WO2009135334A1 - Process and apparatus for measuring the concentration of composition during fermentation process - Google Patents
Process and apparatus for measuring the concentration of composition during fermentation process Download PDFInfo
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- WO2009135334A1 WO2009135334A1 PCT/CN2008/000900 CN2008000900W WO2009135334A1 WO 2009135334 A1 WO2009135334 A1 WO 2009135334A1 CN 2008000900 W CN2008000900 W CN 2008000900W WO 2009135334 A1 WO2009135334 A1 WO 2009135334A1
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- flow path
- reagent
- sample
- measuring
- concentration
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/14—Beverages
- G01N33/146—Beverages containing alcohol
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
- G01N1/20—Devices for withdrawing samples in the liquid or fluent state for flowing or falling materials
- G01N1/2035—Devices for withdrawing samples in the liquid or fluent state for flowing or falling materials by deviating part of a fluid stream, e.g. by drawing-off or tapping
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4005—Concentrating samples by transferring a selected component through a membrane
- G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis
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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
- G01N21/03—Cuvette constructions
- G01N2021/0346—Capillary cells; Microcells
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/775—Indicator and selective membrane
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
-
- 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
- G01N21/03—Cuvette constructions
- G01N21/05—Flow-through cuvettes
Definitions
- the present invention relates to the field of monitoring of fermentation processes, and more particularly to a device for measuring the concentration of components in a fermentation process and a method for measuring the concentration of components in a fermentation process. Background technique
- Fermentation is a very important process in the field of biochemistry, especially in the pharmaceutical analysis, food, beverage, energy and environmental industries.
- FCC fermentation process component concentration
- European Patent Application EP0445675 discloses a flow splitting device based on flow injection analysis (FIA) technology.
- FIA flow injection analysis
- this patent application requires multiple peristaltic pumps and multiple valves when implemented in an FIA system, resulting in a very complicated structure of the flow analysis device, increasing the operational complexity of the entire analysis process, and also resulting in the flow analysis device.
- the working condition is unstable.
- FIA systems are not flexible enough to be used in a variety of chemical reactions.
- the apparatus includes a pump, a multi-channel valve, a storage member, a beaker for use as a reaction vessel, a mass sensor, and a conduit connecting them.
- the pump is first sucked into the storage member through the multi-channel valve, then pushed into the beaker through the multi-channel valve, then the reagent is taken through the multi-channel valve and filled with the multi-channel valve, and then the reagent is passed through the multi-channel valve.
- Another object of the present invention is to provide a method of measuring the concentration of components in a fermentation process.
- the present invention provides an apparatus for measuring the concentration of a component of a fermentation process, the apparatus comprising:
- a sample flow path for flowing a sample of the driven fermentation broth therein; a reagent flow path for flowing the driven reagent therein; the shape of the reagent flow path being designed such that Forming a cavity along the sample flow path at least partially overlapping the sample flow path and the reagent flow path;
- a dialysis membrane positioned between the sample flow path and the reagent flow path to allow analytes in the fermentation broth sample to diffuse from the sample flow path into the reagent flow path for the analyte to The reagent reacts;
- a monitoring chamber is disposed on the reagent flow path for measuring the light absorbance of the reagent after the reaction.
- the sample flow path is a groove formed on an upper part
- the reagent flow path is a groove formed on a lower part
- the upper part and/or the lower part is made of poly Made of decyl acrylate (PMMA), glass or silicon.
- sample flow path and the reagent flow path may each be a half tube which is cut in the longitudinal direction.
- the sample flow path and the reagent flow path are twisted and twisted to extend the flow path in a limited space.
- a downstream end of the sample flow path is provided with a fermentation liquid outlet, and a downstream end of the reagent flow path is provided with a reagent outlet.
- the upper member and the lower member are respectively provided with positioning mechanisms that cooperate with each other, and when the upper member and the lower member are closed, the sample flow path and the reagent flow path are aligned with each other to form a cavity. .
- a temperature measuring element and a temperature control element are disposed in the sample flow path, and the temperature control element is used to adjust the temperature of the fermentation liquid sample in the sample flow path.
- the dialysis membrane may fill the area where the sample flow path is located.
- the monitoring chamber is located downstream of the reagent flow path and outside the portion where the reagent flow path overlaps the sample flow path.
- one side of the monitoring room is connected to one input fiber, and the other side is connected with an output fiber, the input fiber and the output fiber are aligned with each other; the input fiber is connected to a light source, and the output fiber is Connected to a detector.
- the optical path length between the input fiber and the output fiber of the monitoring chamber is adjustable.
- two slots are disposed in the lower member for respectively fixing the input fiber and the output fiber.
- the device is coupled to a sequential injection analysis flow system comprising: a pump, a multi-channel valve, and a reservoir, wherein the pump is coupled to one end of the reservoir
- the multi-channel valve has a plurality of ports respectively connected to the other end of the liquid storage tube, the inlet of the sample flow path, the inlet of the reagent flow path, the outlet of the reagent container, and an outlet of the container of the fermentation liquid to be measured.
- the multi-channel valve further includes: a port connected to the container of the calibration solution and/or a port for discharging the waste liquid.
- the invention also proposes a method for measuring the concentration of components in a fermentation process, comprising: inputting a sample of the fermentation broth into a sample flow path, inputting the reagent into a reagent flow path; utilizing between the sample flow path and the reagent flow path Dialyzing the membrane, passing the analyte in the fermentation broth sample through the dialysis membrane into the reagent flow path, and reacting with the reagent; passing light through the post-reaction reagent in the reagent flow path, and measuring the The light absorbance of the reagent after the reaction;
- the analyte concentration in the fermentation broth sample is calculated based on the light absorbance.
- the method further comprises: adjusting before the measuring the light absorbance of the reagent after the reaction The length of the optical path in the reagent after the reaction.
- the method further comprises: replacing the fermentation broth sample with a calibration solution, and measuring an analyte concentration in the calibration solution; analyzing the standard value and the measured concentration of the analyte concentration in the calibration solution The concentration of the analyte, the concentration of the analyte in the sample of the fermentation broth is calibrated.
- the method further includes: introducing a cleaning fluid into the sample flow path and/or the reagent flow path to clean the sample flow path and/or the reagent flow path.
- the device of the present invention adopts a unique structure, it is not necessary to use a large-capacity container for the reaction, and only a small amount of the fermentation broth sample and the reagent can be used for the measurement. , reducing the adverse effects on the fermentation process.
- the device of the present invention can be directly rinsed with a cleaning solution, and the cleaning process is simple, and the fermentation liquid sample and reagent are not easily left, thereby avoiding the problem that the residual fermentation liquid sample or reagent affects the next measurement.
- the sensitivity of the measurement can be varied by adjusting the length of the light path in the reagent after the reaction. With the technical solution of the present invention, it is not necessary to perform a pretreatment step of separating the fermentation broth sample and the reagent, thereby simplifying the measurement step.
- FIG. 1 is an exploded perspective view of an apparatus for online measurement of a component concentration in a fermentation process according to an embodiment of the present invention
- Figure 2 is a schematic view of the device shown in Figure 1 as a whole;
- Figure 3 is a schematic view of the upper part when the apparatus of Figure 1 further includes a temperature control element and a temperature measuring element;
- Figure 4 is a schematic view showing the structure of an SIA system connected to the apparatus shown in Figure 1. detailed description
- the technical solution proposed by the present invention can be used to measure the concentration of components in the fermentation process during fermentation, such as the concentration of the substrate or the concentration of the product. Since glucose is an important carbon source in many fermentation processes, glucose is used as the analyte in the following description.
