CN114235685A - Shake flask cultured biomass on-line measuring device - Google Patents
Shake flask cultured biomass on-line measuring device Download PDFInfo
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- CN114235685A CN114235685A CN202111294703.6A CN202111294703A CN114235685A CN 114235685 A CN114235685 A CN 114235685A CN 202111294703 A CN202111294703 A CN 202111294703A CN 114235685 A CN114235685 A CN 114235685A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
Abstract
The invention discloses a biomass on-line detection device for shake flask culture, which comprises a fixed bracket, a base, a light source, an optical signal receiver, a control circuit board, an acceleration sensor and the like; the light source, the optical signal receiver and the control circuit board are arranged on the base; light emitted by the light source is scattered through the detected liquid; the optical signal receiver is used for receiving the scattered light and converting an optical signal into an electric signal; the acceleration sensor is arranged on the control circuit board, and the position information of the shake flask is accurately measured by reading corresponding acceleration and angular velocity values; the control circuit board is responsible for automatic adjustment control of the light source and noise reduction, reading, conversion, operation, transmission and the like of signals. The invention can avoid the influence of ambient light, does not need to additionally use a shading sleeve, and is convenient for an experimenter to observe the growth state of the thalli in the shake flask; meanwhile, the light source can be adjusted according to the test value so as to meet the test of the same device on different shake flasks, and the error caused by manually adjusting the shake flask position is avoided.
Description
Technical Field
The invention relates to the technical field of biological experiment equipment, in particular to a biomass online detection device for shake flask culture.
Background
In the microbial cultivation process, a shake flask is often used for cultivation in a shaking table, and in order to monitor the growth condition of the shake flask-cultivated thalli, offline sampling is required for many times to test the concentration. Usually, a spectrophotometer is used for testing the OD600, when the concentration rises, a sample needs to be diluted and then measured, the risk of infecting mixed bacteria by a culture solution is increased by an off-line testing method, a culture device needs to be interrupted frequently, and the accuracy of the test cannot be ensured by manual sampling. The existing non-invasive biomass on-line monitoring device can solve the problems, but needs shading treatment, is relatively complex to operate, and cannot enable an experimenter to visually observe the state of cell culture. In addition, the placing position of the shake flask is required to be fixed.
In bioengineering or fermentation engineering, shaking flasks are often used for cultivation in a shaker. In order to monitor the growth state of microorganisms in a medium, detection by Optical Density (OD) measurement or the like is generally required.
CN 204945038U discloses a non-invasive biomass online detection device for shake flask culture, which comprises a main control unit, a shake flask capable of containing biological suspension, and a fixed sleeve covering the outer side of the shake flask, wherein the fixed sleeve is connected with a detection probe and the like at the outer side of the bottom of the shake flask. The device can realize carrying out on-line measuring to the concentration of biological turbid liquid, need not manually remove the sample or use spectrophotometer.
CN 204945038U discloses a non-invasive biomass online detection device for shake flask culture, which can realize biomass online detection of shake flask culture without manually removing a sample for multiple sampling. In addition, the device uses a fixed sleeve to shade the shake flask. Due to the fact that the bottom of the shake flask is made by the glass process, light scattering at different positions of the same shake flask is different, the shake flask needs to be matched with the shake flask one by one in the testing process, and the position of the shake flask needs to be fixed, so that the experimental operation steps are increased, and the experiment operation is complicated.
At present, the most common off-line Optical Density (OD) measuring method in a laboratory needs scientific research personnel to manually sample for multiple times, is complex to operate and is easy to infect mixed bacteria. The existing biomass on-line detection device needs shading treatment, and cannot avoid the influence of external environment light.
Disclosure of Invention
In view of the above, in order to solve the above problems in the prior art, the present invention provides an online biomass detection device for shake flask culture. The device need not to carry out extra shading and handles, can avoid the influence of external environment light, makes things convenient for the laboratory technician to observe the thallus growth condition in shaking the bottle. In addition, self-adaptive adjustment can be carried out according to the placing positions of different shake flasks.
The invention solves the problems through the following technical means:
an on-line biomass detection device for shake flask culture comprises:
a base;
the fixed bracket is arranged on the base and used for fixing the shake flask;
the light source is arranged on the base and used for emitting light to be scattered through the detected liquid;
the optical signal receiver is arranged on the base and used for receiving scattered light and converting an optical signal into an electric signal;
the acceleration sensor is arranged on the base and used for reading corresponding acceleration and angular velocity values to accurately measure the position information of the shake flask;
the control circuit board is arranged on the base, is respectively and electrically connected with the light source, the optical signal receiver and the acceleration sensor, is used for receiving an external control signal, performs biomass on-line detection by adopting an external environment light deduction method and matching algorithms aiming at different shake flasks according to the external control signal, is responsible for automatic adjustment control of the light source and noise reduction, reading, conversion, operation and transmission of the signal, and sends the measured data to a worker for checking.
