WO1999008096A1

WO1999008096A1 - A detector

Info

Publication number: WO1999008096A1
Application number: PCT/GB1998/002394
Authority: WO
Inventors: Derek Adeyemi Palmer; Martin Thomas French; Si Jung Hu
Original assignee: Kalibrant Limited
Priority date: 1997-08-12
Filing date: 1998-08-10
Publication date: 1999-02-18
Also published as: AU8737898A; JP2001512827A; EP1004018A1; CA2300060A1; GB9717021D0

Abstract

The present invention relates to a fluorimeter. The fluorimeter has emitter means arranged to selectively emit light having a first wavelength predetermined to correspond to the excitation wavelength of a first fluorophore and to selectively emit light having a second wavelength predetermined to correspond to the excitation wavelength of a second fluorophore, i.e. the light sources are paired to respective fluorophores. This simplifies the analysis of the emitted fluorescent signals.

Description

A Detector

The present invention relates to a detector for determining whether or not a particular substance is present in a sample. The invention further relates to a method of operating a detector- In particular, but not exclusively, the present invention relates to a fluorimeter for detecting a fluorophore in the sample.

It is well known to use fluorescence spectroscopy and microscopy to determine the concentration of analytes in a sample by measuring the emission signal for changes in intensity or wavelength shifts. Various methods and apparatus are known for this purpose such as those described in US Patent Nos . 4,877,965 and 5,196,709.

Fluorescence detection is particularly useful in biological tests where sample size is often very small, e.g. in assaying for nucleic acids or proteins. In these circumstances it is very important to minimise interference from background radiation and techniques have been devised as described, for example, in US Patent Nos. 4,006,360, 4,341,957 and 4,791,310.

A great number of different techniques exist for performing assays and the instances in which assay techniques are used are becoming more widespread. An overview of currently used techniques is given in the article "Update on immunoassay automation" Proc. UK NEQAS Meeting 1994:1:163- 170; Wheeler, Michael J. For example, various types of immunoassays are now commonly used to test blood and other samples for a great number of different compounds. Yet, even a large hospital may not have a large number of different assay machines and so the apparatus must be capable of performing a range of different tests on different samples. The versatility of any assay technique must necessarily increase as the number of tests that can be requested on samples increases. The growing desire that assays can be performed directly in places such as Doctors Surgery's, rather than sending the samples away for analysis by a laboratory in a hospital further increases the demand for versatile machinery.

In conjunction with the growth of the assay systems, it has become necessary that any particular assay is performed ever more precisely. The importance of the accuracy of the test will be apparent, as, for example, a patient's treatment may be determined based on the result of the assay and so an inaccurate result may lead to inappropriate treatment of the patient.

The growth in assay techniques being used has led to a large number of systems on the market. Presently, most systems are semi-automated or automated systems, where after loading of the reagents and samples no further input is required from a human operator, unless a breakdown occurs .

As will be apparent from the aforementioned article by Wheeler, the majority of the devices presently on the market comprise a loading tray for loading multiple samples, for example between 20 and 100 samples, which are not necessarily of the same nature or having the same assay performed on them. There is also a reagent input tray which holds a number of reagent cartridges for the various different tests to be performed. In the machine the samples are transferred, normally by pipetting into an assay cell where the sample is combined with the necessary reagent or reagents. The assay cell is then transferred to a part of a machine where it can be held for sufficient time for the reagent and the sample to combine. Thereafter the sample cell is transferred to the detector which detects the presence of a known indicator to determine whether or not the sample contained a particular component and/or how much of that component was present in the assay. Normally a robotic arm is used for transferring the assay cell around the machine, for example from the loading area to the wash station, to the waiting area and onward to the detector. Whilst assay apparatus of this type can offer semi-automated functioning, problems do occur due to the mechanical movement of the samples. Furthermore, it is necessary to have pipettes with replaceable pipette tips or other means to ensure that one sample does not contaminate another sample. As each sample cell must be incubated with the appropriate reagents, a relatively large number of sample cells may have to be incubated at any one time and thus the size of the machine remains relatively large due to the space required for the waiting area.

Recently, it has been suggested to use flow injection technology in assay equipment. A review of this technology is given in an article by Puchades, R. et al_. "A Comprehensive Overview on the Application of Flow Injection Techniques in Immunoanalysis , Critical Reviews in Analytical Chemistry" 23 , ( 4 ) : 301-321 ( 1992 ) . The fundamental difference between this technology and the above described systems is that the samples and the reagents are combined in a fluid stream, which extends to the detector. This dispenses with the need of sample cells and generally reduces the mechanical components of the system. The present invention seeks to provide a detector which allows precise and versatile measurement of multiple analytes in a sample.

According to a first aspect of the present invention there is provided a detector comprising: emitter means arranged to selectively emit light having a first wavelength predetermined to correspond to the excitation wavelength of a first fluorophore and to selectively em t light having a second wavelength predetermined to correspond to the excitation wavelength of a second fluorophore; sensor means arranged to receive fluorescent emissions from the first fluorophore and output a first data signal characteristic thereof and fluorescent emissions from the second fluorophore and output a second data signal characteristic thereof; and analyser means adapted to analyse the first and second data signals. The present invention thus provides a detector which minimises the variables contained in the emission spectrum of the fluorophores . The emitter means provides a signal where the majority (e.g. substantially all) the energy is contained in small (e.g. lOnm) ranges around the two wavelengths. This simplifies data analysis and allows for multiple analyte detection from a single sample .

