GB2258728A

GB2258728A - A fluorimeter

Info

Publication number: GB2258728A
Application number: GB9220976A
Authority: GB
Inventors: James N Miller; Nichola J Seare; Tony E Edmonds
Original assignee: Loughborough Consultants Ltd
Current assignee: Loughborough Consultants Ltd
Priority date: 1988-12-06
Filing date: 1992-10-06
Publication date: 1993-02-17
Anticipated expiration: 2012-10-06
Also published as: GB9220976D0; GB2258728B

Abstract

The fluorimeter includes means (2) for receiving a sample of a fluorescont material having a plurality of separate areas (4, 5) for receiving the sample. A source of electromagnetic radiation (11) is arranged to illuminate each of the sample areas (4, 5). A detector (16) detects electromagnetic radiation emitted by a sample in any one of the sample areas (4, 5). The components are linked by waveguides 6-9. <IMAGE>

Description

A fluorimeter This invention relates to a fluorimeter, and to a method of carrying out a fluorescent assay of a plurality of analytes. The invention is concerned particularly, but not exclusively, with fluorescent immunoassays.

Many types of fluorimeter are known which may be used for carrying out a fluorescent assay in respect of only one analyte at a time. Prior art fluorimeters are generally expensive and can be complicated to use and are not therefore suitable for use by an inexperienced operator, and may require complicated modification when it is desired to change between solid and liquid samples.

According to the present invention, there is provided a fluorimeter including means for receiving a sample of a fluorescent material, a source of electromagnetic radiation arranged to illuminate a sample in the receiving means, a detector for detecting electromagnetic radiation emitted from the sample, at least one filter device having a plurality of filter portions each for interposition in the path of the incident or the emitted radiation to select a respective wavelength of electromagnetic radiation which is allowed to pass through the device, motor drive means for repeatedly positioning selected ones of the filter portions in turn in the path Xf the electromagnetic radiation.

The fluorimeter is capable of being used to carry out a fluorescent assay simultaneously in respect of a plurality of analytes. By selecting the wavelength of the incident and/or the detected radiation, it is possible to detect separately each of the fluorescent dyes present in the sample. For example, if the wavelength of the incident radiation is selected to correspond to the excitation wavelength of just one of the dyes present in the sample, only that dye will be excited by the radiation and will subsequently emit radiation which will be detected. if, on the other hand, the wavelength of the detected radiation is selected to correspond to te emission wavelength of only one of the fluorescent dyes, only radiation emitted by that dye will be detected, even though two or more fluorescent dyes may be emitting radiation. if the wavelength of the incident and/or the detected radiation is repeatedly selected to correspond in turn to the excitation and/or the emission wavelengths respectively of each of the fluorescent dyes present in the sample, each of those dyes may be repeatedly detected in turn, allowing a fluorescent assay to be carried out simultaneously in respect of a plurality of analytes.

The fluorimeter may include a first filter device for selecting the wavelength of the incident radiation and a second filter device for selecting the wavelength of the detected radiation, the two filter devices being arranged to select the different wavelengths of the incident and detected radiation in synchronisation with one another.

Selecting the wavelengths of the incident radiation and the detected radiation simultaneously to correspond to the excitation and emission wavelengths respectively of one of the dyes present in the sample allows the dyes to be distinguished even more clearly from one another.

Each filter device may comprise a rotatable filter wheel having a plurality of filter port cons rate around its circumference. A measuring device ray be included for determining the angular of the or each filter wheel and therefore @ filter portions is interposed in the path of the incident and/or the detected radiation.

The fluorimeter prederably includes an excitation waveguide for transmitting incident electromagnetic radiation from the source to the receiving means arc preferably also an emission waveguide for transmitting emitted radiation from the receiving means to the detector. The receiving means may have two separate areas for receiving a sample of a fluorescent material, excitation waveguides being provided for transmitting incident radiation from the source to each of the sample areas and emission waveguides being prov @ded Cor transmitting emitted radiation from each of the san e areas to the detector.The waveguides may b arrance to transmit radiation in the visible or near in@@-@@@ @@@'s of the electromagnetic spectrum, thereby enab inexpensive light source, such as a quartz halogen bulb, to be used.

An infra-red-absorbing filter may be provided in the path of the incident radiation.