Further, the method for subtracting the external environment light specifically comprises:
the biomass online detection device is matched with a shake flask A, firstly, a default current output is given to a light source to drive the light source to emit light, the light source is in an initial current gear A, an OD original value A0 is read at the moment, the actually tested voltage value obtained by adding light emitted by the light source and external environment light conversion of the A0 is obtained, the light source is turned off, 0D original value B0 is read, B0 is the voltage value obtained by external environment light conversion, and theoretically, A0-B0 is the voltage value obtained by light source conversion; this operation needs to be repeated since the ambient light changes; in addition, the device has different positions of the shaking table and different ambient light, the shaking table can move at different rotating speeds in the culture process, the shaking table position is detected through the acceleration sensor, the corresponding 0D original value is read when the shaking table reaches a specified angle region and is used for deducting the influence of the ambient light, and the OD original value read in the angle region is not influenced by the liquid capacity in the shaking table and the rotating speed of the shaking table; in order to ensure the accuracy of the numerical value and the stability of the light source, a large amount of data needs to be read for algorithm processing, and the original value of the OD after the ambient light is removed is obtained.
Further, the matching algorithm for different shake flasks specifically includes:
the biomass online detection device is matched with the shake flask A, and is calibrated and fitted by an offline OD test method or an existing online OD concentration test device to obtain the relation between an OD original value and the biomass concentration, the OD original value and the OD initial value form a certain exponential relation; after calibrating a biomass on-line detection device, a corresponding fitting parameter is generated; meanwhile, an angle area and an OD initial value C when the biomass on-line detection device is tested need to be recorded; when the shake flask B is matched to start an experiment, the OD original value read in the angle area is also C by adjusting the current of the light source; then, the fitting parameters of the biomass online detection device are called, so that the accuracy of the same biomass online detection device in the concentration tests of different shake flasks is ensured; and the growth condition of the thallus is checked in real time through OD on-line detection in the culture process.
Further, the deduction method of the external environment light is realized by adopting an analog mode, specifically:
two photoelectric sensors are adopted, one photoelectric sensor is used for measuring external environment light, and the other photoelectric sensor is used for measuring a light source and the external environment light;
using an operational amplifier, where V1 is the voltage value of the light source plus the ambient light conversion, and V2 is the voltage value of the ambient light conversion, Vout — V1-V2 are obtained by calculation.
Further, the control of the light source by the matching algorithm for different shake flasks is realized in a simulation mode specifically as follows:
the current-limiting resistor is added on the light source control circuit, the resistor is a common resistor or a variable resistor with higher precision, and the current is changed by adjusting the resistance value, so that the aim of adjusting the brightness of the light source is fulfilled.
Further, the deduction method of the external environment light is realized by adopting a software mode, and specifically comprises the following steps:
for a photoelectric sensor, by controlling the switch of a light source, when a shake flask reaches a specified angle region, reading a corresponding voltage value, namely an OD original value, wherein the OD original value read in the region is not influenced by the volume of liquid in the shake flask and the rotating speed of a shaking table, in order to ensure the accuracy of numbers and the stability of the light source, the switching time of the light source needs to be accurately controlled, the OD original value, which is the voltage value after deducting ambient light, is obtained by multiple times of reading in the angle region, and the OD original value, which is the voltage value after deducting the ambient light, is calculated by algorithm processing.
Further, the control of the light source by the matching algorithm for different shake flasks is realized in a software manner specifically as follows:
the special LED drive circuit is used for controlling the brightness of the light source, and the software adjusts the brightness of the light source when different shake flasks are used according to the fitting parameters and the OD initial values determined by the device, so that the matching use can be met.
Further, the light source and the optical signal receiver follow a certain arrangement: the scattering type light source emits light which passes through the liquid to be detected and is received by the light receiver.
Furthermore, the biomass online detection device adopts an internal battery for power supply or adopts a wired external power supply for power supply.
Further, the acceleration sensor is mounted on the control circuit board.
Compared with the prior art, the invention has the beneficial effects that at least:
the invention can avoid the influence of ambient light, does not need to additionally use a shading sleeve, and is convenient for experimenters to observe the growth state of thalli in the shake flask. Meanwhile, the light source can be adjusted according to the test value so as to meet the test of the same device on different shake flasks, and the error caused by manually adjusting the shake flask position is avoided.