Preferably the control means has a system clock which is arranged to control the emitter means to emit light of the two wavelengths separately and in a predetermined order. This has the corresponding advantage that the fluorescent emissions from the fluorophores are separated in time further simplifying the analysis of the signal. The control means is advantageously arranged so that analyser means analyses data from the first data signal during a first period and from the second data signal during a second period, the first period corresponding to when the light of the first wavelength is being emitted and the second period corresponding to when the light of the second wavelength is being emitted. This control means is normally arranged to control the analyser means to only analyse data from at most one data signal at any given time. Preferably the first and second periods terminate prior to the termination of emissions from the respective first and second fluorophores . In this way, the lack of accuracy associated with decay of the fluorescence signal is substantially minimal.

The first data signal is preferably characteristic only of a first wavelength range and the second data signal is characteristic of only a second wavelength range. In this way, the quantity of the separate fluorophores can be determined even if the emission spectrum of the two fluorophores substantially, but not completely, overlaps as the first and second ranges can be chosen to minimise such overlap. So long as one of the wavelength ranges can be analysed for the quantity of the one of analytes, it will be possible to resolve the signal to quantify the amount of the second fluorophore from the signal. In this way, a continuous monitor of the changing concentration of fluorophores can be made if this is desired.

It is preferable if the emitter means is arranged to emit light on more than one cell where the first or second fluorophore may be present. In this way, a far more efficient analyser can be produced. Preferably the first and second fluorophores may be present in at least one test cell which comprises a flow cell.

Normally the emitter means comprises a first emitter and a second emitter. These are preferably a laser or lasers which provide a very clean light emission. Preferably each laser is a light emitting diode laser which has the advantage of small size. It is particularly advantageous as it provides a narrow band width of emitted light which can be closely paired to the fluorophore. Such a light source also provides a very intense light beam but at relatively low power consumption. Normally the laser will be operating in the range of 400-1200nm. Preferably, the laser operates in the range of substantially 600nm to 900nm as this part of the spectrum has low background radiation and less endogenous compounds emitting fluorescence that may cause interference.

Preferably, the sensor comprises an array which is arranged to receive different wavelengths at different parts of the array. In this way, the emissions signals or parts thereof from the different fluorophores can be easily separated by the sensor. The sensor may further include a polychromator arranged to disperse the fluorescent emissions of the first and second fluorophores over the respective first and second regions of the sensor which regions may overlap to a large extent. This is a particularly simple way to implement the separation in signals. Alternatively these sensor means may further include a monochromator arranged to disperse the fluorescent emissions of the first and second fluorophores on respective first and second regions.

In a preferred embodiment of the invention, the sensor comprises a charged coupled device. The detector may be used where the first fluorophore is tested for in one sample and the second fluorophore is tested for in another sample. Alternatively or additionally the first and second fluorophores may be tested for in the same sample. The emitter means may include several emitters each arranged to emit light corresponding to a particular fluorophore. The detector preferably has: a) three emitters; b) four emitters; c) five emitters; or d) six emitters; wherein each emitter is paired to a respective fluorophore. In this way, multianalyte determination from a single sample can be accomplished due to the advantageously clean signal produced by the sensor. In this way, from a single sample it should be possible to conduct sufficient tests on that sample, e.g. if a particular disease state has several indications, a single sample from an individual possibly suffering from the disease state may be tested to find out if the individual has said disease state. This will greatly simplify testing procedures and significantly reduce costs compared to the multiple sample/testing regimes presently required.

The detector according to the invention is preferably included in an assay apparatus. It is particularly advantageous when used in flow injection assay apparatus.

According to a second aspect of the invention there is provided a method of operating a detector comprising the steps of : operating emitter means arranged either to selectively emit light having a first wavelength predetermined to correspond to the excitation wavelength of a first fluorophore and/or to selectively emit light having a second wavelength predetermined to correspond to the excitation wavelength of a second fluorophore; sensor means arranged to receive fluorescent emissions from the first fluorophore and output a first data signal characteristic thereof and fluorescent emissions from the second fluorophore and output a second data signal characteristic thereof; and analyser means arranged to analyse the first and second data signals to quantify the amount of the first or second fluorophore.

Preferably the method of operating a detector comprises a method adapted to operate the detector of the first aspect of the invention.

The first fluorophore may be tested for in one set of samples, and the second fluorophore may be tested in a second set of samples. Preferably the first and second fluorophores are tested for in the same sample contained in a third set of sample. Preferably there are three, four, five, six or more analytes which are tested for in a single sample .

Normally, a detection pathway forms the fluid pathway downstream of the barrier, the detection pathway including waste-valve means arranged to direct fluid in the detection pathway to either an outlet or past the detector means which prevents the uncomplexed mixture from contaminating the flow cell.

