JPH03503314A - Fluorescence measuring instrument - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Fluorescence measuring instrument The present invention relates to a fluorometer.

Current fluorometers are fairly large laboratory instruments, with 150 watts A high-power light source such as a non-lamp source and a high-voltage photomultiplier y) Use a detector.

Although these instruments are in good condition, they cannot be used in laboratory conditions unless they are used by a trained operator. Restricted to use by pelleters.

The aim of the invention is to provide a fluorometer that is not limited to laboratory conditions. It's there.

The term fluorometer and similar terms used in this text will be understood by those skilled in the art. Analytical standards for fluorescence, phosphorescence and luminescence, and fluorescence that are similar to The invention includes instruments that use at least one of the similar techniques, including a calibration step.

According to the invention, a sample expected to produce a first specific fluorescence emission upon excitation A flash fluorescence analysis method is provided, which ``Blank When performing J analysis (blank test): When excitation, the second A reference fluorophore expected to produce a characteristic reference emission is supported at the test location. , applying a pulse of excitation radiation to said test location and directing radiation from said test location to said first and each detector for a second radiation; From the relative response of ” value, and also during similar implementation of certain sample analyses: upon excitation by the incident radiation. A reference fluorophore was maintained in the sample that was expected to produce a specific emission. supporting in said test position when present; The radiation from the test location is examined by each of the detectors and the relative response of the detectors is determined. , determine the sample value for the radiation from the test position in the presence of the sample, Evaluate the "blank" value and the sample value to determine the sample for the calibration step. A method is provided that includes analyzing.

Advantageously, the reference fluorophore is located within the structure of the cuvette containing the sample. or contained within another sample support format.

The cuvette may be of optical construction.

The sample for analysis contains a reacting sample that can fluoresce to produce the specific radiation mentioned above. Samples and reagents may be used.

One of the samples and reagents is present during a "blank" run, the other is a "blank" ” added after analysis. A reference fluorophore is included in this sample and in one of the reagents. be able to.

The method requires a time interval between reference and sample analysis to ensure stable reference fluorescence emission. This may include limiting.

The instrument for fluorescence analysis of samples is battery powered and includes a control circuit, a flash tube, It is desirable to include an optical path from the flash tube to the photodetector via the sample position. The control circuit is configured to process and determine the sample position with respect to the calibration step. A ratiometric comparison method is used to determine the specific radiation level from a sample at to provide said detector with radiation containing a reference fluorescence upon activation in order to determine the , the flash tube is charged to produce appropriate radiation passing through the sample location, causing it to emit light. act.

According to the invention, an aperture is provided within the defined configuration for receiving and retaining the optical element. An optical structure formed in a material body is provided within the fluorescence, and this material body a sample aperture for receiving and holding the sample in a particular position; A source aperture that is held in a specific positional relationship with respect to the sample aperture, and a source aperture that is Defines an input radiation path aperture that provides a specific path between the material apertures and an output radiation path aperture. a detector aperture providing an output radiation path from the sample aperture to a radiation detector; provide.

The material body may be a single piece. This material body is a beam Output radiation path aperture for receiving and holding a splitter in a specific position within said output path A beam splitter aperture inside the beam splitter aperture and an output radiation product from this beam splitter aperture. an output radiation component aperture defining a branch path; an output radiation component aperture defining a branch path; The body of material may be provided with the sample opening around the opening. Maintaining a defined configuration of the optical element relative to the mouth. The beam splitter is It may be either a partially reflective type or an optical fiber type.

According to the invention, the fluorometer is provided with a material body that retains the optical path configuration; The material body is made of an opaque material and receives and retains the optical system elements within the optical path arrangement. apertures defined therein, and these apertures defined by said body of material, when in use. a sample aperture that receives the sample and holds the sample in a specific position, and a sample aperture that receives the radiation source and A source aperture that holds this source in a specific position relative to the sample aperture, and a source aperture between the source and sample aperture. an input radiation path aperture that provides a specific radiation path from the sample aperture to the radiation detector aperture. and an output radiation path aperture defining a specified output radiation path leading to the material. The body retains a defined configuration of optical elements around the aperture and relative to the sample aperture. Ru.

The aperture has control therein at least one of a radiation intensity and a radiation frequency range. Light control elements can be incorporated.

According to the invention, a monolithic thermally conductive opaque material defining an aperture for the radiation source is provided. The optical core structure of the fluorometer, the sample input radiation path, the sample for inspection, and the sample output a force radiation path, and a material body is provided around the aperture with the aperture and a force radiation path around the sample aperture. The optical element is held in a configuration defined within the periphery of the optical element.

Embodiments of the invention will now be described with reference to the accompanying drawings. In the diagram Then, FIG. 1 shows a part of the optical system of a fluorometer according to the invention, schematically showing the optical elements. Showing the cut plane, FIG. 2 is a block diagram of the fluorometer of FIG. 1 and related electronic circuitry, and FIGS. 3 and 4 are partially cutaway views of the optical system of yet another fluorometer according to the present invention. Shown in squares.

