A photoelectric signal acquisition method for large-volume deep-well plates during bacterial culture

Abstract

The present application discloses a method for collecting photoelectric signals in a large-volume deep-well plate during a bacterial culture process, which relates to the field of data processing technology, including installing a deep-well plate culture tank containing a culture solution to be tested on an oscillation unit of a high-throughput microbial growth instrument; vertically irradiating the culture solution to be tested below the deep-well plate culture tank with a semiconductor laser; detecting photoelectric signals above the deep-well plate culture tank with a photoelectric detector to obtain a detection signal, and during the detection, the photoelectric detector and the semiconductor laser move synchronously along the plane where the deep-well plate culture tank is located; amplifying the detection signal in an exponential function manner to obtain an amplified signal; filtering the amplified signal to obtain a filtered signal; and digitally processing the filtered signal. The present application ensures a larger range in the range of photoelectric signal collection in a large-volume deep-well plate during a bacterial culture process.

Description

Technical Field

[0001] The present application relates to the field of signal processing technology, and in particular to a method for collecting photoelectric signals in a large-volume deep-well plate during bacterial culture. Background Art

[0002] Microbiology is one of the foundations of modern biotechnology and scientific research. Through the study of microbiology, new biological tools, biological drugs, genetic engineering technologies, etc. can be developed to promote scientific research and technological innovation. With the in-depth study of microbial diversity and function, the demand for screening a large number of strains is increasing, and high-throughput screening technology can meet this demand. The demand for efficient strains in various industries is increasing, such as pharmaceuticals, agriculture, environmental protection and other fields, which require faster and more accurate screening of strains with specific functions. High-throughput screening technology can meet these needs. The development of high-throughput screening technology has promoted innovation in the fields of microbiology and biotechnology, and provided stronger support for the development of new products and new processes.

[0003] The optical density detection module is the core part of the microbial growth analyzer. The construction of the optical system and the design of the photoelectric signal detection circuit are the key issues of optical density detection. The accuracy, stability and reliability of optical density detection will directly affect the results of biological experiments. The growth curve obtained by abnormal optical density will cause the experimenter to draw wrong test results and conclusions about the growth performance of microorganisms, so accurate optical density measurement is of great significance.

[0004] In the growth detection of aerobic bacteria, the large volume of oxygen culture leads to a higher optical density value of the bacterial solution. Therefore, how to improve the range of optical density detection becomes a key issue. The optical signal detection module is an important component of the optical density detection system, mainly including the photoelectric conversion module, the signal amplification module and the signal acquisition module. The design performance of these modules is the key factor affecting the range, resolution and accuracy of optical density detection. According to the Lambert-Beer law, the optical density and light intensity are logarithmically related, resulting in a large range of light intensity changes in the optical density detection of microorganisms. The dynamic range of the input signal of the signal amplification module is very large. At high optical density, the signal-to-noise ratio of the system will decrease. The operational amplifier has the characteristics of high gain and low noise, but its dynamic range is limited. The phase-locked amplifier is sensitive to noise and has a relatively narrow dynamic range. None of the above solutions can meet the requirements of high range in optical density detection. Summary of the invention

[0005] The purpose of the present application is to provide a method for collecting photoelectric signals in a large-volume deep-well plate during bacterial culture to solve the technical problems raised in the above background.

[0006] To achieve the above purpose, the present application discloses the following technical solution: a method for collecting photoelectric signals in a large-volume deep-well plate during bacterial culture, the method comprising the following steps:

[0007] Installing a deep-well plate culture tank containing the culture solution to be tested on an oscillation unit of a high-throughput microbial growth instrument, wherein the microbial growth instrument provides a culture environment for the culture solution to be tested, and the oscillation unit is used to oscillate the deep-well plate culture tank during the detection process;

[0008] The culture solution to be tested is vertically irradiated below the culture tank of the deep well plate by a semiconductor laser;

[0009] The photoelectric signal is detected above the deep-well plate culture tank by a photoelectric detector to obtain a detection signal, and during the detection, the photoelectric detector and the semiconductor laser move synchronously along the plane where the deep-well plate culture tank is located;

[0010] Amplifying the detection signal in an exponential function manner to obtain an amplified signal;

[0011] Performing filtering on the amplified signal to obtain a filtered signal;

[0012] The filtered signal is digitized.