- the solution of the present invention is exemplified, but the present invention is not limited to measuring the glucose concentration, and other analytes such as glutamic acid, lactic acid, ethanol and the like can also be measured by selecting the corresponding reagent.
- the detection methods of analytes such as glucose can be roughly classified into two types, one is an enzyme-based electrochemical (EC) method, and the other is a photometric method.
- the present invention mainly relates to photometric methods.
- the photometric method obtains the concentration of glucose by measuring the light absorbance of the solution after the reaction of the glucose and the reagent.
- the photometric method can be very accurate where the temperature and pH of the solution remain stable. Also, at room temperature, a typical measurement can be done quickly in less than a minute, allowing measurements to be used to control the fermentation process in real time.
- glucose is oxidized by glucose oxidase to form gluconic acid and hydrogen peroxide, while hydrogen peroxide is reacted with peroxylase and 4-amino-anthracene.
- Bilin (4-APP) and N-ethyl-N-sulfopropyl-m-mercaptoaniline (TOPS) react to form quinone.
- the quinone imine has a color and has an absorption peak for light having a wavelength of 545 nm.
- A sLC ( 3 )
- A the light absorbance
- the coefficient represents the light absorbance of the solution per unit mole (mol)
- L the optical path length in the solution
- C the concentration of the analyte, in this embodiment C
- A sLC ( 3 )
- an embodiment of the present invention provides a device for measuring the concentration of components in a fermentation process on-line, the device measuring the light absorbance of the reagent solution after the reaction, The concentration of glucose in the fermentation broth is obtained, so that the state of fermentation can be known.
- the apparatus 100 for measuring the concentration of components in a fermentation process includes an upper member 101, a lower member 102, a dialysis membrane 103, an input fiber 106, an output fiber 107, a light source 108, and a detector 109. .
- the upper member 101 includes a sample flow path 104 through which the fermentation broth sample flows.
- the lower member 102 includes a reagent flow path 105 through which the reagent flows.
- the upper member 101 and the lower member 102 are joined together such that the sample flow path 104 and the reagent flow path 105 at least partially overlap each other, thereby forming a cavity along the sample flow path 105.
- the sample flow path may be a groove formed in the upper member 101
- the reagent flow path 105 may be a groove formed in the lower member 102.
- the upper member 101 and the lower member 102 may be respectively provided with positioning mechanisms that cooperate with each other such that when the upper member 101 and the lower member 102 are closed, the sample flow path 104 and the reagent flow path 105 are overlapped with each other. Form a cavity.
- the upper member 101 and/or the lower member 102 may be made of a material such as polymethyl methacrylate (PMMA), glass or silicon.
- the device 100 can be constructed in a more compact configuration.
- the sample flow path 104 and the reagent flow path 105 are respectively a half pipe which is longitudinally cut, thereby eliminating the upper member 101 and the lower member 102.
- sample flow path 105 and the reagent flow path 105 may be arranged in a meandering manner, for example, arranged in a "Z" shape, an "S" shape, a sinusoidal function shape, or a shape as shown in FIG. 1, thereby extending the flow path in a limited space.
- a meandering manner for example, arranged in a "Z" shape, an "S" shape, a sinusoidal function shape, or a shape as shown in FIG. 1, thereby extending the flow path in a limited space.
- the dialysis membrane 103 is located at least between the sample flow path 104 and the reagent flow path 105, and the area of the dialysis membrane 103 may be larger than a portion where the fermentation sample flow path 104 and the reagent flow path 105 overlap, for example, the area where the sample flow path 104 is filled. It is even possible to cover the entire portion where the upper part 101 and the lower part 102 are in contact, which facilitates assembly.
- the dialysis membrane 103 separates the sample flow path 104 from the reagent flow path 105 to form two independent flow paths. The width of the two flow paths can be tens of microns to several centimeters.
- the sample flow path 104 has a sample inlet 110 and a sample outlet 11 1, the sample inlet 1 10 for the fermentation liquid sample to flow in, and the sample outlet 1 11 for the fermentation liquid sample to flow out.
- the reagent flow path 105 has a reagent inlet 1 12 and a reagent outlet 1 13, and a reagent inlet 112 for The reagent flows in, and the reagent outlet 113 supplies the reagent out.
- the dialysis membrane 103 located between the sample flow path 104 and the reagent flow path 105 can allow analytes such as glucose in the sample of the fermentation liquid in the sample flow path 104, and small molecules such as salt ions to pass, that is, glucose is allowed to be
- the analyte diffuses from the fermentation broth sample into the reagent of the reagent flow path 105, and those large or large particles having a diameter larger than the pore diameter of the dialysis membrane 103 are blocked by the dialysis membrane 103.
- the dialysis membrane 103 may be a cellulose membrane, a polycarbonate membrane or the like.
- the pore diameter of the dialysis membrane 103 needs to be larger than the diameter of the analyte such as glucose.
- the diameter of the pores of the dialysis membrane 103 can be less than 0.22 ⁇ m, so that it is possible to block bacteria and the like while transmitting an analyte such as glucose. Since the dialysis membrane 103 separates the fermentation broth sample and the reagents and allows the glucose to permeate and blocks the large particles, the centrifugation of the fermentation broth sample and the reagent pretreatment are not required before the measurement, thereby shortening the measurement time.
- the apparatus 100 of the embodiment of the present invention can inject a calibration solution into the sample flow path and obtain a calibration solution measurement result, and use the measurement result and calibration.
- the standard value of the solution can be used to calibrate the measurement of the actual fermentation broth sample.
- a monitoring chamber 114 is disposed in the reagent flow path 105.
- the monitoring chamber 1 14 can be located anywhere in the reagent flow path 105, preferably downstream of the reagent flow path 105, to ensure that there is a fully reacted post-reaction reagent in the monitoring chamber 114.
- the monitoring chamber 114 may be located within the portion of the reagent flow path 105 that is aligned with the sample flow path 104, or may be located outside of the portion of the reagent flow path 105 that is aligned with the sample flow path 104. In the example shown in the drawings, the monitoring chamber 114 is located outside the portion of the reagent flow path 105 that is aligned with the sample flow path 104.
- the monitoring chamber 114 is connected to an input fiber 106 and an output fiber 107.
- the length L of the optical path in the monitoring chamber 114 i.e., the distance between the input fiber 106 and the output fiber 107, can be designed to be adjustable. It can be seen from the formula (3) that by adjusting the optical path length of the monitoring chamber 144, the sensitivity of the measurement can be adjusted very easily, and thus applied to different fermentation broth samples.
- the input fiber 106 and the output fiber 107 may be misaligned or aligned. In the case of misalignment, the output optical fiber 107 receives less light intensity than that received under alignment, so the present invention preferably employs two The structure of the alignment.
- Two aligned or misaligned guiding grooves can be formed at both ends of the monitoring chamber 114 in the lower member 102, and the input optical fiber 106 and the output optical fiber 107 can be respectively fixed in the guiding groove, so that it can be both accurate and convenient.
- the input fiber 106 and the output fiber 107 are fixed.
- the incident light input from the input fiber 106 passes through the post-reaction reagent solution in the monitoring chamber 114 and is received by the output fiber 107.
- the light absorbance of the reagent solution after the reaction can be calculated based on the input light intensity and the output light intensity, and then the glucose concentration in the fermentation liquid can be calculated according to the formula (3).
- the light source 108 is used to generate incident light.
- a laser diode, a light emitting diode or the like can be used.
- the output spectral range of the light source 108 includes an absorption peak of an analyte such as glucose.
- the incident light generated by the light source 108 is not necessarily a laser, but may be other types of light such as white light.
- the detector 109 can be a photodiode, a photomultiplier tube or the like for measuring the intensity of light passing through the reagent solution after the reaction.