For diagnostic chemical analysis, potable water supply testing and environmental monitoring, “on-site” testing There is an increasing demand for established and centralized research operations. The above three analyzes Although fluorometry is a well-established research method in all areas, Instruments that allow "in situ" or "real twist" measurements to be made using analytical methods are I can not use it. As mentioned above, phosphorescence, delayed fluorescence and luminescence measurements are Very similar to fluorescence to be included in the descriptive term fluorescence.

In current fluorometer designs, high sensitivity is associated with photovoltage photomultiplier tube detectors. Continuously use a high power excitation light source (e.g., a 150 watt xenon lamp) This is achieved by holding the sample in a high-precision quartz oscillator cell. Such a trial Although it is acceptable for instruments in the laboratory, it is not suitable for battery-powered fluorescence measuring instruments. is inappropriate.

Next, we will discuss the ratiometric dual fluorometer and related assay system. Bell. This fluorometer measures two waves from the same pulse of intense UV-V TS radiation. Measure fluorescence at long distances.

Both a portable battery-powered fluorometer and a low-cost laboratory fluorometer structure becomes possible.

An apparatus embodying the invention is capable of measuring successive samples at wavelengths transmitted through the sample. Measurements are performed using the ratiometric method using radiation pulses. these successive A reaction of interest occurs between pulses, and continuous measurements provide information about this reaction. provide. The method incorporates calibration. This makes the device simple. to enable on-site use. Laboratory use is not included and is actually more than this device. Wide range of possibilities due to lower cost and less operator skill required have.

FIG. 1 shows an overview, in partial section, of a fluorometer according to the invention. this In a fluorometer, a suitable frame structure (not shown in detail) is configured to support. Although the details of construction will be clear to those skilled in the art, I will discuss the main points. In one form described below, the frame The system structure is a metal block.

The holder 51 for the cuvette 5 holding the sable or other material is A detector assembly including a specific radiation detector 10 and a reference radiation detector 12 from a radiation source 1 Position the cuvette precisely in the light path or radiation path of the song. In operation In this case, a reference fluorescent radiation source is provided at the cuchette location as described below. .

Between the source 1 and the cuvette 5, the radiation path passes through the aperture 2.3 and the quartz window 4. has. Source 1 is preferably a flash tube filled with high-strength xenon. If necessary, the light shielding filter 6 may be placed between the openings 2 and 3 as shown in the figure or at other locations. to prevent breakdown of materials within the cuvent by flash energy. Fi The router 6 is configured to control the intensity and/or wavelength of the radiation reaching the cuvette. can be placed in

The cuvette 5 may be a high precision quartz cell, or may be made of e.g. acrylic or polystyrene. Made by injection molding from optical quality plastic material. A disposable structure is fine.

The cuvette and holder remain in the same relative position when removed. Marks should be placed to ensure re-insertion of the bet.

Between the cuvette 5 and the detector assembly, the path includes a quartz window 7 and a via. system splitter 8. Beam splitter 8 divides the radiation evenly and is or weight to increase the strength in one channel (e.g. 80 vs. 20) can be attached. Energy not deflected by the beam splitter is to a detector 10 for a particular radiation via a narrow bandpass filter 9 for the radiation of reach The energy deflected by the beam splitter is passed through a narrow bandpass filter. The reference radiation detector 12 for the reference radiation is reached via the detector 11 . These filters are , of the appropriate wavelength or frequency for the expected radiation. If this instrument has special features, For specific applications, it is recommended to maximize optical properties and use individual specific filters Dichroic filters can also be used to eliminate the need for light and emission filters. Ru. The quartz window prevents liquid spills from the cuvettes in the cuvette holder. hold.

An optical link 13, such as an optical conduit, is installed between the reference radiation detector 12 and the specific radiation detector 10. I'm being kicked. This means that the specific radiation detector always receives a constant component of the reference radiation. This is to ensure that The reference radiation is collected from the output side of the filter 11 and detected. It is given to the vessel 10.

The structure of FIG. 1 can be made in a very compact form. In one embodiment The overall structure 53 is such that the various elements fit into cavities formed by planing and drilling. Define the 401rI+ square plan view of the 40m+ aluminum solid that will be accommodated. . The cuvette is basically a standard 10 square square shape (inner dimensions). It is typically 12 mm square on the outside. The holder 51 has a cuvette required. The surface of the cuvette has grooves that allow access only to certain positions. A hole with a diameter slightly smaller than the circumscribed circle is sufficient. Flash tubes are used in pocket cameras. The length may be approximately 35 ohm. Beam splitter is 1 It is readily available from component suppliers such as IEL1ES-Griot. Alternatively, the filter can be formed by polishing available elements. The optical link is A certain length of optical fiber is sufficient.

Mechanical stability is achieved by constructing the device in a solid, thermally conductive block. The solid block is A strong "core" or "heart" with all important components of shock-resistant structure form. This core is equally suitable for laboratory instruments.

The electronic circuit that provides power and controls the fluorometer is shown in Figure 2 below. I will give an overview regarding this. Suitable components for specific requirements will be provided to those skilled in the art. Although this may be obvious, I will mention some special features.

