RU2452937C1 - Apparatus for analysing chemi- and bioluminescence of liquid media - Google Patents

Abstract

FIELD: physics.

SUBSTANCE: apparatus has a cuvette chamber for a sample and a photodetector section with a photomultiplier, connected through a shaping amplifier to a control unit, an interface for connection to a personal computer, a high-voltage source for connecting the photomultiplier and a power supply. The cuvette chamber and the photodetector section are thermo- and light-insulated and are air-tightly separated from each other by a transparent window, have independent apparatus for heating and controlling temperature of the cuvette and cooling the photomultiplier, wherein the cuvette chamber further has a dropping bottle and sample mixer with a drive and a rod. The apparatus for heating and controlling temperature of the cuvette includes a temperature sensor and a heater connected to a first output stage. The apparatus for cooling the photomultiplier is in form of a collar made from heat conducting material connected to the cold junction of a thermoelectric cooler, and the current supply bus of the cooler is connected to a second output stage.

EFFECT: low inherent noise of the photomultiplier while allowing independent heating and stabilisation of temperature of the sample.

5 cl, 3 dwg

Description

The invention relates to means for studying the state of liquid media using bioluminescence methods and can be used to control the content of toxins in domestic and industrial wastewater, liquid products for various purposes, as well as in biotechnology and medicine.

It is known that studies of the characteristics of biological fluids, including blood, using chemo- and bioluminescence methods can expand diagnostic capabilities and improve accuracy, which requires the use of appropriate analyzers (see, for example, cited sources in RU 2366953 C2, Deryabin, Karimov, 10.09. 2009).

A device is known for measuring the chemiluminescence and bioluminescence of a liquid, consisting of a dark chamber with a thermostatic cuvette having a reflective inner wall, a photomultiplier tube (PMT) located above the cuvette and connected to an electric signal amplification and processing unit, in which the cell is rotated to increase the efficiency registration (SU 1400258 A1, BAGAEV and others. G01N 21/76, publ. 10.01.2000). Also known is a device for measuring bioluminescence in liquid media, containing a reaction chamber and a recording system including a photomultiplier, a power supply, amplification and data output units to external recording devices. The power supply is connected to the elements of the registration circuit, and the elements themselves are connected in series (RU 2009466 C1, Ishutin et al., March 15, 1994). However, these devices have disadvantages. With an increase in the emission intensity of the sample, the pulse frequency from the output of the PMT increases. Since the interval between pulses is random in nature, with increasing frequency, the pulses begin to merge due to the finite magnitude of their duration. As a result, the counter registers a smaller number of pulses than there actually is, which leads to errors in estimating the luminosity of the sample.

Known environmental monitoring device "Biotox-10" for converting fluxes of luminescence quanta into a proportional number of electrical pulses for quantitative control of the degree of integrated toxicity of water samples and extracts from various environmental objects in laboratory and field conditions for medical, sanitary and environmental purposes based on bioluminescent analysis (http://www.biotox.ru/instruction).

A device for measuring chemiluminescence and bioluminescence RU49998 U1, Deryabin et al., May 13, 2005, including a cuvette compartment with a dark chamber for placing a test sample, a photoelectric multiplier coupled to it, and a pulse detection system connected to it, characterized in that a cuvette is described the compartment has two identical dark cameras, each of which has a separate photoelectronic multiplier connected to its own pulse registration system, the signal from which is fed to a single interface circuit, allowing th to enter data into the computer and to calculate absolute and relative differences in the intensity of luminescence compared samples. At the bottom of the dark chambers there is a device for rotating cylindrical cells with samples around its own axis, driven by a low-speed electric motor. The cuvette compartment is equipped with a device for thermostating of sample cuvettes located in the dark chambers.

A device is known for analyzing the chemiluminescence and bioluminescence of liquid media, comprising a sample cell and a photodetector with a photomultiplier connected through an amplifier-former to an anode pulse counter and a microprocessor, means for linearizing the output characteristic, an interface for connecting to a personal computer, a high-voltage source, and power supply unit (RU 2200315 C2, Chalkin, Ostrozhinsky, 05.25.2001 - the closest analogue). Means for linearizing the output characteristic contain a table of correction factors entered into the memory unit, calculated from the calibration results on a reference radiation source.

However, this device is characterized by a sufficiently high level of intrinsic noise due to the properties of the PMT. In addition, the device does not provide controlled thermal effects on the test sample, which reduces the operational capabilities of the analysis.

