JP2001512827A - Detector - Google Patents

Abstract

(57) [Problem] To provide a detector capable of performing precise and various multipurpose measurements on a plurality of analytes in a sample. The present invention relates to a fluorometer. The fluorometer selectively emits light having a predetermined first wavelength corresponding to the excitation wavelength of the first phosphor and has a predetermined second wavelength corresponding to the excitation wavelength of the second phosphor. It comprises emitter means arranged to selectively emit light, i.e. the light sources are each paired with a phosphor. This simplifies the analysis of the emitted fluorescent signal.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a detector for determining whether a specific substance is present in a sample. The invention further relates to a method of operating the detector. In particular, but not exclusively, the invention relates to a fluorometer for detecting a phosphor in a sample.

The use of fluorescence spectroscopy and microscopy to determine the concentration of an analyte in a sample by measuring changes in the intensity or wavelength shift of the luminescent signal is well known. Various methods and devices are known for this purpose and are described, for example, in U.S. Patent Nos. 4,877,965 and 5,196,709.

Fluorescence detection is particularly useful for biochemical tests, often with very small sample sizes, for example, for nucleic acid or protein assays. In these circumstances, minimizing interference from background radiation is extremely important and techniques devised include, for example, U.S. Pat. Nos. 4,006,360, 4,341,957 and 4,304.
No. 791,310.

[0003] A wide variety of techniques for performing an assay exist, and cases using the assay technique have become widespread. For a review of currently used technologies, see the literature, “Update on Immunoassay Automation” [Proc. UK NEQAS Meeting 1994: 1:
163-170; Wheeler, Michael Jay
el J)]. For example, various immunoassays are now widely used to test blood and other samples for many different compounds. Since even a large hospital cannot have a large number of different testing devices, the device must be able to perform a wide variety of different types of tests on different samples.
With the increase in the number of tests required for a sample, the diversity of assay techniques must necessarily increase. The demand for versatile instruments is increasing with the increasing demand for physicians to perform the assay directly in the operating room, etc., instead of sending the sample to a laboratory in a hospital for analysis.

[0004] In connection with the increasing number of assay systems, any particular assay has to be performed more accurately than ever. The importance of test accuracy is clear. For example,
Patient treatment is determined based on the assay results, and inaccurate results may lead to inappropriate treatment of the patient.

[0005] The increase in the assay technology used has led to a large number of systems in the market. Currently, most systems are semi-automated or automated, and only require loading of reagents and samples without the need for further input by an operator unless interruption occurs.

As is evident from the above-mentioned Wheeler literature, most devices currently on the market are equipped with loading trays for loading a plurality, for example 20-100 samples, of the same type or of the same type. It is not necessary to perform the same test. There is also a reagent input tray which holds a large number of reagent cartridges for performing various different tests. In such an instrument, the sample is transferred, usually by pipetting, into an assay cell, where the sample is combined with the required reagents or reagents. The assay cell is then transferred to a portion of the instrument and held for a time sufficient for the sample and reagent to bind. afterwards,
The sample cell is transferred to a detector, which detects whether the sample contains a particular component and / or the presence of a known indicator to determine how much that component is present during the assay. Is detected. Typically, a robotic arm is used to transfer the assay cells around the instrument, for example, from the loading area to the washing station, the waiting area, and beyond. This type of assay device can function semi-automatically, but suffers from mechanical movements of the sample. In addition, it is necessary to provide a pipette with a replaceable pipette tip or other means to ensure that one sample does not contaminate another. Because individual sample cells must be incubated with the appropriate reagents, and sometimes a relatively large number of sample cells must be incubated at one time, the size of the instrument is comparable due to the space required for the waiting area. Remains large.

[0007] More recently, it has been proposed to use flow injection (flow injection) technology in the verification facility. A review of this technology can be found in Puchades.
Et al., "A review on the application of flow injection technology in immunoassays, a review of analytical chemistry (" A Comprehensive Overview on "
the Application of Flow Injection T
techniques ", Critical Reviews in Analy
physical Chemistry ", 23, (4): 301-321 (1992). The fundamental difference between this technique and the system described above is that the sample and reagent are combined in a fluid stream, which leads to a detector. This eliminates the need for a sample cell and generally reduces the mechanical components of the system.

[0008] The present invention is directed to providing a detector that enables precise and versatile measurement of multiple analytes in a sample.

According to the first aspect of the present invention, light having a predetermined first wavelength corresponding to the excitation wavelength of the first phosphor is selectively emitted, and the predetermined light corresponding to the excitation wavelength of the second phosphor. Emitter means configured to selectively emit light having a second wavelength of: receiving fluorescence emission from the first phosphor, outputting a first data signal characteristic thereof, and Fluorescent emission from the second phosphor is received, and the second
A detector is provided, comprising: sensor means configured to output a data signal; and analysis means adapted to analyze the first and second data signals. Accordingly, the present invention provides a detector that minimizes variables contained in the emission spectrum of the phosphor. The emitter means provides a signal in which the majority (eg, substantially all) of the energy is contained in a narrow range (eg, 10 nm) around two wavelengths. This simplifies data analysis and allows for multiple analyte detection from a single sample.