A polarising filter may be provided in the path of the incident and/or the emitted radiation, and a motor may be included for rotatably driving the polarising filter to rotate its axis of polarisation, enabling a fluorescent polari-sation assay to be carried out.

The detector may include a photomultiplier tube.

rar code render may be provided in the receiving cans, -or receiving information concerning the sample placed in the receiving means, thereby enabling the fluorimeter to operate automatically.

gleans for measuring the intensity of the incident radiation may be included.

The fluorimeter may include means for analysing signals from the detector, and the analysing means may include a plurality of memory devices, each arranged to receive and store in turn a signal corresponding to the output of the detector when each of the filter portions is positioned in turn in the path of the electromagnetic adiation. The memory devices may be arranged to store a signal in analogue and digital form (for example, they may include sample-and-hold amplifiers), of in a digital @@@@.

The analysing means may further include a processor.

The present invention further provides a fluorimeter including means for receiving a sample of a fluorescent material having a plurality of separate areas for receiving the sample, a source of electromagnetic radiation arranged to illuminate each of the sample areas and a detector for detecting electromagnetic radiation emitted by a sample in any one of the sample areas.

The fluorimeter may therefore be arranged to carry out a fluorescent assay using either a liquid sample or a solid sample, without the need to alter the configuration or the operation of the device.

The present invention further provides a fluorimeter including means for receiving a sample of a fluorescent material, a source of electromagnetic radiation for illuminating a sample in the receiving means, a detector for detecting electromagnetic radiation emitted by the sample, and an excitation waveguide for transmitting incident radiation from the source to the receiving means. An emission waveguide for transmitting emitted radiation from the receiving means to the detector is preferably also provided.

The use of waveguides for transmitting the incident and emitted radiation is particularly advantageous and enables radiation to be transmitted between the different components or the device without the need for complicated and expensive optical components which in known fluori meters have to be positioned very precisely in order to function effectively. in certain applications, especially ones employing only a single sample area, an excitation waveguide without an emission waveguide may be employed.

According to the present invention, there is yet further provided a method of carrying out a fluorescent assay of a plurality of analytes, in which each analyte is chemically labelled with a different fluorescent dye, the analytes are illuminated with incident electromagnetic radiation and emitted electromagnetic radiatn is repeatedly detected from each of the fluoresoert dyes in turn.

A plurality of different optical filters 22y be repeatedly interposed in turn in the path of the incicent radiation, each to select a different wavelength of incident radiation, the selected incident radiation wavelengths corresponding to the excitation wavelengths of the different fluorescent dyes.

Alternatively, or additionally, a plurality of different optical filters may be repeatedly interposed in turn in the path of the emitted radiation, each to select a different wavelength of detected radiation, the selected detected wavelengths corresponding to the emission wavelengths of the different fluorescent dyes.

in the case where optical filters are interposec in the paths of both the incident and the detected radiation.

the different optical filters may be interpose lr.

synchronisation with one another, the wavelengths of the incident and the detected radiation at any one time corresponding to the excitation and emission wavelengths respectively of a particular fluorescent dye.

By way of example, an embodiment of the invention will be described with reference to the accompanying drawings, of which: Figure 1 shows, diagrammatically, the major components of a fluorimeter device, and Figure 2 is @@@ck diagram of an electronic cireur arrargement of the device.

As shown @n Figure 1, the fluorimeter includes a light source , means or receiving a sample fluorescent material 2 and a detector 3 for detecting light emitted from the fluorescent sample. The receiving means 2 has two separate sample areas 4, 5, the first 4 of which is designed for use when carrying out fluorescent assays of solid samples, and the second 5 for use with liquids.

Light is transmitted between the various components of the fluorimeter by four optical waveguides: to excitation waveguides t, , transmit incident light from the source ' to the receiving means 2, and two emission waveguides 8, 9 transmit light emitted by a fluorescent sample to the detector 3. The two excitation waveguides 6, 7 extend @@e@ @@@ source @ to the receiving @e@ns @, one to the first sample area 4 and the other to the second sample area 5. Both sample areas 4, 5 are thereby illuminated with incident light from the source 1.The two emission waveguides 8, 9 extend from the receiving means 2, one from each of the two sample areas 4, 5, to the light detector 3. Light emitted by a fluorescent sample in either of the sample areas 4, 5 is thereby transmitted to the detector 3. The above arrangement enables the fluorimeter to be used to carry out a fluorescent assay of either a solid sample, placed in the first sample area 4, or a liquid sample, placed in the second sample area 5, without making any changes to the configuration or operation of the fluorimeter.

