the invention. Figure 4 shows a perspective view of a spectrophotometric analysis unit of Figure 3.

Referring to Fig. 1 of the drawings, there is indicated generally by reference numeral 1 an apparatus suitable for the analysis of a samples. The apparatus 1 comprises a light transmitting module, indicated generally as 3. The light transmitting module 3 comprises a light receiving portion 5, a light transmitting portion 6 and a light emitting portion 7. The light transmitting portion 6 of the module is formed by reflective parabolic wall 9. The light receiving portion 5 is formed by a truncation of parabolic wall 9 such that there is a flat end and an opening 11. Dichroic filter 13 is positioned at the light emitting portion 7 at 45 degrees to the axis of light transmitting portion 6. Dichroic filter 13 is characterized in that light of the emitted wavelength of interest is transmitted whilst light of the wavelength for excitation is reflected. The apparatus further comprises an LED 15 arranged to shine light on the dichroic filter and along light transmitting portion 6 and to light receiving portion 5.

In Figure 2 there is shown an apparatus 1 as depicted in Figure 1 with sample tube 17 in place in optical communication with light transmitting module 3. Sample tube 17 contains sample 19 and it is tubular in shape with a transparent tip 21.

In use, with reference to light transmitting module 3, with sample tube 17 in place, excitation wavelength light (495nm) is emitted from LED 15 and it is reflected from dichroic filter 13. The light travels through light transmitting portion 6 and light receiving portion 5 to sample tube 17. In sample tube 17, the excitation light excites fluorescent marker molecules (SYBR Green marker molecules or other fluorophore, e.g. fluorescein) present in sample 19 resulting in fluorescent light emission by the fluorescent marker molecules. The emitted light, with a wavelength of approximately 520nm, that leaves the sample tube through tip 21 passes into the light receiving portion. By virtue of the reflective internal walls of light transmitting portion 6, light emitted at a variety of angles from the tip 21 of sample tube 19 is gathered and transmitted through light transmitting portion 6 to light emitting portion 7. The light passes light emitting portion 7 and through dichroic filter 13 towards detector 23 (not shown in Figures 1 or 2).

In Figure 3 there is shown a spectrophotometric analysis unit indicated generally with reference numeral 25. Unit 25 comprises a light transmitting module 3 as described above in relation to Figures 1 and 2 and, in addition, a hght detection module indicated generally by reference numeral 27. Light detection unit 27 comprises an optical filter 29 located so as to receive light emitted from the light transmitting module 3. Light detection unit 27 further comprises two lenses 31 and 33 arranged in series to receive light from the optical filter 29. A photomultiplier tube 35 is located so as to receive light from lens 33.

In use, with a sample tube 17 in place, excitation light passes from LED 15 to the sample as described above in relation to figure 2. Light emitted from the sample passes into the light receiving portion 5, is gathered in light transmitting portion 6 and is emitted from light emitting portion 7 through dichroic filter 13 and into the light detection module 27. After passing through optical filter 29 (with light of undesired wavelengths being filtered out), the light is focused by lenses 31 and 33 onto photomultiplier tube 35.

In Figure 4 there is shown a three dimensional view of a device 37 of the type shown in Figure 3 suitable for the simultaneous analysis of a plurality of samples. Device 37 is arranged for the simultaneous analysis of 12 samples and it comprises a light transmitting module block 39 of the type shown in Figures 1 and 2 comprising 12 light transmitting modules. Device 37 further comprises a light detection unit 41 comprising lenses 31 and 33 and a photomultiplier tube 35. Lenses 31 and 33 serve to focus the light from each sample onto the same area of photomultiplier tube 35.

In use, with one or more sample vessels in place, each sample is illuminated by its respective LED light source in turn. Emitted light passes from the sample through the light transmitting module to lenses 31 and 33 which focus the light into photomultiplier tube 35. Each sample is illuminated in turn for a period of around 20ms to 60ms. The light received in the photomultiplier tube is accordingly from a single sample at a time and, as it is known which sample is illuminated at a particular time, the sample from which the emitted light is derived can be deduced.

The following example illustrates the invention further:

Example 1: Demonstration of Optical Sensitivity

In order to be successfully used in typical spectrophotometric analysis of samples, a spectrophotometric detection device needs to be capable of detecting a fluorochrome across a wide range of concentrations. For typical PCR applications the sensitivity of such a device should be such that 0.3nM to 0.5nM of fluorescein is detectable in a 20μl sample. The data presented in this example demonstrates that when used in the spectrophotometric analysis unit of Figure 3 and Figure 4, the light transmitting module of the invention allows detection of fluorescein at concentrations of about 0.03nM in 20μl of sample, a sensitivity that is more than ten times that which is required for most spectrophotometric applications.