Preferably, the control means includes a system clock, and is configured to control the emitter means to emit light of two wavelengths separately in a predetermined order. this is,
The fluorescence emission from the phosphor is separated in time, with the attendant advantage of further simplifying signal analysis. Conveniently, the control means is configured such that the analyzing means analyzes data from the first data signal during a first time period and data from the second data signal during a second time period; The first period corresponds to a period during which the light of the first wavelength is emitted, and the second period corresponds to a period during which light of the second wavelength is emitted. The control means is typically configured to control the analysis means to analyze only data from at most one data signal at each predetermined time.
Preferably, the first and second periods end before the light emission from the first and second phosphors ends, respectively. In this way, the loss of precision associated with the decay of the fluorescent signal is substantially minimized.

Preferably, the first data signal is characteristic only in a first wavelength range, and the second data signal is characteristic only in a second wavelength range. According to this method, even if the emission spectra of the two phosphors substantially, but not completely, overlap, the first and second ranges are minimized so as to minimize such overlap. Can be selected, so that the amounts of the different phosphors can be determined. As long as one wavelength range can be analyzed for one analyte quantity, it is possible to resolve the signal from the signal to quantify the second fluorophore. According to this method, the change in the concentration of the phosphor can be continuously monitored as desired.

Preferably, the emitter means is configured to emit light on one or more cells where the first or second phosphor may be present. According to this method, a more effective analyzer can be created.

[0013] Preferably, the first and second phosphors may be present in at least one test cell constituting a flow cell.

Usually, the emitter means has a first emitter and a second emitter. These emitter means are preferably lasers or lasers emitting very clear light. Preferably, each laser is a light emitting diode laser which has the advantage of being compact. This is particularly advantageous because it emits light with a narrow band that can be closely paired with the phosphor. Such a light source provides a very bright light beam, but consumes relatively little power. Typically, lasers operate in the 400-1200 nm range. Preferably, the laser is in the spectral region with low background emission and low endogenous compounds that emit interfering fluorescence, substantially 600-90.
Operates in the 0 nm range.

[0015] Preferably, the sensor has an array and is configured to receive different wavelengths at different portions of the array. According to this method, the light emission signals from different phosphors or a part thereof can be easily separated by the sensor. The sensor further comprises a polychromator configured to distribute the fluorescent emission of the first and second phosphors to corresponding first and second regions of the sensor over a wide area and overlapping regions, respectively. . This is a particularly simple way of implementing signal separation. Alternatively, these sensor means may further comprise a monochromator configured to distribute the fluorescence emission of the first and second phosphors to corresponding first and second regions, respectively.

In a preferred embodiment of the invention, the sensor comprises a charge transfer device (charge coupled device).

The detector is used when the first phosphor is inspected in one sample and the second phosphor is inspected in another sample. According to another or additional method,
The first and second phosphors are tested in the same sample. The emitter means may have several emitters each configured to emit light corresponding to a particular phosphor. The detector preferably has (a) three emitters, (b) four emitters, (c) five emitters, or (d) six emitters, each emitter having a corresponding phosphor. To each other. According to this method, multiple analyte measurements from a single sample can be achieved with a favorable fining signal generated by the sensor. This method should allow sufficient testing of a single sample to be performed, for example, if a particular disease state indicates several indicators, then the individual is afflicted with the disease. A single sample from a suspected individual can be tested to determine if the individual is in the disease state. This greatly simplifies the testing procedure and significantly reduces costs compared to the currently required multiple sample / testing regime.

The detector according to the invention is preferably incorporated in an assay device. It is particularly advantageous when used in a flow injection assay device.

According to the second aspect of the present invention, light having a predetermined first wavelength corresponding to the excitation wavelength of the first phosphor is selectively emitted, and / or corresponding to the excitation wavelength of the second phosphor. Operating emitter means configured to selectively emit light having a predetermined second wavelength; receiving fluorescence emission from the first phosphor and outputting a first data signal characteristic thereof; Operating the sensor means configured to receive the fluorescent emission from the second phosphor and output a characteristic second data signal characteristic thereto; and analyze the first and second data signals. Operating the analysis means configured to quantify the amount of the first or second phosphor by using the method of operating the detector.

Preferably, the method of operating the detector is a method adapted to operate the detector according to the first aspect of the present invention.

The first phosphor is inspected in a first set of samples, and the second phosphor is a second phosphor.
It can be tested in a set of samples. Preferably, the first and second phosphors are the third phosphor.
The same sample contained in the set of samples is tested. Preferably, there are 3, 4, 5, 6, or more analytes tested on a single sample.