[0013] Preferably, the photodetector is a silicon photocell.

[0014] Preferably, the amplifying the detection signal in an exponential function manner specifically includes:

[0015] A logarithmic amplifier is used as a signal amplification device.

[0016] Preferably, the model of the logarithmic amplifier is AD8304.

[0017] Preferably, the digital processing of the filtered signal specifically includes:

[0018] The transfer function between the output voltage of the logarithmic amplifier and the measured optical density value OD is: Among them, V out is the output voltage of the logarithmic amplifier, G is the post-stage gain coefficient of the logarithmic amplifier, V y is the slope voltage of the logarithmic amplifier, I z is the intercept current of the logarithmic amplifier, ρ is the responsivity of the logarithmic photodiode, P opt is the optical power generated by the photodiode of the logarithmic amplifier;

[0019] Calculate the attenuated power P2, Wherein, P1 is the power of the semiconductor laser when it is emitted, P2 represents the attenuated power corresponding to the propagation distance d of P1, d is the propagation distance, and the semiconductor laser is aligned, indicating that the laser propagation distance corresponding to each deep hole culture tank is the same;

[0020] The attenuated power P2 is used as the amplified signal.

[0021] Preferably, the digital processing of the filtered signal further comprises:

[0022] Calculate the measured optical density value OD, Among them, I trans is the transmitted light intensity, I inc is the incident light intensity;

[0023] The dimensions of each deep-hole culture tank are configured to be the same so that the area reaching the photodetector is the same. V trans is the transmitted light voltage value;

[0024] Based on the output voltage of the logarithmic amplifier and the photocurrent generated by the photodetector satisfying in,

[0025] Then OD=K·V trans +C, where K and C are constants;

[0026] Generates the value corresponding to OD = K·V trans +C growth curve.

[0027] Preferably, the digital processing of the filtered signal further comprises: performing error analysis on the amplified signal; the error analysis specifically comprises:

[0028] According to the definition of differential Define V0 as the reference voltage, then

[0029] make Define the measurement error δ, then make but

[0030] 0<Α<1, then Reverse the connection to obtain the relationship between a and A, δ, and δ0, and we get

[0031] Based on the relationship between δ and A, the relationship between the relative error and the relative measured voltage of the permeated solution was obtained;

[0032] Complete the error analysis.

[0033] Preferably, the filtering process of the amplified signal specifically includes:

[0034] The amplified signal is filtered using a Kalman filter algorithm.

[0035] Beneficial effects: The photoelectric signal acquisition method of the present application for a large-volume deep-well plate during bacterial culture realizes the conversion of weak light signals into transmitted light voltage values, and finally into optical density values. In addition, a larger measuring range is ensured through the signal amplification, filtering processing and digital processing of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 A flowchart of a method for collecting photoelectric signals in a large-volume deep-well plate during bacterial culture provided in an embodiment of the present application.

[0038] Figure 2 A schematic diagram of the hardware circuit of the high-throughput microbial growth analyzer system provided in an embodiment of the present application;

[0039] Figure 3 A peripheral resistor circuit diagram of a logarithmic amplifier provided in an embodiment of the present application;

[0040] Figure 4 A schematic diagram of the relationship between the relative error and the ratio of the measured voltage to the reference voltage provided in an embodiment of the present application. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0042] In this article, the term "comprising" is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, the elements defined by the sentence "comprising..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0043] This embodiment discloses Figure 1 A method for collecting photoelectric signals in a large-volume deep-well plate during bacterial culture is shown, the method comprising the following steps:

[0044] Installing a deep-well plate culture tank containing the culture solution to be tested on an oscillation unit of a high-throughput microbial growth instrument, wherein the microbial growth instrument provides a culture environment for the culture solution to be tested, and the oscillation unit is used to oscillate the deep-well plate culture tank during the detection process;