Table 1 below shows data obtained from a fluorescein standard dilution curve duplicated across twelve sample positions (Al to A12) of a spectrophotometer utilizing light transmitting modules of the invention. Each datum in the table is based on an average of four data points, equivalent to the typical averaging time available during the extension phase of a PCR cycle.

Table 2 and table 3 show respectively the standard deviation and % coefficient of variance for each datum.

Table 4 shows the sensitivity achieved in each sample position as calculated from the data in table 1 and shows the lowest concentration of fluorescein that can be reliably detected, that is to say the concentration that is expected to give a signal that is more than two standard deviations above the background noise level. As is seen in Table 4, the present data show that sensitivity is such that fluorescein is detectable down to a concentration of from 0.02nM to 0.05nM. Table 1

Table 2

Table 3

Table 4

Claims

1. A light transmitting module for use in an analytical apparatus comprising:

- a light receiving portion for receiving light from a sample vessel, - a light emitting portion,

- a light transmitting portion located between the light receiving portion and the light emitting portion, the light fransmitting portion comprising an internally reflective cavity with an internal cross sectional width in a direction perpendicular to a line joining the light receiving and emitting portions, which increases from the end proximate to the light receiving portion to its end proximate to the light fransmitting portion, the light receiving portion being formed by an opening at the light receiving end of the cavity.

2. A light transmittmg module as claimed in claim 1 wherein the light transmitting portion comprises an internally reflective cavity with an internal cross section, in a plane containing the light receiving and emitting portions, which is curved for at least a part of its length.

3. A light transmitting module as claimed in claim 1 or claim 2 wherein the light fransmitting portion comprises an internally reflective cavity with an internal cross sectional width in the direction perpendicular to a line joining the light receiving and emitting portions, which increases from its end proximate to the light receiving portion to its end proximate to the light transmitting portion with a decreasing rate with respect to the displacement along the line joining the light receiving and emitting portions for at least a part of its length.

4. A light transmitting module as claimed in any one of claims 1 to 3 wherein the light transmitting portion comprises an internally reflective cavity with an internal cross section, in a plane containing the light receiving and emitting portions, which has the shape of a truncated parabola for at least a part of its length.

5. A light transmitting module as claimed in any one of claims 1 to 4 in which the light transmitting portion comprises an internally reflective cavity with the internal shape of a truncated paraboloid of revolution for at least a part of its length.

6. A light transmitting module as claimed in any one of claims 1 to 5 in which the light fransmitting portion comprises an internally reflective cavity comprising two or more sections of different cross sectional width shapes.

7. A light transmitting module as claimed in claim 6 in which the light transmitting portion comprises an internally reflective cavity with a section with an internal cross sectional width, in the direction perpendicular to a line joining the light receiving and emitting portions, which has a truncated conical shape at the end proximate to the light receiving portion and a section with an internal cross sectional width, in the direction perpendicular to a line joining the light receiving and emitting portions, which is parabolic at the end proximate to the light emitting portion.

8. A light transmitting module as claimed in any one of claims 1 to 7 in which the opening of the light receiving portion is of a suitable size for receiving a sample vessel.

9. A light transmitting module as claimed in any one of claims 1 to 8 in which the locating means for positioning a sample vessel is so arranged that, in use, a sample vessel is held in a position whereby an end of the sample vessel is within the cavity.

10. A light transmitting module as claimed in any one of claims 1 to 9 in which the locating means for positioning a sample vessel is so arranged that, in use, a sample vessel tube is so positioned by the locating means that its end is in optical communication with the light receiving portion.

11. A light fransmitting module as claimed in any one of claims 1 to 10 in which the locating means comprises a sample vessel locating cup to guide or hold the sample vessel in a favourable position near or in the light receiving portion.

12. A light transmitting module as claimed in any one of claims 1 to 11 in which the light transmitting module is further arranged to transmit illuminating light from a light source towards the location for a sample vessel.

13. A light transmitting module as claimed in claim 12 in which the light source is an LED.

14. A light transmitting module as claimed in claim 12 or claim 13 further comprising a filter positioned so as to direct hght from the light source towards the light transmitting portion and the location of the sample vessel.

15. A light fransmitting module as claimed in claim 14 in which the filter is dichroic.

16. A light transmitting block comprising a plurality of light fransmitting modules as claimed in any one of claims 1 to 15.

17. A spectrophotometric device comprising a light transmitting module as claimed in any one of claims 1 to 15 or a light transmitting block as claimed in claim 16.

18. A spectrophotometric device as claimed in claim 17 comprising a means for controlling the temperature of a sample.

19. A thermal cycler apparatus comprising a light fransmitting module as claimed in any one of claims 1 to 15 or a light fransmitting block as claimed in claim 16.

20. Use of a light fransmitting module as claimed in any one of claims 1 to 15 or a light transmitting block as claimed in claim 16 in a spectrophotometric analysis experiment.