Typically, the sensing path forms a fluid path downstream of the barrier, and the sensing path includes a waste that is configured to direct fluid in the sensing path either to an outlet or to pass through detector means. Valve means to prevent uncomplexed mixture from contaminating the flow cell;

Preferably, the fluid path is divided into a plurality of the detection paths by splitter valve means configured to guide each aliquot to a selected one of the detection paths. Multiple channels can extend from any point after the incubation loop. As a result, the sample can be analyzed in another flow path while one flow path is being washed, and the performance of the apparatus can be improved.

Preferably, one detector has detection means for all the detection paths using the same detection principle.

For certain applications, the detector can be provided with an additional detection channel after the sample and reagent (s) are mixed, for example, in an incubation loop. The other flow path is configured to branch the sample from the fluorescence detector and the sample is directed to another detector that can be located in the additional flow path. Another detector is selected from one or more detectors that utilize spectrophotometry and / or electrochemical principles. In these situations, the method is simpler than the immunoassay based on fluorescence detection, so that an immobilized reagent and a barrier separation device can be eliminated. Suitably, the analysis is performed on a single sample and different types of tests are applied with at least two different detectors measuring the analytical signal from the target analyte in the sample, and under these conditions additional detectors may be used. Is preferably incorporated in a flow path leading to a fluorescence detector. Many chemical tests rely on the principle of absorbance, for example, as an analytical measurement in which a color change occurs in the reagent in the presence of the target analyte. One variation in which this approach can be applied is the determination of endogenous enzyme or other protein levels, which involves a colorimetric absorbance measurement of the substrate indicating the relative amount of the enzyme or protein in the sample. . In the flow system described below, the analysis can be performed by keeping the reagent / sample mixture in an incubation loop and producing a colored product. Thereafter, the mixture is released into the fluid stream and directed into a flow path to a spectrophotometer where the absorbance measurement is made.

[0026] In addition to measuring endogenous proteins, it is often necessary to determine the level of electrolytes in a blood sample, which can be accomplished using an ion selective electrode. The operation of these devices is well known, and since electrolyte measurements are made as the sample passes over the electrodes, it is a simple matter how to incorporate the device into the flow.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.

FIG. 1 shows the major parts (units) of a preferred embodiment of a multiple analyte detector 30 of the present invention. Control unit 34 is connected to a user interface via a personal computer or other device. The concatenation usually takes the form of a software interface. The software interface receives information from the user or upstream of the detector 30 regarding the intervals between tests performed on samples and / or individual samples. The control unit 34 operates the components of the detector 30 according to the information received, as described in more detail below.

In some situations, it may also be possible to qualitatively measure simply whether a target substance has been detected, ie whether there is some or a certain amount of fluorescence that can be assigned to the phosphor. However, more accurate measurements of the amount of target substance are often required.

The present invention relates to a detection system, which is designed to capture fluorescence from a phosphor, including a solution, or a solid contained in a cuvette or retained on a solid surface, but with a flow The solution passed through the flow cell 27 connected to the injection system is preferred.

1. Utilization of Two or More Excitation Wavelengths The system preferably has more than one wavelength selectivity with a laser diode 31. Laser diodes have several advantages over conventional pump sources, including: a. Does not require a high voltage power supply and consumes less power b. Fixed wavelength and constant light output c. Digitally controlled compact unit d. Includes easy exchange for wavelength selection.

In this detection system, two or more laser diodes 31, 32, 33 (or other light sources) are used as excitation sources, each of which is controlled by a laser driver and sequentially as shown in FIG. Light pulses are generated at various intervals. The laser is configured to shine on the test cell 27 in which the fluorescent molecule (fluorescent substance) is embedded,
The obtained fluorescent light is guided to the sensor array 35. These phosphors have their spectral properties matched to the specific laser properties, and each of the lasers 31, 32
, 33 each having at least one specific and individual phosphor partner.

The pulse speed (rate) is calculated and controlled via a programmable microprocessor or control software so that the speed of the laser driver and the time interval of the pulses meet the requirements of the sensor array, and the fluorescence from each phosphor is In the appropriate data channel. The lasers can be operated sequentially or one laser can be operated repeatedly (pulsed) before activating another laser.

[0034] 2. A multi-channel time-gated approach The use of a pulsed laser source means that the sensor array 35 will be able to detect the fluorescence from the phosphors in the flow cell 27, unspecified fluorescence from any other molecules present, and any scattered light Means receiving a pulse light signal from the flow cell 27 including In conventional fluorimeters, unspecified fluorescence or background fluorescence is removed or greatly reduced using filters or monochromators with scattered light. The fluorimeter described herein can make measurements of specific fluorescence without the need for filters or monochromators.