The light source 1 comprises a quartz halogen bulb 10 having a power rating of 50 to 150w, connected to a 10 to 15 Volt power supply 11. A lens 12 located in front of the bulb 10 focusses light on to the near ends of the two excitation waveguides 6, 7, which transmit the light to the two sample areas 4, 5. An infra-red absorbing filter 13 is located between the lens 12 and the near ends of the excitation waveguides 6, 7 to prevent undesirable heating of a sample in either of the receiving areas 4, 5. The excitation waveguides 6, 7 are designed to transmit radiation in the visible or near 5nfra-red parts of the electro-magnetic spectrum. A motor-driven fan 14 provides forced air cooling for the bulb 10.

The light detector 3 comprises a photomultiplier tube 15, having high-tension power supply 16, and arranged to receive light transmitted from the sample areas 4, 5 by the emission waveguides 8, 9. The photomultiplier tube 15 has an output 17 for delivering a signal to a computer (not shown) which analyses the signal. A photodiode 18 is provided adjacent the light source 1 to measure the intensity of the incident light.

Located between the light source 1 and the near ends of the excitation waveguides 6, 7 is a motor-driven filter wheel 19 having a plurality of optical filters mounted around its perimeter. Each of the filters transmits light of a different wavelength so that, as the filter wheel 19 rotates, a fluorescent sample in one of the sample areas 4, 5 is illuminated sequentially by incident light of various different wavelengths, as selected by the filters on the filter wheel. The filter wheel 19 may have two, three or more optical filters mounted upon it, and it is rotated sufficiently fast for the sample in the receiving means 2 to receive light through each of the different filters between 50 and 100 times per second.

A second motor-driven filter wheel 20 is located between the detector 3 and the near ends of the emission waveguides 8, 9 and operates in the same way to select the wavelength of the fluorescent light detected by the detector. The two filter wheels 19, 20 are rotated in synchronisation with one another so that whenever an o particular optical filter is located in front of the light source 1 to select the wavelength of the incident light, a corresponding optical filter is located between the emission waveguides and the detector 3 to select the wavelength of the detected light.Each optical filter on the first filter wheel 19 is therefore matched to a respective filter on the second filter wheel 20, and the filters of each matched pair are chosen to correspond respectively to the optimum excitation and fluorescence wavelengths of a particular fluorescent dye. The fluorimeter is therefore arranged to detect, sequentially, the presence or a plurality of different fluorescent dyes in a sample, each dye being detected in turn 50 to 100 times per second. While a rate of detecting to 50 to 100 times per second is preferred, a slower rate even as lo as once per second might be employed.A phase-measuring device (not shown) is connected to the filter wheels 19, 20 to provide a signal concerning the angular position of the wheels at any particular time, thereby enabling a computer connected to the light detector 3 to determine which fluorescent dye is being detected.

A motor-controlled rotatable polarising filter 21 is provided adjacent each sample area , 5, between the sample area and the associated waveguides 6, 7, 8, 9.

The polarising filters 21 crit fluorescence polarisation assays to be carried out, and rotation of the filters may be controlled by a microprocessor (the microprocessor 27 referred to below).

A bar code reader (not shown) is provided in each of the sample areas 4, 5 for reading a bar code attached to a sample, thereby enabling the fluorimeter to obtain automatically information about the sample as it is placed in the receiving means 2. The information contained in the bar code may identify, for example, the analytes which are to be detected and the nature of the fluorescent dyes that have been used to label an immunoassay system.

Figure 2 is a d acram of an electronic circuit arrangement for aralysrg signals from the photomultiplier tube 15. The photomultiplier tube 15 produces an electrical current which is related to the intensity of the light falling upon it, and this current is converted externally by means not shown in the drawing into a voltage signal on line 17. The output signal is delivered to an analogue multiplexer 22, which also receives a digital signal indicating the instantaneous angular position of the filter wheels 19, 20 from the phase measuring device 23.

