RU2200315C2 - Facility analyzing liquid media by luminescent method - Google Patents

Abstract

FIELD: study of aqueous media by physical methods, by bioluminescence method in particular. SUBSTANCE: facility includes light-insulated dish compartment for check and measured samples, register of weak glows based on photoelectron multiplier working under mode of count of anode pulses. Output of multiplier is connected to amplifier, comparator to which other input unit setting threshold value is connected. Output of comparator is connected to one of inputs of 2 AND gate, to which another input of generator of meander. 2 AND gate is connected to counting input of counter and its output is linked to parallel port. Parallel port is coupled to controlling input of counter. Data and address bus of parallel port is coupled to data and address bus of microprocessor. In addition parallel port is coupled to storage and series interface and through the latter to computer. EFFECT: linearization of characteristic " output signal-content of toxins ", enhanced noise immunity and reliability and reduced mass of facility. 2 cl, 4 dwg

Description

 The invention relates to means for studying aqueous media by physical methods, in particular by 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.

 Methods of biological testing of waters are very promising when solving environmental issues, not only through the use of living biological objects (microorganisms, algae, invertebrates, fish) as indicators, but also through the use of modern optical-physical means and instruments that allow monitoring in current time mode and objectify registration (Ecological chemistry. Fundamentals and concepts / Edited by F. Corte. M .: Mir, 1997) [1]. This distinguishes between the fluorescence method and the bioluminescence method, to which the present invention relates.

Known methods for testing media to quench the bioluminescence of luminous bacteria. To do this, a predetermined amount of a suspension of specially prepared luminous bacteria is introduced into the studied liquid medium and the control medium. Then, with the help of a luminometer, the luminosity intensity and the dynamics of its change in time are recorded, according to which the environmental toxicity indicator is calculated, for example, according to the degree of survival of bacteria LC ₅₀ (see Gil T.A. et al. Biotesting method for quenching luminescence of luminous bacteria // Biotesting methods vod. Chernogolovka, 1988, p.13-15) [2]. The luminometer includes a dark chamber for placing the test medium, optically coupled to a photodetector and a registration system. As a photodetector, a highly sensitive photomultiplier tube (PMT) is usually used.

 Thus, it is known a device for measuring chemiluminescence and bioluminescence of a liquid, consisting of a dark chamber with a thermostatically controlled cell having reflective internal walls, a PMT located above the cell and connected to the amplification and processing unit of the electrical signal, in which the cell is rotated to increase the detection efficiency (SU 1400358 A1, BAGAEV et al. G 01 N 21/76, publ. 10.01.2000) [3].

 A known apparatus for measuring bioluminescence in liquid media, containing a reaction chamber and a registration system, including PMT, power supply, amplification units and output data 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, G 01 N 15/02, 03/15/1994) [4] (the closest analogue).

 However, the apparatus [4], as well as other devices of a similar purpose [2, 3], has disadvantages. With an increase in the luminescence 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 value 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.

The objective of the invention is to provide a device with an extended dynamic range and with a linear characteristic, providing registration in the mode of counting photons in the range of 10 ³ -10 ⁷ impulses / s, as well as weight reduction and increased reliability of the device.

 The technical result, which consists in linearizing the characteristic “output signal - toxin content”, increasing the noise immunity and reliability of the device and reducing its weight, is achieved by the fact that the device for analyzing liquid media by the luminescent method contains a reaction chamber and a luminescence recording system including a photoelectron multiplier connected to an amplifier and a control unit and outputting data to external recording devices, a power supply unit of a photoelectronic multiplier.

 The luminescence recording system contains means for linearizing the output characteristic. The control unit and the data output to external recording devices contains a comparator and a 2-I logic circuit connected in series with the signal inputs, to the other input of which a meander generator is connected, with an output connected to the counter input of the counter, the output of which is connected via a parallel port via the data and address bus to microprocessor, memory unit and serial interface for connecting to a personal computer. A threshold level setting unit is connected to the other input of the comparator, while the parallel port is connected to the counter control input, input unit, and display panel. Means for linearizing the output characteristic represent a table of correction factors entered into the memory unit, calculated from the calibration results on a reference radiation source.