One of the main advantages of measuring fluorescence in the near-infrared (NIR) region (approximately in the range from 600 to 900 nm) is that there is relatively little endogenous NIR fluorescent molecule. Therefore, background interference due to unspecified endogenous fluorescence is greatly reduced. Scattered light from a broadband excitation source in a conventional fluorometer is often problematic. This is because in order to obtain maximum detection sensitivity, it is often necessary to use a broadband path setting up to 20 nm. This ensures that the maximum amount of light is collected on the analytical sample, but at the cost of a significant increase in Rayleigh scattering, surpassing certain fluorescent signals. Increase. By using a laser diode source, a large amount of light power can be transferred to a very tight (tight) bandwidth (
2-4 nm), thereby minimizing the effect of Rayleigh scattering on the short spectral range. Further, since the intensity of this scattered signal varies with the fourth power of the reciprocal of the wavelength, operating in the NIR region significantly reduces the magnitude of the scattered light. Despite the advantages of operating in the NIR region, a multi-channel time gate (MCTG), as some unspecified signals are still detected at the sensor, including from co-excitation from some other phosphors An approach is used with a sensor array to select the fluorescence of interest. With the MCTG, the fluorescence signal from each laser pulse is collected from the sensor 35 and stored in a specific data channel for the laser 31. In this manner, if desired, two or more emission pulses are
By using two or more laser diodes, it is possible to monitor from two or more phosphors in the test cell. Each of the laser diodes 31, 32, and 33 emits a pulse sequentially, and sequentially excites a phosphor having matching spectral characteristics. Each laser pulse has its own specific time interval, which is shared by the associated emission pulses. Therefore, a light emitting pulse generated by a specific laser can be time gated into a channel selected for data acquisition, and data can be fed back to prepare a sensor array. With this method, the fluorescence measurement can be performed as follows. The overall timing of all events is controlled by the system clock (FIG. 3a), and the laser 1 emits a light pulse of a specified time and frequency (FIG. 3b). Phosphor 1 responds with fluorescence over a similar period of time at an intensity according to its concentration (FIG. 3c). The other laser / phosphor pairs operate sequentially in the same manner (FIGS. 3d-3h), and the emission pulses are collected from the sensors into the respective data channels (FIG. 3i). Data collection should be terminated shortly before the end of the laser pulse to minimize variable fluorescence decay.

In addition to utilizing the laser in a sequential pulse mode, the laser can be driven in a constant wave mode, in which each laser operates continuously to illuminate the sample throughout the measurement period. In this way, multiple analyte measurements can be performed if more than one laser is operating, and these lasers are paired with the appropriate phosphor and have a recognizable fluorescence response with respect to the laser source. FIG. 4 shows the excitation of three phosphors in a stylized manner, with peaks 1, 2 and 3 being the phosphor emission after being excited by fixed wavelength lasers 1, 2 and 3, respectively. The scattered laser light has been omitted for clarity. The mixed emission signal can be processed in the following two ways. First, a method based on evaluation of a pulsed emission signal as described below can be used to generate a pure emission spectrum using multivariate analysis software. According to another method, the sensor is programmed
It is possible to collect data over a narrow wavelength range specific to each label, namely Δλ ₁ , Δλ ₂ and Δλ ₃ , thereby avoiding scattered light, and the intensity of each peak reduces the fluorescence intensity in solution. As shown, this can be used as a quantitative measurement.

The use of a constant-wave laser and a two-dimensional CCD detector, and a spectrometer to collect the fluorescence spectrum from two or more laser diodes provides an alternative to laser pulse driving. Time gating, or spectral separation, of detection is achieved spatially with the introduction of a spectrometer using fiber optic coupling of the fluorescence signals obtained from different parts of the flow cell. Liquid light guides offer another approach to coupling because they have a much higher throughput (throughput) than flux.

While it is preferred to use separate light sources, filters designed to pass only the wavelength of light emission from the desired phosphor can be used. A tunable filter can be used to modify the excitation light, or used for emitted fluorescence. However, variable or fixed filters are not preferred because they use a large amount of energy to generate comparable signals as compared to separate light sources.

[0039] 3. Wavelength Dispersion on Sensor Array Device for Data Acquisition Since the fluorescence emission from the flow cell covers a wide spectral range, the device is required to disperse this spectrum into the sensor array. As a preferred option, the fluorescent emission pulses are focused on a polychromator and then dispersed across the sensor array 35 to develop an emission spectrum. Alternatively, a monochromator is used to rapidly scan the emission beam to generate an emission spectrum at the sensor. Since the spectral properties of each phosphor are known, a pure reference sample is first used to perform wavelength correction of the spectral data stored in the sensor array for each laser / phosphor pair and the associated data channel of the computer. Do. Once the sample measurements are obtained, the emission profiles collected in the data channel can be verified against the expected profiles. In addition, multivariate analysis software is used to remove unspecified components, including spectral overlap, from the co-excited phosphor. The sensor array 35 can be programmed to collect data over a narrow wavelength range specific to each label, such that each signal intensity indicates the fluorescence intensity in solution, which can be used as a quantitative measurement. it can.

For this application, it is preferable to use a high-sensitivity, high-speed sensor to capture the fluorescence from the solution in the flow cell. A suitable sensor is a CCD optoelectronic device, which operates in a rapid self-scan mode, which allows quantitative capture of emitted photons with excellent signal to noise characteristics, mainly due to the extremely low dark current in the device. Done. The CCD has its own driver assembly, which allows the scan time to be set to match the emission pulse. A programmable microprocessor and interface digitally controls data acquisition from the sensor array.