The signal from the phase measuring device 23 is used to route the output voltage of the photomultiplier tube 15 to a plurality of sample and hold amplifiers 24, each amplifier being dedicated to a particular matched pair of filters on the filter wheels 19, 20. The voltage signal stored in each of the amplifiers 24 is therefore related to the intensity ot emitted light detected using a particular pair of filters, and is indicative of the concentration in the sample being tested of the fluorescent dye to which those filters correspond.

The output signals of the amplifiers 24 are smoothed if necessary, and fed to a multi-channel voltage to frequency converter 25 which digitises the signals. The converter 25 has an output range of 1Hz to 1MHz, thereby permitting analogue to digital conversion of a signal having an amplitude range covering several orders of magnitude, and the digitised signals thus produced are delivered to a set of conventional counter chips 26 which log the signals, before passing them on to an on-board microprocessor 27 for analysis.

The microprocessor 27 also receives a signal from the photodiode 18, via an analogue to digital converter 28, indicative of the intensity of the light source 1.

This signal can be used to correct the photomuliplier output signal for fluctuations in the intensity of the source 1. Signals are also received from the polarising filter control motor 29, indicating the angular position of the polarising filters 21 and enabling a fluorescent polarisation assay to be carried out, and from the bar code readers 30 in the sample areas 4, 5. Redundant information from the photomultiplier tube 15, resulting from times when there is situated in the light path either no filter, or else only a filter which does not correspond to an active spectrochemical region for any of the dyes used in the assay, may be used to correct the tube dark current, and to compensate for variations in the transmittance of the optical system. The output of the microprocessor 27 is made available to an external processor via a USART and RS232c link 31.

When a fluorescent immunoassay is to be carried out, a solid or liquid sample is first prepared using a suitable known chemical process for a competitive fluoroimmunoassay. if two or more analytes are to ce detected, different fluorescent dyes are used to label each of the analytes. The prepared sample @@ then placedc in either the first or the sc-con sample are 4, 5 3.3' 3 t, whether it is in the solid cr liquid state, relating to the analytes to be detected and or escent dyes used to label the @mmunoassay system are entered into the fluorimeter, either by providing the sample device with a suitable bar code, or by entering the data on a keyboard.

The filter wheels 19, 20 are then set into rotation, causing the sample to be illuminated repeatedly by light of different wavelengths in succession, as selected by the filters of the first filter wheel 19. whenever a matched pair of filters, corresponding to th optimum excitation and emission wavelengths of one cf the fluorescent dyes used in the immunoassay, is located in the path of the transmitted light, the fluorescent dye will be exceted and the emitted light will be by the photomultiplier tube 15.A signal representing the intensity of light reaching the photomultiplier 15 at that instant is received by the microprocessor 27, thereby providing, with the signal from the phasemeasuring device 23 and the incident-light photodiode 18, an indication of the concentration of that dye in the sample. Due to the speed of rotation of the filter wheels 19, 20, each fluorescent dye is excited and the emitted light detected 50 to 100 times per second, and by providing 2 or more pairs of matched filters on the wheels 19, 20, it is possible to detect the concentration of two or more analytes in the sample simultaneously.

various modifications of the fluorimeter are possible: for example, instead of using a quartz halogen bulb in the light source i, a light-emitting diode or a oboe laser may be used. The photomulitplier tube in the light detector 3 may be replaced by a photo diode, an avalanche photodiode, a charge injection device or a charge coupled device. Although the use of visible or near infra-red radiation is particularly advantageous due to the relatively low cost of suitable technology, it is of course possible to use radiation of different wavelengths. Further, although the use of different matched pairs of filters for each fluorescent dye is comtemplated, it may be possible to provide just one excitation filter or just one emission filter where each of the fluorescent dves being used has substantially the same optimum excitation wavelength or emission wavelength respectively.

Claims

1. A fluorimeter including means for receiving a sample of a fluorescent material having a plurality of separate areas for receiving the sample, a source of electromagnetic radiation arranged to illuminate each of the sample areas and a detector for detecting electromagnetic radiation emitted by a sample in any one of the sample areas.

