JPS62115348A - Device and method of measuring luminous intensity by multiple beam - 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 This patent application is a companion application to U.S. patent application Ser. Application No. 782.635 is a related application of U.S. Patent Application No. 597.135, dated April 5, 1984, which is a related application of U.S. Patent Application No. 597.135, dated March 1, 1984. This is a related application of No. 534.

[Industrial application field]

The present invention generally relates to a photometric measuring device for measuring the density of a semi-transparent area. More particularly, the present invention relates to automatic scanning devices for analyzing the contents of a plurality of translucent samples by measuring their optical density using endpoint or dynamic measurement techniques.

[Conventional technology]

A variety of techniques and devices are commercially available for detecting and measuring substances present in a fluid sample or other translucent sample by determining its optical transmittance/). In particular, many photometric devices have the ability to simultaneously perform separate analyzes on multiple fluid samples or other translucent samples.

These devices commonly use "microplates" to collect multiple samples. This "microplate" has a standard array of wells (8 x 12), which are made of a material that is transparent to light. The optical density of a translucent sample contained in the wells of a microplate is measured by passing light through the sample, receiving the light with conventional light detection means, and determining the attenuation of the received light.

This microplate is suitable for oxygen-linked immunosorbent analysis (E
widely used in LISA) technology. This technology is used in academic research, biotechnology, and clinical testing to detect and quantify a wide range of substances and biological cells. In such an analysis, molecules of a labeled enzyme (such as alkaline phosphatase) are deposited on the bottom and part of the sides of each well of the microplate 1-. Each well is pre-interacted, directly or indirectly, with a sample containing an analyte. The number of labeled enzyme molecules in each well of the microplate is a function of the concentration of analyte in the analysis sample. Therefore, by determining the activity of the labeled enzyme, the analyte can be detected or quantified.

In determining the activity of enzymes in the liquid phase, research and clinical applications currently use dynamic analysis. This kinetic analysis measures the chromogenic reaction triggered by an enzyme in the presence of an excess of enzymatic material. It is well known that this dynamic analysis has several advantages over "endpoint analysis".

In end point analysis, an enzyme and a color-forming substance are allowed to react for a certain period of time, and then the enzyme reaction is inhibited and optical density measurement is performed once. In dynamic analysis, on the other hand, multiple measurements are taken within an initial reaction period (generally linear), and the time between each measurement is necessarily short (generally 30 seconds or less). By using dynamic analysis, (a) the initial optical density difference, (b
) errors due to loss of independence from substance concentration are substantially prevented.

Currently available automatic optical density measurement devices for microplates accommodate multiple independent sample storage locations by mechanically moving a microplate with multiple wells or by mechanically moving an optical system. Analyze the samples in sequence. This need for mechanical movement severely limits the time to measure the permeability of all wells in the microplate, and large-scale dynamic analyzes require too much sampling time. cannot be executed. For these reasons, end point analysis is performed in enzyme-linked immunosorbent analysis using conventional equipment.

Wer is a measurement method that can sequentially measure multiple sample storage locations without relative movement between the microplate and the optical system.
No. 4,4013,534 to tz et al. This U.S. patent discloses a method in which a single light source is sequentially coupled to a plurality of optical fibers by utilizing optical fiber transmission means, and the plurality of optical fibers transmit light from the light source to a measurement point. ing. but,
The apparatus described in this patent uses extremely inefficient equipment to couple light from a light source into multiple optical fibers, which poses various problems for dynamic measurements of enzyme reactions. arise. For example, the device of the above-mentioned US patent requires a high power light source, and this high intensity light adversely affects chemical reactions at the sample receiving site. This is because the operating temperature of the measuring device is raised non-uniformly and the reaction rate in each well is varied. Furthermore, the apparatus is very complex, and the signal level generated by the photodetector that receives the light that has passed through the sample storage area fluctuates greatly. For this reason, the signal amplifier of the measurement device cannot efficiently utilize its entire amplification dynamic range.

Other limitations of conventional microplate measurement devices include:
In enzyme-linked immunosorbent analysis performed using microplates with bottom filters, effective quantitative measurements cannot be made. The basic problem is that the initial optical density for each well space in the microplate is different, so that endpoint measurements are subject to significant errors.

However, dynamic analysis is not susceptible to such problems.

Another problem with conventional microplate measurement devices is that when dynamically analyzing a color reaction, the redistribution of the color product is unstable, leading to errors. This is due to phase separation or uncontrolled lump movement of the aqueous phase of the sample during dynamic analysis. That is, in the case of enzyme-linked immunosorbent analysis, the enzyme binds to some plastic surfaces at the bottom and sides of the wells of the microplate, but this bound enzyme interacts with the unstirred aqueous layer. This generally produces local phase separation of the colored products of the enzymatic reaction.

This is due to the high local concentration of color product. This phase separation causes unquantifiable errors and introduces nonlinearity into the dynamic measurements.

Even when the colored product is in true solution, unstable agglomeration of the aqueous phase results in non-uniform redistribution of the compacted product, leading to unquantifiable errors.

[Problem that the invention seeks to solve]

The main object of the present invention is to provide an automatic photometric device for dynamic concentration analysis or end point concentration analysis at multiple locations, which is capable of accurately performing a large number of measurements in a relatively short period of time. be.

Another object of the present invention is to provide a photometric measurement device that uses a single light source of relatively low power and provides reduced ambient temperature and long life.

Another object of the present invention is to provide a photometric measurement device that can easily control the processing accuracy of detection signals.

Another object of the present invention is to provide a photometric measuring device that does not require mechanical relative movement between a microplate and an optical system when continuously analyzing multiple locations.

Another object of the invention is to provide a photometric device capable of performing dynamic analysis and allowing quantitative measurement of enzymatic reactions in microplates with bottom filters.

Another object of the present invention is to provide a photometric measuring device which significantly enhances the color distribution in a sample undergoing a color reaction and eliminates measurement errors due to non-uniform distribution of color products in the color reaction.

Another object of the invention is to provide a photometric measurement device that is efficient, economical and easy to use.

[Means for solving problems]

The above objects of the invention are achieved by a photometric device according to the invention. The device includes an optical assembly. The forward assembly includes a single light source of modulated light, a filter assembly that passes only a desired band of wavelengths, and an optical system that appropriately focuses the light from the light source. Light from a light source is received by a plurality of light transmission means via an efficient light distribution means, and is separately transmitted by the plurality of light transmission means to a plurality of sample storage locations arranged on a sample plate. Ru. The optical transmission means consists of a plurality of optical fibers, which are evenly distributed around the axis of the optical fiber distributor.

An optical fiber rotor is provided to sequentially couple parallel light from the light source to each of the plurality of optical fibers disposed in the distributor. This fiber optic rotor effectively connects only one of the multiple sample receiving locations to the light source at a time.

The light passing through the wells of the sample plate is received by separate photodetectors arranged in one-to-one correspondence with the wells of the sample plate. A reference fiber is provided that connects the independent photodetector and the light source and provides a reference optical signal. This reference optical signal is compared with detection signals based on signal outputs from other photodetectors. The output signal from the photodetector is then reduced or eliminated from variations due to changes in equipment conditions such as lamp degradation and temperature effects on the photodetector. The signals from the photodetectors are appropriately multiplexed and processed by special circuitry. This special circuit configuration allows control of signal processing accuracy. The signal processing is performed on the basis of the instantaneous strength of the signal and reference measurements at the initialization of the device. Various transmittance measurements are provided externally by suitably arranging multiple optical fibers to quickly sample each specimen site and processing the resulting detected signals, which are not possible using conventional digital It is executed under the control B of the signal processing device.

Automatic stirring of the reactants in the sample wells is performed to promote color distribution resulting from the color reaction. This stirring is performed before taking measurements in each sampling period of dynamic measurements. Such a configuration ensures uniform distribution of color products resulting from chemical reactions, promotes uniformity of reactants, improves transmittance measurement accuracy, and improves overall device efficiency. Furthermore, stirring increases the pl+ equilibrium ratio of the enzyme to the substrate solution. For example, in enzyme-linked immunosorbent assays, the immobilized enzyme is washed with a wash solution (typically ρ117) before the substrate solution is added.

If the pH of the substrate solution is significantly different from the p)l of the wash solution (e.g., the commonly used substrate solution, alkaline phosphatase, is p119 or pHlO), there will be a slow phase in the color reaction due to the slow pH equilibration of the enzyme. arise. Automatic stirring of the reactants promotes pH equilibrium and significantly reduces slow phases.

Other objects, features and advantages of the invention will be explained in detail below with reference to the accompanying drawings.