Alternatively, an acousto-optic tunable filter (acousto-optic tun)
A tunable filter, such as an able filter, can be used as a wavelength selection element instead of a spectrometer. A single detector element, such as a photomultiplier or photodetector, can replace a CCD array. In this case, the system clock is usually tuned to a voltage ramp (eg, an oscilloscope, a transient digitizer (transient digitizer), a multi-channel scaler (multi-channel counter), etc.) that drives the light source in pulses and drives a tuning filter. Then, by switching the detector output between different data channels of the data storage device, the fluorescence spectra are sequentially accumulated. A suitable tuning filter is available from Brimros.
e Corp. ) Is commercially available. Such devices have somewhat lower production costs than CCD-based systems. Alternatively, the filters can be switched.

[0042] 4. Thermoelectric Cooling Unit Since the wavelength of the light emitted by the laser diode changes with temperature, the operating temperature of the laser diode is controlled by a fan or a thermoelectric cooling unit to secure a constant excitation wavelength. Furthermore, since the sensitivity of the sensor array varies with temperature due to changes in dark current, lower detection limits are possible if low operating temperatures are maintained consistently. Similarly, fluorescence emission is temperature dependent, and temperature control of the flow cell is important.

A single thermoelectric cooling unit is used for temperature control of the laser, sensor array and flow cell. This unit is a reversible solid state heat pump and precision controller for thermoelectric temperature stabilization. The cooling unit is composed of a doped semiconductor, bismuth telluride. The unit is controlled via a software package,
The temperature range at each control point can be set, for example, as follows. Radar diode: -25 ° C to + 15 ° ± 0.5 ° C Sensor array: -50 ° C to-+ 50 ° ± 0.1 ° C Flow cell: + 10 ° C to + 40 ° ± 0.1 ° C.

[0044] 5. PC Window with Operation Package The detection system including the laser driver, flow cell holder and sensor array is connected to a microprocessor (interface) and linked to a PC based operation software package. The package operates the laser diode driver, monochromator driver (if specified), thermoelectric cooling unit and sensor array, feeds back signals to each control point, and automatically acquires data.

The detector of the present invention can be used for analysis of a test sample held on a solid support, in a cuvette or otherwise. However, the detector according to the invention is notably described in PCT Application No. PCT / GB97 / 00334 (filing date February 6, 1997).
(Priority date (February 9, 1996)) as a detector of the flow analysis system described above. This PCT application is incorporated herein by reference, in particular as part relating to the type of flow analysis system employed.

The present invention provides a detector with a large number of different inspections enabled by multiple light sources compared to a single light source device. For example, in a flow analysis system where individual samples flow through the system, the present invention provides a detector that allows multiple tests for each sample via different fluorophores. The higher number of phosphors makes it possible to construct a device capable of performing almost all the tests necessary for surgery, for example.
Inspection time can be reduced. It is also economical to perform very large numbers of tests, since most tests need not be commissioned to specialized laboratories with many different analyzers for the different tests required. This is particularly evident when utilizing a flow analysis system.

Hereinafter, the detector of the present invention will be described in a preferred means of the flow analysis system described in PCT / GB97 / 00334, which is incorporated herein by reference.

FIG. 7 illustrates a particularly preferred embodiment of the present invention, a flow injection immunoassay analyzer, as one integrated system. This system works using the principle of flow injection analysis. That is, a continuous stream of liquid is used to transport a discrete amount of sample or reagent injected into this stream. These substances are mutually
Or it can be brought into contact with other substances immobilized in solution or on the surface and interact in a measurable way. Thus, the flow injection process is a procedure performed in conventional immunoassays using microtiter plates or tubes, except that the injection loop or syringe and precise control of the flow rate replace the use of pipettes, washers, shakers (shakers). Is similar to

The carrier buffer flow is generated from the run buffer 12. A plurality of samples are held in the sample processing unit 14, where a sample for analysis is prepared. Analysis of a particular target molecule (product) is performed by injecting a known amount of sample suspected of containing the product into the carrier buffer stream and mixing with the reagent from the reagent cartridge 15.

The sample processing (processor) unit 14 has a volume for holding about 100 samples,
It has the usual various tube sizes. Unit 14 can perform accurate and precise pipetting to produce dilutions of the sample as required. This can be done by transferring the appropriate volume to a separate tube on the processor body (bed) or by mixing a fixed volume of sample and a variable volume of diluent (or vice versa) before collecting a fixed volume for analysis. This is a conventional method that uses a flow system that mixes the components.
Unit 14 can also carry a sample probe (not shown) using a robotic arm (not shown). The robot arm is usually three-plane movable, and the probe can be cleaned at the on-board (on-board) cleaning station during sample manipulation. The sample is loaded onto the processor unit 14, preferably in its own tube of various dimensions, in a pre-prepared tube rack or in a pre-prepared microtiter plate. The identification and tracking of the sample can be performed by applying a barcode to the individual tubes, i.e., tube racks or microtiter plates, and reading the barcode with an on-board reader, or using other tracking systems.