The main switch has two series of requirements that turn on the device and automatically zero the device. It has elements 14A and 1413. Switch element 14A is configured such that voltage regulator 16 A tie that allows power to be supplied to other circuits for a set period of time from the closing of switch 14A. 15, which results in an off operation at the end of this set period.

When the switch element 14B is closed, the flash tube drive circuit 18 controls the voltage regulator 16. A monostable multivibrator configured to time a period sufficient to charge through The data controller 17 is made available for use.

The output of the monostable multivibrator 17 is further output from another monostable multivibrator 3. 0 "enable" terminal, and an isolation diode "Enable" of flash tube drive circuit 18 and monostable multivibrator 20 through connected to the terminal. With the above configuration, the flash tube 1 emits light and the associated tie The timing signal is obtained from a monostable multivibrator 17.20.30.

Specified radiation detector ■0 and reference radiation detector 12 must be photodiodes. is preferably connected to each operational amplifier 24,25. Gates 22 and 23 are between each gate operational amplifier 24, 25 and each storage capacitor 26, 27 . Analog divider 28 receives the voltages at capacitors 26, 27 and divides these voltages. The output is configured to produce an output representative of a ratio of pressures.

For this purpose, the monostable multivibrator 20 1 and the gates 22 and 23 respond to the operation of the monostable multivibrator 17. generates an open period timing signal. During this period, flash tubes were first fired. A short time later the monostable multivibrator 17 causes the photodiode to The voltage outputs of the operational amplifiers 24 and 25 resulting from the radiation incident on the samples are respectively and stored in capacitors 26 and 27.

The optical link 13 (FIG. 1) ensures that the specific radiation detector always receives a constant portion of the reflected radiation. We guarantee that This stabilizes the behavior of the detector in the absence of specific radiation. The dual purpose of ensuring consistent readings of specific emissions as they occur has. The monostable multivibrator 30 has a gate 31 on the output side of the divider 28. connected to operate. The output of the divider 28 is connected to the gate 31 and the difference operation. applied to both first inputs of amplifier 36. The output of gate 31 is a buffer It is connected to capacitor 32 at one input of operational amplifier 33. gate 3 1 is opened by the operation of the monostable multivibrator 30, the light enters the detector. The output of divider 28, representing the proportion of the radiation Due to the operation of the gate 33, the value of this output changes even if the gate 31 is again a monostable multi-byte transistor. This is maintained even when the circuit is closed by the operation of the breaker 30. Output of operational amplifier 33 is connected to the second input of the difference operational amplifier 36. The output of the operational amplifier 36 is is applied to the potential divider 40, and the tapped-off portion of this output is displayed on the display 37. It will be done. The display on display 37 represents the difference between the inputs of differential operational amplifier 36.

The switch 38 enables the tli stable multivibrator 39 when closed. This times the charging of the flash tube drive circuit 18, which in turn turns the flash tube into a monostable It emits light in the same way as the vibrator 17. Monostable multivibrator 20 and 21 is a monostable multivibrator for the monostable multivibrator 17 as described above. The output is sampled in response to the regulator 39 and stored in capacitors 26 and 27. I can do it. The specific radiation detector 10 and the reference radiation detector 12 are responsive to the radiation received. The output of the voltage divider 28 determines the difference between the values of the ratio of the specified and reference radiation to the operational amplifier 36. is applied only to the ``first'' input of . These inputs to the difference operational amplifier 36 The difference in price opening is shown on the display.

Timer 15 serves to limit the time for actuation of switch 38. If If 1 switch 38 is not actuated quickly enough, this cycle will cause switch +4A, 14B. It takes approximately 5 minutes for the flash tube to first emit light. with an appropriate time limit for the buffer operational amplifier 33 to hold the value obtained at the time. be.

Fluorometer operation for typical measurements is discussed after the cuvette discussion. I decided to go to bed.

As mentioned above, the cuvette 5 is a disposable component made of a suitable plastic material. It can be modularized as an element.

The optical requirements of the modular cuvette for the fluorometer according to the invention are: , not as stringent as in the disposable cuvettes used today. Only two toru Clear faces are required and these faces are usually adjacent. If the cuvette is removed If so, it is important not to turn it over when putting it back together.

Therefore, it is necessary to mark or give a shape so as not to conflict with this. . For example, the holder 51 can be placed one It is possible to have protrusions at two intervals.

For example, by including a fluorophore in the material from which the cuvette is modularized, An important feature is that the reference fluorophore can be built into the cuvette itself.

Although the inclusion of a fluorophore in the cuvette is a very convenient measure, other techniques It can also be used. For example, a reference fluorophore can be included in a reagent placed in a cuvette. You can also

The important point is that a stable fluorophore material is present within the optical path of the fluorometer. . The material, including, among other things, the intensity of the flash, the position of the flash and the quality of the cuvette. Provides a dynamic optical “reference” that can normalize the variables of all fluorometers.