[0045] The culture solution to be tested is vertically irradiated below the culture tank of the deep well plate by a semiconductor laser;

[0046] The photoelectric signal is detected above the deep-well plate culture tank by a photoelectric detector to obtain a detection signal, and during the detection, the photoelectric detector and the semiconductor laser move synchronously along the plane where the deep-well plate culture tank is located;

[0047] Amplifying the detection signal in an exponential function manner to obtain an amplified signal;

[0048] Performing filtering on the amplified signal to obtain a filtered signal;

[0049] The filtered signal is digitized.

[0050] The light required for microbial growth analysis is monochromatic light, and because there is a good correlation between the OD600 value and the cell concentration in the culture medium, the relationship between OD600 and the number of cells is relatively stable during the logarithmic period of cell growth, which can be used as an effective indicator for estimating cell growth rate and growth stage. Therefore, the commonly used light wavelength for microbial growth curve detection is 600nm. Considering that the system needs to achieve high-throughput measurement, the miniaturization of the optical system is very important. Semiconductor lasers can produce high-brightness light beams with good directionality and a very narrow wavelength range, which is almost monochromatic. The light emitted by semiconductor lasers has a very narrow wavelength range, which is almost monochromatic. This monochromaticity is very beneficial for applications such as spectral analysis and interferometry that require precise control of light wavelengths. Small size, low energy consumption, fast start-up, and the light source uses a semiconductor laser with a wavelength of 650nm.

[0051] Secondly, as a device for converting light signals into electrical signals, photodetectors are commonly used, including photoresistors, photodiodes, photocells, phototransistors, photomultiplier tubes, and photodetector modules. In this embodiment, the photodetector is a silicon photocell. A silicon photocell is a PN junction photoelectric device that can convert light energy into electrical energy without adding a bias voltage. Its advantages are a wide linear response range, high sensitivity, suitable spectral response, good stability, and long service life. It is widely used in photometric, chromatic, optical precision measurement and testing equipment. Silicon photocells are favored because of their wide photosensitive area, large junction capacitance, and low frequency response. In contrast, the photoelectric conversion linearity of photoresistors is poor, the dynamic range is narrow, and the time response is slow, so it is not suitable as a first choice. Compared with photodiodes, silicon photocells have a wider photosensitive area, show higher photoelectric conversion efficiency and a wider dynamic range, and thus become the best choice for system photodetectors. Specifically, this embodiment uses 2DU6 silicon photocells.

[0052] In addition, the high-throughput microbial growth analyzer can be any one of the prior art, and in particular, it is adapted to the deep-well plate culture tank. In this embodiment, the high-throughput microbial growth analyzer generally includes two parts: hardware and software:

[0053] The hardware part is mainly divided into three parts including motion control part, temperature control part and light density detection part.

[0054] The motion control part of the detection module is used to realize the synchronous movement of the photoelectric detector and the semiconductor laser along the plane where the deep-well plate culture groove is located.

[0055] Temperature control part: The main control board is used to control the working time of the heating plate to adjust the temperature of the incubator (composed of multiple deep-hole culture tanks). At the same time, the temperature sensor is used to monitor the ambient temperature in real time to achieve closed-loop control of the incubator temperature and ensure that the sample is in a constant temperature environment during the culture process.

[0056] Optical density detection system: including an optical system and a photoelectric signal detection system corresponding to the optical system. The optical system is the aforementioned semiconductor laser with a wavelength of 650nm. The silicon photocell collects the optical signal attenuated by the culture solution, and then logarithmically amplifies the photocurrent generated by the silicon photocell. After AD conversion, the voltage value is read and the optical density value is calculated.

[0057] The software part mainly includes the upper computer human-computer interaction interface design, data processing module, growth curve display and control.