The instrument's flow system comprises a transmission tube 10 made of a chemically and biologically inert material, such as commercially available nylon or PEEK, with a standard inner diameter of 0.8 mm. But it can change depending on the situation. The pump system (not shown) consists of several low pressure pumps, most of which are peristaltic pumps (peristaltic pumps) of different size, sophistication and performance, delivering a highly reliable flow rate. Can be. The central pump is used to move carrier buffers, samples and reagents through the system, while other pumps, which are less sophisticated than the central pump, perform other operations, such as reagent transfer, barrier cleaning and conjugates. Used for elution. The operation of each pump is controlled by a central computer (not shown) to ensure optimal performance and effective tuning. The computer also controls a number of automatic switching valves (described in detail below),
At appropriate times, the sample or reagent is directed into and out of the main carrier stream. Since these valves are operated electrically or pneumatically and are used many times on any working day, they must be extremely reliable and robust. These valves are simple in design and require only the switching of liquid flow between one or two or a limited number of channels. The valve must have a chemically and biologically inert surface, which is in contact with the liquid stream.

In general, the reagents required for each assay are specific to the analyte of interest, but in each case the same principles apply, and usually only two components are required. It is preferred to utilize microbeads for all assays, where the beads have a defined diameter and intermediate density to remain in suspension and may be made of cellulosic material with low non-specific binding properties However, other suitable materials are also suitable. The surface of these beads can be coated via a covalent bond, but other methods such as adsorption with ligand binding substances (ligand binders) such as antibodies or other compounds that specifically bind only to the analyte of interest in the assay. Is also possible. In a more sophisticated assay format, two or more ligand binders of different specificities can be coated on the bead surface, which allows the measurement of multiple analytes from the same sample. The second reagent is a labeled substance, which may be the class of analyte of interest, or a binding agent specific for the analyte depending on the assay format required. Labels are often fluorophores whose spectral properties can be detected in the near infrared region of the electromagnetic spectrum, but other labels such as liposomes, enzymes and chemiluminescence can also be used.

A fixed volume of reagent and sample are mixed in the mixing coil 17 and incubated together for a fixed time. Incubation is preferably performed by removing an aliquot from the main fluid into one of the incubation loops 19, but can also be performed in the main stream in some cases, for example, when the incubation time is short. Access to the incubation loop 19 is controlled by valve means 20. The loop 19 is made of a fixed length transmission tube having an appropriate inner diameter,
The total volume should be carefully chosen to ensure accurate repetition of the incubation conditions. During incubation, any product contained in the sample interacts with the reagent to form a complex that binds to the microbeads. The complex that binds to the microbead must contain a detectable moiety. After a predetermined amount of time, the computer switches the mixture aliquot back to the mainstream, if it was diverted from the mainstream, and carries it to the membrane barrier 22, where the microbeads are retained but other materials are All flows through and proceeds to disposal. The barrier 22 is composed of a porous membrane made of a chemically and biologically inert material such as nylon,
The beads are sized and arranged so that not all of the beads pass into the flow cell. The porous structure of the membrane depends on the size of the microbeads, but the membrane is less likely to bind non-specifically with excess reagent, and substantially all (eg,> 95%) of the beads are retained. It's important to. The channel is then washed for a period of time with the carrier buffer flow to wash away all unbound reagent, but during this period the flow rate is increased as much as possible, so that the barrier 22 must have good flow characteristics. Subsequently, various valves are tuned and switched to divert the main buffer flow from the barrier 22 while the container 24
To the elution buffer through the barrier 22. Opening and closing of the switch can be determined by monitoring the unbound reagent flowing from the flow cell 27 to the waste. In this way, the label (detectable portion) from the microbeads is released and flows through the barrier 22. This time, the flow is directed to a downstream flow cell 27 for measurement. Flow cell 2
7 is often a silica silica cylinder, but other materials and shapes are also suitable, the total volume rarely exceeds 200 μl, illuminated by a light source and, as will be described in more detail below, a detector 30 Monitored by Further switching of the valve following elution allows the membrane to be backflushed with the appropriate buffer from vessel 28, removing the beads to waste via valve 25, cleaning the membrane and Prepare a sample aliquot of

The detector 30 is shown in more detail in FIG.
32, 33, mirror optical paths and beam splitters 38-42, two identical flow cells 27 and a single detector 35 already described. Two flow cells 27
However, an instrument equipped with a detector according to the present invention may have one, two, three or more flow cells depending on the capabilities required for the instrument.
A single flow cell simplifies valve operation upstream of the detector and simplifies control software, thus reducing equipment costs when only low capabilities are required. On the other hand, as the number of flow cells increases, the equipment capacity increases, but an additional flow path in the equipment is required to achieve the fuller detection capability.