The device can realize online real-time adjustment of the brightness of the light source, the device does not need to be matched with the shake flasks in a one-to-one correspondence manner, the position of the shake flasks does not need to be fixed, and the test accuracy is improved;
the invention does not need to additionally add a light shield, can directly observe in the culture process and does not need to interrupt experimental equipment;
the invention does not need to interrupt the test and adjusts the brightness of the light source.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic view of a usage scenario of an online biomass detection device for shake flask culture according to the present invention;
FIG. 2 is a schematic flow chart of a method for subtracting ambient light according to the present invention;
FIG. 3 is a schematic diagram of an analog circuit deduction method for deducting ambient light according to the present invention.
In the figure:
1. shaking the flask; 2. fixing a bracket; 3. a base.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. It should be noted that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work based on the embodiments of the present invention belong to the protection scope of the present invention.
Examples
The biomass online detection device for shake flask culture mainly comprises a fixed bracket and a base (an online experimental device containing biomass inside). The biomass online experimental device comprises: light source, light signal receiver, control circuit board, acceleration sensor etc.. The light source and the optical signal receiver are arranged on the base. The light emitted by the light source is scattered by the liquid to be detected. The optical signal receiver is used for receiving scattered light, converting an optical signal into an electrical signal and measuring optical density. The acceleration sensor is arranged on the control circuit board, and the position information of the shake flask is accurately measured by reading corresponding acceleration and angular velocity values, so as to monitor the position state of the shake flask. The control circuit board is responsible for automatic adjustment control of the light source and noise reduction, reading, conversion, operation, transmission and the like of signals. The use scene is shown in figure 1: (in the figure: 1, shake flask, 2, fixed support, 3, base).
The light source of the invention can be a common LED, or an infrared LED, or an optical fiber, as long as the light source can be provided; the optical signal receiver can be a voltage output, a frequency output, or a digital signal output, and is not limited to a digital type and an analog type; the driving circuit of the light source is not limited to the PWM signal adjustment inside the controller, or an external special driving chip is used, as long as the brightness of the light source can be adjusted by software; the acceleration sensor may be any chip capable of measuring acceleration and angular velocity.
The invention provides a biomass on-line detection device for shake flask culture, which mainly comprises a light source, an optical signal receiver, an acceleration sensor, a control circuit board, a fixed support and a base. The light source and the optical signal receiver realize the measurement of a sample to be measured, the acceleration sensor realizes the position measurement of the shake flask, the control circuit board realizes the related control function, the base is used for fixing the light source, the optical signal receiver and the control circuit board, and the support is used for fixing the shake flask.
The light source and the light signal receiver follow a certain arrangement mode, and the light is emitted by the light source in a scattering mode, passes through the liquid to be detected and is finally received by the light receiver.
The device can be powered by an internal battery or a wired external power supply.
The device can realize the receiving/sending of external control signals and measured data, and can adopt a wireless signal transmission mode (Wifi, RFID, Bluetooth and the like) and a wired signal transmission mode.
The fixing bracket has no fixing form as long as the arrangement mode followed by the above is satisfied.
The control circuit board realizes related control functions and comprises an automatic dimming circuit part. The microprocessor controls the on-time interval of the light source and can control the current of the light source to achieve the function of regulating the light brightness.
The biomass on-line monitoring device for the shake flask culture is detailed as follows:
1. the biomass online experimental device A is matched with the shake flask A, firstly, a default current output is given to the light source to drive the light source to emit light, if the initial current is in the gear A, the OD original value A0 is read at the moment. The OD original value refers to a voltage value of the corresponding photoelectric conversion in the current OD concentration state. The actually tested voltage value of the A0 light emitted by the light source is added with the voltage value obtained by converting the external environment light, the light source is turned off, 0D original value is read to be B0, B0 is the voltage value obtained by converting the external environment light, theoretically, A0-B0 obtains the voltage value obtained by converting the light source, and therefore the phenomenon that the voltage converted by the photoelectric sensor is changed due to the change of the external environment light can be avoided, and the biomass concentration online detection cannot be accurately realized. This operation needs to be repeated since the ambient light changes. In addition, the device has different shaking table positions and different ambient light, during the culture process, the shaking table can move at different rotating speeds, the shaking table position is detected through the acceleration sensor, the corresponding 0D original value is read when the shaking table reaches a specified angle region and is used for deducting the influence of the ambient light, and the OD original value read in the angle region is not influenced by the liquid capacity in the shaking table and the rotating speed of the shaking table. In order to ensure the accuracy of the values and the stability of the light source, a large amount of data is read and processed by an algorithm to obtain the original value of OD after ambient light is removed, as shown in fig. 2.