Preferably, the fluid pathway is divided into a plurality of the said detection pathways with splitter-valve means arranged to direct each respective aliquot into a respective selected one of the detection pathways. The multiple pathways may extend from any point after the incubation loops. This improves the capacity of the apparatus as one pathway can be washed whilst another is analysing a sample.

Advantageously, one detector comprises the detection means for all the detection pathways where the same detection principle is being utilised.

For certain applications the detector may include additional detection flow-paths after the sample and reagent (s) have mixed e.g. in the incubation loops. The other flow-paths are arranged to divert the samples from the fluorescence detector so that they travel to alternative detectors which may be placed in the additional flow-paths. The alternative detectors would be selected from one or more of the detectors utilising spectrophotometric and/or electrochemical principles. In these circumstances it is possible that the analysis will be more simple than immunoassays relying on fluorescence detection and so the use of immobilised reagents and the barrier separation device may be avoided. It may be appropriate to carry out analysis on a single sample where different types of test are applied together with at least two different detectors measuring the analytical signal from the target analytes in the sample and under these conditions it may be beneficial to incorporate the additional detectors into the flow-path leading to the fluorescence detector. Many chemical tests rely on the principle of absorbance as the analytical measurement where for example a colour change occurs in a reagent in the presence of the target analyte. One variation where this approach can be applied is in the determination of endogenous enzyme or other protein levels with absorbance measurements of a colorimetric substrate indicating the relative amount of enzyme or protein in the sample. In the flow system described hereinafter the analysis could be carried out by holding the reagent/sample mixture in the incubation loops to allow for the generation of the coloured product. After this time the mixture would be released into the flow stream and directed to the flow-path that leads to the spectrophotometer where the absorbance measurement is made.

In addition to measuring endogenous proteins there is often a requirement to determine the levels of electrolytes in blood samples and this may be carried out through the use of ion selective electrodes. The operation of these devices is well known and their incorporation into the flow stream at any point would be a simple matter with electrolyte measurements made as the sample passes over the electrode .

Preferred embodiments of the present invention will now be described with reference to the accompanying drawings, in which: Fig. 1 is a block diagram of a preferred embodiment of the detector of the present invention; Fig. 2 illustrates a sequential time control of the lasers of a preferred embodiment of the present invention; Fig. 3 illustrates data acquisition of fluorescence emissions of the embodiment of Fig.2; Fig. 4 shows a sample spectrogram illustrating use of a continuous wave laser source;

Fig. 5 is a block diagram showing control of a preferred embodiment of the present invention; Fig. 6 is a block diagram showing temperature control of a preferred embodiment of the present invention. Fig. 7: is a schematic representation of an assay apparatus according to the present invention; Fig. 8: illustrates a preferred embodiment of the detector of the present invention for use in the apparatus of Fig. 7;

Fig. 9: depicts the overall construction of a flow injection apparatus incorporating the detector of the present invention. Fig. 10: is a schematic representation of another assay apparatus according to the present invention.

Fig. 1 illustrates the main units of a preferred embodiment of the multiple analyte detector 30 of the present invention. A control unit 34 is linked to a user interface via a personal computer or other device. The linkage will normally take the form of a software interface. The software interface will receive from the user or from upstream of the detector 30 the information on the interval between samples and/or the tests being performed on each of the samples. The control un t 34 operates the components of the detector 30 in accordance with the received information as described in more detail hereinafter.

In some circumstances the qualitative measurement may be simply whether or not the target suostance was detected i.e. was any or more than a certain amount of fluorescence attributable to the fluorophore present. Often however a more precise measurement of the amount of target substance will be required.

This invention relates to a detection system designed to capture fluorescence from a fluorophore containing solution or solid which may be contained in a cuvette or held on a solid surface, but preferentially is a solution passed through a flow cell 27 connected to a flow injection system.

1. UBΘ of two or more excitation wavelengths

The system provides two or more wavelength options, preferably provided by laser diodes 31. A laser diode has several benefits when compared with a conventional excitation source including: a. No requirement for a high power supply and small power consumption b. Fixed wavelength and constant light output c. Compact unit w th digital control d. Easy replacement for wavelength options.

In this detection system two or more laser diodes 31, 32, 33 (or other light source) are employed as the excitation source each of which is controlled by a laser driver to produce sequential interval pulses of light as shown in Fig. 2. The lasers are arranged to shine on a test cell 27 where fluorescent molecules (fluorophores) may be present in the test cell 27 and the resulting fluorescence emission is guided to the sensor array 35. These fluorophores are chosen because they have spectral properties, which match those for a specific laser and each laser 31, 32, 33 has at least one specific and separate fluorophore partner.

The pulse rate can be calculated and controlled through a programmable microprocessor or the control software so that the laser driver speed and the interval time of the pulses match the requirements of the sensor array so as to capture the fluorescence from each fluorophore into the appropriate data channel. The lasers may be operated sequentially or one laser may be operated (pulsed) repeatedly before another laser is operated.

2. Multichannel Time Gating Approach

The use of a pulsed laser source means that the sensor array 35 receives a pulsed light signal from the flow cell 27 that consists of the fluorescence from the fluorophores in the flow cell 27, non specific fluorescence from any other molecules present and any scattered light from the laser source. In traditional fluorimeters the non-specific or background fluorescence, along with the scattered light, is removed or greatly reduced by the use of filters or monochromators . The fluorimeter described here can make specific fluorescence measurements without the need of filters or monochromators.