In the illustrated embodiment, there are two detector channels. This is particularly advantageous in that one of the channels can analyze the sample, while the other channel can be washed with the wash buffer 28 via one of the wash valves 29. Thus, the number of samples that can be analyzed within a predetermined time is greatly increased. This design has two (or more) flow cells 2
7 is particularly advantageous when used in conjunction with the detector of the present invention, which can be analyzed with a single radiation generator / emitter (emitter), with increased capacity at a small additional cost. I can do it.

The choice of laser is highly dependent on the available phosphor, as the laser wavelength and the optimal excitation wavelength of the phosphor need to be well matched. The speed of development in the field of solid state lasers (solid state lasers) and suitable phosphors is fast, and a final selection of these components is currently not possible. However, the laser has an output of at least 1 milliwatt (preferably 10 milliwatts) and needs to operate above 400 nm, while the phosphor is water-soluble when an aqueous solution is used, is stable in the solution, and It would be necessary to be able to emit fluorescence above 600 nm without being affected by a pH change and to have the general properties required for a good phosphor. Various phosphors are known in the art, and more phosphors have been developed. See, for example, Fabian, J. et al., Chemical Rev.
review), 1992, 92, 1197-1226, detailing many different types of available phosphors along with spectral details. Similarly, various suitable light sources are known (eg, laser diode modules) or have been developed. One skilled in the art can then pair the light sources and their respective phosphors, depending on the requirements of the pair.

The laser modules 31, 32, 33 can include one or more lasers, each of which is sequentially switched to an optical path, while simultaneously collecting data from the detector on separate channels. By computer controlling this switching, two or more carefully selected lasers with different excitation wavelengths are driven in rapid pulse mode one after another,
By monitoring the corresponding emission from the paired phosphors, the possibility of multi-label detection is created. According to this method, specific measurements can be made in a mixture of phosphors, leading to the possibility of measuring multiple analytes from the same eluting peak. If more than one pair or more of the analyte is measured in this manner, the sample can be supplemented using mixed specific beads, thereby significantly increasing instrument throughput and reducing sample usage. The amount is reduced. The obtained signal is plotted as a peak, and the sample concentration is determined from the curve created from the standard solution using the calculated area.

The system operates in a random access mode, but has a built-in (in-built) capacity for immediate analysis of emergency samples and is mounted on a separate rack of an automatic sampler (autosampler). The timing and schedule of operation is precisely controlled by icon operated and software that operates in an intuitive Windows ™ environment. The software is designed to run on a notebook computer and can be closed and stored on the base of the device when not needed.
Communication with the instrument is bi-directional and feedback from off-scale results can be used to initiate appropriate dilution and re-analysis. The equipment and software are a laboratory information management system (Laboratory Information Management)
Mentor System (LIMS) and includes assay control and quality control monitoring of reagent cartridge performance.

The analyzer is designed to measure over 20 clinically important substances, each of which has its own reagent cartridge, which is held in a reagent carousel mounted on the instrument Approximately 200 analyzes can be performed. Due to the cartridge design, the reagent can be agitated if necessary, and the temperature can be kept constant through control of the carousel chamber and the cartridge itself. Each cartridge is
It holds its own information, for example in a bar code.

It will be appreciated that detector 30 may be used in or with other assay / analysis systems to take advantage of it. Further, the detector may be an advantageous single detector and / or
Alternatively, other methods besides a laser operating with a light source and having one or several flow cells for detecting the detectable part may be used.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a preferred embodiment of a detector according to the present invention.

FIG. 2 is a diagram illustrating continuous time control of a laser in a preferred embodiment of the present invention.

FIGS. 3 (1/2) and 3 (2/2) are diagrams showing the data acquisition of the fluorescence emission of the embodiment shown in FIG.

FIG. 4 is a spectral diagram of a sample illustrating the use of a continuous wave laser source.

FIG. 5 is a block diagram illustrating control of a preferred embodiment of the present invention.

FIG. 6 is a block diagram illustrating temperature control according to a preferred embodiment of the present invention.

FIG. 7 is a schematic diagram of an assay device according to the present invention.

8 shows a preferred embodiment of the detector according to the invention for use in the device shown in FIG.

FIG. 9 is a diagram showing the overall structure of a flow injection device incorporating a detector according to the present invention.

FIG. 10 is a schematic diagram of another assay device according to the present invention.