The present invention is aimed at increasing the sensitivity of registration of fluorescence by lowering the temperature of the photocathode of the PMT, as well as stabilizing the temperature of the sample to a working value (for example, for blood + 37 ° C) at small volumes of biological fluid. It is known that cooling photocathodes using thermoelectric cooling for this purpose allows us to reduce the dark current of photomultipliers and, accordingly, increase their sensitivity.

A patented device for the analysis of chemiluminescence and bioluminescence of liquid media contains a cuvette chamber for sampling and a photodetector compartment with a photomultiplier connected through an amplifier-former to a control unit, an interface for connecting to a personal computer, a high-voltage source for a photomultiplier, and a power supply.

The invention is characterized in that the cuvette chamber and photodetector are thermally, light-insulated and hermetically separated from each other by a transparent window, have independent means of heating and thermostating the cuvette and cooling the photoelectronic multiplier, the cuvette chamber additionally containing a dropper and a sample mixer with a drive and a rod.

Means for heating and thermostating the cuvette include a holder of heat-conducting material with a cavity for accommodating the cuvette, a first temperature sensor installed in the holder body and a heater connected to the first output stage, a dropper is installed in the body of the casing, and its drip tip is open in the cuvette.

The cooling means of the photoelectronic multiplier are made in the form of a cage of heat-conducting material installed with a thermal contact around the zone adjacent to the working area of the photocathode of the multiplier and connected with the cold junction of the thermoelectric cooler, the hot junction of the refrigerator is communicated with a cooling radiator, a second temperature sensor is installed in the body of the said casing, and the current bus power supply of the refrigerator is connected to the second output stage. The inputs of both output stages connected to the power supply are connected to the control outputs of the control unit, to the inputs of which the aforementioned temperature sensors are connected.

The device may be characterized in that the control unit includes a microcontroller with a storage device and associated controls and indication of operating modes. The first and second signal inputs of the microcontroller are the switched inputs of an analog-to-digital converter for connecting temperature sensors, the third is the input of the counter of the anode pulses of the photoelectronic multiplier, the first and second control outputs of the microcontroller are outputs of the integrated PWM controllers, made with the possibility of generating a periodic pulse signal with an adjustable duty cycle in depending on the signal from the temperature sensors to power the heater and the refrigerator, and the microcontroller is connected bus th data exchange and management interface.

The device can be characterized by the fact that the dropper and the drive of the sample mixer are connected to the power supply, as well as the fact that the transparent window is made in the form of a vacuum glass.

The device can also be characterized by the fact that the control unit contains a switch of operating modes: “LUMINOMETER”, “RESET”, “CONTROL”, “SAMPLE”, “TOXICITY”, “MODE”, “FEU COOLING”, “SAMPLE HEATING”.

The technical result of the invention is to reduce the intrinsic noise of the photomultiplier tube while allowing independent heating and stabilization of the sample temperature.

The invention is illustrated in the drawings, where:

figure 1 shows a schematic diagram of a patented device for analyzing liquid media; figure 2 is a structural diagram of a control unit; figure 3 is a functioning algorithm.

The device includes a thermo- and light-insulated casing 1, in which independent cuvette 2 and photo-receiving 3 compartments are formed, sealed from each other by a transparent window 4 (see Fig. 1). It is advisable to make window 4 in the form of a vacuum double-glazed window to improve the thermal insulation of the compartments.

The cuvette compartment 2 contains a holder 21 for the cuvette 22, a heater 23, a temperature sensor 24, a stirrer 25 with a drive, a dropper 26 with a drive for introducing reagents into the cuvette 22. The holder 21 is made of heat-conducting material, mainly aluminum or copper, in the body of which a temperature sensor 24 is installed .

Thermostating can be provided in two ways: from the control unit 5 by resistively heating the heater 23 and monitoring the temperature of the holder by the temperature sensor 24, as well as from the laboratory liquid thermostat. In the first case, the control output of block 5 is connected to the output stage 6 connected to the power block 7 (see figure 1). In the second case, an autonomous thermostating system can be used, provided by a thermostat and a heat exchanger in contact with the holder 21. This design does not change the essence of the technical solution for regulating and stabilizing the sample temperature and the design of the device as a whole. The control range is + (27-42) ° С, accuracy ± 0.1 ° С.