[0058] The input and output of the logarithmic amplifier are in a logarithmic relationship, and the output signal it generates is proportional to the logarithm of its input. Due to the compression characteristics of the function, the logarithmic amplifier can be used to process wide dynamic range signals, such as signals with amplitude changes exceeding 100dB. Some aerobic bacteria in microorganisms require large volumes of aerobic culture, and the optical density often reaches above 5. At the same time, according to Lambert-Beer's law, the optical density is proportional to the logarithm of the input light intensity. The use of a logarithmic amplifier can directly convert the optical density and the output signal into a linear relationship, simplifying the logarithmic operation. In this regard, in this embodiment, the detection signal is amplified in the manner of an exponential function, specifically including:

[0059] A logarithmic amplifier is used as a signal amplification device, and the model of the logarithmic amplifier is AD8304.

[0060] Based on the above, this embodiment adopts Figure 2 The hardware circuit of the high-throughput microbial growth analyzer system is shown in the figure. Among them, a 24V switching power supply is used to power the system; STM32F103C8T6 is used as the main control chip; STM32F103C8T6 embedded microcontroller is used as the lower computer to communicate through the serial port; most of the experiments of the high-throughput microbial growth analyzer are motion control, and the sample addition motion control mainly includes three stepper motors for the XY axis motion of the detection module and the circular motion of the sample box; DS18B20 is used as the temperature acquisition unit.

[0061] In order to determine the growth curve of microorganisms, it is necessary to measure the change in the intensity of the light signal, and the stability of the light source and the accuracy of the monochromatic light source will directly affect the measurement results of the instrument. In the measurement of light signals, photoelectric conversion must be performed to convert the light signal into an electrical signal for measurement. As mentioned earlier, this system uses photocells as photoelectric conversion devices. A photocell (also called a photovoltaic cell or solar cell) is a device that can convert light energy into electrical energy. Its working principle is based on the photoelectric effect, which realizes the conversion of light energy to electrical energy. The typical working principle of a photoelectric conversion device is based on the photoelectric effect, the most common of which is the photoelectric effect under light irradiation and the photovoltage effect of semiconductor materials. The following are its basic principles:

[0062] (1) Photoelectric effect under light irradiation: When light irradiates the surface of a specific material, the energy of the photon is transferred to the electrons in the material, causing the electrons to jump from the valence band to the conduction band, forming electron-hole pairs. These electrons and holes can move inside the material to form current, thereby realizing the conversion of light energy into electrical energy.

[0063] (2) Photovoltaic effect of semiconductor materials: In a PN structure semiconductor device, when light is irradiated to the PN junction area, the energy of the photon excites the electron-hole pair. Due to the built-in electric field of the PN junction, the electrons move to the N region and the holes move to the P region, generating photovoltage. By connecting an external circuit, the photovoltage can be converted into usable electrical energy. In order to collect the signals generated by the photoelectric conversion device, it is usually necessary to use appropriate circuits and equipment. The following are general signal collection methods:

[0064] (1) Amplification circuit: The output signal generated by the photoelectric conversion device is often very small, so an amplification circuit is usually required to increase the signal amplitude. The amplification circuit can be implemented by an amplifier (such as an operational amplifier) ​​to improve the measurability and stability of the signal.

[0065] (2) Filter circuit: Photoelectric conversion devices may be affected by noise. In order to reduce the impact of noise on the signal, a filter circuit can be used for filtering. The filter circuit can select a suitable frequency band to filter out unnecessary frequency components.

[0066] (3) Data acquisition system: The collected signals can be digitized by the data acquisition system. The data acquisition system usually includes an analog-to-digital converter (ADC) to convert the analog signal into a digital signal, which is further processed and analyzed by a microcontroller or computer.

[0067] In order to solve the problem of deep liquid level, low transmitted light intensity and weak output signal of sensor in large volume deep well plate during bacterial culture, the logarithmic amplifier can amplify the input signal in the form of exponential function, so that the smaller input value can be amplified to a larger range while maintaining the distinguishability of the larger input value.