[Procedure for Amendment] Submission of translation of Article 19 Amendment of the Patent Cooperation Treaty

[Submission date] February 10, 2000 (2000.2.10)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor French, Martin Thomas Kent T.N. Oldbury Villas 1 (72) Inventor Fu, Chee Jung, Leicestershire, UK 11 3 T. Laughboro Ashby Road Laughboro University Department of Chemistry F-term (Reference) 2G043 AA04 BA16 CA03 DA02 DA05 EA01 GA07 GB21 JA03 KA09 KA08 LA03 MA01 2G058 BA04 BB10 CB08 EA04 FB02 GA01 GE03

Claims (34)

[Claims] 1. A method for selectively emitting light having a predetermined first wavelength corresponding to an excitation wavelength of a first phosphor and emitting light having a predetermined second wavelength corresponding to an excitation wavelength of a second phosphor. Emitter means configured to selectively emit light; receiving the fluorescent light emission from the first phosphor, outputting a first data signal characteristic thereof; and emitting the fluorescent light emission from the second phosphor. Sensor means configured to receive and output a second data signal characteristic thereof, and analysis means configured to analyze the first and second data signals. Detector. 2. The control system according to claim 1, further comprising control means comprising a system clock, wherein said control means is configured to control said emitter means to emit light of two wavelengths separately in a predetermined order. Detector. 3. The control means is configured such that the analyzing means analyzes data from the first data signal during a first period and data from the second data signal during a second period. 3. The detector according to claim 1, wherein a first period corresponds to a period during which light having the first wavelength is emitted, and the second period corresponds to a period during which light having the second wavelength is emitted. . 4. The detector of claim 3, wherein said control means is configured to control said analysis means to analyze only data from at most one data signal at each predetermined time. 5. The detector according to claim 3, wherein the first and second periods end before emission of light from the first and second phosphors ends. 6. The detector according to claim 1, wherein said first data signal is characteristic only in a first wavelength range, and said second data signal is characteristic only in a second wavelength range. . 7. Detection according to any of the preceding claims, wherein the emitter means is arranged to emit light on one or more test cells in which the first or second phosphor may be present. vessel. 8. The detector according to claim 1, wherein the first and second phosphors can be present in at least one test cell constituting a flow cell. 9. The sensor according to claim 1, wherein the first or second phosphor is present.
Detector according to any of the preceding claims, adapted to receive fluorescence emission from one or more test cells. 10. A detector according to claim 1, wherein said emitter means has a first emitter and a second emitter, which can be lasers. 11. The detector according to claim 10, wherein said laser is a light emitting diode laser. 12. The detector according to claim 10, wherein the laser operates substantially in a range from 400 nm to 1200 nm. 13. The detector of claim 12, wherein said laser operates substantially in the range 600 nm to 900 nm. 14. A detector according to any of the preceding claims, wherein the sensor has an array and is configured to receive different wavelengths at different portions of the array. 15. The sensor means further comprising a polychromator configured to distribute the fluorescence emission of the first and second phosphors to first and second regions of the sensor, respectively. The detector of claim 14. 16. The sensor means further comprises a monochromator configured to distribute the fluorescence emission of the first and second phosphors to first and second regions of the sensor, respectively. The detector of claim 14. 17. The detector according to claim 1, wherein the sensor has a charge transfer element. 18. The detector according to any of the preceding claims, wherein the first phosphor is tested in one sample and the second phosphor is tested in another sample. 19. The detector according to claim 1, wherein the first and second phosphors are inspected in the same sample. 20. A detector according to claim 1, wherein said emitter means has a plurality of emitters each configured to emit light corresponding to a particular phosphor. 21. The detector comprising: a) three emitters, b) four emitters, c) five emitters, d) six emitters, or e) more than six emitters. 21. The emitter according to claim 20, wherein each emitter is paired with a phosphor.
Detector. 22. An assay device incorporating the detector according to any one of the above claims. 23. The test device according to claim 22, further comprising a flow injection test device. 24. The method of claim 1, further configured to analyze a sample.
24. The assay device according to claim 22 or 23, comprising at least one further detector which does not depend on any of the above. 25. The or each further detector is selected from the group consisting of: a spectrophotometric detector, an electrochemical detector, an electrolyte detector such as an ion selective electrode. An assay device according to claim 24. 26. The assay device according to claim 24, wherein each detector is associated with a separate test cell. 27. The assay device of claim 26, wherein each test cell is in a separate flow path. 28. A method of operating a detector, comprising: selectively emitting light having a predetermined first wavelength corresponding to an excitation wavelength of a first phosphor; Operating emitter means configured to selectively emit light having a corresponding predetermined second wavelength; receiving a fluorescent emission from the first phosphor and generating a first data signal characteristic thereof; Operating the sensor means configured to output and receive a fluorescent emission from the second phosphor and output a characteristic second data signal thereto; and analyzing the first and second data signals. Operating the analysis means configured to quantify the amount of the first or second phosphor. 29. The method of claim 28 adapted for operation of a detector according to any of the preceding claims. 30. The first phosphor is inspected in a first set of samples,
26. The method of claim 24 or claim 25, wherein the second phosphor is tested in a second set of samples, and the first and second phosphors are tested in the same sample in a third set of samples. 31. The method according to claim 28, further comprising the step of detecting one or more analytes with different types of detectors. 32. The assay method described herein with reference to and / or by way of example in the accompanying drawings. 33. A detector as described herein with reference to and / or by way of example in the accompanying figures. 34. An assay device as described herein with reference to and / or by way of example in the accompanying drawings.