It is well known that one of the major advantages of measuring fluorescence in the near infra red (NIR) region (approximately over the range 600-900nm) is that there are relatively few endogenous NIR fluorescent molecules. Therefore background interference from non-specific endogenous fluorescence is greatly reduced. Scattered light from the broad band excitation source in traditional fluorimeters is often a problem because to obtain maximum detection sensitivity it is necessary to use a wide band pass setting, often up to 20nm. This ensures the maximum amount of light falls on the sample being analysed but at the expense of much increased Rayleigh scatter, which can often swamp the specific fluorescent signal. With a laser diode source it is possible to deliver a large amount of light power in a very tight bandwidth (2-4nm) thereby minimising the influence of Rayleigh scatter to a short spectral range. Furthermore the intensity of this scatter signal varies with the inverse of the wavelength to the fourth power and so operation in the NIR region greatly diminishes the magnitude of the scattered light. In spite of the advantages of working in the NIR region some non specific signal will still be detected at the sensor, including that from co-excitation of some of the other fluorophores and so a multi channel time gating (MCTG) approach is used with the sensor array to select out the fluorescence of interest. With MCTG the fluorescent signal from each laser pulse is collected from the sensor 35 and stored in a data channel specific for that laser 31. In this way two or more emission pulses can be monitored from two or more fluorophores in the test cell by the use of two or more laser diodes if this is desired. Each laser diode 31, 32, 33 pulses in turn to sequentially excite the fluorophore with matched spectral properties. Each laser pulse has its own specific interval time and this is shared by the associated emission pulse. It is therefore possible to time gate the emission pulse generated by a specific laser into a selected channel for data acquisition and feedback the data to regulate the sensor array. In this way fluorescence measurements can be made as follows. The overall timing of all events is controlled by the system clock (Fig. 3a), at a given time frequency laser 1 fires a light pulse of defined time and intensity (Fig. 3b). Fluorophore 1 responds with fluorescence over a similar time period and at an intensity dependent on its concentration (Fig. 3c). The other laser/fluorophore pairs operate in sequence in a similar fashion (Figs. 3d-3h) and the emission pulses are collected from the sensor into their respective data channels (Fig. 3i). In order to minimise the influence of variable fluorescence decay the data collection ceases fractionally before the laser pulse finishes . In addition to using the lasers in sequential pulsed mode it is possible to drive them in constant wave mode, where each laser operates continuously and illuminates the sample for the whole time it is being measured. If two or more lasers are operated in this way and paired with suitable fluorophores so that there is a recognisable fluorescent response for each laser source then multiple analyte measurements can be made. Figure 4 shows a stylised representation of the excitation of three fluorophores where peak 1, peak 2 and peak 3 are the fluorophores emission after excitation by the fixed wavelength lasers 1, 2 and 3 respectively. The scattered laser light has been omitted for clarity. The mixed emission signal can now be dealt with in two ways, firstly as described below in the evaluation of pulsed emission signals, where multivariate analysis software can be used to generate the pure emission spectra. Alternatively the sensor can be programmed to collect data over a narrow wavelength range specific for each label i.e. λ_{ι r} Δλ₂ , and Λλ₃, thereby avoiding the scattered light and the intensity of each peak indicates the strength of fluorescence in the solution which can then be used as a quantitive measure.

Use of constant wave lasers and two dimensional CCD detectors and spectrographs to collect the fluorescence spectra from two or more laser diodes can provide an alternative to pulsing the laser. Time gating the detection, i.e. spectral separation, could be achieved spatially at the input of the spectrograph using optical fibre coupling of the fluorescence signals taken from different parts of the flow cell. Liquid light guides offer an alternative approach to coupling because of their much higher throughput compared to bundles. Although it is preferred to use separate light sources, it is possible to use filters designed to allow passage only to the wavelengths of the emissions from the desired fluorophores. A variable wavelength filter can be used to modify the excitation light, and this can also be used on the emitted fluorescence. However, filters, variable or fixed, are not preferred as more energy is used to cause the same degree of signal compared to the separate light sources .

3. Wavelength dispersion onto the sensor array device for data acquisition

The fluorescence emission from the flow cell is over a wide spectral range and so a device is required to disperse this spectrum over the sensor array. A preferred option is where the emitted pulse of fluorescence is focused onto a polychromator and then dispersed across the sensor array 35 to reveal the emission spectrum. Alternatively a monochromator is used to rapidly scan the emission beam in order to generate the emission spectrum at the sensor. Because the spectral properties of each fluorophore is known a pure reference sample is first used to wavelength calibrate the sensor array for each laser/fluorophore pair and the spectral data stored in the relevant data channel in the computer. When sample measurements are taken the emission profile gathered in the data channel can be examined against the expected profile and multivariate analysis software used to remove the non-specific components, including that of spectral overlap from co- excited fluorophores. The sensor array 35 can be programmed to collect data over a narrow wavelength range specific for each label and the intensity of each signal indicates the strength of fluorescence in the solution, which can then be used as a quantitative measure.