〔Example〕

Although the present invention will be described based on preferred embodiments, the present invention is not limited to these preferred embodiments.

The invention includes all modifications, alternative constructions, and analogous constructions that come within the scope of the appended claims.

FIG. 1 is a schematic diagram illustrating a dynamic measurement device 10 according to an embodiment of the invention. Apparatus 10 includes an optical assembly 12 for coupling finely focused reflected light to an examination or analysis site. This optical assembly IJ 12 is
A single light source 14 of uniformly focused reflected light is provided. This light source 1
4, which in the preferred embodiment has a tungsten crystal ball, produces light at a wavelength in the range of approximately 300-900 nanometers. The output of light source 14 is passed through chopper 16 where it is modulated at a fixed frequency. This fixed frequency is 800)1z in the preferred embodiment.

The modulated light from the chopper 16 is made into parallel light by the lens 1, and is directed toward the filter disk 19.

The filter disk 19 extracts light having a selected wavelength and bandwidth, the selection being made corresponding to the wavelength at which the sample exhibits optical absorption. The light that has passed through the filter disk 19 passes through the lens 20. This lens 20 is
It receives parallel light and focuses it on a fine point 22. Light from this fine point 22 is coupled into selected fibers and transmitted to multiple analysis locations.

The dynamic measurement device 10 uses an optical coupling and transmission mechanism that is highly efficient and simple in construction.

As shown in FIG. 1, collimated light exiting optical assembly 12 is directed to optical coupling means. This means consists of a cylindrical rotor 2
Contains 4. The rotor 24 rotates about its axis and is accurately positioned. Further, the rotor 24 is
An optical fiber 26 is provided. The optical fiber 26 is the input end 2
8, the input end 28 of which is located at the center of the rotor 24 and converges with the focal point 22 of the output light from the optical assembly 12. On the other hand, the output end 30 of the optical fiber 26 is
It is located at the edge of the rotor 24. Therefore, the rotor 24
As the optical fiber rotates, the input end 28 of the optical fiber remains stationary with respect to the optical assembly 12, but the output end 30 moves in a circular trajectory. Thus, rotor 24 can efficiently couple light from optical assembly 12 to a selected one of a plurality of optical transmission means. These light transmission means transmit the light to corresponding analysis points.

Output light from optical fiber 26 within rotor 24 is received by optical fiber splitter 32 having a plurality of optical fibers 34. The input ends of these optical fibers 340 are arranged in a circle. The radius of this circular array is the same radius as the position of the output end of the optical fiber 26 of the rotor 24. Thus, as the rotor 24 is rotated for positioning about its axis, the output end of the optical fiber 26 is sequentially aligned with the optical fiber 34 of the distributor 32. The optical fiber 34 connects to the distributor 3
On the output side of 2, an optical fiber bundle 36 is formed leading to an optical fiber manifold 38. This manifold 38 is
Each optical fiber 34 is aligned with each analysis location described below.

The rotor 24 is positioned and rotated by a suitable stepper drive or controllable displacement means. For this reason, the focal point 22
The parallel light from the optical fiber bundle 36 is directed sequentially to each optical fiber of the optical fiber bundle 36.

The optical fiber coupling configuration couples the light from the optical assembly 12 to each optical fiber 34 in the distributor 32, and since the transmission efficiency of this configuration is high, the use of this configuration provides great convenience. is obtained. In other words,
Optical fiber 26 reliably couples substantially all collimated light from light source 14 to a selected optical fiber 34.

At this time, almost no light diffusion occurs at the coupling points at both ends of the optical fiber 26. Light from light source 14 is coupled through rotor 24 to only a selected one of optical fibers 34, with very little undesired optical coupling to optical fibers adjacent to the selected one. In this way, a single light source of relatively low power can be used because of the highly efficient coupling. This is because most of the light emitted from the light source is transmitted to the measurement location.

Although the light source shown in FIG. 1 is a preferred form, any light source that can provide light with uniform intensity and a desired wavelength range may be used.
Any single or multiple light sources may be used. In this connection, alternative methods of photometric measurement will be described below with reference to FIGS. 8-13. In this alternative, multiple light sources are operated sequentially.

At the measurement location, light emitted from each optical fiber 34 of fiber optic manifold 38 passes through lens array 42 to a respective analysis location on sample plate 40 .

Lens array 42 is positioned between manifold 38 and sample plate 40.

Sample plate 40 is a conventional microplate with a series of wells. This series of wells typically consists of 8 rows, each row having 12 wells, for a total of 96 wells. The sample plate 40 is preferably mounted at a location under isothermal conditions, with the ambient temperature controlled using a fan or the like. This is important in order to prevent a temperature gradient on the sample plate, since the reaction rate will vary depending on the sample location, which will reduce accuracy.

The photodetectors 44 are provided on a detector board 46 and arranged in a shape that corresponds to each position of the wells placed on the sample plate 1-40. A second lens array 48 is positioned below the sample plate 40 and focuses the light onto each well of the sample plate 40. Lens arrays 42 and 48 are of conventional type and include gaps (not shown). This gap serves to direct light to each lens and reduce the spread of light to adjacent lenses. The sample plate 40, or at least the bottom of each well within the sample plate, is translucent or transparent. The light is thereby coupled into a particular optical fiber within the fiber optic manifold 38 and paralleled into the corresponding sample well;
It can be transmitted through the well and its contents, through a corresponding focusing lens located in lens array 48, and to a corresponding photodetector 44 located directly below the sample well.

Each photodetector 44 detects the intensity of light passing through its corresponding sample well and generates an electrical output signal proportional to the light intensity. Each photodetector 44 on detector board 46 functions in a similar manner, providing a signal that is proportional to changes in the intensity of light impinging on each photodetector. The light intensity changes as the transmittance of the sample changes in accordance with the progress of chemical reactions in the sample and in accordance with changes in the components of the sample. The electrical signal from photodetector 44 is sent to analysis and display device 50. This device 50 processes the received signals and processes the sample play 1-40.
The transmittance or optical 4 degrees of each sample contained in the plurality of wells is externally displayed.

The measuring device shown in FIG. 1 includes one reference fiber 52. The measuring device shown in FIG. This reference fiber 52 is placed adjacent to the focal point 22 and continuously receives light each time the light source 14 is activated, ie each time any of the sample locations are examined. Light emitted from the output end of reference fiber 52 is coupled through the air or empty sample well to a separate photodetector 45 on detector board 46 to provide a reference photodetector signal. The meaning of this reference signal will be described in detail later.

The optical fiber distributor 32 has one optical fiber called the home fiber. This home fiber serves as a reference for determining the current position of distributor 32 relative to rotor 24. The details of the home fiber function will be described later.

A color reaction occurs in the sample to be measured, and in order to promote uniform distribution of color caused by this reaction, the measuring device shown in FIG. Means are provided for stirring the chemical solution. That is, as shown in FIG. 1, the sample plate 40 is
It is attached to the stirring mechanism 54. This stirring mechanism 54 is
The sample plate 40 is vibrated to thoroughly mix the chemical solutions contained in the sample wells.

When performing dynamic analysis of enzyme-linked immunosorbent analytes using conventional microplate measurements, a common problem and problem is that the color distribution generated as the color reaction develops is not uniform across the width of the sample well. . Dynamic measurement of optical density is
Since the transmission of light passing through the central portion of the sample well is utilized, non-uniform distribution of color, for example due to color development on the walls or corners of the sample well, significantly reduces the accuracy of the measurement of light transmittance. If the color distribution in the sample is non-uniform, the measured values for each well will vary even if the same chemical reaction is occurring in each well, and the reliability of the transmittance measurement will be lost.

Some conventional measurement techniques suggest manually shaking the microplate before reading. However, dynamically measured optical density, which originally varies linearly with time, undergoes unpredictable and non-reproducible changes because the color diffuses arbitrarily as the reaction progresses.

That is, the transmittance of the sample becomes dependent on the non-uniform color distribution that develops in the sample. This non-regenerative change is
This cannot be overcome by vibrating the microplate at the beginning of the dynamic measurement cycle. This is because, unlike conventional end point measurement in which the reaction is suppressed before measurement, in dynamic measurement the optical density is measured while the progress of the reaction is not suppressed. Color continues to be generated as the reaction progresses, so even if some degree of linearity is achieved by applying vibration at the beginning, the reaction will continue. 8? It is insufficient to provide a uniform distribution of color throughout &, resulting in low measurement accuracy.