2. The biomass online experimental device A is matched with the shake flask A, and an offline OD test method or an existing online OD concentration test device is used for calibrating and fitting the shake flask A to obtain the relation between an OD original value (after external environment light is deducted) and the biomass concentration, wherein the biomass concentration, the OD original value and the OD initial value form a certain exponential relation. The OD initial value is a voltage value of corresponding photoelectric conversion when the OD concentration is 0. After calibration of an online biomass experimental setup, there will be a corresponding fitting parameter. Meanwhile, the angular region and the initial value C of OD (the value corresponding to the OD being 0) of the biomass online experimental device A during the test need to be recorded. When the experiment was started with shake flask B, the current of the light source was adjusted so that the OD original value read in this angle region is also C. And then the fitting parameters of the biomass online experimental device are called, so that the accuracy of the same biomass online detection device for testing different shake flask concentrations is ensured. The growth condition of the thalli can be checked in real time through OD on-line detection in the culture process.
Several possible embodiments
A. Realized by adopting an analog mode
Two photosensors may be used, one for measuring ambient light and the other for measuring light source plus ambient light. The subtraction of the two or the implementation of an analog circuit is realized by software.
As shown in fig. 3 below, using an operational amplifier, where V1 is the voltage value of the light source plus the ambient light conversion, and V2 is the voltage value of the ambient light conversion, Vout — V1-V2 are obtained by calculation (in the figure, R1, R2, R3, and R4 have the same resistance values).
The control of the light source can be controlled by an analog circuit, a current limiting resistor is added on a light source control circuit, the resistor can be a common resistor with higher precision or a variable resistor, and the current is changed by adjusting the resistance value, so that the aim of adjusting the brightness of the light source is fulfilled.
B. Using software algorithms
For a photoelectric sensor, by controlling the switch of a light source, when a shake flask reaches a specified angle region, reading a corresponding voltage value (OD original value), wherein the OD original value read in the region is not influenced by the liquid capacity in the shake flask and the rotating speed of the shaking table, in order to ensure the accuracy of numbers and the stability of the light source, the switch time of the light source needs to be accurately controlled, the OD original value which is the voltage value after deducting ambient light is calculated through algorithm processing, the deduction method is always carried out under the working state of the biomass on-line detection device, and during the first test, the OD needs to be tested by using a spectrophotometer and other off-line methods or existing on-line OD concentration testing equipment for fitting and calibration. And obtaining the relation between the subtracted original OD value and the concentration.
The special LED drive circuit is used for controlling the brightness of the light source, and the software adjusts the brightness of the light source when different shake flasks are used according to the fitting parameters determined by the shake flask control device and the initial value of OD (the value corresponding to OD being 0), so that the matching use can be met.
The biomass on-line detection device for shake flask culture also comprises a matching algorithm aiming at different shake flasks besides the subtraction of ambient light.
Because the requirement of light on a reflecting surface is very high, the voltage values read by the photoelectric sensors are different due to different differences, in addition, due to the problem of a glass process, the bottoms of the shake flasks cannot be completely consistent, the light reflection at different positions at the same bottom of each shake flask is different, and the different shake flasks are different, so that each biomass online detection device is required to be matched with one shake flask, and meanwhile, the position of each shake flask needs to be fixed, so that the complexity of an experiment is increased, and the test accuracy is not accurate.
According to the biomass online detection device for shake flask culture, the brightness of a light source is adjusted through the judgment of an algorithm according to the primarily read photoelectric conversion value (OD initial value), so that the shake flask can be matched with the detection device.
The invention can realize automatic brightness adjustment so as to facilitate the installation of different shake flask tests; the influence of external environment light can be avoided; the biomass on-line detection of the sample can be realized without sampling; biomass on-line detection can be carried out in a non-contact mode; can be combined with other common tools for biological experiments, and has convenient use and strong universality.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. An online biomass detection device for shake flask culture is characterized by comprising:
a base;
the fixed bracket is arranged on the base and used for fixing the shake flask;
the light source is arranged on the base and used for emitting light to be scattered through the detected liquid;
the optical signal receiver is arranged on the base and used for receiving scattered light and converting an optical signal into an electric signal;
the acceleration sensor is arranged on the base and used for reading corresponding acceleration and angular velocity values to accurately measure the position information of the shake flask;
the control circuit board is arranged on the base, is respectively and electrically connected with the light source, the optical signal receiver and the acceleration sensor, is used for receiving an external control signal, performs biomass on-line detection by adopting an external environment light deduction method and matching algorithms aiming at different shake flasks according to the external control signal, is responsible for automatic adjustment control of the light source and noise reduction, reading, conversion, operation and transmission of the signal, and sends the measured data to a worker for checking.