For this application it is preferred to use a highly sensitive and fast speed sensor to capture the fluorescence from the solution in the flow cell. A suitable sensor would be a CCD photo-electric device which operates in a rapid self scan mode so that there is quantitative capture of the emission photons with excellent signal to noise characteristics, due mainly to very low dark current in the device. The CCD has its own driver assembly through which the scan time can be set to match the speed of the emission pulse. A programmable microprocessor and an interface digitally control the data acquisition from the sensor array.

Alternatively, it would be possible to use a tunable filter, such as an acousto-optic tunable filter, as the wavelength selection element in place of a spectrograph.

A single detector element such as a photomultiplier tube or photo detector could replace the CCD array. In this case it would be normal for the light source to pulse and fluorescence spectra to be accumulated in sequence by means of switching the detector output between the different data channels of a data storage device with the system clock synchronised to the voltage ramp (e.g. an oscilloscope, transient digitiser, multichannel sealer, etc.) driving the tunable filter. Suitable tunable filters are available from Brimrose Corp. Such an apparatus would cost somewhat less to produce than a CCD-based system. Alternatively, the filter could be switched.

4. Thermoelectric Cooling Unit

The wavelength of the light emitted from a laser diode varies with temperature, so to ensure a consistent excitation wavelength the operation temperature of the laser diode is controlled by a fan or a thermoelectric cooling unit. In addition, the sensitivity of the sensor array varies with temperature due to changes in dark current and so much lower detection limits are possible if a consistent low operating temperature is maintained. Similarly fluorescence emission is temperature dependent and so a temperature controlled flow cell is important.

A single thermoelectric cooling unit is used to control the temperature in the laser, sensor array and flow cell. This unit is a reversible solid-state heat pump and a precision controller for thermoelectric temperature stabilisation. The cooling unit is constructed from a doped semiconductor, bismuth telluride. The unit is controlled via the software package and can be set at a range of temperatures at each control point for example as follows: Laser diode -25°C - + 15°C ± 0 . 5°C Sensor array -50^αC - + 50°C ± 0 . 1°C Flow cell + 10°C - + 40°C ± 0 . 1°C

5. PC Window with Operational Package

The detection system including the laser driver, flow cell holder and sensor array is connected to a microprocessor (interface) and then linked to a PC based operational software package. The package operates the laser diode driver, the monochromator driver (if specified), the thermoelectric cooling unit and the sensor array, feeds back the signals to each control point, and also automatically acquires the data.

The detector of the present invention may be used to analyse test samples on a solid support, in a cuvette or otherwise. However, the present detector is particularly advantageous when used as the detector in a flow analysis system as described in PCT application No. PCT/GB97/00334 filed on 6th February, 1997 and claiming a priority date of 9th February, 1996, the contents of which are hereby incorporated by reference, in particular in relation to the types of flow analysis system which may be adopted.

The present invention provides a detector which allows a much greater number of alternative tests due to the multiple light sources compared to single source devices. For example, in a flow analysis system where a stream of separate samples flow through the system, the present invention provides a detector that allows multiple tests on each sample via different fluorophores. The greater number of fluorophores also allows the analyser to form a device which can perform nearly all the tests necessary for e.g. a surgery and thus speed up the time for testing and make it economical for a far larger number of tests to be conducted as it will not be necessary to send most tests to specialist laboratories having many different analysers for the various different tests that are required. This is particularly apparent when used in a flow analysis system.

The detector of the present invention will now be described in the preferred instrumentation of a flow analysis system as described in PCT/GB97/00334 which is hereby incorporated by reference.

Figure 7 shows a flow injection immunoassay analyser as an integrated system which is a particularly preferred embodiment of the invention. The system operates using the principles of flow injection analysis, that is a continuous stream of liquid is used to transport discrete volumes of sample or reagents that are injected into the stream. These materials can then be brought into contact with one another or with other materials that may be in solution or fixed to a surface so that they interact in a way that can be measured and thus the flow injection process is directly analogous to the manipulations that take place in traditional immunoassays using microtiter plates or tubes except that injection loops or syringes and precise control of flow rate replace the use of pipettes, washers and shakers .

A carrier buffer stream is generated from run buffer 12. A plurality of samples are held in a sample processing unit 14 which also prepares each sample for analysis. Analysis for a particular target molecule (a product) takes place by injecting a known volume of a sample that possibly contains the product, into the carrier buffer stream and mixing it with reagents from a reagent cartridge 15.

The sample processor unit 14 has the capacity to hold approximately 100 samples and a normal variety of tube sizes. The unit 14 is capable of carrying out accurate and precise pipetting to generate a sample dilution as required. This may be in a traditional manner with appropriate volumes transferred to a separate tube on the processor bed or by using the flow system where a fixed volume of sample and a variable volume of diluent (or vice versa) are merged in a mixing coil before a fixed volume is taken for analysis. The unit 14 may employ robot arm (not shown) carrying a sample probe (not shown) . The robot arm would normally be capable of movement in three planes and the probe can be washed between sample manipulations at an on board wash station. Samples can be loaded onto the processor unit 14, preferably in their original tubes, of varying dimensions, in pre-prepared racks of tubes or in pre-prepared microtiter plates. Sample identification and tracking is made possible through bar codes which may be placed on the individual tubes, or the tube racks or the microtiter plates and the bar codes are read by the on board bar code reader, though other tracking systems can be used.