In accordance with the present invention, in multiple readings during a dynamic measurement cycle, stirring the reactants immediately before each reading ensures uniform distribution of the color developed throughout the sample where the chemical reaction is progressing, ensuring measurement accuracy. was found to be significantly improved. In the embodiment shown in FIG. 1, a uniform distribution of color is achieved by shaking the entire microplate before each measurement. This vibration agitates the reactants in each sample well and prevents local separation of dye products due to enzymatic reactions. In this way, the variation in the transmittance of the sample within the sample well is maintained approximately linear with time throughout the dynamic measurement cycle.

In the embodiment shown in FIG. 1, sample plate 40 is connected to a stirring mechanism 54. In the embodiment shown in FIG. This stirring mechanism 54 includes a motor configuration for applying gentle vibrations of a desired period to the sample plate 40. Based on the preferred embodiment, the stirring mechanism 5
4 utilizes a reversible motor used to control the position of the sample plate and vibrates the sample plate by repeatedly rotating this motor in both directions at a desired rate of vibration. Approximately 17 at a frequency of approximately 2011z
Vibrating the sample plate over a distance of 16 inches gives satisfactory results. The measuring device is programmed to vibrate the sample plate before each reading. A short time delay is placed after this oscillation and before reading. This is to calm down the reaction solution in the sample.

This delay time prevents errors in reading from occurring due to reflection or refraction caused by waves generated during the stirring process. Typical time intervals are 3 seconds for agitation and 1 second for delay.

Stirring does not necessarily have to be performed by mechanical means, as long as the purpose of achieving uniform color distribution is achieved. Other suitable agitation means such as ultrasonic vibrations may also be used. These means are used depending on the desired efficiency, the composition of the reactants, etc. Furthermore, the vibratory motion of the sample plate is not limited to horizontal motion parallel to the plane of the sample plate or rotational motion; longitudinal vibration (vertical motion) can also be used as long as leakage of the sample is prevented.

FIG. 2 shows details of the fiber optic manifold 38 and the placement of each optical fiber on the underside of the manifold. This example is designed to sequentially analyze samples contained in a conventional microplate with 96 test sites or wells arranged in 12x8 columns. therefore,
As shown in FIG. 2, fiber optic manifold 38 has a total of 96 optical fibers. These optical fibers are A
.

They are arranged in 8 rows of B, C, D, E, F, G, H, and each row has 12 optical fibers. For example, row A has 12 optical fibers.
．． It has 12 optical fibers from A3 to A12.

The output end of the reference fiber 52 and the output end of the home fiber are arranged in the manifold 38 . 1 of A-H
The arrangement of the two rows and the arrangement of the gaps between adjacent optical fibers correspond exactly to the arrangement of the sample locations in the sample plate used for analysis. Therefore, the light that has passed through a particular optical fiber is directly parallel incident on the corresponding sample well, transmitted through the sample, and reaches the corresponding photodetector, where the intensity of the light incident on this photodetector is Generating an electrical signal having a proportional magnitude.

FIG. 3 is a schematic diagram showing an optical fiber distributor 32,
The input ends of each optical fiber disposed within optical fiber manifold 38 are shown arranged in a circular manner. As shown in this figure, the distributor 32 is connected to a fiber optic manifold 38.
Each input end of 98 optical fibers is disposed within the optical fiber. These 98 optical fibers are arranged in a circle in the distributor 32. The input end of the home fiber is located at a location preceding the first A row of optical fiber rows.

Three opaque points connect the home fiber and the first fiber A
It is located between 1 and 1. This fiber A1 corresponds to the sample location to be analyzed first. The opacity point is referenced at the start of a new analysis process, as described below.

FIG. 4 is a block diagram illustrating the processing circuitry used to analyze the signal generated by photodetector 44. In the illustrated embodiment, detector board 46 actually includes 98
Of these, 96 photodetectors correspond to the 96 sample locations on the sample plate 40 (Fig. 1), one receives light from the reference fiber 52, and the remaining One of them receives light from the home fiber.

If a single optical fiber serves both optical reference and home reference functions, only one reference detector is required. The signals generated by the 96 photodetectors for each sample location are converted to voltages corresponding to the signals by current/voltage converter 11O. These signals are then passed from band-limiting low-pass filter 111 to multiplexer 112 . Multiplexer 112 controls selection of a desired one of the 96 signals according to a preprogrammed order, and the selected signal is then further analyzed.

The signals generated in response to the light from the reference fiber 52 and the light from the home fiber are converted into equivalent voltages by current/voltage converters 113 and 114, respectively, and passed through band-limiting low-pass filters 115 and 116. be done.

The output of the multiplexer 112 and the two low-pass filters 1
15 and 116 are the outputs of the second multiplexer 1
18, where one of these three outputs is controlled and selected, and the selected signal is then further analyzed.

The home fiber is connected to the optical fiber distributor 32.
A suitable means for determining the angular position of optical fiber 26 within rotor 24 is provided. In other words, the home fiber serves to ensure accurate positioning of the rotor 24, to direct the output light from the coupling optical fiber 26 to the appropriate optical fiber in the distributor 32, and to initiate the analytical sequence. .

Since all light entering the home fiber is transmitted directly to a dedicated home photodetector 44, this home photodetector 44
If a peak signal is present in the output of the rotor 24, the rotor 24 is located at the so-called home position, and in this position the coupling optical fiber 26 is aligned with the home fiber.

Each time a new analysis is started, the measuring device first positions the rotor 24 in the home position. To do this,
While monitoring the output of the home photodetector, rotate the rotor 2 until a peak output signal is detected from this home photodetector.
Rotate 4. Since the position of the home fiber with respect to the first optical fiber AI is a known function (in this example, separated by three opaque points), the coupling fiber 26 and Alignment with fiber AI is easily accomplished by moving rotor 24 three consecutive positions.

The reference fiber 52 acts as a calibration means, whereby only the difference between each measurement of the detected light intensity is used to analyze the transmittance of the sample, thereby accounting for variations in the light output of the light source and variations in the instrument. The effect is removed. That is, each time an analysis is performed, the signal generated by the reference fiber is measured before the start of the read cycle. All signals generated by the photodetector for each analysis site are then measured relative to the signal originally generated by the reference fiber.

In this way, only the actual change in the transmittance of the liquid sample as a result of the chemical reaction occurring at the sample location is measured. Over the life of a light source, its light intensity will change, but local differences in light intensity due to these changes and readings taken before the light source or other equipment has stabilized are ignored.

Since the home fiber is directly coupled to its corresponding photodetector, the home fiber can be used as a reference optical readout source, and as described above, the fiber rotor 24 is connected to the fiber splitter 32. In addition to providing a reference for positioning, all functions of a reference fiber can be effectively provided.

Returning to FIG. 4, the signal selected by multiplexer 118 is passed through a conventional bandpass filter 119. This bandpass filter 119 has a reasonably sized band approximately centered at the frequency at which chopper 16 modulates the light passing through it (eg, 800 Hz). The bandwidth of bandpass filter 119 is selected to be large enough to accommodate variations in signal settling time caused as a result of the high speed readings performed on the sample plate. The band limited output from filter 119 is sent to variable gain amplifier 120. The function of this amplifier will be described later.

The output of amplifier 120 is sent to a two-way switch 122. This switch 122 is normally in the closed position and provides a direct electrical connection from the amplifier 120 to a subsequent portion of the processing circuit. When the two-way switch 122 assumes the open position,
Said part of the measuring device is disconnected and the switch 122 serves to effect a virtual short circuit and to take measurements only of the signal generated by the remaining part of the processing circuitry. The switch 122 therefore serves as a means for determining the dark current, which is the amount of light that passes through the fiber optic splitter 32.
If the signal does not pass through at all, it flows through the processing circuitry. This dark current measurement is not affected by noise or distortion from previous parts of the two-way switch 122. This is because the part of the processing circuitry that measures the dark current is completely isolated from the rest of the measurement circuitry. It makes sense to take such readings while the circuit is isolated from the light source. This will be discussed later.

The output signal from two-way switch 122 passes through high pass filter 124. This high-pass filter 124, together with the low-pass filters 111, 115, 116, provides a second band characteristic for the processing circuitry. High pass filter 12
The output of 4 is sent to phase detector 126.

This phase extractor 126, together with the reference input from the light source chopper 16 and the low pass filter 128, functions to extract a DC signal corresponding to the AC due to the modulation effect of the chopper 16. That is, the phase detector 126 directs the negative portion of the AC output signal from the detector board 46 to the chopper 16.
is inverted based on the timing input provided by . The average value of this resulting signal represents the DC equivalent of an AC signal. To extract this average value, the output of phase detector 126 is passed through a low pass filter 128.