To operate a fluorometer using a cuvette containing a fluorophore, it is necessary to have pure water. Place a cuvette like this in the holder 51 as a blank material, and press the switch 14. A and 14B are activated. The automatic operation of this circuit makes it possible to A "zero" analysis is performed first to yield a "zero" value. Monostable multivibrator 17 operates to time the charging of the flash tube circuit, and then its output goes "low"; Trigger the flash tube drive circuit 18 to generate monostable power along with the monostable multivibrator 30. Start the multivibrator 20. Radiation from the flash tube passes through the aperture 2.3 and the quartz The fluorescence from the cuvette passes through window 4 and enters the beam splitter. reaches 8. A portion of the reference fluorescence radiation from this beam splitter is transmitted to the detector. a bandpass filter 11 suitable for the reference frequency from the fluorophore for measurements by 12; At the same time, the pond part is applied to a bandpass filter 9 suitable for a specific frequency. and therefore this is the reference frequency emission from the fluorophore for measurement by the detector 10. prevent the shooting. of several microseconds set by the monostable multivibrator 20. After a delay, the monostable multivibrator 21 is activated and a sampling period of several milliseconds is generated. gates 22.23 are opened, and the outputs of operational amplifiers 24.25 are connected to each capacitor 26.23. Save up to 27. As mentioned above, the bandpass filter 9 is configured so that the reference radiation is detected by the specific radiation detector 1. 0, but as mentioned earlier the optical link provides some radiation. child The delay of can be selected according to the type of measurement being performed. Like this, So-called "short period" fluorescence is measured within the first 200 microseconds after the flash; As one skilled in the art will appreciate, "temporally resolved" fluorescence lasts approximately 200 microseconds or longer. The phosphorescence is measured after a delay of approximately 1000 microseconds or more after the flash. need Depending on the .

The voltage stored on the capacitor is used by divider 28 to determine the specific and reference frequency. It produces an output value representing the proportion of detected fluorescent radiation in wavenumbers. monostable multivibe When the output of the regulator 17 becomes low and starts the monostable multivibrator 30, the monostable multivibrator 30 is activated. Stable multivibrator 30 opens gate 31 for a short period of time to indicate the rate of radiation. The output of divider 28 is applied to capacitor 32. This release with blank cuvettes The value representing the ratio of morphism is thus held in the capacitor 32 by the operational amplifier 33. and is applied to the second input of the difference operational amplifier 36. Minute representing the value of the radiation ratio The output of the divider 28 is the capacitor 26.27 charged again via the gate 22.23. is applied directly to the first input of the difference operational amplifier 36 until the difference is applied. The cuvette When "blank", only the reference fluorescence is produced, resulting in a differential operational amplifier 36. The inputs for can be obtained. A suitable reset circuit may be required for indicator 37.

Next, the “blank” in the cuvette is placed so that sample analysis can be performed. Replaced with sample for analysis. The switch 38 is opened and the timer 15 starts the time. If not, the flash tube will fire as part of the circuit's continued automatic operation. .

At this time, the excitation radiation passing through the cuvette is at least 2 Two fluorescences are produced, one produced by the sample and one produced by the reference material. These emanations A portion of the light passes through a quartz window 7 and then passes to a beam splitter 8.

Fluorescence from the sample is collected by a detector 10.12 and its ratio is determined by a differential operational amplifier. 36:1 added to the first j force. The optical link again directs a portion of the radiation to the detector 10 give.

The ratio value for the blank cuvette is already at the second input of the difference operational amplifier 36. so that the display 37 shows the value of the sample ratio in the cuvette. 1Blank” and the ratio value for the “Blank” cuvent in the analysis. It will be shown. Knowing the calibration value of the instrument will result in fluorescence in the sample The amount of material can be determined.

The scale length of the display of the display 37 can be set by the potential divider 40. can.

The timer 15 is set to "zero" due to instability of the instrument or reference fluorophore. The “zero” reading on the blank cuvette is no longer true. to ensure that it is not invalidated by delays prior to testing.

Fluorescence analysis, such as blocks doped with fluorophores of known concentration for instrument calibration The use of reference values is well known in the art and this step can be It is assumed that this will be done, and there is no special addition.

Since the flash of light is very short (1-5 ms), photodegradation of the sample occurs. Although unlikely, it is possible to limit the excitation wavelength to a wavelength shorter than that of the specified and reference radiation. A light blocking filter 6 may be built in to limit the amount of light.

A very important feature is that +Huvette incorporates a fluorescent polymer into the thermoplastic material into which it is modularized. It is important to include the key. Using such a cuvette, we can emit fluorescence at two different wavelengths. The group can be scaled (i.e. normalized) in sync with the test as described above. Ru.

To make such cuvettes, conventionally known injection molding tools can be used without modification. It is possible to simply coat the feedstock particles with the reference fluorescent marker.

This marker includes phenol or indole, fluorescein, and quinine (quinine). ), anthracine, naptha D7 one), ovalene, p-terphenyls (p-te) rphenyl), terphenyl-butadiene (terphenyl-butadiene) odiene), o-damine B (rhodamine B), compound 610, or other similar compounds.