- the light absorbance A in the formula (3) can be calculated by using the intensity of the light output from the light source 108 and the intensity of the light measured by the detector 109.
- the combination of the structures shown in Fig. 1 can form a device 100 as shown in Fig. 2, which may be referred to as a small on-line photometer (MOP) integrated chip.
- the apparatus 100 can be fabricated using a fine processing or microelectromechanical system (MEMS) processing process, and the material can be made of polymethyl methacrylate, glass or silicon or the like according to various needs.
- MEMS microelectromechanical system
- the process of measuring the concentration of components in the fermentation process using the apparatus shown in Figures 1 and 2 can include the following steps:
- Step S1 1 a sample of the fermentation broth is extracted from a fermentation broth container such as a fermenter, and a sample of the fermentation broth is introduced into the sample flow path 104 through the sample inlet 110.
- step S12 the reagent is introduced into the reagent flow path 105 through the reagent inlet 112.
- steps S1 1 and S12 do not have a strict sequence, and may be performed simultaneously, or step S11 or S 12 may be performed first.
- step S13 since the concentration of the analyte in the fermentation liquid sample in the sample flow path 104 and the reagent flow path 105 is different, the analyte in the fermentation liquid sample passes through the pore of the dialysis membrane 103 into the reagent. And chemically reacting with related substances in the reagent, for example, the reactions shown by the chemical formulas (1) and (2), forming a post-reaction reagent including the reaction product, and the reagent is colored after the reaction.
- Step S14 it is possible to wait for a period of time so that the reaction proceeds sufficiently. Then, light is output by the light source 108, and the light enters the monitoring chamber 113 through the input fiber 106. After absorption in the monitoring chamber 113, the reagent is absorbed by the output fiber 107 and transmitted to the detector 109. In step S15, the detector 109 measures the intensity of the light absorbed by the reagent after the reaction. Step S16, the light absorbance A of the reagent after the reaction is calculated based on the intensity of the light output from the light source 108 and the intensity of the light measured by the detector 109.
- Step S17 based on the calculated light absorbance and the optical path length L in the coefficient solution, the concentration C of the analyte is calculated according to the formula (1), so that the fermentation state can be known.
- Step S18 the fermentation broth sample is output from the sample outlet 1 1 1 , and the reagent is output from the reagent outlet 1 13 .
- the method may further include: step S19, inputting the cleaning liquid from the sample inlet 110 and outputting from the sample outlet 111, and inputting the cleaning liquid from the reagent inlet 112 and outputting from the reagent outlet 113, thereby cleaning the device 100 for the next Used for secondary measurements.
- the method may also include a calibration process: performing the above steps S11 to S18 with the calibration solution instead of the fermentation liquid, thereby measuring the concentration of the analyte in the calibration solution; using the standard value of the analyte concentration in the calibration solution and The resulting analyte concentration is measured to calibrate the measured concentration in the measured fermentation broth sample.
- the above method can also adjust the optical path length L in the detection chamber 114 to change the sensitivity of the measurement. For example, increasing the length L of the optical path reduces the sensitivity of the measurement; reduces the length L of the optical path to increase the sensitivity of the measurement.
- Fig. 3 is another embodiment of the apparatus for online measurement of the concentration of components in a fermentation process in the present invention.
- the apparatus shown in FIG. 3 adds a temperature control for controlling the temperature of the fermentation broth sample in the sample flow path 104 in the sample flow path 104 of the upper member 101 on the basis of the apparatus 100 shown in FIGS. 1 and 2.
- a component such as a Peltier line 201. By applying a current in a different direction to the Peltier line 201, the temperature of the fermentation broth sample in the sample flow path 104 can be controlled.
- the Peltier line 201 absorbs heat, thereby lowering the temperature of the fermentation liquid sample, and when the current is applied in the other direction, that is, in the opposite direction, the Peltier line 201 emits heat, thereby increasing the fermentation liquid. The temperature of the sample.
- the apparatus shown in Fig. 3 can further provide a temperature measuring element for measuring the temperature of the fermentation liquid sample in the medium sample flow path 104 in the sample flow path 104 of the upper member 101, such as a temperature sensor 202, a thermometer, or the like.
- a temperature measuring element for measuring the temperature of the fermentation liquid sample in the medium sample flow path 104 in the sample flow path 104 of the upper member 101
- closed loop control of the temperature of the fermentation liquid in the sample flow path 104 can be achieved.
- the temperature of the fermentation broth sample is measured by the temperature sensor 202.
- the temperature of the fermentation broth sample is lowered by applying a current to the Peltier line 201.
- the temperature of the fermentation broth sample is increased by applying a current to the Peltier line 201. In this way, the temperature of the fermentation broth sample can be controlled within a suitable range.
- Figure 4 shows an embodiment in which a device for measuring the concentration of components in a fermentation process is combined with an SIA flow system 300.
- the apparatus for measuring the concentration of components in the fermentation process on-line in Fig. 4 may employ the apparatus 100 shown in Fig. 1, Fig. 2 or Fig. 3, and will not be described again.
- the SIA flow system 300 shown in Figure 4 includes a pump 301, "- a multi-channel valve 302, a reservoir 303, and a line connecting them.
- the pump 301 is coupled to one end of the reservoir 303.
- the reservoir 303 The other end, the inlet 110 of the sample flow path 104, the inlet 112 of the reagent flow path 105, the fermentation liquid container (e.g., fermentor, not shown), and the reagent container 304 are respectively connected to one port of the multi-channel valve 302.
- the end to which the pump 301 and the liquid storage tube 303 are connected is also connected to a cleaning liquid container 305.
- the multi-channel valve 302 has a discharge port for discharging waste liquid.
- the above measurement fermentation can be completed by the SIA flow system 300.
- the pump 301 is connected to the fermentation liquid container through the liquid storage tube 303, the multi-channel valve 302, and the sample of the fermentation liquid in the fermentation liquid container is pumped into the liquid storage tube 303 by the pump 301, and then the pump 301 is passed through the storage.
- the liquid tube 303, the multi-channel valve 302 is connected to the sample inlet 110 of the sample flow path 104, and the fermentation liquid sample in the liquid storage tube 303 is pushed to the sample inlet 110 by the pump 301, thereby inputting the fermentation liquid sample into the sample flow path 104.
- the reservoir 303 is purged by the pump 301 to purge the reservoir 303, and the purge is discharged from the discharge port of the multi-channel valve 302.
- the pump 301 is connected to the reagent container 304 through the reservoir 303, the multi-channel valve 302, the reagent in the reagent container 304 is pumped into the reservoir 303 by the pump 301, and then the pump 301 is passed through the reservoir 303, the multi-channel valve 302 is connected to the reagent inlet 112 of the reagent flow path 104, and the reagent in the liquid storage tube 303 is pushed by the pump 301 to the reagent inlet 112 of the reagent flow path 105, thereby inputting the reagent into the reagent flow path 105.
- the fermentation broth sample and the reagent can be separately input into the sample flow path 104 and the reagent flow path 105 of the apparatus 100.
- the analyte such as glucose in the fermentation liquid sample is diffused into the reagent of the reagent flow path 105 through the dialysis membrane 103, and reacts with the reagent. If you need to, you can wait a while for the reaction to be more adequate.
- the light absorption rate A is obtained, and then the glucose-glucose concentration can be calculated according to the formula (3), so that the glucose concentration in the fermentation liquid can be known. .
- the cleaning liquid in the cleaning liquid container 305 is pumped by the pump 301, and the cleaning liquid is pushed to the sample flow path 104 and the reagent flow path 105 through the liquid storage tube 303 and the multi-channel valve 302, respectively, to clean the two flow paths.