The output signal from low pass filter 128 is sent to a second variable gain amplifier 130. This amplifier is a precision amplifier to provide a good range of gain settings. The variable gain amplifier 130 is the first variable gain amplifier 120
In conjunction with this, the processing circuitry of the present invention provides controllable precision and high dynamic range, as will be discussed below. The output of the amplifier 130 is passed through the low-pass filter 13.
2 and sent to the AD converter 134. The AD converter 134 functions to convert the amplified analog signal into a corresponding digital signal. The digital signal generated by AD converter 134 is sent to a conventional digital microprocessor device. The microprocessor device performs a series of mathematical calculations and comparisons to determine the optical density of the sample based on a predetermined algorithm. The microprocessor device also performs sequential scanning of multiple test locations;
Provides control of different multiplexing arrangements and all associated processing circuitry.

Based on one of the features of the invention, the measuring device can be made more efficient and economical by effectively increasing the dynamic range of the AD converter. The AI) converter is used to represent an analog signal that defines the processed value of the various signals generated by the photodetector. Variable gain amplifiers 120 and 130 adjust the range over which the processed signal is amplified, since a significant portion of the quantization level of the AD converter also changes over a wide amplification range. , done in such a way that it is used without exceeding its fundamental dynamic range. The actual operation of these amplifiers is that the AD converter 134 shown in FIG.
2-bit capacity, that is, the quantization output level is 0 to 409
The explanation will be made assuming that the number is 4096 in the range of 5. When the light emitted from the light source is extracted by the filter disk 19, as the wavelength of the light changes, the intensity of the light also changes. This is a signal that varies in amplitude over a wide range, along with changes in the light transmission properties from one optical fiber to another, changes in the frequency response characteristics of the photodetector itself, and changes in transmittance due to chemical changes in the sample. to occur.

In order to prevent the signal with the maximum amplitude from having an output value that exceeds the range of the AD converter, a low gain amplifier is conventionally used. However, a drawback of such a configuration is that when the intensity of the light received by the photodetector generates a relatively weak detection signal, the low gain amplifier generates a low level digital output, which reduces the dynamic output of the AD converter. This resulted in extremely inefficient use of the microwave. In such cases, ADs with high bit processing performance have conventionally been used.
This problem has been addressed by upgrading the equipment by using a converter, but this significantly increases the cost of the entire equipment.

In accordance with the present invention, variable gain amplifiers 120 and 130
First, process the input signal at a very low gain setting. The resulting digital output is compared with the maximum value of the AD converter output to determine the maximum gain for which the input signal is processed without exceeding the maximum digital output value of the AD converter.

For example, if the input signal produces an AD converter output of approximately 50 counts at the initial gain setting (usually 1), the device will output this count value and the maximum count value of the AD converter (4095 for a 12-bit AD converter). ) and
The digital output falls within the maximum count value to determine the range within which the signal can be safely amplified.

A safety factor of approximately 10% is incorporated into this dynamic range processing. This is achieved by not basing the comparison on the maximum output value of the AD converter, but comparing it with approximately 90% of its maximum output. For a 12-pin 1-AD converter, the actual 50 counts are divided into 3686 counts, which is the maximum output considering the safety factor, which is approximately 7
A desired amplification factor of 4 is obtained. After this decision has been made,
The gain settings of the variable gain amplifier are adjusted to approximate the desired gain factor. The configuration therefore allows maximum use of the dynamic range of the measuring device, regardless of changes in the relative strength of the signals generated by the photodetector.

According to the invention, the gain G1 of the first variable gain amplifier 120 is adjusted based on the signal obtained from the light transmitted through space. On the other hand, the gain G2 of the second variable gain amplifier 130 is adjusted based on the signal obtained from the photodetector at the sample location. The actual operating sequence of gain setting adjustment for the variable gain amplifier and the operating method of the entire measuring device will be explained with reference to FIG.

FIG. 5 is a flowchart illustrating the general sequence of operations used in a typical sequential scan based on the apparatus of the present invention. The sequence begins at step 150, where various device variables are initialized, such as the number of reads performed within a dynamic read cycle, the agitation time, and the post-agitation delay time. Spatial calibration is performed at step 152 by reading each empty sample well. The measurements made during the spatial calibration stage are performed using the second variable gain amplifier 130.
The gain G1 of the first variable gain amplifier 120 is optimized for the maximum dynamic range of the AD converter. Details of the various steps and measurements performed in the spatial calibration step 152 are discussed below.

Spatial calibration step 152 is followed by step 154, where dynamic read recycling is performed in step 150.
The measurement is started based on the initialization data given to the measuring device. In this dynamic read cycle, agitation, delay, and reading are performed at each discrete preprogrammed time interval.

In each of these time intervals, for a particular measurement sample,
An optical density reading is taken. For conventional endpoint analysis, the steps are performed only in a single time interval during a read cycle.

Step 156 includes a series of three steps, beginning with an agitation step 158. During this stirring step, the sample plate is vibrated for a predetermined period of time. Next, in step 160, a settling phase is performed for a predetermined delay time during which the vibration mechanism is stopped and the reactants in all sample wells of the sample plate are allowed to settle for a predetermined time interval. , then the actual signal reading is performed. In step 162, the measurement device takes transmission readings for all wells of the sample plate. In this step, the gain setting G2 of the second variable gain amplifier 130 is optimized, while the gain setting G1 of the first variable gain amplifier 120 is maintained at the optimal value determined during spatial calibration. Details of the procedure of reading step 162 will be described later.

After the read step 162, a check is made in step 164 as to whether the device has completed a predetermined dynamic read cycle. If the result of step 164 is No, step 166 continues the dynamic read cycle. The measurement device repeats the reading step 156 at each predetermined time interval of the dynamic reading cycle until the stirring step and the associated delay step and reading step are performed.
In step 164, the measurement device is stopped once the readings at each predetermined time interval are completed. This marks the end of the dynamic read cycle.

The read recycling described above is performed by the illustrated photometric device to obtain the various optical readings necessary to calculate the optical density at a sample location. A microprocessor device, forming part of the analysis and display device shown in FIG. It calculates based on an algorithm and calculates the time interval for performing the desired measurement.

The following definitions and symbols will be used to describe the various operations performed by the measuring device of the invention during the spatial calibration and reading stages.

ODn: Calculated optical density of a given sample well (n is 1
~96, designating each of the 12 sample wells each placed along each of the eight columns A-H of the microplate).

Wn: the output signal of the photodetector corresponding to a given sample well for containing the reaction sample.

G1: Adjustable gain of the first variable gain amplifier 120 (controllable by multiplication factors including 1, 2, 4, 8, 16, 32, 64, 128).

G2: Adjustable gain of second variable gain amplifier 130 (controllable by multiplication factor including 1, 10, 100)
.

Dn: Dark current reading in the open position of two-way switch 122 shown in FIG. 4 for a given sample well. This reading is obtained with the first gain setting G1 equal to 1 and the same second gain setting G2 used to obtain the corresponding signal output Wn.

W, AlRn: Signal reading in spatial calibration of a given sample well, obtained with the gain setting of the second variable gain amplifier G2=1.

Dair: Spatial calibration dark current reading obtained with second gain setting 02=1.

L, REFair: Reference signal reading obtained during spatial calibration with second gain setting 02=1.

L, REFread: Set the second variable gain setting to 02
Reference signal obtained at the start of read recycling as =1.

Dread: Dark current reading taken at the beginning of a read cycle.

FIG. 6 is a flowchart illustrating the sequence of operations involved in the spatial calibration step shown in FIG. first step 20
At 0, the measuring device positions the rotor 24 in the home position. This is done by sequentially displacing the rotor to a position where a peak signal is detected from the output of the home photodetector. At this stage, the first
And the gain settings of the second variable gain amplifiers 120 and 130 are set to 1.

The next step in the spatial calibration sequence is step 202, where the measurement device switches to the photodetector H for the reference fiber or the photodetector H for the home fiber and measures the optical reference signal REFair. This reading is performed by setting the gain G2 of the second variable gain amplifier to 1 and optimizing the gain G1 of the first variable gain amplifier to obtain the maximum count value at the output of the AD converter 134 (as described above). (following a safety factor adjusted dynamic range procedure). Also, in step 202, the measured reference signal value REFair and the optimized first gain setting G I L, air are stored in the memory of the microprocessor device and used to calculate the optical density. .

In the next step 204, the rotor is displaced a predetermined number of positions relative to the home position and the fiber optic distributor 32
Upper opaque point X1. X2. It is moved to a position corresponding to one of X3. According to the preferred embodiment, the rotor is displaced two positions relative to the home position and comes to rest at a position corresponding to the opaque point X2. In this position, the opacity point prevents the coupling of light from the coupling optical fiber 26 to all optical fibers in the fiber optic manifold 32;
Isolate the photodetector from the light source.