If the marker is available as a fine powder, it may be Ren, polymethyl methacrylate (polymethyl methacrylate), PTX or “Dusting” optical grade thermoplastic materials You can also do it. Alternatively, this marker can be dissolved in a solvent that is not miscible with the thermoplastic. When the granules are washed in a doped solvent, each granule is covered with a small amount of fluorescent marker. make it covered.

This modified charge is then used to injection mold a cuvette.

Perfect uniformity of fluorescence is not required; the methodology, as described elsewhere, % patch-to-patch variation is acceptable and ensures that each cuvette is or at least found for zeroed cuvettes. .

This is a standard fluorescent particle, but this reference fluorescence is a variable throughout the system. Since it is a normalized level, it is optically stable during the analysis period (1 to 10 minutes). Must.

For small-volume production, a simple mold is sufficient; cuvettes may be made of acrylic or other polyester. in which the monomer is doped with a small amount of fluorescent marker . This technique is effective in marker optimization and testing. cuvette 5 For example, in a solvent-resistant high-precision silicon cell, the reference fluorophore is one of the reagents. or doped into silica or borosilicate materials. Wear. Quinine sulfate is a suitable fluorophore for use in certain reagents.

Specific and reference wavelengths share a common path to the surface of the beam splitter, allowing short-term During the excitation flash (1 to 5 milliseconds), the detectors of the specific and reference radiation It can be seen that the response is synchronous. There is no need to provide secondary filters 9 and 11. (and specific and reference radiation detectors (with the same amount of heat sink)) There is also no drift difference between the (Specific radiation) Fluorescence/(Reference radiation) Fluorescence remains constant regardless of any changes in subsequent flashes of the excitation source. For this reason, In fluorescence analysis, the channel in which the specific radiation for the test is measured is called the reference is synchronously normalized (i.e., standardized) with respect to the channel. For phosphorescence analysis The optical layout is the same, but the signal from a particular radiation channel is Acquisition is delayed by a few milliseconds in obtaining phosphorescence measurements.

In luminescence analysis (luminoIIetry), high power excitation Neither off-axis nor off-axis luminosity is required, but low-power on-axis light emitting diodes (L ED) can serve as an internal reference for such measurement groups.

The device described above is particularly suitable for battery-powered field use. This device is It can be a compact and robust structure with a built-in battery. Power consumption is Relatively low. Power is only needed to charge the flash tube emitting circuit for a short period of time; requires only small power to operate the semiconductor control circuit and display 37 .

This equipment is particularly suitable for use in the American Society for Testing and Materials (ASTM) revised agreement. are doing.

In Figure 3, this means placing the optical element in the inner hole of the solid material block. By doing this, the cross-sectional plane of the optical system of another fluorescence meter is attached to the A view is shown in FIG. 3a, and a cross-sectional elevation thereof is shown in FIG. 3b. In Figure 3b Some elements have been omitted for clarity.

For convenience, the block indicated by 353 has dimensions of 50 x 55 mm on a plane and a thickness of It is a block of easily machined material such as 35mm aluminum. exemplified In the construction, the inner hole is cut into a solid block, but other methods, e.g. High-precision Guicasting can also be used and the material remains stable and accurate. can be selected for specific purposes as long as possible.

As in the previous example, the boulder is, in this example, a 16 mm square cuvette or A roughly circular inner hole 351 with a groove cut therein to receive another test piece or sample holder 305 is formed towards the center of the block. Yet another inner hole 352 is connected to the inner hole 351. In this example, a xenon flash tube (in particular, a Mapl in type FS 77J). This flash tube has 03 watt seconds per flash. It flashes at a high flash rate, typically 60 flashes per minute, with an energy input of . This flash tube , is fitted into bore 352 by an insulating bushing (not shown) and suitable electrical connections are made. I get kicked.

Internal holes 354, 355, 356 are provided in a plane that intersects internal holes 351, 352. . These bores receive tubes fitted with various optical elements, which are then placed in place. Optical arrangement of key points, such as a stop such as 358.359 threaded into a hole in the block. It is held securely in place by internal threads. These inner holes have a diameter of 14 mm. It is desirable that

Considering the bore 354 first, this provides a good slip fit into the bore. A filter and collimator assembly is provided in the form of a tube, the bore having a diameter at each end. The aperture disks (302, 303) are fitted, but the ultraviolet transmission filter 306 is It is held at the end closest to the holder 351 inside the tube. Opening disc 30 2.303 has a circular hole or slit or other shaped opening, and the radiation source 3 Perform the required standardization and intensity control of the ancient light. Ultraviolet filter of this example 306 has a passband of 300 to 390 nanometers. This filter is For use in diagnostic instruments such as those mated to TECIINICON® analyzers. It is of the type used, with a peak of about 356 nanometers, 396 and The transmittance is reduced to about 10% at 296 nanometers and 428.260 nanometers. It has a very well defined passband with minimum transmission at the meter.

This assembly is within 0.5+n+n of the cuvette in holder 351. It is placed in the inner hole 354 as shown in FIG.