The flow system of the instrument consists of transmission tubing 10 made from chemically and biologically inert material such as commercially available nylon or PEEK with an inner diameter of typically 0.8mm, although this may change to suit the circumstances. The pumping system (not shown) consists of several low pressure pumps, most likely peristaltic pumps which may be of differing size, sophistication and performance and will be capable of delivering a highly reliable flow rate. A central pump will be used to move the carrier buffer, samples and reagents through the system whilst other pumps which are likely to be less sophisticated, will be used to carry out other manipulations, such as reagent transport, barrier washing and conjugate elution. The operation of each pump will be controlled by the central computer (not shown) to ensure optimum performance and effective synchronisation. The computer will also have control over the many automated switching valves (described in more detail hereinafter) , which at the appropriate times direct samples or reagents into or out of the main carrier stream. These valves may be electronically or pneumatically operated and must be extremely reliable and robust as they will be used many times in any working day. They will be very simple in design needing only to switch the liquid flow between one of two channels or limited number of channels. The valves must have chemically and biologically inert surfaces where they come into contact with the liquid stream.

Generally, the reagents required for each assay are specific for the analyte of interest, however the same principles are applied in each case and only two components are normally required. It is preferred for all assays to utilise micro-beads of a defined diameter and with the property of neutral density so that they remain in suspension and it is likely that the beads will be made of a cellulose material with low non-specific binding properties although other suitable materials may be preferred. The surface of these beads is coated, probably through covalent conjugation, although other procedures such as adsorption may be possible with a ligand binder material such as an antibody or other compound that specifically binds only the analyte of interest in the assay. In a more sophisticated assay format, it is possible to coat two or more ligand binders with different specificity's onto the bead surface and this allows for multianalyte determination from the same sample. The second reagent is a labelled material which may be an analogue of the analyte of interest or binder with specificity for the analyte depending on the assay format required. The label is often a fluorophore with spectral characteristics that allow it to be detected in the near infra red region of the electro-magnetic spectrum, however other labels such as liposomes, enzymes and chemiluminescent materials are also possible.

The fixed volumes of the reagents and the sample are mixed together in a mixing coil 17 and allowed to incubate together for a fixed time. The incubation is preferably accomplished by removing the aliquot out of the main flow strea and into one of the incubation loops 19 but may take place in the main flow in some cases, e.g. when the incubation time is short. The access to the incubation loops 19 is controlled by valve means 20. The loop 19 is made from a fixed length of transmission tubing of an appropriate internal diameter, however the overall volume should be carefully chosen to ensure precise replication of incubation conditions. During incubation any of the product which is contained in the sample should interact with the reagent to form a complex which is bound to a microbead. The complex bound to the microbead must include a detectable moiety. After a given time the computer switches the aliquot of mixture back into the main stream if it has been diverted therefrom and it is carried down to the membrane barrier 22 where the micro beads are retained whilst all other materials flow through and are passed to waste. The barrier 22 consists of a porous membrane made from a chemically and biologically inert material such as nylon and the barrier 22 is sized and arranged to prevent all beads from passing through to the flow cell. The pore structure of the membrane is governed by the size of the micro beads, however it is important that the membrane has a low non-specific binding of excess reagents and that substantially all (eg>95%) of the beads are retained. The flow-path is then washed by a period of flow with carrier buffer to wash off any unbounα reagents and possibly the flow rate is raised during this period, so the barrier 22 should have good flow properties. Subsequently, the various valves are switched in synchronisation to divert the main buffer flow from the barrier 22 whilst introducing an elution buffer from vessel 24 to flow through the barrier 22. The turning of the switch can be gauged from monitoring the unbound reagent flowing through the flow cell 27 to waste. This releases the label (detectable moiety) from the microbead to flow through the barrier 22. The flow is now directed to a flow cell 27 for measurement downstream. The flow cell 27 is likely to be a quartz silica cylinder, although other materials and shapes may be preferred, with a total volume unlikely to exceed 200μl and which is normally illuminated by a light source and monitored by a detector 30 as explained in more detail below. Following elution further valve switching allows the membrane to be back flushed with an appropriate buffer from vessel 28 which removes the beads to waste through valve 25 and cleans the membrane ready for the next sample aliquot .

The detector 30 is shown in greater detail in Figure 2 and consists of a laser diode module 31, 32, 33, a light path of mirrors and beam splitters 38 to 42, identical duplicate flow cells 27 and a single detector 35 as described hereinbefore. Although two flow cells 27 are shown, an instrument employing the detector of the present invention may include one, two, three or more flow cells depending upon the capacity required for the machine. If there is only one flow cell then the valving upstream of the detector can be simplified as can the controlling software which can reduce the cost of the apparatus for situations where only a low capacity apparatus is required. On the other hand a greater number of flow cells may increase the capacity of the apparatus, but additional flow-paths through the apparatus may be required to fully exploit the greater detector capacity.