2. The device for the online detection of biomass in shake flask culture according to claim 1, wherein the subtraction method of the ambient light is specifically as follows:
the biomass online detection device is matched with a shake flask A, firstly, a default current output is given to a light source to drive the light source to emit light, the light source is in an initial current gear A, an OD original value A0 is read at the moment, the actually tested voltage value obtained by adding light emitted by the light source and external environment light conversion of the A0 is obtained, the light source is turned off, 0D original value B0 is read, B0 is the voltage value obtained by external environment light conversion, and theoretically, A0-B0 is the voltage value obtained by light source conversion; this operation needs to be repeated since the ambient light changes; in addition, the device has different positions of the shaking table and different ambient light, the shaking table can move at different rotating speeds in the culture process, the shaking table position is detected through the acceleration sensor, the corresponding 0D original value is read when the shaking table reaches a specified angle region and is used for deducting the influence of the ambient light, and the OD original value read in the angle region is not influenced by the liquid capacity in the shaking table and the rotating speed of the shaking table; in order to ensure the accuracy of the numerical value and the stability of the light source, a large amount of data needs to be read for algorithm processing, and the original value of the OD after the ambient light is removed is obtained.
3. The device for the online detection of biomass in shake flask cultivation according to claim 1, wherein the matching algorithm for different shake flasks is specifically:
the biomass online detection device is matched with the shake flask A, and is calibrated and fitted by an offline OD test method or an existing online OD concentration test device to obtain the relation between an OD original value and the biomass concentration, the OD original value and the OD initial value form a certain exponential relation; after calibrating a biomass on-line detection device, a corresponding fitting parameter is generated; meanwhile, an angle area and an OD initial value C when the biomass on-line detection device is tested need to be recorded; when the shake flask B is matched to start an experiment, the OD original value read in the angle area is also C by adjusting the current of the light source; then, the fitting parameters of the biomass online detection device are called, so that the accuracy of the same biomass online detection device in the concentration tests of different shake flasks is ensured; and the growth condition of the thallus is checked in real time through OD on-line detection in the culture process.
4. The device for the online detection of biomass in shake flask culture according to claim 2, characterized in that the subtraction method of ambient light is implemented in an analog manner, specifically as follows:
two photoelectric sensors are adopted, one photoelectric sensor is used for measuring external environment light, and the other photoelectric sensor is used for measuring a light source and the external environment light;
using an operational amplifier, where V1 is the voltage value of the light source plus the ambient light conversion, and V2 is the voltage value of the ambient light conversion, Vout — V1-V2 are obtained by calculation.
5. The biomass on-line detection device for shake flask culture according to claim 3, characterized in that the matching algorithm for different shake flasks is implemented in an analog manner for the control of the light source, specifically as follows:
the current-limiting resistor is added on the light source control circuit, the resistor is a common resistor or a variable resistor with higher precision, and the current is changed by adjusting the resistance value, so that the aim of adjusting the brightness of the light source is fulfilled.
6. The device for the online detection of biomass in shake flask culture according to claim 2, wherein the subtraction method of the ambient light is implemented by software, and specifically comprises the following steps:
for a photoelectric sensor, by controlling the switch of a light source, when a shake flask reaches a specified angle region, reading a corresponding voltage value, namely an OD original value, wherein the OD original value read in the region is not influenced by the volume of liquid in the shake flask and the rotating speed of a shaking table, in order to ensure the accuracy of numbers and the stability of the light source, the switching time of the light source needs to be accurately controlled, the OD original value, which is the voltage value after deducting ambient light, is obtained by multiple times of reading in the angle region, and the OD original value, which is the voltage value after deducting the ambient light, is calculated by algorithm processing.
7. The biomass on-line detection device for shake flask culture according to claim 3, characterized in that the control of the light source by the matching algorithm for different shake flasks is implemented in software as follows:
the special LED drive circuit is used for controlling the brightness of the light source, and the software adjusts the brightness of the light source when different shake flasks are used according to the fitting parameters and the OD initial values determined by the device, so that the matching use can be met.
8. The on-line biomass detection device for shake flask culture according to claim 1, characterized in that the light source and the light signal receiver follow a certain arrangement: the scattering type light source emits light which passes through the liquid to be detected and is received by the light receiver.
9. The on-line biomass detection device for shake flask culture according to claim 1, wherein the on-line biomass detection device is powered by an internal battery or a wired external power supply.
10. The on-line biomass detection device for shake flask culture according to claim 1, wherein the acceleration sensor is installed on a control circuit board.
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