[0068] By using a logarithmic amplifier, the peripheral resistor circuit of the logarithmic amplifier is as follows Figure 3 As shown, in this embodiment, the digital processing of the filtered signal specifically includes:

[0069] The transfer function between the output voltage of the logarithmic amplifier and the measured optical density value OD is: Among them, V out is the output voltage of the logarithmic amplifier, G is the post-stage gain coefficient of the logarithmic amplifier, V y is the slope voltage of the logarithmic amplifier, I z is the intercept current of the logarithmic amplifier, ρ is the responsivity of the logarithmic photodiode, P opt is the optical power generated by the photodiode of the logarithmic amplifier;

[0070] Based on the law of light intensity attenuation in light propagation, as the laser propagation distance increases, the power of the laser will decay, which is represented by the attenuated power P2. Wherein, P1 is the power of the semiconductor laser when it is emitted, P2 represents the attenuated power corresponding to the propagation distance d of P1, d is the propagation distance, and the semiconductor laser is aligned, indicating that the laser propagation distance corresponding to each deep hole culture tank is the same;

[0071] The attenuated power P2 is used as the amplified signal.

[0072] Furthermore, the digital processing of the filtered signal further includes:

[0073] Calculate the measured optical density value OD, Among them, I trans is the transmitted light intensity, I inc is the incident light intensity; the light intensity is expressed as the light power value per unit area, and the power is expressed as follows:

[0074]

[0075] The dimensions of each deep-hole culture tank are configured to be the same so that the area reaching the photodetector is the same. V trans is the transmitted light voltage value;

[0076] Based on the output voltage of the logarithmic amplifier and the photocurrent generated by the photodetector satisfying in,

[0077] Since the incident power of the semiconductor laser is a constant value, OD = K·V trans +C, where K and C are both constants; the final calculation results are K=-5.27615, C=7.59756.

[0078] Generates the value corresponding to OD = K·V trans +C growth curve.

[0079] Furthermore, the digital processing of the filtered signal further includes: performing error analysis on the amplified signal; the error analysis specifically includes:

[0080] According to the definition of differential Define V0 as the reference voltage, then

[0081] make Define the measurement error δ, then make but

[0082] 0<Α<1, then Reverse the connection to obtain the relationship between a and A, δ, and δ0, and we get

[0083] Based on the relationship between δ and A, the relationship between the relative error and the relative measured voltage of the permeated solution is obtained, as follows: Figure 4 As shown;

[0084] Complete the error analysis.

[0085] Preferably, the filtering process of the amplified signal specifically includes:

[0086] The amplified signal is filtered using a Kalman filter algorithm.

[0087] It is known that Kalman filtering is one of many filtering technologies. Its algorithm does not need to retain historical observation values. It only needs to use the current observation value and the estimated value of the previous moment to generate a new estimation result through iterative calculation to complete the filtering process. Kalman filtering is suitable for dynamic systems containing uncertain information, can effectively resist noise interference, and give the best estimate at the current moment.

[0088] In particular, in order to display the growth curve, this embodiment uses Qt Charts, which is a module officially provided by Qt for creating interactive charts and data visualization. It is an extension module based on the Qt framework and can be easily integrated into Qt applications to display various types of charts, such as line charts, bar charts, pie charts, etc. It provides a wealth of functions and style customization options, and users can flexibly customize the appearance and interactive behavior of charts to meet the needs of different application scenarios. With Qt Charts, developers can quickly create attractive and interactive charts to help users understand data more intuitively. The Qt Charts module is easy to use, well-documented, supports a variety of common chart types and data series, and can effectively simplify the development process and improve development efficiency. Whether it is used for data visualization in enterprise applications or for data display in scientific research, Qt Charts is a powerful and practical tool. The theme of the display control module is QChart objects, which are placed in the QT main window as controls. A 6*8 grid is created using the QGridLayout grid layout to create a Qchart for the growth curve of each hole and place it in the QGridLayout. When new data is detected, the data in QSpineLine is refreshed to achieve real-time detection of the growth curve.