- the cleaned liquid can be directly discharged from the sample outlet 111 of the sample flow path 104 and the reagent outlet 113 4 of the reagent flow path 105.
- the multi-channel valve 302 in the SIA flow system 300 can also have a port that is coupled to a calibration solution container 306 containing a calibration solution.
- concentration of the analyte in the calibration solution is measured by the above process, and the analyte concentration of the measured fermentation broth sample can be calibrated using the measured analyte concentration and the actual analyte concentration of the calibration solution.
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Abstract
An apparatus (100) for measuring the concentration of composition during fermentation process comprises a sample line (104), a reagent line (105), the configuration of which is so designed that the both lines at least partly overlap each other to form a chamber, a dialysis membrane (103) mounted between the sample line (104) and the reagent line (105), allowing the sample to be analysed in the fermentation liquor sample to diffuse from said sample line (104) to said reagent line (105), to make said sample to be analysed and said reagent reaction, and an operating device (114) mounted in said reagent line to measuring optical absorption degree of reagent after response. A process for measuring the concentration of composition during fermentation process can be finished only by a small quantity of fermentation liquor sample and reagent.
Description
用于测量发酵过程组分浓度的装置和方法 技术领域 Apparatus and method for measuring component concentration in a fermentation process
本发明涉及发酵过程的监测领域, 特別是一种用于测量发酵过程 组分浓度的装置和一种测量发酵过程组分浓度的方法。 背景技术 The present invention relates to the field of monitoring of fermentation processes, and more particularly to a device for measuring the concentration of components in a fermentation process and a method for measuring the concentration of components in a fermentation process. Background technique
发酵是生物化学领域一种非常重要的过程, 尤其是在药物分析、 食品、 饮料、 能源和环境工业方面。 为了优化发酵, 需要在发酵过程 中对发酵进行监测和控制。 通常, 通过测量发酵过程中的发酵过程组 分浓度(FCC ) 来监测发酵过程。 Fermentation is a very important process in the field of biochemistry, especially in the pharmaceutical analysis, food, beverage, energy and environmental industries. In order to optimize the fermentation, it is necessary to monitor and control the fermentation during the fermentation process. Typically, the fermentation process is monitored by measuring the fermentation process component concentration (FCC) during the fermentation process.
传统的测量 FCC的方法包括化学滴定、 高效液相色谱等。 这些传 统方法都是提取大量的发酵液进行离线测量, 因此不能够实时得到测 量结杲并应用于对发酵过程的控制。 另外, 由于这些传统方法需要从 发酵罐中提取大量的发酵液, 所以这些方法对发酵过程产生非常不利 的影响。 Traditional methods for measuring FCC include chemical titration, high performance liquid chromatography, and the like. These traditional methods all extract a large amount of fermentation broth for off-line measurement, so it is not possible to measure the crusting in real time and apply it to the control of the fermentation process. In addition, since these conventional methods require a large amount of fermentation broth to be extracted from the fermenter, these methods have a very adverse effect on the fermentation process.
鉴于上述传统方法的缺点, 人们研究出了在线测量 FCC的技术。 例如, 欧洲专利申请 EP0445675公开了一种基于流动注射分析( FIA ) 技术的流动分折装置。 但是, 该专利申请在采用 FIA 系统实现时, 需 要采用多个蠕动泵和多个阀, 导致流动分析装置的结构非常复杂, 增 加了整个分析过程的操作复杂性, 并且也导致该流动分析装置的工作 状态不稳定。 另外, FIA系统也不能灵活应用于多种化学反应。 In view of the shortcomings of the above conventional methods, techniques for measuring FCC online have been developed. For example, European Patent Application EP0445675 discloses a flow splitting device based on flow injection analysis (FIA) technology. However, this patent application requires multiple peristaltic pumps and multiple valves when implemented in an FIA system, resulting in a very complicated structure of the flow analysis device, increasing the operational complexity of the entire analysis process, and also resulting in the flow analysis device. The working condition is unstable. In addition, FIA systems are not flexible enough to be used in a variety of chemical reactions.
在 FIA 的基础上, 于 20世纪 90年代提出了一种顺序注射分析 ( SIA )技术。 美国专利申请 US6,096,274也公开了一种基于 FIA技术 的用于在线滴定的流动分析装置。 该装置包括一个泵、 一个多通道阀、 一个存储件、 一个用作反应容器的烧杯、 一个^量传感器以及连接它 们的管道。 在测量过程中, 利用泵先通过多通道阀将样品吸到存储件 中, 再通过多通道阀推送到烧杯中, 然后通过多通道阀吸取试剂并充 满多通道阀, 接着通过多通道阀将试剂推送到烧杯中, 样品和试剂在
烧杯中被搅拌进行反应, 而测量传感器则在烧杯中进行测量。 该方案 与 EP0445675中的方案相比,提供了一种可用于多种场合的简单结构。 但是, 该方案中使用的反应容器是烧杯, 因此每次测量都需要大量的 样品和试剂。 另一方面, 在使用该流动分析装置进行在线测量时难以 彻底清洗, 残留的样品或试剂会影响下一次测量。 发明内容 Based on the FIA, a sequential injection analysis (SIA) technique was proposed in the 1990s. A flow analysis device for in-line titration based on FIA technology is also disclosed in U.S. Patent No. 6,096,274. The apparatus includes a pump, a multi-channel valve, a storage member, a beaker for use as a reaction vessel, a mass sensor, and a conduit connecting them. During the measurement process, the pump is first sucked into the storage member through the multi-channel valve, then pushed into the beaker through the multi-channel valve, then the reagent is taken through the multi-channel valve and filled with the multi-channel valve, and then the reagent is passed through the multi-channel valve. Pushed into the beaker, samples and reagents are in The beaker is stirred for reaction and the measuring sensor is measured in the beaker. Compared to the solution in EP 04 4 5675, this solution provides a simple structure that can be used in a variety of applications. However, the reaction vessel used in this protocol is a beaker, so a large amount of sample and reagent is required for each measurement. On the other hand, it is difficult to thoroughly clean the on-line measurement using the flow analysis device, and residual samples or reagents may affect the next measurement. Summary of the invention
有鉴于此, 本发明的目的在于提出一种用于测量发酵过程组分浓 度的装置。 本发明的另一个目的在于提出一种测量发酵过程组分浓度 的方法。 In view of this, it is an object of the present invention to provide an apparatus for measuring the concentration of components in a fermentation process. Another object of the present invention is to provide a method of measuring the concentration of components in a fermentation process.
因此, 本发明提供了一种用于测量发酵过程组分浓度的装置, 该 装置包括: Accordingly, the present invention provides an apparatus for measuring the concentration of a component of a fermentation process, the apparatus comprising:
一个样品流路, 用来使受驱动的发酵液样品在其中流过; 一个试剂流路, 用来使受驱动的试剂在其中流过; 所述的试剂流 路的形状这样设计, 使得所述样品流路与所述试剂流路至少部分彼此 重叠沿所述样品流路形成一条腔体; a sample flow path for flowing a sample of the driven fermentation broth therein; a reagent flow path for flowing the driven reagent therein; the shape of the reagent flow path being designed such that Forming a cavity along the sample flow path at least partially overlapping the sample flow path and the reagent flow path;
一个透析膜, 位于所述样品流路和试剂流路之间, 允许所述发酵 液样品中的被分析物从所述样品流路扩散到所述试剂流路中, 以便所 述被分析物与所述试剂发生反应; a dialysis membrane positioned between the sample flow path and the reagent flow path to allow analytes in the fermentation broth sample to diffuse from the sample flow path into the reagent flow path for the analyte to The reagent reacts;
一个监测室, 设置在所述试剂流路上, 用于测量反应后试剂的光 吸收度。 A monitoring chamber is disposed on the reagent flow path for measuring the light absorbance of the reagent after the reaction.