The photodetector compartment 3 includes a photomultiplier tube PMT 31, the cathode part of which is mounted in a flange 32 made of heat-conducting material, and in the body of which there is a thermal sensor 33. The flange 32 is in thermal contact with a thermoelectric single or multi-stage cooler 34 on a Peltier element. The heat sink from the hot junction is carried out by a radiator 35 with water (shown in figure 1) or air cooling. The refrigerator 34 is connected to the output stage 8, also connected to the power supply unit 7. The PMT 31 is powered from a high-voltage source 13 connected to a divider 36. The microcontroller-based control unit 5 is connected to temperature sensors 24 and 33, which generate a voltage proportional to current temperatures, which ensures the generation of corresponding signals in the output stages 6.8 connected to the heater 23 and the refrigerator 34. The output of the PMT 31 is connected to the input of the amplifier-former 9, the output of which is connected to the control unit 5. Block 5 through the communication interface 10 by a two-way bus is connected to the computer 11. The power supply unit 7 from the 220 V network is connected to the output stages 6.8. In addition, if necessary, the unit 7 can be connected to the drives of the mixer 25 and dropper 26.

Figure 2 presents a block diagram of a control unit 5. It contains a microcontroller 51, which includes an analog-to-digital converter (ADC) 52 with two switched inputs for connecting temperature sensors 24.33. As the microcontroller 51, for example, an Atmega 8535 chip and the like can be used. The control outputs of the microcontroller 51 are the outputs of the integrated PWM controllers 53, 54, providing the generation of a periodic pulse signal with an adjustable duty cycle depending on the signal from the temperature sensors 24.33. The reliability of the 53.54 controllers depends on the difference between the signals from the temperature sensor and the expected signal for the set temperature. The output stages 6.8 are connected to the heater 23 and the refrigerator 34, respectively. The third input of the microcontroller 51 is designed to connect the output of the amplifier-former 9 to the counter 55 of the anode pulses of the photoelectronic multiplier 31. The microcontroller 51 is connected by a bi-directional communication line for data exchange and control with an interface 10 for connecting to a personal computer 11. The microcontroller 51 contains a memory unit 56 and is connected to located in the block 5 of the bodies 57 of the display and the switch 58 of the choice of operating modes. The program in the computer 11 draws a graph, processes and saves data in a file, controls the operation of the device.

The device operates as follows. The cuvette 22 with the analyzed biological fluid, for example, blood, is charged into the holder 21 and, by means of a temperature control system controlled by the microprocessor 51, is heated to the temperature set by the experiment, recorded by the temperature sensor 24. The necessary reagents are introduced by means of a dropper 26, and the fluid is mixed with a stirrer 25. The temperature sensor 24 generates a voltage proportional to the temperature. Further, this voltage is supplied to the ADC 52, the code from the output of which is compared with a code corresponding to a given temperature. The difference in codes determines the value of the control code for the output stage 6. The control code is converted into a pulse signal by the pulse width modulation controller (PWM controller) 54 in the microcontroller 51, the duty cycle of which depends on the value of the control code. This signal is fed to the output stage 6 connected to the heater 23.

In the same way, PMTs 31 are also prepared for measurements. The temperature sensor 33 generates a voltage proportional to the temperature. Further, this voltage is supplied to the ADC 52, the code from the output of which is compared with a code corresponding to a given temperature. The difference in codes determines the value of the control code for the output stage 8 of the thermoelectric refrigerator 34. The control code is converted into a pulse signal by the PWM controller 53 in the microcontroller 51, the duty cycle of which depends on the value of the control code. This signal is fed to the output stage 8, connected to a thermoelectric refrigerator 34. So, when using a photomultiplier tube 3 PMT -101 in the photodetector, when the temperature drops to -3 ° C, the level of intrinsic noise drops by about three times while stabilizing the sample temperature in the range + (27-42) ° С.

Indication of operating modes: “LUMINOMETER”, “CONTROL”, “SAMPLE”, “TOXICITY”, “MODE”, “FEU COOLING”, “HEATING OF THE SAMPLE” is carried out by the LEDs of the indication body 57, they are selected using the switch 58 - buttons for selecting the operating modes, and in addition to the closest analogue - “incl. cooling ”, indicators on the LEDs“ incl. cooling ”and reaching the set temperature (“ normal ”). Additional controls are connected to the microcontroller.

The block diagram of the algorithm for the operation of the control unit is presented in figure 3.