In the next step 206, the two-way switch 122 is activated and the dark current is read Dair. At this time, the first and second gains Gl and G2 are set to 1. The dark current Dair reading is representative of the residual current flowing in the portion of the processing circuitry that follows the two-way switch 122 in FIG.

This value is subtracted from the signal reading for each sample well so that it represents the actual value of transmittance for the sample well at the specified time. Furthermore, in step 206,
The measured dark current reading Dair is stored in the device's memory and used to calculate the optical density.

At this stage, the measurement device is ready to perform spatial calibration readings of each sample well. Therefore, step 2
At 08, the rotor 24 advances to position A1 corresponding to the first sample well of the sample plate, and the photodetector corresponding to sample well AI is turned on. In the next step 210, the spatial calibration signal value for sample well Al−, Al
Rn is read. At this time, the gain G2 of the second variable gain amplifier is set to 1, and the gain G1 of the first variable gain amplifier is optimized to the value GIAI. This GIAI
represents a gain setting value that can obtain the maximum output of the AD converter in consideration of the safety factor within a range that does not exceed the rated dynamic range of the AD converter. Step 210
At the end of , the measured signal reading W, AlRn (n = A I in this example) and the optimized gain setting GJAI
is stored in memory.

In step 212, the microprocessor device:
Check whether spatial calibration was performed in each of the 96 sample wells on the sample plate. Step 21
If 2 is No, step 214 advances the rotor to a position corresponding to a subsequent sample well before returning to spatial calibration step 210. Step 212 is YES, that is 9
If spatial calibration has been performed in six sample wells, the spatial calibration sequence ends at step 216.

The entire spatial calibration sequence consists of the sample plate retracted position,
That is, at a location remote from the photodetector board, the light from the fiber optic manifold 38 is transmitted directly to the photodetector.

FIG. 7 is a flowchart showing the operational sequence of the reading stage by the measuring device. It should be noted that before taking actual readings on a given sample plate, the measurement device performs a spatial calibration step while leaving the sample plate in the retracted position. Prior to taking a reading, the sample plate is advanced and prepared for the reading step.

Step 300 of FIG. 7 is the beginning of the reading phase, in which the measuring device positions the rotor in the home position. For this purpose, the signal from the photodetector for the home fiber is tracked by the processing circuitry, with the gains Gl, G2 of the first and second variable gain amplifiers both set to 1.

Next, the process advances to step 302. The reference signal is now measured. In other words, the measurement device switches to the photodetector corresponding to the reference fiber (if the home fiber also functions as the reference fiber, the photodetector corresponding to the home fiber), and measures the optical reference signal REFread. do.

At this time, G2 is 1, and Gl is optimized to the maximum value GIL,air based on the dynamic range procedure described above. As part of step 302, the measured or
The REFread value is stored in the device's memory and used to calculate optical density.

In the next step 304, the rotor 24 is displaced by a specified number of positions and the three opaque points X1 . X2. It is located in one of X3.

In this example, the rotor 24 is moved two positions relative to the home position and is located at the second opaque point X2.

In the next step 306, the two-way switch 122 is activated and a series of dark currents Dair,x are measured. At this time, the first variable gain Gl is set to 1. Second
One reading is taken at each of the possible gain settings of G2 of the variable gain amplifier (t, io, 1oo in this example). The measured value of Dair,x is determined in step 30
6 in the device's memory.

In the next step 308, the rotor 24 advances to the first sample well location Al. The device also switches to the photodetector corresponding to the first sample well A1 and begins the actual sequential read recycling.

In the next step 310, a stirring mechanism is activated for a predetermined time interval TI to promote uniform color distribution as described above.

Following the agitation phase of step 310 is the calming phase of step 312. During this calming phase, the stirring mechanism is stopped and the device is stationary for a time interval T2, allowing the stirred sample to settle and become ready for transmittance readings. The agitation step of step 310 moves and vibrates the sample plate between the fiber optic manifold and the photodetector board.

The calming step is therefore carried out during the time during which the sample plate is moved from the stirring position to the reading position. Immediately after the stirring phase and subsequent settling phase of the reaction sample in the sample plate, optical density readings are taken.

In step 314, a measurement of the signal WAn for the first sample well A1 is made. At this time, G1 is set to the gain value GIAI determined and stored as part of the spatial calibration in step 210 of FIG. Optimized to a value such that a regulated maximum output is generated. Also, as part of step 314, the measured signal value WAl is stored in instrument memory and used to calculate the optical density for the sample well.

In the next step 316, the microprocessor device checks whether signal readings have been taken for all 96 sample wells. If step 316 is No, then
The microprocessor device advances the rotor to a position corresponding to the next sample well.

At the same time, the processing circuitry switches to monitor the photodetector corresponding to the selected sample well.

Step 310 is then returned to and the agitation, sedation, and read steps 310, 312, and 314 are performed again. These three steps are repeated until step 316 is YES and all signal readings for the sample wells are completed. This ends the read recycling in step 320.

According to the above description, for a given sample well denoted by n, the gain G of the first variable gain amplifier 120
It can be seen that ln is determined separately during the spatial calibration phase and then this value is kept constant during dynamic read recycling. The value of G1 is not adjusted until the next spatial calibration step. The gain G2n of the second variable gain amplifier 130 is set to 1 for spatial calibration and optical reference readings, so the dynamic range of the second amplifier 130 is only used when the actual transmission readings are taken, and the spatial Not used for calibration readings.

Once the optical parameters defined and described in connection with FIGS. 6 and 7 have been obtained by the measuring device for a given sample well, the calculation of the optical density ODn of the reaction sample contained in this sample well is as follows. It is done like this.

00,,=LOG+. ((W,AIR,1-D,i,)
/(-7-〇,) book (L, REFr--a D,
--a) /(L, REFair Da+r) book (G2.)) The above formula represents the logarithm value of the product of three independent quantities. 1st
Amount of (W, AIRn-Dair) / (Wn-On)
is the ratio of adjusted signal readings for a given sample well (1) without sample and (1) with sample. All readings taken by the microprocessor device's processing circuitry are adjusted for offset voltages generated by the AD converter or other system offsets and drifts. At this time, the corresponding dark current reading is taken into account. These adjustments are as shown in the above equation for ODn. For example, the signal reading W
.

AlRn is normalized for dark current effects by subtracting the Dair of the corresponding dark current reading. Similarly, this signal reading Wn is adjusted by subtracting the corresponding dark current reading Dn.

Second quantity (L, REFread-Dread) / (
L, REFair-Dair) is the ratio of the optical reference readings for a given sample well obtained during the spatial calibration stage and the reading stage. These two readings are also normalized based on the corresponding dark current reading.

The third 1l(G2n) in the above equation takes into account the effect of the dynamic range procedure described above.

In other words, this quantity neutralizes the amplification effect of the signal reading by the processing circuitry.

Applying the above equation to the parameters measured during dynamic read recycling provides highly accurate optical density measurements. This means that the above equation accounts for effects due to instrument offset voltage, effects due to local differences in light intensity, effects of measurement conditions from one dynamic read cycle to another, and changes from one sample well to another. This is because the influence of measurement conditions on the sample well is taken into account.

In accordance with another feature of the invention, the logarithmic operations required to calculate the optical density readings are performed by storing all necessary logarithmic values in the microprocessor unit in the form of a look-up table and using the digital output of the processing circuitry. used as an indicator,
This can be done by searching for the appropriate logarithm value. In conventional equipment, the output signal of the photodetector on the photodetector board is
It was sent to a logarithmic amplifier, where the logarithmic value of the output signal was obtained. This technique has many problems and limitations.

This is because the device always needs to be adjusted for the offset gain of the logarithmic amplifier. There is also a need to accurately track and appropriately compensate for temperature drift in the computing device to maintain computational accuracy. According to the invention, the calculation of logarithmic values is performed with substantially higher precision and is obtained independently of the parameters of the device. This is accomplished by storing in the memory of the microprocessor device all the logarithmic values required by the device and calculating the optical density readings.

That is, the lookup table has logarithmic values corresponding to each output quantization level for the AD converter of the device shown in FIG. For example, for a 12-bit AD converter, possible quantization levels range from 0 to 4095. This means that there are 4096 different values, and when a signal is detected, processed, and digitized, it will have one of these values. 409
The logarithm values corresponding to each of the six values are stored in a logarithm lookup table. This table is contained in the ROM portion of the microprocessor device. A logarithmic lookup table is defined in such a way that the digital output from the AD converter becomes an address or index to the corresponding logarithmic value stored in the lookup table.