在上述技术方案中, 所述样品流路是成型在一个上部部件上的槽, 所述试剂流路是成型在一个下部部件上的槽, 所述上部部件和 /或所述 下部部件由聚甲基丙烯酸曱酯 (PMMA )、 玻璃或者硅材料制成。 In the above technical solution, the sample flow path is a groove formed on an upper part, the reagent flow path is a groove formed on a lower part, and the upper part and/or the lower part is made of poly Made of decyl acrylate (PMMA), glass or silicon.
另一方面, 所述样品流路和所述试剂流路也可以分别是一条沿纵 向剖开的半管。 Alternatively, the sample flow path and the reagent flow path may each be a half tube which is cut in the longitudinal direction.
优选地, 所述样品流路和所述试剂流路迂回曲折希置, 在有限的 空间内延长流路。 Preferably, the sample flow path and the reagent flow path are twisted and twisted to extend the flow path in a limited space.
所述样品流路的下游端设有一个发酵液出口, 所述试剂流路的下 游端设有一个试剂出口。
所述上部部件和所述下部部件上分别设置有彼此相配合的定位机 构, 在所述上部部件与下部部件合拢时, 使所述样品流路和所述试剂 流路彼此对准形成一条腔体。 A downstream end of the sample flow path is provided with a fermentation liquid outlet, and a downstream end of the reagent flow path is provided with a reagent outlet. The upper member and the lower member are respectively provided with positioning mechanisms that cooperate with each other, and when the upper member and the lower member are closed, the sample flow path and the reagent flow path are aligned with each other to form a cavity. .
所述样品流路中设置有一个温度测量元件和一个温度控制元件, 所述温度控制元件用来调节所述样品流路中发酵液样品的温度。 A temperature measuring element and a temperature control element are disposed in the sample flow path, and the temperature control element is used to adjust the temperature of the fermentation liquid sample in the sample flow path.
优选地, 所述透析膜可布满所述样品流路所在区域。 Preferably, the dialysis membrane may fill the area where the sample flow path is located.
所述监测室位于所述试剂流路的下游, 并位于所述试剂流路与所 述样品流路重叠部分之外。 The monitoring chamber is located downstream of the reagent flow path and outside the portion where the reagent flow path overlaps the sample flow path.
进一步, 所述监测室的一侧连接有一条输入光纤, 另一侧连接有 一条输出光纤, 所述输入光纤和所述输出光纤彼此对准; 所述输入光 纤与一个光源相连, 所述输出光纤与一个探测器相连。 Further, one side of the monitoring room is connected to one input fiber, and the other side is connected with an output fiber, the input fiber and the output fiber are aligned with each other; the input fiber is connected to a light source, and the output fiber is Connected to a detector.
较佳地, 所述监测室位于所述输入光纤和输出光纤之间的光路长 度可调节。 Preferably, the optical path length between the input fiber and the output fiber of the monitoring chamber is adjustable.
进一步, 所述下部部件中设置有两个槽, 分别用于固定所述输入 光纤和输出光纤。 ' Further, two slots are disposed in the lower member for respectively fixing the input fiber and the output fiber. '
为了结合 SIA系统,该装置与一个顺序注射分析流动系统相连接, 所述顺序注射流动系统包括: 一个泵、 一个多通道阀和一个储液管, 其中, 所述泵与储液管的一端连接, 所述多通道阀具有复数个端口, 分别连接所述储液管的另一端、 样品流路的入口、 试剂流路的入口、 试剂容器的出口、 以及待测量发酵液的容器的一个出口。 To incorporate the SIA system, the device is coupled to a sequential injection analysis flow system comprising: a pump, a multi-channel valve, and a reservoir, wherein the pump is coupled to one end of the reservoir The multi-channel valve has a plurality of ports respectively connected to the other end of the liquid storage tube, the inlet of the sample flow path, the inlet of the reagent flow path, the outlet of the reagent container, and an outlet of the container of the fermentation liquid to be measured.
所述多通道阀还包括: 一个与定标溶液的容器相连接的端口和 /或 一个用于排放废液的端口。 The multi-channel valve further includes: a port connected to the container of the calibration solution and/or a port for discharging the waste liquid.
本发明还提出了一种测量发酵过程组分浓度的方法, 包括: 将发酵液样品输入一个样品流路, 将试剂输入一个试剂流路; 利用位于所述样品流路和试剂流路之间的透析膜, 使发酵液样品 中的被分析物透过透析膜进入所述试剂流路, 并与所述试剂发生反应; 将光穿过所述试剂流路中的反应后试剂, 并测量所述反应后试剂 的光吸收度; The invention also proposes a method for measuring the concentration of components in a fermentation process, comprising: inputting a sample of the fermentation broth into a sample flow path, inputting the reagent into a reagent flow path; utilizing between the sample flow path and the reagent flow path Dialyzing the membrane, passing the analyte in the fermentation broth sample through the dialysis membrane into the reagent flow path, and reacting with the reagent; passing light through the post-reaction reagent in the reagent flow path, and measuring the The light absorbance of the reagent after the reaction;
根据所述光吸收度计算所述发酵液样品中的被分析物浓度。 The analyte concentration in the fermentation broth sample is calculated based on the light absorbance.
该方法在所述测量反应后试剂的光吸收度之前进一步包括: 调节
反应后试剂中的光路长度。 The method further comprises: adjusting before the measuring the light absorbance of the reagent after the reaction The length of the optical path in the reagent after the reaction.
该方法进一步包括: 利用定标溶液代替所述发酵液样品, 并测量 所述定标溶液中的被分析物浓度; 根据所述定标溶液中被分析物浓度 的标准值和测量得到的被分析物浓度, 校准所述发酵液样品中的被分 析浓度。 The method further comprises: replacing the fermentation broth sample with a calibration solution, and measuring an analyte concentration in the calibration solution; analyzing the standard value and the measured concentration of the analyte concentration in the calibration solution The concentration of the analyte, the concentration of the analyte in the sample of the fermentation broth is calibrated.
该方法进一步包括: 将清洗液输入所述样品流路和 /或所述试剂流 路, 以清洗所述样品流路和 /或所述试剂流路。 The method further includes: introducing a cleaning fluid into the sample flow path and/or the reagent flow path to clean the sample flow path and/or the reagent flow path.
从上述方案中可以看出, 由于本发明的装置采用了独特的结构, 相对于现有技术来说, 不需要使用大容量容器来进行反应, 只需要少 量的发酵液样品和试剂就可以完成测量, 减少了对发酵过程的不良影 响。 本发明的装置可以利用清洗液直接冲洗, 并且清洗过程简单, 不 易残留发酵液样品和试剂, 从而避免了残留发酵液样品或试剂影响下 一次测量的问题。 通过调节光在反应后试剂中的光路长度, 可以改变 测量的灵敏度。 采用本发明的技术方案, 不需要进行分离发酵液样品 和试剂等预处理步骤, 从而简化测量步骤。 附图说明 As can be seen from the above scheme, since the device of the present invention adopts a unique structure, it is not necessary to use a large-capacity container for the reaction, and only a small amount of the fermentation broth sample and the reagent can be used for the measurement. , reducing the adverse effects on the fermentation process. The device of the present invention can be directly rinsed with a cleaning solution, and the cleaning process is simple, and the fermentation liquid sample and reagent are not easily left, thereby avoiding the problem that the residual fermentation liquid sample or reagent affects the next measurement. The sensitivity of the measurement can be varied by adjusting the length of the light path in the reagent after the reaction. With the technical solution of the present invention, it is not necessary to perform a pretreatment step of separating the fermentation broth sample and the reagent, thereby simplifying the measurement step. DRAWINGS
图 1 为本发明实施例中一种用于在线测量发酵过程组分浓度的装 置的分解示意图; 1 is an exploded perspective view of an apparatus for online measurement of a component concentration in a fermentation process according to an embodiment of the present invention;
图 2为图 1所示装置组成整体时的示意图; Figure 2 is a schematic view of the device shown in Figure 1 as a whole;
图 3为当图 1所示装置进一步包括一个温度控制元件和一个温度 测量元件时上部部件的示意图; Figure 3 is a schematic view of the upper part when the apparatus of Figure 1 further includes a temperature control element and a temperature measuring element;
图 4为一个与图 1所示装置相连接的 SIA系统的结构示意图。 具体实施方式 Figure 4 is a schematic view showing the structure of an SIA system connected to the apparatus shown in Figure 1. detailed description
为使本发明的目的、 技术方案和优点更加清楚, 以下举实施例对 本发明进一步详细说明。 In order to make the objects, technical solutions and advantages of the present invention more comprehensible, the present invention will be further described in detail below.