 The device can be characterized in that the power supply unit of the photoelectronic multiplier is made according to the blocking generator circuit and is additionally equipped with a voltage multiplier, a choke and a compensation winding, the beginning of which is connected via a choke to the positive power terminal and the first capacitor, the other tap of which is connected to the ground, and the end to the end of the transformer winding connected to the transistor and the second capacitor, the other tap of which is connected to ground. The output winding of the transformer through a voltage multiplier is connected to a terminal to connect to the cathode of the photomultiplier tube and to one tap of the resistive voltage divider, which has taps for connecting to the dyno of the photomultiplier tube, while another tap of the resistive divider is connected to the ground and the housing of the voltage multiplier.

 The device can also be characterized by the fact that the control unit and outputting data to external recording devices contains a switch of operating modes: "LUMINOMETER", "CONTROL", "SAMPLE", "TOXICITY", "MODE".

 The analysis shows that, in the present form, the patented device satisfies the condition of patentability "novelty", since no means are found in the prior art that are identical to the patented combination of features.

 An analysis of the compliance of the patented device with the condition "inventive step" showed that a light signal recorder is known that contains a photomultiplier connected to a scan unit and to a pulse registration system (SU 1343249 A1, G 01 J 1/44, 07.10.87) [5]. The pulse registration system includes a series-connected amplifier, an amplitude discriminator, a pulse shaper, an AND-NOT logic element connected to counters, an adder, and to a computing unit.

 However, the device [5] has a different logic of operation, namely, it is designed to measure the difference of only two practically coincident pulses, and at the same time it is more complicated circuitry.

 The patented device has no restrictions on the number of pulses. In other words, the popularity of the causal relationship “distinctive features - technical result” has not been established, which indicates the presence of an inventive step.

The invention is disclosed in the following description and illustrative graphic materials, where:
figure 1 presents a block diagram of a device;
figure 2 is an electrical diagram of a high voltage source;
figure 3 - characteristic "output signal - luminosity";
figure 4 shows the algorithm of the device.

 The device includes (see Fig. 1) a light-insulated cuvette compartment 10 for control and measured samples. In optical communication with the cuvette compartment 10, there is a dimmer recorder 11 based on a photoelectronic multiplier 12 operating in the anode pulse counting mode.

 The output of the photoelectronic multiplier 12 is connected to an amplifier 14, the output of which is connected to one of the inputs of the comparator 16, to the other input of which a threshold level setting unit 17 is connected. The output of the comparator 16 is connected to one of the inputs of the logic circuit 18 2-I, to the other input of which a meander generator 19 is connected.

 The output of the logic circuit 18 2-I is connected to the counter input of the counter 20. The output of the counter 20 is connected via a bus (1) to the parallel port 21, while the parallel port 21 is connected via the bus (2) to the control input 23 of the counter 20. The parallel port 21 the data and address bus (5) is connected to the data bus and the microprocessor 22 address, via the bus (3) with the input unit 24 and via the bus (4) with the display panel 26. In addition, the parallel port 21 via bus (5) is connected to the memory unit 28 and the serial interface 30 (RS 232), through which it is connected to the computer 32. The computer 32 functions as a means for statistical processing and calculation of complex parameters of the studied environments, protocol design measurements, creation of databases on the toxicity of waste water, fixing graphs and files.

 The display panels 26 may be implemented on LED or liquid crystal type elements. The power of the photomultiplier tube (PMT) 12 is provided from a high voltage source 34.

 Figure 2 presents the electrical circuit of the high-voltage source 34. In the source 34 to obtain a high voltage, a reverse pulse is used in the circuit of a standard blocking generator, which reduces the overall dimensions of the source.

The transformer 300 includes windings 301, 302, 303 and a core 304. The beginning of the winding 302 is connected to the base of the transistor 306, and the end is connected to the resistor 307 and the output of the feedback unit 308, the housing of which is connected to ground. The beginning of the coil 301 is connected to the emitter of the transistor 306, the collector of which is connected to the ground. The end of the winding 301 through a capacitor 309 is connected to the ground, and is also connected to the end of the winding 310, the beginning of which through the inductor 312 is connected to terminal 314 (+ U _PIT ). Terminal 314 is connected through capacitor 316 to ground.