2. A fluorimeter including means for receiving a sample of a fluorescent material, a source of electromagnetic radiation arranged to illuminate a sample in the receiving means, a detector for detecting electromagnetic radiation emitted from the sample, at least one filter device having a plurality of filter portions each for interposition in the path of the incident or the emitted radiation to select a respective wavelength of electromagnetic radiation which is allowed to =ro through the device, and motor drive means for repeatedly positioning selected ones of the filter portions in turn in the path of the electromagnetic radiation.

3. A fluorimeter according to claim 2, including a first filter device for selecting the wavelength of the incident radiation and a second filter device for selecting the wavelength of the detected radiation, the two filter devices being arranged to select the different wavelengths of the incident and detected radiation in synchronisation with one another.

4. A fluorimeter according to claim 2 or claim 3, in which each filter device comprises a rotatable filter wheel having a plurality of filter portions arranged around its circumference.

5. A fluorimeter according to claim 4, including a measuring device for determining the annular dls.lacement of the or each filter wheel.

6. A fluorimeter according to any preceding claim, including an excitation waveguide for transmitting incident electromagnetic radiation from the source to the receiving means.

7. A fluorimeter according to claim 6, also including an emission waveguide for transmitting emitted radiation from the receiving means to the detector.

8. A fluorimeter according to claim r, in which the receiving means has two separate areas for receiving a sample of a @lucrescent material, excitation waveguides being provided for transmitting incident radiation from the source to each or t sample areas and emission waveguides being provided for transmitting emitted radiation from each o the sample areas to the detector.

9. A fluorimeter according to any one of claims 6 to 8, in which the or each waveguide is arranged to transmit radiation in the visible or near infra-red parts of the electromagnetic spectrum.

10. A fluorimeter according to any preceding claim, in which an infra-red-absorbing filter is provided in the path of the incident radiation.

11. A fluorimeter according to any preceding claim, In which a polarising filter is provided in the path of the incident and/or the emitted radiation.

12. t fluorimeter according to claim 11, Including ably driving the te polarizing filter te rotate its axis o polarisation.

13. A fluorimeter according to any preceding claim, in which the detector includes a photomultiplier tube.

14. A fluorimeter according to any preceding claim, in which a bar code reader is provided in the receiving means.

15 A fluorimeter according to any preceding claim, including means for measuring the intensity of the incident radiation.

16. A fluorimeter according to any preceding claim, including means for analysing signals from the detector.

17. A fluorimeter according to claim 16, in which the analysing means includes a plurality of memory devices, each arranged to receive and store in turn a signal corresponding to the output of the detector when each of the filter portions is positioned in turn in the path of the electromagnetic radiation.

1etc. A fluorimeter according to claim 16 or claim 17, in which the analysing means includes a processor.

19. A fluorimeter including means for receiving a sample of a fluorescent material, a source of electromagnetic radiation for illuminating a sample in the receiving means, a detector for detecting electromagnetic radiation emitted by the sample, and an excitation waveguide for transmitting incident radiation from the source to the receiving means.

20. A fluorimeter according to claim 1o, including an emission waveguide for transmitting emitted radiation from the receiving means to the detector.

21. A fluorimeter substantially as deserl herein, with reference to and as illustrated b, accompanying drawings.

22. A method of carrying out a fluorescent as of a plurality of analytes, in which each analvte is chemically labelled with a different fluorescent dyne, the analytes are illuminated with incident electromagnetic radiation and emitted electromagnetic radiation is repeatedly detected from each of the fluorescent dves in turn.

23. A method according to claim 22, in which a plurality of different optical filters are repeated lv interposed in turn in the path of the incident radiation, to select a different wavelength of incident radiation, the selected incident radiation wavelengths corresponding to the excitation wavelengths of the differed dyes.

24. A method according to claim 22 or claim 23, in which a plurality of different optical filters are repeatedly interposed in turn in the path of the emitted radiation, to select a different wavelength of detected radiation, the selected detected wavelengths corresponding to the emission wavelengths of the different fluorescent dyes.

25. A method according to claim 2 when dependent on claim 23, in which the different optical filters are interposed In the paths of the incicent and the emitted raiticn in synchronisation with one another, the wavelengths of the incident and the detected radiation at any one time corresponding to the excitation and emission wavelengths respectively of a particular fluorescent dye.

26. A method for carrying out a fluorescent assay of a plurality of analytes, the method being substantially as described herein with reference to, and as illustrated by, the accompanying drawings.