本发明所提出技术方案可以用来测量发酵过程中发酵过程组分浓 度, 例如底物的浓度或产物的浓度。 由于葡萄糖是众多发酵过程中的 重要碳源, 因此在下面的描述过程中主要以葡萄糖作为被分析物来说
明本发明的方案, 但是本发明并不局限于测量葡萄糖浓度, 还可以通 过选择相应的试剂来测量其它被分析物, 例如谷氨酸、 乳酸、 乙醇等。 The technical solution proposed by the present invention can be used to measure the concentration of components in the fermentation process during fermentation, such as the concentration of the substrate or the concentration of the product. Since glucose is an important carbon source in many fermentation processes, glucose is used as the analyte in the following description. The solution of the present invention is exemplified, but the present invention is not limited to measuring the glucose concentration, and other analytes such as glutamic acid, lactic acid, ethanol and the like can also be measured by selecting the corresponding reagent.
葡萄糖等被分析物的探测方法大致可以分为两类, 一类是基于酶 的电化学 (EC ) 方法, 另一类是光度测量方法。 本发明主要涉及光度 测量方法。 The detection methods of analytes such as glucose can be roughly classified into two types, one is an enzyme-based electrochemical (EC) method, and the other is a photometric method. The present invention mainly relates to photometric methods.
由于葡萄糖和试剂反应之后的溶液的光吸收度与葡萄糖的浓度成 正比, 因此, 光度测量方法通过测量葡萄糖和试剂反应之后的溶液的 光吸收度来获得葡萄糖的浓度。在溶液的温度和 pH值保持稳定的情况 下, 光度测量方法可以非常准确。 并且, 在室温条件下, 一次典型的 测量可以在一分钟内迅速完成, 使得测量结果可以用于实时控制发酵 过程。 Since the light absorbance of the solution after the reaction of the glucose and the reagent is proportional to the concentration of glucose, the photometric method obtains the concentration of glucose by measuring the light absorbance of the solution after the reaction of the glucose and the reagent. The photometric method can be very accurate where the temperature and pH of the solution remain stable. Also, at room temperature, a typical measurement can be done quickly in less than a minute, allowing measurements to be used to control the fermentation process in real time.
如化学式 ( 1 ) 和 (2 ) 所示, 葡萄糖在葡萄糖氧化酶的作用下被 氧化, 生成葡萄糖酸和过氧化氢, 而过氧化氢在过氧化物酶的作用下, 与 4-氨基安替比林( 4-APP )以及 N-乙基 -N-磺丙基 - m-曱基苯胺( TOPS ) 反应生成醌亚胺。 醌亚胺具有颜色, 对于波长为 545 纳米的光存在一 个吸收峰。 As shown in the chemical formulas (1) and (2), glucose is oxidized by glucose oxidase to form gluconic acid and hydrogen peroxide, while hydrogen peroxide is reacted with peroxylase and 4-amino-anthracene. Bilin (4-APP) and N-ethyl-N-sulfopropyl-m-mercaptoaniline (TOPS) react to form quinone. The quinone imine has a color and has an absorption peak for light having a wavelength of 545 nm.
葡萄糖 + H2O + 02 —— > 葡萄糖酸 + H202 ( 1 )Glucose + H 2 O + 0 2 —— > Gluconic acid + H 2 0 2 ( 1 )
2 H202 + 4-AAP + TOPS —— > S昆亚胺 + 4 H20 ( 2 ) 由于反应后试剂溶液在 545 纳米处具有吸收峰, 而且反应后试剂 溶液对于光的吸收度与发酵液中的葡萄糖浓度成正比, 因此本发明实 施例可以利用这一特点来测量发酵液中的葡萄糖浓度。 光吸收度与葡 萄糖浓度的关系遵循如公式(3 )所示的朗伯 -比尔 (Lambert-Beer ) 定 律。 2 H 2 0 2 + 4-AAP + TOPS —— > S ketimine + 4 H 2 0 ( 2 ) Since the reagent solution has an absorption peak at 545 nm after the reaction, and the absorption of the reagent solution for light after the reaction The concentration of glucose in the fermentation broth is directly proportional, and thus embodiments of the present invention can utilize this feature to measure the concentration of glucose in the fermentation broth. The relationship between light absorbance and glucose concentration follows the Lambert-Beer law as shown in equation (3).
A = sLC ( 3 ) 其中, A表示光吸收度, 系数 表示单位摩尔 (mol )溶液的光吸 收度, L为溶液中的光路长度, C为被分析物的浓度, 在本实施例中 C 即为葡萄糖浓度。 根据公式(3 ), 当 和 L确定时, 通过测量 A, 可以 计算得到葡萄糖浓度 (:。 A = sLC ( 3 ) where A represents the light absorbance, the coefficient represents the light absorbance of the solution per unit mole (mol), L is the optical path length in the solution, and C is the concentration of the analyte, in this embodiment C For the glucose concentration. According to formula (3), when and L are determined, by measuring A, the glucose concentration can be calculated (:.
基于上述原理, 本发明实施例提出了一种用于在线测量发酵过程 组分浓度的装置, 该装置通过在线测量反应后试剂溶液的光吸收度,
得到发酵液中的葡萄糖浓度, 从而可以获知发酵状态。 Based on the above principle, an embodiment of the present invention provides a device for measuring the concentration of components in a fermentation process on-line, the device measuring the light absorbance of the reagent solution after the reaction, The concentration of glucose in the fermentation broth is obtained, so that the state of fermentation can be known.
如图 1 所示, 在线测量发酵过程组分浓度的装置 100包括一个上 部部件 101、 一个下部部件 102、 一个透析膜 103、 一条输入光纤 106、 一条输出光纤 107、 一个光源 108以及一个探测器 109。 As shown in FIG. 1, the apparatus 100 for measuring the concentration of components in a fermentation process includes an upper member 101, a lower member 102, a dialysis membrane 103, an input fiber 106, an output fiber 107, a light source 108, and a detector 109. .