 The beginning of the winding 303 through the capacitor 318 and the resistor 320 is connected to the ground and the control input 322 of the feedback unit 308. The end of the winding 303 through a voltage multiplier 324 is connected to a terminal 326 for connecting to the cathode of the PMT 12 and to one tap of the resistive voltage divider 328, having taps 330 for connecting to the PMT dynodes. Another tap of the resistive divider 328 is connected to the ground and via bus 325 to the housing of the voltage multiplier 324.

 It is known (see, for example, Gusev V.V. et al. Fundamentals of pulsed and digital technology. M.: Sov. Radio, 1975, p. 247) [5] that the reverse voltage in a standard blocking generator is determined by the load and the energy stored in the winding 301. The energy, in turn, depends on the magnitude of the current in the winding 301 and the inductance of the winding 301. The maximum value of the current at the end of the forward stroke (when the transistor 306 is open) is reached when the core 304 of the transformer 300 begins to saturate. the larger the cross section of the magnetic core (core 304), the later we come core saturation. If there is a constant current component in the coil 301, core 304 saturates earlier. The compensation winding 310 has the same number of turns as the winding 303, but the current flows in opposite directions. The inductor 312, due to its large inductance, eliminates the influence of the compensation winding 310 on the functioning of the blocking generator, and the capacitors 309 and 316 prevent the occurrence of ripples in the power circuit. As a result of using such a circuit in the reverse pulse generation cycle, saturation of the core 304 occurs much later. This allows you to reduce the core cross-section by 2 times at the same power source. Although an additional element (inductor 312) is introduced, the dimensions of the high voltage power supply 34 are significantly reduced.

A voltage U _oc proportional to the current through the resistive divider 328 is allocated to the resistor 320 and capacitor 318. The feedback unit, responding to this voltage U _oc and adjusting the base current of the transistor 306, keeps U _oc constant. Block 34 provides a PMT supply voltage of about 1000 V with a high stability of 0.1%.

 The device operates as follows.

 The objects of research (water samples or extracts prepared with the necessary test objects — luminescent bacteria in accordance with the recommendations [1]) are placed in the cell compartment 10. Due to the change in bioluminescence, the signal from the registrar 11 of low light also changes in proportion to the toxins in the samples, what is fixed PMT 12.

 The signal from the PMT 12 in the form of pulses of negative polarity is fed to the input of amplifier 14, and after amplification, to the information input of comparator 16. Comparator 16 generates pulses with standard levels set by the threshold level device 17.

 These pulses are fed to the counting input of the counter 20. Simultaneously, a pulse signal is supplied to the second input of the logic circuit 18 2-I, the repetition period of which is equal to the expected duration (0.5-2 μs) of a single pulse from the output of the comparator 16. As a result, the merging pulses which is typical at high luminosity of the test object) are separated in time, and the counter 20 registers the true number of pulses, which ensures the linearity of the output characteristic.

 The counter 20 counts the number of pulses for a specific, short time interval specified by the microprocessor 22, and then is reset to zero by the control signal supplied to its control input 23. The measurement interval Δt in the LUMINOMETER mode is 1 s, in the CONTROL mode - 10 from. During this interval, the counter 20 is turned on repeatedly at Δt, the total number of pulses is calculated by the microprocessor 22, during which the total sum of pulses for the time interval Δt is calculated. At the beginning of each interval Δt, the counter 20 is reset to zero.

 Further, after counting the number of pulses, correction coefficients are introduced into the result from the memory unit 28 to linearize the amplitude characteristic "output signal - luminosity", which is uniquely related to the characteristic "luminosity - toxin content".

 The principle of introducing correction factors is illustrated in figure 3, which shows the ideal (linear) and real characteristics of the "output signal - luminosity". It can be seen that the real characteristic (shown by a dotted line) is significantly nonlinear, therefore, the determination of correction coefficients and subsequent linearization is a reserve for increasing the reliability of registration.

In the process of calibrating the device from a reference source, the entire range of possible values of the number of pulses (in the realized case, 10 ³ -10 ⁷ pulses / s) is divided into n ranges. At calibration points (in FIG. 3, these are: 10, 20, 30, 50, 75, 100, etc. luminosity units, relative units), the values of U _n corresponding to the beginning of the nth section, the values of b _n showing the deviation real characteristics from ideal at calibration points, as well as K _n values showing the deviation of the slope of the real characteristics from 45 ^o for the n-th section. The mentioned correction factors (U _n , b _n , K _n ) and the corresponding interval numbers are entered into the memory unit 28. These coefficients are taken into account later in the process of linearizing the characteristics of the output signal.