The main mode is the LUMINOMETER mode. In the “CONTROL” mode, the “LUMINOMETER” mode is repeated several times, and the measurement results are averaged. The number of cycles of the LUMINOMETER mode is determined by the constant n set in the device memory in the tuning mode in the range N = 10-1280 (usually N = 10). The “TEST” mode is similar to the “CONTROL” mode, but the measurement result is stored in another memory cell. In the “TOXICITY” mode, the results obtained in the “CONTROL” and “SAMPLE” modes are compared, which is described below. In the “PERIODIC CONTROL” mode, the “CONTROL” mode is periodically repeated until this mode is removed.

The operation algorithm is as follows.

With the beginning of work (p. 1.0), ports, timer, emulation of constants t, q, z, s, N, PC (correction sign) are set. The functioning of the memory unit is monitored and a message is issued to the indication body 57 in case of failure (Section 2.0).

At the step (p. 3.0) of the program, the LUMINOMETER mode is first set. The value of the number of pulses I ₀ measured in a given time is transmitted to the organ 57 and to the computer.

The following is an analysis of the status of the sign of correction PC. If the result is negative (PC = 0), the program analyzes the specified mode and, if it has not changed, returns to step (Section 3.0). If the result is positive (PC = 1), the calculated value of I ₀ is transferred to the result correction subroutine (4.11-4.13). The value of I _{0 is} adjusted taking into account the correction factors contained in the block 56 of the memory (p.5.21). To do this, first determine the number n of the value range into which the value of I ₀ fell, by comparing I ₀ with a number of values of U _n from the block 56 of the memory. Then, the coefficients U _n , b _n , K _n are read from block 56 for the found value of n. After that, the adjusted value of I ₀ ^{1 is} calculated by the formula: I ₀ ^I = K _n (I ₀ -U _n ) + (b _n + U _n ). The adjusted value of I ¹ ₀ is transmitted to the body 57 and to the computer. Next, the program analyzes the specified mode and, if it has not changed, returns to step (Section 3.0).

In accordance with the selected scheme of the patented device, it is provided that only one button out of 6 available can be activated: "LUMINOMETER", "RESET", "CONTROL", "SAMPLE", "TOXICITY", "MODE". The buttons "COOLING PMT", "HEATING SAMPLES" are included independently. The "RESET" button provides the output of the algorithm to the beginning and is not conventionally shown in Fig. 3.

If the "LUMINOMETER" button is activated (section 5.1), then the program returns to step (section 3.0).

If the "CONTROL" button is activated (Section 5.2), then the "LUMINOMETER" mode is switched on N times (by default N = 10), after which the "LUMINOMETER" mode is turned off. In the process of executing the "CONTROL" mode, I is calculated _{to the} total number of pulses I ₀ for N cycles (Section 5.21).

Further, the program works similarly to the steps of 4.0, 4.11-4.13. If PC = 1, then the correction of the value of I _{K is} carried out taking into account the correction factors contained in the block 56 of the memory (p.5.22-5.23). If PC = 0, correction is not performed. The result obtained I _K or I _K ¹ is transmitted to the body 57 and to the computer.

If the “TEST” button is activated (Section 5.3), then operations 5.31, 5.32, 5.33 are carried out similar to operations 5.21, 5.22 and 5.23. The result obtained I _P or I _P ¹ is transmitted to the body 57 and to the computer.

If the "TOXICITY" button is activated (Section 5.4), then the value of the characteristic of the mode q =? (Clause 5.41). At q = 0 (Section 5.42), the toxicity P _t is calculated by the formula: P _t = (I _K ¹ -I _P ¹ ) / I _K ¹ · 100, where t is the reference number P.

Then, the reference number t is increased by 1 (if t> 3, then t = 0). Next, the value P _{t is} transmitted to the body 57 (Section 5.43) and the operation of assigning the attribute q = 1, setting the coefficient c = 1 (Section 5.44).

When q = l (clause 5.45), the gamma function G is calculated by the formula G = (I _K ¹ -I _P ¹ ) / I _P ¹ and the calculated value of G is output to organ 57 (clause 5.46). The coefficients are set: z = 1, c = 1, as well as zeroing the q value (Section 5.47).

If the “MODE” button (p. 6.0) is activated simultaneously with the “RESET” button, then the analysis of the previous states and the change of signs of the t, z, s, N modes (p. 6.1) are carried out.

The “MODE” command is used simultaneously with the “RESET”, “CONTROL,“ TOXICITY ”commands.