In the illustrated embodiment there are two detector flow- pathways. This is particularly advantageous as one of the two pathways can be analysing a sample whilst the other pathway is being washed from wash buffer 28 via one of the wash valves 29. This greatly increases the number of samples which can be analysed in a given time period. This design is particularly advantageous when used in conjunction with the detector of the present invention which allows for the two (or more) flow cells 27 to be analysed from a single radiation generator/emitter and so the increased capacity is provided at little extra cost.

The choice of lasers will be very much dependent on the available fluorophores since the lasing wavelength and optimum fluorophore excitation wavelength need to be well matched. The rate of development in the field of solid state lasers and appropriate fluorophores is rapid and the final choice for these components cannot be made now. However it is likely that the lasers should have at least a 1 milliwatt output (preferable 10 milliwatt) and operate above 400nm, whilst the fluorophores should be water soluble if an aqueous solution is used, stable in solution, unaffected by pH changes, emit their fluorescence above 600nm and have the general properties required of a good fluorophore. Various fluorophores are known in the art and more are being developed. For example see the review article by Fabian, J., et al Chemical Reviews, 1992, 92, 1197-1226 which gives details of a number of different types of available fluorophores with some spectral details. Similarly, various suitable light sources are known (i.e. laser diode modules) or are being developed. The skilled person can pair light sources with respective fluorphores depending upon the particular requirements for the pair required at the time.

The laser module 31, 32, 33 can contain more than one laser, each of which can in turn be switched into the light path whilst at the same time collecting data from the detector into a separate channel. Computer control of this switching allows the potential for multi-label detection by operating 2 or more carefully chosen lasers of different excitation wavelength in a rapid pulse mode, one after the other, and monitoring the associated emission from its paired fluorophore. In this way specific measurements can be made in mixtures of fluorophores and this leads to the possibility of multi analyte determinations from the same elution peak. If pairs or more of analytes are measured in this way the throughput of the instrument is greatly increased and the usage of sample greatly reduced since mixed specificity beads can be used for the sample capture. The resulting signal is plotted as a peak and the calculated area used to determine the concentration of the sample from a curve generated from standard solutions.

The system operates in random access mode but has the inbuilt capacity for immediate analysis of emergency samples, which are placed in separate rack on the autosampler. The timing and scheduling of operations are precisely controlled by the software which is icon driven, intuitive to use and which operates in a Windows™ environment. The software is designed to run on a notebook type computer which can be closed and stored in the base of the instrument when not required. Communication with the instrument is bi-directional, allowing feedback from off- scale results to initiate appropriate dilution and sample re-analysis. The instrument and software are fully configured for operation within a Laboratory Information Management System (LIMS) environment, including quality control monitoring of assay controls and reagent cartridge performance.

The analyser is designed to be capable of measuring greater than 20 clinically important substances, each of which will have a dedicated cartridge of reagents, capable of approximately 200 analyses held in the reagent carousel on board the instrument. The cartridge design will ensure that reagents can be stirred if required and kept at constant temperature through control of either the carousel compartment or the cartridge itself. Each cartridge holds information about itself, possibly on a bar code.

Of course, the detector 30 can be used in or with other assay/analysis systems where its advantages can also be utilised. Furthermore, other types of detectors could be used other than lasers that still operate with the advantageous single detector and/or source but have one or several flow cells for detection of the detectable moiety.

Claims

Clai s :

1 . A detector comprising : emitter means arranged to selectively emit light having a first wavelength predetermined to correspond to the excitation wavelength of a first fluorophore and to selectively emit light having a second wavelength predetermined to correspond to the excitation wavelength of a second fluorophore; sensor means arranged to receive fluorescent emissions from the first fluorophore and output a first data signal characteristic thereof and fluorescent emissions from the second fluorophore and output a second data signal characteristic thereof; and analyser means arranged to analyse the first and second data signals.

2. The detector according to claim 1, further including control means having a system clock and being arranged to control the emitter means to emit light of the two wavelengths separately and in a predetermined order.

3. The detector of claim 1 or claim 2, wherein the control means is arranged so that the analyser means analyses data from the first data signal during a first period and from the second data signal during a second period, the first period corresponding to when the light having a first wavelength is being emitted and the second period corresponding to when the light having the second wavelength is being emitted.

4. The detector of claim 3 , wherein the control means is arranged to control the analyser means to only analyse data from at most one of the data signals at any given time.

5. The detector of claim 3 or claim 4, wherein the said first and second periods terminate prior to the termination of emissions from the respective first and second fluorophores .

6. The detector of any one of the preceding claims, wherein the first data signal is characteristic of only a first wavelength range and the second data signal is characteristic of only a second wavelength range.

7. The detector according to any one of the preceding claims, wherein the emitter means is arranged to emit light on more than one test cell where the first or second fluorophore may be present.

8. The detector according to any one of the preceding claims, wherein the first and second fluorophores may be present in at least one test cell which comprises a flow cell .

9. The detector according to any one of the preceding claims, wherein the sensor means is arranged to receive fluorescent emissions from more than one test cell where the first or second fluorophore may be present.

10. The detector according to any one of the preceding claims, wherein the emitter means comprises a first emitter and a second emitter which may be a laser.