 It should be noted that the accuracy of the correction depends on the number of stored coefficients, i.e. of the number of plots that represent the entire characteristic. It is established that for practical purposes n = 17 is sufficient.

 Correction factors are set individually for each device experimentally using a calibrated light source and are entered into the microcontroller program in the form of a table stored in the memory unit 28. The above algorithm allows not only to linearize the output characteristic, but also to standardize the sensitivity of the device as a whole, despite the variation in the parameters of the PMTs used.

 The microprocessor program 22 operates as follows (see Fig. 4).

 With the beginning of work (p. 1.0), ports, a timer are emulated, signs of PC modes are set (monitoring frequency), t, q, z, s constants; setting n = 10 is performed. The functioning of the memory unit 28 is monitored and a message is sent to the indicator 26 in case of failure (Section 2.0).

 At the step (item 3.0) of the program, the "LUMINOMETER" mode is set. The measured value of the number of pulses is transmitted to the indicator 26 through the parallel port 21.

Further, in the next step (Section 4.0), it is determined whether any of the buttons of the input unit 24 is pressed, and also what is the status of the periodic monitoring indicator (PC). If the result is negative and PC = 0, the program returns to step (Section 3.0). If the result is negative and PC = 1, the "LUMINOMETER" mode is turned off. The total number of pulses I ₀ and N cycles is calculated (paragraph 4.11). Next, the correction of the value of I _{0 is} carried out taking into account the correction coefficients contained in the block 28 of the memory (section 5.12).

First, the number n of the interval of values into which the value of I ₀ falls is determined by comparing I ₀ with a series of values of U _n from the block 28 of the memory. Then, the coefficients U _n , b _n , K _n are read from the memory unit 28 for the found value n.

After that, the adjusted value of I ^{10 is} calculated by the formula:
I ₀ ^I = K _n (I ₀ -U _n ) + (b _n + U _n ) (1)
and the corrected value I ₁ ¹ is sent to the serial interface 30 and through the parallel port 21 to the indicator 26. In addition, the signs of the modes and coefficients q = 0, c = 1 are set (section 4.13).

 If the result of step p.0.0 ("YES") is positive, the program proceeds to the next step (p.5.0), which analyzes the appearance of the pressed button.

 In accordance with the selected scheme of the patented device, it is provided that only one button out of 5 available can be activated: "LUMINOMETER", "CONTROL", "SAMPLE", "TOXICITY", "MODE".

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

 If the "CONTROL" button is activated (Section 5.2), then the clock pulses are issued N times (by default N = 10) to start the "LUMINOMETER" mode, as a result of which the "LUMINOMETER" mode is turned off.

Further, the program works similarly to steps 4.11-4.13. The total number of pulses I ₀ for N cycles is calculated (Section 5.21). Next, the correction of the value of I _{0 is} carried out taking into account the correction factors contained in the block 28 of the memory (p.5.22).

First, the number n of the range of values is determined, into which the value of I ₀ falls by comparing I ₀ with a series of values of U _n from the block 28 of the memory. Then, the coefficients U _n , b _n , K _n are read from the memory unit 28 for the found value n.

After that, the corrected value of I ₀ ¹ no is calculated by formula (1) and the corrected value of I ₀ ¹ is output to the serial interface 30 and through the parallel port 21 to the indicator 26. In addition, the setting of mode signs, coefficients q = 0, c = 1 (Clause 5.23).

If the “TEST” button is activated (Section 5.3), then synchronization pulses are issued for the “LUMINOMETER” mode N times (by default N = 10). The total number of I _P pulses is calculated for N cycles (Section 5.31). Next, the correction of the value of I _{P is} carried out taking into account the correction coefficients contained in block 28 (p.5.32), according to the formula
I _P ¹ = K _n (I _P -U _n ) + (b _n + U _n ) (2)
and the issuance of the adjusted value of I ¹ _P to the serial interface 30 and through the parallel port 21 to the indicator 26. The coefficients q = 0, c = 1 are set (paragraph 5.33).