上部部件 101中包括一个样品流路 104 ,发酵液样品在这个样品流 路 104中流过。 下部部件 102中包括一个试剂流路 105, 试剂在这个试 剂流路 105中流过。 上部部件 101和下部部件 102接合在一起, 使得 其中的样品流路 104与试剂流路 105至少部分彼此重叠, 从而沿样品 流路 105 形成一条腔体。 例如, 样品流路可以是成型在上部部件 101 上的槽, 而试剂流路 105可以是成型在下部部件 102上的槽。 在这种 情况下, 上部部件 101和下部部件 102可以分别设有彼此相互配合的 定位机构, 从而使得在上部部件 101和下部部件 102合拢时, 样品流 路 104和试剂流路 105彼此对准重叠形成一条腔体。 上部部件 101和 / 或下部部件 102可以由聚曱基丙烯酸甲酯(PMMA )、 玻璃或者硅等材 料制成。 The upper member 101 includes a sample flow path 104 through which the fermentation broth sample flows. The lower member 102 includes a reagent flow path 105 through which the reagent flows. The upper member 101 and the lower member 102 are joined together such that the sample flow path 104 and the reagent flow path 105 at least partially overlap each other, thereby forming a cavity along the sample flow path 105. For example, the sample flow path may be a groove formed in the upper member 101, and the reagent flow path 105 may be a groove formed in the lower member 102. In this case, the upper member 101 and the lower member 102 may be respectively provided with positioning mechanisms that cooperate with each other such that when the upper member 101 and the lower member 102 are closed, the sample flow path 104 and the reagent flow path 105 are overlapped with each other. Form a cavity. The upper member 101 and/or the lower member 102 may be made of a material such as polymethyl methacrylate (PMMA), glass or silicon.
另外, 装置 100可以采用更筒单的结构。 例如, 样品流路 104和 试剂流路 105 分别是一条沿纵向剖开的半管, 从而省却上部部件 101 和下部部件 102。 Additionally, the device 100 can be constructed in a more compact configuration. For example, the sample flow path 104 and the reagent flow path 105 are respectively a half pipe which is longitudinally cut, thereby eliminating the upper member 101 and the lower member 102.
进一步, 样品流路 105和试剂流路 105可以迂回曲折布置, 例如 布置成 "Z" 形、 "S" 形、 正弦函数形状或图 1所示的形状, 从而在有 限的空间内延长流路, 以便于被分析物和试剂的反应能够充分进行。 Further, the sample flow path 105 and the reagent flow path 105 may be arranged in a meandering manner, for example, arranged in a "Z" shape, an "S" shape, a sinusoidal function shape, or a shape as shown in FIG. 1, thereby extending the flow path in a limited space. In order to facilitate the reaction of the analyte and the reagent.
透析膜 103至少位于样品流路 104和试剂流路 105之间, 透析膜 103 的面积可以大于发酵样品流路 104与试剂流路 105之间重合的部 分, 例如布满样品流路 104所在区域, 甚至可以布满整个上部部件 101 和下部部件 102相接触的部分, 这样可以便于组装。 透析膜 103将样 品流路 104和试剂流路 105隔开, 从而形成两个独立的流路。 这两个 流路的宽度可以是数十微米到几个厘米。 The dialysis membrane 103 is located at least between the sample flow path 104 and the reagent flow path 105, and the area of the dialysis membrane 103 may be larger than a portion where the fermentation sample flow path 104 and the reagent flow path 105 overlap, for example, the area where the sample flow path 104 is filled. It is even possible to cover the entire portion where the upper part 101 and the lower part 102 are in contact, which facilitates assembly. The dialysis membrane 103 separates the sample flow path 104 from the reagent flow path 105 to form two independent flow paths. The width of the two flow paths can be tens of microns to several centimeters.
样品流路 104具有一个样品入口 110和一个样品出口 11 1,样品入 口 1 10供发酵液样品流入, 而样品出口 1 11 供发酵液样品流出。 试剂 流路 105具有一个试剂入口 1 12和一个试剂出口 1 13,试剂入口 112供
试剂流入, 而试剂出口 113供试剂流出。 另外, 位于样品流路 104和 试剂流路 105之间的透析膜 103可以允许样品流路 104中发酵液样品 中的葡萄糖等被分析物、 以及盐离子等小分子物质通过, 即允许葡萄 糖等被分析物从发酵液样品扩散到试剂流路 105 的试剂中, 而那些直 径大于透析膜 103孔直径的大分子或大颗粒就被透析膜 103阻挡。 The sample flow path 104 has a sample inlet 110 and a sample outlet 11 1, the sample inlet 1 10 for the fermentation liquid sample to flow in, and the sample outlet 1 11 for the fermentation liquid sample to flow out. The reagent flow path 105 has a reagent inlet 1 12 and a reagent outlet 1 13, and a reagent inlet 112 for The reagent flows in, and the reagent outlet 113 supplies the reagent out. In addition, the dialysis membrane 103 located between the sample flow path 104 and the reagent flow path 105 can allow analytes such as glucose in the sample of the fermentation liquid in the sample flow path 104, and small molecules such as salt ions to pass, that is, glucose is allowed to be The analyte diffuses from the fermentation broth sample into the reagent of the reagent flow path 105, and those large or large particles having a diameter larger than the pore diameter of the dialysis membrane 103 are blocked by the dialysis membrane 103.
透析膜 103 可以采用纤维素膜、 聚碳酸酯膜或者类似产品。 透析 膜 103 的孔直径需要大于葡萄糖等被分析物的直径。 优选地, 透析膜 103的孔直径可以小于 0.22微米, 从而可以在透过葡萄糖等被分析物 的同时还能够阻止细菌等。 由于采用透析膜 103将发酵液样品和试剂 相互分离, 并且让葡萄糖透过而阻止了大颗粒, 那么在测量之前无需 进行发酵液样品的离心分离与试剂预处理, 从而缩短了测量时间。 如 果长时间使用, 有可能降^ ί氏透析膜 103 的透析效率, 本发明实施例的 装置 100 可以向样品流路中注入定标溶液并得到定标溶液测量结果, 利用这个测量结果和定标溶液的标准值可以校准实际发酵液样品的测 量结果。 The dialysis membrane 103 may be a cellulose membrane, a polycarbonate membrane or the like. The pore diameter of the dialysis membrane 103 needs to be larger than the diameter of the analyte such as glucose. Preferably, the diameter of the pores of the dialysis membrane 103 can be less than 0.22 μm, so that it is possible to block bacteria and the like while transmitting an analyte such as glucose. Since the dialysis membrane 103 separates the fermentation broth sample and the reagents and allows the glucose to permeate and blocks the large particles, the centrifugation of the fermentation broth sample and the reagent pretreatment are not required before the measurement, thereby shortening the measurement time. If it is used for a long period of time, it is possible to reduce the dialysis efficiency of the dialysis membrane 103. The apparatus 100 of the embodiment of the present invention can inject a calibration solution into the sample flow path and obtain a calibration solution measurement result, and use the measurement result and calibration. The standard value of the solution can be used to calibrate the measurement of the actual fermentation broth sample.
在试剂流路 105中设置有一个监测室 114。监测室 1 14可以位于试 剂流路 105的任何部位, 优选地位于试剂流路 105的下游, 以保证监 测室 114中存在充分反应的反应后试剂。 另外, 监测室 114可以位于 试剂流路 105 中与样品流路 104对准的部分之内, 也可以位于试剂流 路 105 中与样品流路 104对准的部分之外。 在附图所示的例子中, 监 测室 114位于试剂流路 105中与样品流路 104对准的部分之外。 A monitoring chamber 114 is disposed in the reagent flow path 105. The monitoring chamber 1 14 can be located anywhere in the reagent flow path 105, preferably downstream of the reagent flow path 105, to ensure that there is a fully reacted post-reaction reagent in the monitoring chamber 114. Alternatively, the monitoring chamber 114 may be located within the portion of the reagent flow path 105 that is aligned with the sample flow path 104, or may be located outside of the portion of the reagent flow path 105 that is aligned with the sample flow path 104. In the example shown in the drawings, the monitoring chamber 114 is located outside the portion of the reagent flow path 105 that is aligned with the sample flow path 104.