If the “MODE” button (item 6.1) is activated simultaneously with the “TOXICITY” button, then at step step 6.11 of the program with c = 1, t = 3, the average toxicity P _cf is calculated by the formula P _cf = (P ₁ + P ₂ + P ₃ ) / 3. Then, the value of P _{cr is} output to the indication body 57 and to the serial interface 10 (item 6.12) and the setting z = 0, q = 0, t = 0 (item 6.13).

If the “MODE” button is activated (clause 6.2) simultaneously with the “CONTROL” button, then at step 6.3, the transition to the “PERIODIC CONTROL” mode is performed. In this case, the "CONTROL" mode is continuously switched on and the operations of items 5.21-5.23 are performed until the "PERIODIC CONTROL" mode is removed by setting another mode.

If the button "COOLING PMT" (p.7.0) is activated, then the refrigerator 34 (p.7.1) is turned on, the LED of the block 57 (p.7.2) is connected, the corresponding input of the ADC 52 is connected (p.7.3), the code is read (p. 7.4), error calculation (clause 7.5). Next, a PWM controller 53 with a frequency of 10 KHz is connected (clause 7.6) and the “Normal” state is determined (clause 7.7). If “yes”, then the LED is turned on (item 7.8), if “no” - then the LED turns off (item 7.9). In these cases, as well as in the case (clause 7.10), the program returns to the beginning (clause 7.0).

In the “HEATING OF SAMPLES” mode (p.8.0), which provides stabilization of the sample temperature 22, a control algorithm similar to that described in p.7.0–7.9 is used (p.8.0 is shown schematically in FIG. 3), which operates simultaneously with cooling the PMT.

The data and experimental studies show that the device provides enhanced functionality and the achievement of a technical result — reduction of the intrinsic noise of the photomultiplier tube while allowing independent heating and stabilization of the sample temperature. So, when using a photomultiplier tube PMT -101 and lowering its temperature to -3 ° C, the level of intrinsic noise drops by about three times, while at the same time it is possible to stabilize the sample temperature in the range + (27-42) ° С.

Claims (5)

1. A device for analyzing the chemiluminescence and bioluminescence of liquid media, comprising a sample cell and a photodetector with a photomultiplier connected through an amplifier-former to a control unit, an interface for connecting to a personal computer, a high-voltage source for a photomultiplier and a power supply,
characterized in that
the cuvette chamber and photodetector compartment are thermo-, light-insulated and hermetically separated from each other by a transparent window, have independent means for heating and thermostating the cuvette and cooling the photoelectronic multiplier, the cuvette chamber additionally containing a dropper and a sample mixer with a drive and a rod, means for heating and temperature control of the cuvette include holder of heat-conducting material with a cavity for placement of the cuvette, the first temperature sensor installed in the body of the holder and the heater connected to the first outlet to the bottom of the cascade, a dropper is installed in the body of the aforementioned body, and its drip guide tip is open in the cuvette,
cooling means of the photomultiplier tube are made in the form of a cage of heat-conducting material installed with a thermal contact around the zone adjacent to the working area of the photocathode of the multiplier and connected with the cold junction of the thermoelectric cooler, the hot junction of the refrigerator is communicated with a cooling radiator, a second temperature sensor is installed in the body of the said casing, and the current bus power supply of the refrigerator is connected to the second output stage,
the inputs of both output stages connected to the power supply are connected to the control outputs of the control unit, to the inputs of which the aforementioned temperature sensors are connected. 2. The device according to claim 1, characterized in that the control unit includes a microcontroller with a storage device and associated controls and indication of operating modes, while
the first and second signal inputs of the microcontroller are switched inputs of an analog-to-digital converter for connecting temperature sensors, the third is the input of the counter of anode pulses of the photoelectronic multiplier, the first and second control outputs of the microcontroller are outputs of the integrated PWM controllers, made with the possibility of generating a periodic pulse signal with an adjustable duty cycle in depending on the signal from the temperature sensors to power the heater and the refrigerator, and the microcontroller is connected bus th data exchange and management interface. 3. The device according to claim 1, characterized in that the dropper and drive of the sample mixer are connected to the power supply. 4. The device according to claim 1, characterized in that the transparent window is made in the form of a vacuum glass. 5. The device according to claim 1, characterized in that the control unit contains a switch of operating modes: “LUMINOMETER”, “RESET”, “CONTROL”, “SAMPLE”, “TOXICITY”, “MODE”, “COOLING OF THE FEU”, “HEATING” SAMPLES. "

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