Next, consider the inner holes 355,356. The inner hole 355 is directly connected to the inner hole 354. It forms a corner and receives the radiation emanating from the holder 351 area.

The filter 371 that blocks ultraviolet rays of 400 nanometers or more has an inner hole 355 that is It is fitted in a position where it meets the driver 351. This filter is BALZER8 ( A UV blocking filter (registered trademark) is preferable. Quartz that extends to the UV range Beam splitter 308 has a 10001111 hole at one end and an inner hole 356. 14wm OD tube 360 with ioam holes on the sides placed in matching positions installed on. This beam splitter is located at the center of the axis of the 10-stroke side hole. is positioned within the tube so as to have a reflective output surface located at and at right angles thereto. tube 360 is the inner hole 355 with the axis of the side hole of l(bnm aligned with the axis of the inner hole 356) fitted inside.

Two detector assemblies are installed in block 353, one in each of bores 355, 356. It is fitted. Each detector assembly is a tube that mates with an inner bore and is provided by a sleeve. It has an inner diameter to receive each supported photodiode 310,312. This frame The photodiode has a response range of 320 to 1000 nanometers. Must be fflamatsu (registered trademark) type SL 226-448K is desirable. Each interface filter 309.311 (as noted with respect to FIG. (as shown) in front of each diode, for example, a beam splitter as shown. The detector assembly is fitted onto the 3D surface of the beam splitter or onto the diode itself, and the detector assembly is assembled into each bore so as to just contact the cutter assembly. Electrical connection is photo die It is provided for ord.

Figure 4 is similar to Figures 3a and 3b in Figures 4a and 4b. , using an optical fiber instead of a beam splitter to direct the radiation from the holder region. The optical system for branching is shown. In Figure 4, elements similar to those in Figure 3 are shown. The prime is indicated by replacing the first digit "3" with "4".

A beam splitter, indicated generally by 460, is in the plane of bore 454. is attached to an inner hole 457 that is perpendicular to the inner hole 454 and intersects with the holder 451. It will be done. Filter 471 has the same function as filter 371 (described above). This bit A beam splitter consists of a neat optical fiber with one end exposed to the holder area. It is a row of rows. This fiber is distributed between two or more bundles in the usual manner and is Each photodetector and each filter are placed for each branch at the other end of the optical fiber. . In one embodiment, a 5 ttva x 3 m orderly 0.25 cm diameter The rectangular array of fibers has alternating columns (dimension 6 m) assigned to each bundle. (50150 branches).

This results in two bundles 408.418, each approximately 4 m in diameter. light fu Place an appropriate amount of water around one end and the other end to hold the fiber in the desired position. Opaque potting compound with ferrule and epoxy base placed. Optical fiber and filter 471 at one end glued into tube 460 Preferably, the tube is located within the bore 457 of the cuvette in the holder area. ．． It can be fitted and held within 5m+n. The sun ring at the other end fits inside the pipe. These tubes can be connected to each interference filter and photodetector (of the types mentioned above). and hold it against the end of the optical fiber.

In yet another configuration, not shown, on the opposite side of bore 454 (or 354) Two internal holes are provided to allow examination of the radiation transmitted through the holder area. Receive the detector.

Electronic control and measurement of the radiation source shown in Figure 2 and the resulting optical detection Another configuration that uses a microprocessor to control the flash tube's emission and Controls processing of detector signals. The general steps above still apply, but Superior control and performance by using technology that improves signal/noise ratio It is possible.

The signal from the detector can be processed digitally, so the microprocessor The sensor can quickly examine the "signal/noise ratio" output. If this is full If the size is not large enough to display an acceptable signal/noise ratio, use a flash tube (e.g. This may cause the light to emit again (within 300 ms, for example) to improve the signal/noise ratio. signals can be statistically aggregated.

This may continue at a flash rate of e.g. 3Hz until a sufficient signal is obtained. can.

From the foregoing details, a suitable microprocessor configuration will be readily apparent to those skilled in the art. Dew.

Comparisons of radiation intensities may account for flash variations and other lack of optical reproducibility. effects are removed and the response of the device is normalized. Two from one light pulse The measurement at different wavelengths of the optical and electronic drift in the measurement system compensation for soft and noise. In this way, one of the wavelengths is the sample to be analyzed. test i.e. active at a particular wavelength, which is expected from the test, but other wavelengths are selected to be unaffected by changes in the intensity of fluorescence at a particular wavelength. is the reference wavelength produced by a fluorophore in a defined optical path. into a small flash tube Excitation by silicon P, which is a good detector but requires rather high emission levels Provides sufficient strength for TN photodiodes. Specifically, it contains a built-in fluorophore. A disposable cuvette then greatly facilitates the use of the device. Electronic, This is possible by ensuring that the above-mentioned "drift" in optical or chemical properties is not large. It is Noh.

The signal from the detector can be processed digitally, so the microprocessor The sensor can quickly examine the "signal/noise ratio" output. If this is full If the size is not large enough to display an acceptable signal/noise ratio, use a flash tube (e.g. This may cause the light to emit again (within 300 ms, for example) to improve the signal/noise ratio. signals can be statistically aggregated.