If the "TOXICITY" button is activated (Section 5.4), then the value of the characteristic of the mode q =? (Clause 5.41). When q = 0 (Section 5.42), the toxicity P _t is calculated by the formula:
P _t = (I ₀ ¹ -I _P ¹ ) / I ₀ ¹ • 100, (3)
where t is the reference number P.

Then, the reference number t is increased by 1 (if t> 3, then t = 0). Then, the value П _t is output to the indicator 26 and to the serial interface 30 (item 5.43) and the operation of inverting the attribute q, setting the coefficients c = 1, 2 = 0 (item 5.44).

At q = 1 (Section 5.45), the gamma function G is calculated by the formula
G = (I ₀ ¹ -I _P ¹ ) / I _P ¹ (4)
and the issuance of the calculated value of G to the indicator 26 and to the serial interface 30 (section 5.46). The coefficients are set: z = 1, c = 1, as well as the inversion of the q value (Section 5.47).

 If the "MODE" button is activated (Section 6.0), an analysis of previous states and analysis of the signs of the t, z, s modes (Section 6.1) are performed.

At 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 _{cf is} output to indicator 26 and to the serial interface (clause 6.12) and setting z = 0, q = 0, t = 0 (clause 6.13).

 In step 6.2 of the program, with c = 0, an increase in the number N is performed by a factor of 2 (for N = 1280, then N = 10) and the value N is displayed on indicator 26.

 In step 6.3 of the program, with the values of the signs of the modes c = 1, z = 0, t = 1 and with PC = 1, the system switches to the "PERIODIC MONITORING" mode and returns the program to step 4.11.

 As a result of testing the patented invention in the BIOTOX series luminometers, it was found that as a result of the correction, the output characteristic of the device approaches linear with high accuracy (no worse than 1.0% in the range up to 10,000 units according to the testimony of the LKB device (Finland)), and The luminometer is characterized by increased noise immunity, reliability and lower weight.

 Industrial applicability. As a photomultiplier tube, a PMT 86 type device can be used; an amplifier can be implemented, for example, on a K574UD1A type microcircuit. As a microprocessor, a Zilog type Z80 microcontroller or another similar one with the following parameters can be used: clock frequency ≥4 MHz, number of bits of the data bus ≥8, number of bits of the address bus ≥16.

 The memory unit 28 can be implemented on chips of type 2764 or similar. As the computer 32, any IBM compatible computer with a COM port (RS232 interface) can be used.

Claims (3)

 1. A device for analyzing liquid media by the luminescent method, comprising a reaction chamber and a luminescence recording system, including a photoelectronic multiplier connected to an amplifier and a control unit and outputting data to external recording devices, a photoelectric multiplier power supply unit, characterized in that the luminescence recording system contains means for linearization of the output characteristic, the control unit and data output to external recording devices contains series-connected via comparator and 2-I logic circuit, to the other input of which the meander generator is connected, with an output connected to the counter input of the counter, the output of which is connected via a parallel port via the data and address bus to the microprocessor, memory unit and serial interface for connecting to a personal computer, a threshold level setting unit is connected to the other input of the comparator, while the parallel port is connected to the control input of the counter, the input unit and the display panel, and the means for linearizing the output characteristics contain a table of correction coefficients entered into the memory unit, calculated according to the results of calibration on a reference radiation source.  2. The device according to claim 1, characterized in that the power supply unit of the photoelectronic multiplier is made according to the blocking generator circuit and is additionally equipped with a voltage multiplier, a choke and a compensation winding, the beginning of which is connected via a choke to the positive power terminal and the first capacitor, the other tap of which is connected to the ground, and the end to the end of the transformer winding connected to the transistor and the second capacitor, the other tap of which is connected to the ground, and the output winding of the transformer through the voltage multiplier is connected It is connected to the terminal connecting to the cathode of the photomultiplier tube and to one tap of the resistive voltage divider, which has taps for connecting to the dynodes of the photomultiplier tube, while the other tap of the resistive divider is connected to the ground and the housing of the voltage multiplier.  3. The device according to p. 1 or 2, characterized in that the control unit and outputting data to external recording devices contains a mode switch: "LUMINOMETER", "CONTROL", "SAMPLE", "TOXICITY", "MODE".

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