11. The detector according to claim 10, wherein the laser is a light emitting diode laser.

12. The detector according to claim 10 or claim 11, wherein the laser operates substantially in the range of 400nm to 1200nm.

13. The detector according to claim 12, wherein the laser operates substantially in the range of 600nm to 900nm.

14. The detector according to any one of the preceding claims, wherein the sensor comprises an array which is arranged to receive different wavelengths at different parts of the array.

15. The detector according to claim 14, wherein the sensor means further includes a polychromator arranged to disperse the fluorescent emissions of the first and second fluorophores on respective first and second regions of the sensor.

16. The detector according to claim 14, wherein the sensor means further includes a monochromator arranged to disperse the fluorescent emissions of the first and second fluorophores on respective first and second regions.

17. The detector according to any one of the preceding claims, wherein the sensor comprises a charge coupled device.

18. The detector according to any one of the preceding claims, wherein the first fluorophore is tested for in a one sample and the second fluorophore is tested for in another sample.

19. The detector according to any one of the preceding claims wherein, the first and second fluorophores are tested for in the same sample.

20. The detector according to any one of the preceding claims, wherein the emitter means includes a plurality of emitters each arranged to emit light corresponding to a particular fluorophore.

21. The detector according to claim 20, wherein the detector has: a) three emitters; b) four emitters; c) five emitters; d) six emitters; or e) more than six emitters wherein each emitter is paired to a respective fluorophore.

22. An assay apparatus incorporating the detector according to any one of the preceding claims.

23. The assay apparatus according to claim 22, comprising a flow injection assay apparatus.

24. The assay apparatus according to claim 22 or claim 23, further including at least one further detector arranged to analyse a sample where the detector is not according to any one of claims 1 to 21.

25. The assay apparatus according to claim 24, wherein the or each further detector is selected from the group consisting of: a spectrophoto-metric detectors; electrochemical detectors; electrolyte detectors such as ion selective electrodes .

26. The assay apparatus according to claim 24 or claim 25, wherein each detector is associated with a separate test cell.

27. The assay apparatus according to claim 26, wherein each test cell is in a separate flow-path.

28. A method of operating a detector, comprising the steps of: operating emitter means arranged either to selectively emit light having a first wavelength predetermined to correspond to the excitation wavelength of a first fluorophore and/or to selectively emit light having a second wavelength predetermined to correspond to the excitation wavelength of a second fluorophore; sensor means arranged to receive fluorescent emissions from the first fluorophore and output a first data signal characteristic thereof and fluorescent emissions from the second fluorophore and output a second data signal characteristic thereof; and analyser means arranged to analyse the first and second data signals to quantify the amount of the first or second fluorophore.

29. The method of claim 28 adapted to operate the detector of any one of claims 1 to 21.

30. The method of claim 24 or claim 25, wherein the first fluorophore is tested for in a one set of samples, the second fluorophore is tested for in a second set of samples and the first and second fluorophores are tested for in the same sample in a third set of samples.

31. The method according to any one of claims 28 to 30, wherein the method includes the steps of detecting more than one analyte in different types of detector.

32. An assay method as hereinbefore described with reference to, and/or as illustrated by, the accompanying drawings.

33. A detector apparatus as hereinbefore described with reference to, and/or as illustrated by, the accompanying drawings .

34. An assay apparatus as hereinbefore described with reference to, and/or as illustrated by, the accompanying drawings.

AMENDED CLAIMS

[received by the International Bureau on 12 January 1998 (12.01.98); original claims land 28 amended; remaining claims unchanged ( 2 pages )]

1. A detector comprising: emitter means arranged to selectively emit a first light pulse having a first wavelength predetermined to correspond to the excitation wavelength of a first fluorophore and to selectively emit a second light pulse having a second wavelength predetermined to correspond to the excitation wavelength of a second fluorophore, said emitter means being arranged to leave an interval between pulses of light; sensor means arranged to receive fluorescent emissions from the first fluorophore and output a first data signal characteristic thereof and fluorescent emissions from the second fluorophore and output a second data signal characteristic thereof; and analyser means arranged to analyse the first and second data signals.

3. The detector of claim 1 or claim 2 , wherein the control means is arranged so that the analyser means analyses data from the first data signal during a first period and from the second data signal during a second period, the first period corresponding to when the light having a first wavelength is being emitted and the second period corresponding to when the light having the second wavelength is being emitted.

4. The detector of claim 3, wherein the control means is arranged to control the analyser means to only analyse data from at most one of the data signals at any given time. — oo —

wherein each detector is associated with a separate test cell.

28. A method of operating a detector, comprising the steps of: operating emitter means arranged either to selectively emit a first light pulse having a first wavelength predetermined to correspond to the excitation wavelength of a first fluorophore and/or to selectively emit a second light pulse having a second wavelength predetermined to correspond to the excitation wavelength of a second fluorophore, said emitter means being arranged to leave an interval between pulses of light; sensor means arranged to receive fluorescent emissions from the first fluorophore and output a first data signal characteristic thereof and fluorescent emissions from the second fluorophore and output a second data signal characteristic thereof; and analyser means arranged to analyse the first and second data signals to quantify the amount of the first or second fluorophore.

31. The method according to any one of claims 28 to 30, wherein the method includes the steps of detecting more