The lookup table is stored in the memory of the microprocessor device, so optical density calculations use the digital output, extract the corresponding logarithmic value stored in the lookup table, and perform simple mathematical subtraction. This can be easily done by executing

In another feature of the invention, the process of selecting a particular photodetector from a plurality of photodetectors on a photodetector board is facilitated. This is achieved using a hierarchical parallel addressing method for the photodetectors. That is, the photodetectors are divided into a selected number of blocks, each block having multiple photodetectors. Corresponding photodetectors within each block are connected in parallel. For example, when the first photodetector of a first block is addressed, the measurement device automatically addresses the first photodetectors of the remaining blocks.

For example, considering a microplate with 96 wells, the photodetector board comprises 97 photodetectors (96 for the sample wells and one for the home reference and light reference). In accordance with a preferred embodiment of the invention, the 96 sample well photodetectors are divided into blocks of 16. The first block includes photodetectors A1 to A4. B1-B4. C1-C
4° D1 to D4, and the second block is a photodetector A5.
~A8,135~B8. C5-C8, D5-D8, and the third block includes photodetectors A9-A12, 89-B1
2. C9-C12, D9-DI2, and the fourth block includes photodetectors E1-E4. Fl~F4. Gl~G4. H
1 to H4, and the fifth block includes photodetectors E5 to E8.
, F5~F8° C5~C8, H5~H8, and the sixth
The blocks are photodetectors E9~E12D, F9~F
12,09~G12. H9-I]12. In such a block configuration, the first photodetector of every block, namely AI, A5, A9, El,
E5.

E9 is connected in parallel and the other remaining photodetectors are connected in a similar manner.

7Dressing to each photodetector is greatly simplified. This is because the same address applies to any selection of the six photodetectors. Even if six photodetectors are turned on each time, only one of them is actually monitored. The photodetectors in each block are spaced far enough apart on the photodetector board that a desired photodetector is affected by scattered light from the other five photodetectors in the block. Never.

A preferred embodiment of a high-speed solid-state multi-beam photometer for microanalysis at multiple locations will be described with reference to FIGS. 8 to 13. This device is capable of performing a large number of readings in a short period of time, without mechanically moving the optical system, the microplate, or the sample to be analyzed at multiple locations. Multiple light sources are arranged so that each separate light source illuminates each sample well or sample location. At least one monolithic photodetector is used so that light coming from different analysis points can be detected at different points on the surface of the photodetector. The light sources are turned on sequentially so that only one light source emits light at a time. The signal detected by the photodetector corresponds to the particular sample well under the light source that is turned on at this time.

In the preferred embodiment, 96 light emitting diodes (LEDs)
) are arranged over 96 wells on a microplate or 96 sample locations on a microplate filter. At least one silicon photodetector is placed below the microplate or filter. This photodetector has an area corresponding to the area of a plurality of wells on a microplate. Connection lines are soldered to the top layer of the photodetector. These lines are arranged in a cross-shape corresponding to the areas between the sample locations so as not to obstruct the light.

When a large photodetector is used, there is a problem of increased noise. This noise can be overcome by a number of properties. Using brighter LEDs increases the signal strength against noise. The main noise signal is the 6011z line power signal.

The effect of this noise signal can be addressed by taking 10 readings for each well, each reading synchronized to 36° from the previous reading in a phase space of 60 tlz. For these 10 readings, the time between readings is:

(10n+1)/600 seconds, where n is an integer. Thus, the 6011z noise encountered in one reading, averaged over 10 readings, is canceled by the noise encountered in other readings in opposite phase.

To reduce noise, do not leave the light source on all the time, but use an LED.
It can be reduced by sequentially turning on at high cycles. Next, a filter is used to remove noise that occurs at a frequency equal to or higher than the cycle of sequentially turning on the LEDs. Measurement is done at the LED O
This high frequency is performed for both the N and OFF states.

Measurements in the OFF state provide a reference level corresponding to noise within the device at frequencies lower than the sequential ON period. Since the measurement in the OFF state is performed immediately after the measurement in the ON state, the noise level is approximately the same in both states and cancels out.

A disadvantage of using LEDs and large photodetectors is that their properties vary with temperature. These properties can change over a period of minutes. AH, which uses a tungsten light source and a small photodetector, does not suffer from the same magnitude of variation problem. The variation problem can be overcome by the following steps.

First, a reading is taken without inserting the sample to obtain a light signal value corresponding to the light transmission through the space between the LED and the photodetector. Immediately after this, the microplate is filled with water (blank plate), buffer, or other base solution in which the chemical reaction takes place, and a second measurement is performed. Alternatively, a second measurement is performed on a blank filter. This provides a blank optical signal level. This signal level corresponds to light transmission through the blank plate. In sequential readings of a chemical sample, each reading is immediately preceded by a second reading through space. Next, the chemical sample reading and the blank reading are compared, and the difference between the two spatial readings is further adjusted. Differences in readings through space are due to variations in photodetector and LED characteristics due to temperature. Using spatial readings eliminates the need to insert a blank reading before each sample.

FIG. 8 is a perspective view of a preferred embodiment of a multi-beam photometry device according to the present invention. The optical measurement device 60 includes a circuit board 70, which will be described in detail below, a monolithic photodetector 80, an LED array 90, and a drawer-type sample holder 100. Microplate or sample plate 140 is typically placed within an opaque mask 160. This opaque mask 160 covers the sample plate 140.
well walls while allowing light to pass from the top to the bottom of each sample well or vice versa. This assembly is placed within the open container 180 of the sample holder 100. In this measuring device, when the sample holder 100 is returned to the closed position, a microswitch is activated, indicating that the sample can be measured.

FIG. 9 is a side cross-sectional view of a sample plate well inserted into mask 160 and positioned between photodetector 80 and LED array 90. Although not shown, a microplate configuration filter can also be placed within the holder 100 to perform measurements. This holder 100 isolates each sample site from each other while allowing light to pass through each sample site.

FIG. 10 is a block diagram showing an electronic control circuit configuration based on the present invention. The central processing unit (CPU) 400 is
This circuit is controlled, and for each LED that is lit,
Data signals are sent on data bus 420 to decoders 440 and 460. The decoded signals are routed to current drive array 50 via the appropriate lines of buses 480 and 520.
0 and current sink array 540, respectively. This signal also travels through drive bus 560 and sink bus 600 to activate a particular LED in LED array 90 . The light passes through sample 140 to photodetector 80 .

The signal from photodetector 80 is transferred to current/voltage converter 660, sample and hold circuit 680, and AD converter 700.
CP (J400
and will be analyzed there.

CPU 400 is a standard CPU board, including a microprocessor, random access memory (RAM), and read only memory (ROM), such as a Microsystems 5323. When this is operated and a specific LED is turned on, a digital code corresponding to this LED is transmitted to the decoder 4 along the data bus 420.
40 and 460. Decoder 440 decodes the data portion that indicates the correct column of LEDs to be lit and energizes the appropriate line of data bus 480 to current drive array 500. The decoder 460 is
The data portion corresponding to the appropriate row of LE', D to be lit is decoded 1 and the data line of data bus 520 connected to current sink array 540 is energized. next,
The current is passed through the appropriate lines of the current drive bus 560;
It passes through the appropriate diodes of 90% of the 8x12 LED array and through the energized lines of sink bus 600 to the corresponding current sinks of current sink array 540.

Light from a particular LED in LED array 90 passes through a well in sample tray 140 and is detected by photodetector 80 . The current generated by photodetector 80 is converted to voltage by current/voltage converter 660. Sample and hold circuit 680 converts this analog voltage level to CP
Sample at time intervals controlled by 0400.

This analog value is converted to a digital value by AD converter 700 and sent along bus 720 to CP 0400. Data from the CPU 400 is sent to the printer 740.
can be sent to.

CPU2O5 is controlled by an operating program. This program determines the timing of data analysis, printing, and transmission to decoder 440 or 460 for lighting the LEDs. A software clock on line 760 dictates when the LEDs are lit. The timing of this software clock is 60H.
Varies with respect to z-line noise. This is because when a program is interrupted to perform a read, the number of steps in the program to perform the associated operation is not the same. To avoid this variation, software clock 760 is gated by hardware clock 780 at gate 800. Therefore, each LED
The absolute time of lighting is precisely controlled. gate 800
The outputs of are coupled to clock inputs of decoders 440 and 460.