This may continue at a flash rate of e.g. 3f(z) until a sufficient signal is obtained. I can do it.

From the foregoing details, a suitable microprocessor configuration will be readily apparent to those skilled in the art. Dew.

Comparisons of radiation intensities may account for flash variations and other lack of optical reproducibility. The effects are removed and the response of the device is "normalized". Two from one light pulse The measurement at different wavelengths of the optical and electronic drift in the measurement system compensation for soft and noise. In this way, one of the wavelengths is the sample to be analyzed. test i.e. active at a particular wavelength, which is expected from the test, but other wavelengths are selected to be unaffected by changes in the intensity of fluorescence at a particular wavelength. is the reference wavelength produced by a fluorophore in a defined optical path. small flash tube Excitation by silicon is a good detector but requires somewhat higher emission levels. Provides sufficient strength for PIN photodiodes. Especially those with built-in fluorophores. If so, disposable cuvettes greatly facilitate the use of the device. Electronic, To ensure that said "drift" in optical or chemical properties is not large, Therefore, once the instrument is normalized, the sample must be analyzed within a set time.

The installation is clearly still in use due to the mains power supply and other related changes. It can be constructed in the same general form as a "field" device, with the advantages of use described above. I can do it.

Dual-wavelength fluorometry (DWF) can be performed as long as the detector is operating under optimal conditions. Let's bring you satisfying performance. Too little light emission reduces the signal/noise ratio and this Therefore, reproducibility also decreases. Excessive excitation energy can lead to saturation and non-saturation. This will result in linearity.

The use of fiber optic beam splitters allows further expansion. beam sp By using a liter, the light is separated from the flash tube and passed through different optical fibers to the next The resulting light is then applied to the cuvette through a similar fiber optic device. combined at selected frequencies to detect any content in a well-known manner. frequency can be excited simultaneously. Thus, lower than 300 nanometers Excitation in the UV range is possible. Suitable fiber optic and UV enhanced light detection equipment can be used.

In another configuration of optical fibers, all three branching cuvette faces are combined. One bundle carries the light to the surface of the cuvette, and the other two Light can be transported from the same plane to each detector. In particular, this configuration Insert the ingredients into a suitably shaped cuvette holder or adapter and place them on one side. It is effective for dry reagents that can be used for measurements. Such reagents are Inside, Kodak Ektachem.

Miles Laboratories (^mes Division), B oelwinger Mannheim.

Provided by Fuji Film (all registered trademarks).

In some systems, optical measurements are performed using a supporting layer or a dispersive reflective layer (TiO□ , BaO, etc.) through a transparent support layer that may contain the reference fluorescence. be exposed. In systems where the support is a reflective layer, the measurement is taken from above; Either the diffuse reflective support or the reagent region may incorporate a reference fluorophore.

Such an approach avoids the problems associated with the use of liquids in fluorometers and It is possible to use "dry" samples.

For this reason, some form of "level measuring rod" can be used. transparent plastic base The plate probably contains a fluorophore as described above with respect to cuvettes, and the substrate The strips are coated with matrix-supported reagents as is well known in the art.

To perform the test, this coated substrate piece is immersed or wiped in sample material. It is applied by well known methods such as wiping and then placed in a holder in a fluorometer. Ru. Make sure that the board pieces are in the correct position and do not touch the holder to avoid contamination. Adapters or spacing devices can be used to ensure this.

Some general guidance regarding the types of tests performed by this equipment. I will explain next. The effective analytical range must match the optimal performance range of the photodetector. Ideally, the exact analyte level (e.g. clinical action level or saturation contamination limits) should occur at the peak performance level of the instrument. excitation pulse reduce the radiation energy or optically attenuate the radiation reaching the detector. Therefore, a method with high intrinsic sensitivity is possible.

The two main types of fluorometric assays are: 1. Fluorescence increases with 1 degree of analytical tlfl. 2. The quenching method, in which the fluorescence decreases with the concentration of the analyte (at the time of quenching), It is well suited for DWF when high precision is required. This method is used to test fluorescent or a specific fluorescence is quenched to the point where instrument performance begins to deteriorate. Adjustments must be made so that a bell occurs. In the extinction method, specific radiation An optical link providing a reference radiation to the detector would not be necessary.

For assays where high levels of analyte concentration are expected or required, fluorescence enhancement is recommended. Additive is preferred. Precision at low levels is required but extinction methods are not practicable. In some assays, a fixed amount of analyte is exposed to a small amount of specific radiation during the initialization step. By incorporation into the resulting reagent, fluorescence enhancement methods can be used. This person The method is such that even at zero concentration of the analyte, the specific radiation detector is still sufficiently Guaranteed to receive luminescence.