[0089] In the embodiments provided in the present application, it should be understood that the embodiments described herein can be implemented in hardware, software, firmware, middleware, code or any appropriate combination thereof. For hardware implementation, the processor can be implemented in one or more of the following units: application specific integrated circuit (ASIC), digital signal processor (DSP), digital signal processing device (DSPD), programmable logic device (PLD), field programmable gate array (FPGA), processor, controller, microcontroller, microprocessor, other electronic units designed to implement the functions described herein or their combination. For software implementation, part or all of the flow of the embodiment can be completed by instructing the relevant hardware through a computer program. When implemented, the above program can be stored in a computer-readable storage medium or transmitted as one or more instructions or codes on a computer-readable storage medium. Computer-readable storage media include computer storage media and communication media, wherein the communication medium includes any medium that is convenient for transmitting a computer program from one place to another. The storage medium can be any available medium that a computer can access. The computer-readable storage medium can include but is not limited to RAM, ROM, EEPROM, CD-ROM or other optical disk storage, disk storage medium or other magnetic storage device, or any other medium that can be used to carry or store the desired program code in the form of an instruction or data structure and can be accessed by a computer.

[0090] Finally, it should be noted that the above is only a preferred embodiment of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for collecting photoelectric signals in a large-volume deep-well plate during bacterial culture, characterized in that: The method comprises the following steps: The deep-well plate culture tank containing the culture solution to be tested is installed on the shaking unit of the high-throughput microbial growth instrument, the microbial growth instrument provides a culture environment for the culture solution to be tested, and the shaking unit is used to shake the deep-well plate culture tank during the detection process; The culture solution to be tested is vertically irradiated below the culture tank of the deep well plate by a semiconductor laser; The photoelectric signal is detected above the deep-well plate culture tank by a photoelectric detector to obtain a detection signal, and during the detection, the photoelectric detector and the semiconductor laser move synchronously along the plane where the deep-well plate culture tank is located; Amplifying the detection signal in an exponential function manner to obtain an amplified signal; Performing filtering on the amplified signal to obtain a filtered signal; Digitally processing the filtered signal; The amplifying the detection signal in an exponential function manner specifically includes: using a logarithmic amplifier as a signal amplification device; the model of the logarithmic amplifier is AD8304; The digital processing of the filtered signal specifically includes: The transfer function between the output voltage of the logarithmic amplifier and the measured optical density value OD is: ,in, is the output voltage of the logarithmic amplifier, is the post-stage gain coefficient of the logarithmic amplifier, is the slope voltage of the logarithmic amplifier, is the intercept current of the logarithmic amplifier, is the logarithm of the photodiode responsivity, is the optical power generated by the photodiode of the logarithmic amplifier; Calculate the power after attenuation , ,in, is the power of the semiconductor laser when it is emitted, Indicated by The propagation distance is The corresponding attenuated power is, is the propagation distance, and the semiconductor laser is aligned and aligned, indicating that the laser propagation distance corresponding to each deep-well plate culture slot is the same; The attenuated power , as the amplified signal; Furthermore, the step of digitally processing the filtered signal further comprises: Calculate the measured optical density value OD, ,in, is the transmitted light intensity, is the incident light intensity; The dimensions of each deep-well plate culture tank are configured to be the same so that the area reaching the photodetector is the same. , is the transmitted light voltage value; Based on the output voltage of the logarithmic amplifier and the photocurrent generated by the photodetector satisfying ,in, ; Then there is ,in, and are all constants; Generates the corresponding Growth curve of The photodetector is a silicon photocell.

2. The photoelectric signal acquisition method for a large-volume deep-well plate during bacterial culture according to claim 1 is characterized in that: The digital processing of the filtered signal further includes: performing error analysis on the amplified signal; the error analysis specifically includes: According to the definition of differential ,definition is the reference voltage, then ; make , defining the measurement error ,but ,make ,but ; , ,but ; Reverse connection to get a and A, as well as The relationship between , , ; based on The relationship between the relative error and the relative measured voltage of the permeated solution was analyzed; Complete the error analysis.

3. The photoelectric signal acquisition method for a large-volume deep-well plate during bacterial culture according to claim 1 is characterized in that: The filtering process of the amplified signal specifically includes: The amplified signal is filtered using a Kalman filter algorithm.

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