The photodetector 80 is a rectangular silicon cell with an N
It is made of doped material, and the bottom part is made of P-doped material. N
The doped layer is thin enough to allow light to pass through. The natural reverse bias of the silicon cell due to the identification and electron diffusion
No reverse bias voltage is applied as this is sufficient for the purposes of the present invention. Due to the resistance of the N-doped region, N
A grid of solder lines is crisscrossed across the top of the doped layer. These lines are placed between the wells of the microplate so that the light transmitted from the LEDs is not blocked by the lines.

Current drive array 500 receives current supply from constant current H820. The amount of current can be set depending on the desired amount and the intensity of light emitted by each LED.

11A and 11B are detailed circuit diagrams showing a part of the block diagram shown in FIG. 10. The circuit shown in Figure 11B is preferably located on the same circuit board as the 8x12 LED array. The circuit shown in FIG.
It can be placed on a separate substrate separate from the ED array.

In FIG. 11A, current/voltage (1/V) converter 66
0 is used coupled to a photodetector 80 shown in FIG. Resistor 840 and capacitor 850 can be adjusted to provide high frequency filtering of the signal from photodetector 80. The output voltage signal of I/V converter 660 is applied to sample and hold circuit 680.

The sampling and holding function of this sample and hold circuit 680 is activated by a trigger input 860.

This trigger input 860 is connected to the CPU U2O shown in FIG.
5 from one of the data lines of bus 880. The output 900 of sample and hold circuit 680 is A.D.
Provided as an input to converter 700. AD converter 7
The maximum voltage that 00 can accept is 10 volts. I/
The resistor 840 of the V converter 660 can be adjusted to generate a voltage such that the maximum signal expected to be detected from the photodetector 80 is slightly less than 10 volts.

The digital output of the AD converter 700 is connected to the data bus 920.
appears in Data bus 920 serves to provide digital values from AD converter 700 to CPU U2O5, and from CPU U2O5 to decoders 440 and 46.
Pass data to 0. When CPU 2O5 transmits data to decoders 440 and 460, the tristate output of AD converter 700 is in a high impedance state. The output of AD converter 700 is controlled by chip enable signal 940.

The digital output from AD converter 700 consists of two 8-bit bytes. Byte address/short cycle input A
The digital signal on O first activates the first byte and then the second byte of the output of AD converter 700. To this end, one or more outputs of AD converter 700 are coupled to each data line.

Decoder 960 provides an enable signal 980 to decoders 440 and 460 of FIG. 11B. This signal is
It also serves as a software clock input to the synchronization circuit 800. The output of the synchronization circuit 800 is the inhibition signal 1000
It is.

In FIG. 11B, data signal Do-D6 is the input to decoders 440 and 460. Constant current power supply 82
0 provides current from current drive 500 to LED array 90. LED array 90 is coupled to current sink 540. Current sink 540 is an inverter with an open collector output.

When a particular LED is selected, a 7-bit digital code appears on data lines DO-D6.

When the enable signal is provided and the inhibit signal is removed,
Decoder 440 activates one output line and decoder 460 also activates one output line. The line activated by decoder 440 is connected to current drive transistor 50
Switch one of the 0s. The current drive transistor 500 is
The current from the current source 820 is applied to the LE of one column A-H.
D Supply all.

The line activated by decoder 460 is connected to inverter 540
ONL to a low power state, thereby causing columns 1 to 1
2, the selected one is grounded. This causes current to flow through a selected one of the selected rows of LEDs in rows A-H.

The front panel display 120 shown in FIG. 11A has switches for "Ready,""Load," a power on indicator light, "Blank Set,""PlateReady," and "Optical Density (OD) Range." These switches and lamps are controlled by signals from the CPU as described below.

The operation of the circuit shown in FIGS. 10 and 11 will be explained based on the timing diagram of FIG. 12. The sample and hold trigger signal is signal 86 shown in FIG. 11A, and the decoder inhibit signal is signal 1 shown in FIGS. 11A and 11B.
000, and the AD chip enable signal is the 11th A
This is the signal 940 shown in the figure.

Two independent readings are taken for each sample.

One of these times is done with the LED ON, and the other time is done with the LED ON.
This is done when D is OFF. The transmission level is the difference between these ON and OFF readings. The ON and OFF reading orders are shown separately.

The 96 LEDs are energized in sequence and the reading sequence is repeated 10 times across evenly spaced phase positions of the 60 Hz line noise signal.

The sample and hold trigger signal on line 860 is 2
One pulse is generated every 50 m5. This pulse is
It is generated in the CPU by a hardware clock from a crystal oscillator. The decoder inhibit signal is generated by a combination of hardware and software clocks.

The software clock signal is generated after an interrupt signal in the CPU program if the operation of the CPU program is interruptible. The amount of time at this time varies between 4 and 20 clock periods, depending on the state of the program operation. When such a software clock signal is generated on line 980, this signal is combined with the hardware clock in synchronization circuit 800 to determine time 104.
At , a downwardly directed pulse is generated on the decoder inhibit signal. By this, as mentioned above, a specific LE
D is turned on.

Sample and hold circuit 680 is enabled to sample at time 106. This sampling is held at time 108. This retention time is
is delayed beyond the time 104 when the switch is turned on. The signal is then processed through I/V converter 660 and filtered through resistor 840 and capacitor 850. The tri-state output of the AD converter 700 is at time 110.
and two chip enable pulses 11
Data is read in 2 bytes under control of 2.

Similarly, if the LED was turned off, the sequence of operations for recording samples is initiated with the reappearance of the inhibit signal at time 114, inhibiting decoder interrupts and switching on the current drive to turn the LED off. Power is cut off. This continues the operations of sampling, holding, starting the AD converter, and reading the signal of the AD converter.

For the first 0N10FF reading, 96 LEs
Each of D is 0N10FF as above for 10 times.
The reading is performed sequentially, and then the second 0N is read in the same way.
A 10FF read is made and the read continues in this manner. Since the main noise signal is the 60Hz line voltage, these 10 readings are equally spaced along different phases of the 6011z cycle. Therefore, the first reading is taken along the rising edge of the first 60Hz cycle, the remaining 95 LEDs are read sequentially, and the second reading is taken along the corresponding later 6
It takes place at a high point on the 011z cycle. LED
Because there is a time difference between reading that the LED is ON and reading that the LED is OFF, the ground voltage or reference voltage that actually changes due to the 6011z noise signal will move slightly, so
An error occurs.

This movement is slightly upward for readings made during part of the 60 Hz cycle, and 6011z
There is a slight downturn for reads made in other parts of the cycle. Therefore, by equally spaced readings over 6011z cycles, the error in the 10 readings is canceled out by taking the average value.

Before inserting the measurement microplate or blank microplate, pull out the drawer 10 and insert the LED.
Measurement is performed using the space between the sensor and the photodetector, and after the measurement is completed, the "load" indicator light on the front panel 120 is turned on. Before measuring the microplate containing the sample, at time zero (1=0), a microplate containing a reference fluid such as water or a reactant is inserted and a blank measurement or t=Q measurement is performed. Once the plate is inserted and the instrument is ready to respond to the "Blank Set" or "Measure" switch, the "Ready" indicator light will illuminate. The reading on the printer 740 will be several digits or a partial number depending on the control by the "Optical Density C0D) Range" switch.

Once the blank value has been determined and the drawer 10 is completely extracted, a sample plate can be placed in the drawer. Immediately before reinserting the drawer 10, the space between the LED and the photodetector is measured again.

The different values of the optical signal for each well of the sample plate are compared to the values for the blank plate to adjust for variations in spatial measurements. For this reason, the blank plate is
There is no need to insert it initially and reinsert it for sample measurements taken over several hours or days. Adjustment of the drift of the LED and photodetector with respect to temperature over such a time period is performed by comparing the spatial measurements just before inserting a blank plate with the spatial measurements just before inserting a particular sample plate.

FIG. 13 is a flowchart illustrating an on-board software program for controlling the operation of the microplate measurement device. The plate measuring device is first turned on and a software preparation function is executed (step A). Next, the operator pulls out the drawer IO. Then, the "outside drawer" state is detected (step 13), and the L
The ED is scanned (step C) and a series of optical signal values corresponding to the light transmitted through space is generated (step D). These values are stored in table 3 in memory.

When the drawer 10 is reinserted (step E), the front panel switch is checked to see if the drawer contains a blank plate (step F).

For the blank plate, the LEDs are scanned (step G) and the light signal values are stored in table 1 in memory (step H).

Then the spatial values of table 3 are transferred to another table 2 (
Step ■), a series of spatial measurements close to the time of the blank plate measurement that generated the values of table l is provided. Spatial measurements are carried out continuously while the drawer 10 is out and before the drawer 10 containing the plates is inserted.
Table 3 is updated continuously.