Thus, in both the extinction method and the proportional increase method, a blank value is read out. The ratio established when (specific radiation) fluorescence / (reference radiation) fluorescence may subsequently change only due to actual changes in the analyte concentration.

international search report -M-・Sakakichi ^-*mms++, PCT/GB 8910OL42 2+n -ms drunkenness-ultcNb-+n, PCT/GB 89100142Page 　　　　2 international search report GB 8900142 SA 26930

Claims (23)

[Claims] 1. A sample that is expected to produce a first specific fluorescence emission upon excitation of the test location. flash fluorescence spectrometry, in which when performing a "blank" analysis, a reference fluorophore that is expected to produce a second characteristic reference emission upon excitation; a test position, applying a pulse of excitation radiation to the test position, and applying a pulse of excitation radiation to the test position; each detector for the first radiation and the second radiation investigated by each detector for From the relative response of the detectors, the radiation from the test position when no sample is present Determine a "blank" value for the strength ratio, and When performing a similar sample analysis, During the maintained presence of the reference fluorophore, the test location is exposed to the incident radiation upon excitation. supporting a sample expected to produce the first specific radiation; each of the detectors examines radiation from the test location; From the relative responses of the detectors, the intensity of radiation from the test location when a sample is present is determined. Determine the value of the sample for the ratio of Evaluate the "blank" value and the sample value to analyze the sample for a calibration step. Ru, A method that includes steps. 2. providing a cuvette supporting the sample at the test location; The method according to claim 1, comprising: 3. Claim 1 comprising the step of incorporating a reference fluorophore within said cuvette. The method described in Section 2. 4. A specimen for analysis as a sample and a fluorescent material to produce said specific radiation. 2. The method according to claim 1, further comprising the step of supplying a reagent capable of reacting with the method. Method. 5. During the "blank" analysis, a step supplying one of the sample and the reagent 5. The method of claim 4, comprising step. 6. providing one of the sample and the reagent with a reference fluorophore; a step of adding the other of said sample and said reagent after said analysis. The method described in item 4 of the scope of the request. 7. Limit the time between the reference and sample analyzes to maintain stable reference fluorescence emission. 2. The method of claim 1, including the step of verifying. 8. subtracting the "blank" value from the sample value to analyze the sample; The method according to claim 1, comprising: 9. During the "blank" analysis, a portion of the first radiation reaches both detectors. Claim 1 for non-quenching fluorescence analysis comprising the step of ensuring that How to put it on. 10. Ratiometric fluorescence analysis with two wavelengths. 11. Ratiometric analysis method with two wavelengths. 12. In a battery-powered instrument for fluorescence analysis of a sample, a control circuit; a flash tube and a light path from the flash tube through the sample position to the photodetector; A control circuit charges and fires the flash tube to produce appropriate radiation through the sample location. and during operation provide radiation containing the reference fluorescence to the detector for calibration. Ratiometry the specific radiation level from the sample at the sample position with respect to the positive step the output of the detector for processing by the control circuit to determine by comparison An instrument characterized by causing the occurrence of 13. 13. The instrument of claim 12, including a reference fluorophore at the sample location during operation. 14. 14. The meter of claim 13, wherein the reference fluorophore is present in the sample cuvette. vessel. 15. Sample support containing reference fluorophores for ratiometric fluorescence analysis. 16. 16. Sample support according to claim 15, in the form of a cuvette. 17. 16. The method according to claim 15, having at least one reagent in dry chemical form. Sample support part. 18. A body of material with an aperture that receives and holds the optical elements in a defined configuration. In an optical structure for a fluorometer formed of a sample aperture that holds the sample in a specific position; and a sample aperture that receives a radiation source and directs the source to the aperture. a radiation source aperture held in a specific positional relationship by a An input radiation path aperture providing a specific path between and an output radiation path aperture defined in front of the an output radiation path aperture that provides an output radiation path from the sample aperture to the radiation detector; structure to provide. 19. Claim 18, wherein the material body is all made of one piece. structure. 20. The body of material, together with each detector aperture in the radiation component path, is connected to the output path. an output radiation path aperture that receives and holds the beam splitter in a specific position in the a beam splitter aperture at and the output radiation from the beam splitter aperture output radiation component apertures defining component paths, thereby Claims wherein said body of material maintains a defined configuration of optical elements relative to said sample aperture. The structure according to item 18. 21. In a material body for a fluorometer that includes an optical path configuration, the material body is an opaque material. an aperture for receiving and retaining an optical element within the optical path arrangement; The port includes a sample opening that receives the sample and holds the sample in a specific position during use; a radiation source aperture holding the radiation source in a specific positional relationship with respect to the sample aperture; an input radiation path aperture providing a specific radiation path between the radiation source and the sample aperture; an output radiation path that defines a specific output radiation path from the sample aperture to the radiation detector aperture; an aperture, whereby the body of material around the aperture is aligned with the sample aperture. structure that maintains a defined configuration of optical elements. 22. the aperture is an optical 22. A structure according to claim 21, incorporating a control element. 23. A monolithic thermally conductive opaque material defining an aperture to the radiation source and a sample entrance. a force radiation path, a sample for inspection, and a sample output radiation path, and A surrounding material body maintains the aperture and the optical element in a defined configuration around the sample aperture. The structure of the optical core of the fluorescence measuring instrument.