If the drawer 10 is inserted and the "set blank" switch is not engaged, the front panel switch is checked to determine whether to take a sample measurement (step J). When the "measurement" switch is turned on, the LEDs are scanned (step K) and the transmittance value is stored in table 4 in memory (step K). The optical density for the sample is then calculated (step M) and printed (step N). .

The optical fourth degree (OD) calculation is performed based on the following formula:

ODn=log (nl)(n3)/(n4)(n2
) Here, the value of the nth optical signal in table X is nX, and the optical density of the sample at location n is ODn.

Each LED is scanned as described above, starting with the first LED and ending with the 96th LED. Ten measurements are repeated at equal intervals across the phase of the 60 Hz cycle. The final optical signal value used in the calculations described above is given as the sum or difference of the 10 signals. As can be seen from the above formula, the optical density is determined by converting the blank value into
It is the logarithm of the sample value divided by the sample value adjusted for differences in spatial measurements.

The measurement of the optical signal according to the flowchart shown in FIG. 13 is initiated by an interrupt signal to the microprocessor within CPU2O5. During a measurement initiated by this interrupt signal, a program in the CPU can calculate the data, transfer the data to one of the tables, and print the data on the printer. Actual measurements are performed in between these functions in accordance with the instructions of the interrupt signal.

Because an interrupt signal interrupts program operation, some steps are required after the interrupt signal. This is basically to hold the interrupt signal.
The time between measurements of ED varies (generally 4 to 2
To prevent this from causing jitter in the measurements (changed by 0 clock cycles), the interrupt is given at least 20 clock cycles before the time the measurement is expected to be taken, as shown in Figures 3 and 3. As shown in FIG. 4, a synchronization gate 800 performs an AND with the actual time clock. This ensures precise control of the position of each measurement relative to the phase of the 60Hz noise signal.

Instead of manually operating switches on control panel 120,
The operation of the measuring device can also be controlled by another computer, such as a microcomputer, coupled to the measuring device. By attaching such a main computer,
This computer operates the switch on your behalf.

The above-described high-speed solid-state multi-beam photometry device reliably measures the transmittance of a fixedly supported analytical sample at multiple locations in about 2 seconds or less without moving the sample or its holder. It has performance. this is,
This is achieved by using at least one monolithic photodetector having an area at least equal to the area of a plurality of fixedly supported analysis sites.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of a photometric device according to the invention; FIG. 2 is a diagram of an optical fiber splitter, with designated positions for coupling light from a light source into each of a plurality of optical fibers; FIG. 3 is a diagram showing the arrangement of each optical fiber corresponding to the sample storage location in the optical fiber manifold. FIG. 4 is a block diagram showing an outline of the processing circuit configuration based on the present invention. 5 is a flowchart showing the sequence of operations in a typical sequential scan of a sample plate by the measurement device shown in FIG. 1; FIG. 6 is a flowchart showing the sequence of operations carried out by the measurement device as part of the spatial calibration stage. , FIG. 7 is a flowchart showing the sequence of operations when measuring a sample plate, FIG. 8 is a perspective view showing a preferred embodiment of the multi-beam photometry device based on the present invention, and FIG. FIG. 8 is a side view showing a cross section of a well of a microplate inserted between the LED array and the photodetector of the photometric device shown in FIG. 8; FIG. 10 is an electronic control circuit for the photometric device shown in FIG. 11A and 11B are block diagrams showing the configuration. FIGS. 11A and 11B are circuit diagrams showing part of the controlled dissociation configuration shown in FIG. 1iJ12 is a timing diagram for a portion of the circuitry shown in FIGS. 1A and 11B, and FIG. 13 is a flowchart showing a computer program for controlling the control circuitry shown in FIG. (Explanation of symbols) 10: Dynamic measurement device, 12: Optical assembly, 14:
Single light source, 16: chopper, 18, 20: lens, 19; filter disk, 24: rotor,
26: Optical fiber, 32: Distributor,
34: Optical fiber, 36: Optical fiber bundle, 38: Optical fiber manifold, 40: Data plate, 42, 48: Lens array,
44: Photodetector, 46: Detector board, 50: Analysis and display device, 52: Reference fiber, 54: Stirring mechanism. Margin below

Claims (1)

[Claims] 1. By performing a series of measurements at a plurality of sample storage locations arranged at a plurality of analysis locations on an analysis plate,
A multi-beam dynamic light intensity measuring device for measuring the optical density of a reactive sample contained in the plurality of sample storage locations, the device comprising: , a single light source that emits light that is selectively focused in accordance with a predetermined sequence at selected locations of the sample receiving location; a plurality of light detection means for receiving light transmitted through each of the sample accommodation locations; means for vibrating the analysis plate over time; and means for analyzing the output of the light detection means to determine and display the optical density at the sample receiving location.
Dynamic luminous intensity measurement device using multiple beams. 2. The optical transmission means includes a plurality of optical fibers, and the coupling means includes: means for modulating the light from the light source at a predetermined ratio; and filtering light of a desired wavelength from the light from the light source to a desired point. a fiber rotor having means for focusing; an optical fiber for receiving substantially all of the filtered and focused light at an input end and transmitting the received light to an output end; and the optical fiber of the fiber rotor. a fiber distributor including the plurality of optical fibers arranged to be selectively coupled to the light emitted from the output end of the fiber rotor, the fiber rotor being displaceable, and the displacement causing the The optical fibers of the fiber rotor sequentially couple light from the light source to the plurality of optical fibers of the fiber distributor, thereby sequentially coupling light from the light source to the sample receiving location. The dynamic measuring device according to item 1. 3. Further comprising a first reference optical fiber for directly coupling the focused light to a first reference photodetector disposed on the photodetector board and also serving as an optical reference means. Dynamic measuring device according to scope 2. 4. further comprising a second reference optical fiber for directly coupling the focused light to a second reference photodetector disposed on the photodetector board, the second reference optical fiber being an optical reference; serving as means and reference means for relative positioning of the fiber rotor and the fiber distributor;
A dynamic measuring device according to claim 3. 5. The dynamic measuring device according to claim 1, further comprising a drive motor that moves the analysis plate forward and backward with respect to the photodetector board and applies the vibration to the analysis plate. 6. A method for measuring the transmittance of a plurality of sample accommodation locations on an analysis plate, the method comprising transmitting light from a single light source to selected sample accommodation locations of the plurality of sample accommodation locations through a plurality of light transmission means. measuring the intensity of light passing through the reactive sample contained in the sample storage area by using a light detection means at successive time intervals for each of the plurality of sample storage areas; , before the start of each successive measurement, vibrating the analysis plate for a first fixed period of time to stir the sample, and allowing the stirred sample to settle for a second fixed period of time; measuring the intensity of the light when the light from the light is directly coupled to the light detection means, and when the reactive sample is not accommodated in the sample accommodation location, the light from the light transmission means is coupled directly to the sample accommodation location. A method for measuring transmittance, comprising the steps of: measuring the intensity of the light when coupled through the light to the light detection means; and using all of the measurements to calculate the optical density of the sample receiving location. 7. A multi-beam photometer for measuring the transmittance of a plurality of sample receiving points arranged at a plurality of analysis points on a solid support, each of which is arranged at substantially only one of said sample receiving points. a plurality of light transmission means for transmitting light to the material storage location; a light detection means disposed in relation to the solid support and receiving light at a plurality of different locations on the surface; and the solid support; A multi-beam photometer comprising: support means for orienting the light detection means in a fixed direction; and means for sequentially arranging the plurality of light transmission means. 8. Claim 10, wherein the light detection means comprises at least one silicon photodetector having a surface area at least equal to the area occupied by the plurality of sample receiving locations on the solid support. The device described in. 9. A method for measuring the transmittance of a plurality of sample storage locations on an analysis support, the method comprising: measuring the optical signal generated by light passing through the space in the absence of the sample; providing an optical signal value; and providing a second spatially transmitted optical signal value by measuring an optical signal generated by light passing through space in the absence of the analytical support; A method for measuring transmittance, comprising steps of making adjustments to compensate for differences between the second spatially transmitted optical signal value and the second spatially transmitted optical signal value. 10. A method for performing colorimetric analysis using a multibeam photometer having light detection means disposed in relation to a solid support and receiving light at different locations on the surface of the sample support, the method comprising: providing a first spatially transmitted optical signal value by measuring the optical signal generated by the light passing through the space in the absence of said support; providing a second spatially transmitted optical signal value by measuring, and each step of making an adjustment to compensate for the difference between the first spatially transmitted optical signal value and the second spatially transmitted optical signal value. A colorimetric analysis method.