监测室 114连接有一条输入光纤 106和一条输出光纤 107。监测室 114中的光路长度 L, 即输入光纤 106和输出光纤 107之间的距离, 可 以设计成可调的。 由公式 (3 ) 可以看出, 通过调节监测室 1 14的光路 长度, 可以非常容易地调节测量的灵敏度, 从而应用于不同的发酵液 样品。 输入光纤 106和输出光纤 107可以不对准, 也可以对准, 在不 对准的情况下输出光纤 107 所接收的光强度要低于在对准情况下所接 收的光强度, 因此本发明优选采用两者对准的结构。 可以在下部部件 102中监测室 114的两端形成两个对准的或不对准的导引槽,将输入光 纤 106和输出光纤 107分别固定在导引槽中, 从而可以既精确也方便
地固定输入光纤 106和输出光纤 107。 从输入光纤 106输入的入射光, 穿过处于监测室 114中的反应后试剂溶液后, 被输出光纤 107接收。 基于输入光强度和输出光强度就可以计算出反应后试剂溶液的光吸收 度, 然后根据公式(3 )就可以计算出发酵液中的葡萄糖浓度。 The monitoring chamber 114 is connected to an input fiber 106 and an output fiber 107. The length L of the optical path in the monitoring chamber 114, i.e., the distance between the input fiber 106 and the output fiber 107, can be designed to be adjustable. It can be seen from the formula (3) that by adjusting the optical path length of the monitoring chamber 144, the sensitivity of the measurement can be adjusted very easily, and thus applied to different fermentation broth samples. The input fiber 106 and the output fiber 107 may be misaligned or aligned. In the case of misalignment, the output optical fiber 107 receives less light intensity than that received under alignment, so the present invention preferably employs two The structure of the alignment. Two aligned or misaligned guiding grooves can be formed at both ends of the monitoring chamber 114 in the lower member 102, and the input optical fiber 106 and the output optical fiber 107 can be respectively fixed in the guiding groove, so that it can be both accurate and convenient. The input fiber 106 and the output fiber 107 are fixed. The incident light input from the input fiber 106 passes through the post-reaction reagent solution in the monitoring chamber 114 and is received by the output fiber 107. The light absorbance of the reagent solution after the reaction can be calculated based on the input light intensity and the output light intensity, and then the glucose concentration in the fermentation liquid can be calculated according to the formula (3).
光源 108 用来产生入射光, 可以采用一个激光二极管、 一个发光 二极管或者其它类似的器件, 光源 108 的输出光谱范围包括葡萄糖等 被分析物的吸收峰。 需要注意的是, 光源 108 所产生的入射光不一定 是激光, 也可以是白光等其它类型的光。 探测器 109可以是一个光电 二极管、 一个光电倍增管或者类似器件, 用来测量穿过反应后试剂溶 液的光的强度。 利用光源 108所输出的光的强度和探测器 109所测量 得到的光的强度, 可以计算得到公式 (3 ) 中的光吸收度 A。 The light source 108 is used to generate incident light. A laser diode, a light emitting diode or the like can be used. The output spectral range of the light source 108 includes an absorption peak of an analyte such as glucose. It should be noted that the incident light generated by the light source 108 is not necessarily a laser, but may be other types of light such as white light. The detector 109 can be a photodiode, a photomultiplier tube or the like for measuring the intensity of light passing through the reagent solution after the reaction. The light absorbance A in the formula (3) can be calculated by using the intensity of the light output from the light source 108 and the intensity of the light measured by the detector 109.
图 1所示结构组合之后可以形成如图 2所示的装置 100,不妨称为 小型在线光度计 (MOP ) 集成芯片。 该装置 100可以利用精细加工或 微型机电系统 (MEMS )加工工艺来制造, 其材料根据不同的需要可 以采用聚甲基丙烯酸曱酯、 玻璃或者硅等等。 The combination of the structures shown in Fig. 1 can form a device 100 as shown in Fig. 2, which may be referred to as a small on-line photometer (MOP) integrated chip. The apparatus 100 can be fabricated using a fine processing or microelectromechanical system (MEMS) processing process, and the material can be made of polymethyl methacrylate, glass or silicon or the like according to various needs.
采用图 1和图 2所示装置来测量发酵过程组分浓度的过程可以包 括如下步骤: The process of measuring the concentration of components in the fermentation process using the apparatus shown in Figures 1 and 2 can include the following steps:
步骤 S1 1 , 从发酵罐等发酵液容器中提取发酵液样品, 并将发酵液 样品通过样品入口 110输入样品流路 104。 Step S1 1 , a sample of the fermentation broth is extracted from a fermentation broth container such as a fermenter, and a sample of the fermentation broth is introduced into the sample flow path 104 through the sample inlet 110.
步骤 S12, 将试剂通过试剂入口 112输入试剂流路 105。 In step S12, the reagent is introduced into the reagent flow path 105 through the reagent inlet 112.
上述步骤 S1 1和 S12不存在严格的先后顺序, 可以同时执行, 也 可以先执行步骤 S11或 S 12。 The above steps S1 1 and S12 do not have a strict sequence, and may be performed simultaneously, or step S11 or S 12 may be performed first.
步骤 S13 ,由于样品流路 104中的发酵液样品和试剂流路 105中的 试剂存在葡萄糖等被分析物的浓度差, 所以发酵液样品中的被分析物 穿过透析膜 103 的孔进入试剂中, 并且与试剂中的有关物质发生化学 反应, 例如化学式 ( 1 )和 (2 ) 所示的反应, 形成包括反应产物的反 应后试剂, 并且反应后试剂带有颜色。 In step S13, since the concentration of the analyte in the fermentation liquid sample in the sample flow path 104 and the reagent flow path 105 is different, the analyte in the fermentation liquid sample passes through the pore of the dialysis membrane 103 into the reagent. And chemically reacting with related substances in the reagent, for example, the reactions shown by the chemical formulas (1) and (2), forming a post-reaction reagent including the reaction product, and the reagent is colored after the reaction.
步骤 S 14, 可以等待一段时间, 以便反应充分进行。 然后, 利用光 源 108输出光, 光经过输入光纤 106进入监测室 113 , 经过监测室 113 中反应后试剂的吸收之后由输出光纤 107接收, 并传送给探测器 109。
步骤 S 15 , 探测器 109测量经反应后试剂吸收后的光的强度。 步骤 S 16,根据光源 108输出的光的强度和探测器 109测量的光的 强度, 计算反应后试剂的光吸收度 A。 Step S14, it is possible to wait for a period of time so that the reaction proceeds sufficiently. Then, light is output by the light source 108, and the light enters the monitoring chamber 113 through the input fiber 106. After absorption in the monitoring chamber 113, the reagent is absorbed by the output fiber 107 and transmitted to the detector 109. In step S15, the detector 109 measures the intensity of the light absorbed by the reagent after the reaction. Step S16, the light absorbance A of the reagent after the reaction is calculated based on the intensity of the light output from the light source 108 and the intensity of the light measured by the detector 109.
步骤 S 17, 根据计算得到的光吸收度、 以及系数 溶液中光路长 度 L, 按照公式( 1 ), 计算得到被分析物的浓度 C, 从而可以获知发酵 状态。 Step S17, based on the calculated light absorbance and the optical path length L in the coefficient solution, the concentration C of the analyte is calculated according to the formula (1), so that the fermentation state can be known.
步骤 S 18 , 将发酵液样品从样品出口 1 1 1输出, 以及将试剂从试剂 出口 1 13输出。 Step S18, the fermentation broth sample is output from the sample outlet 1 1 1 , and the reagent is output from the reagent outlet 1 13 .
该方法还可以包括: 步骤 S 19 ,将清洗液从样品入口 110输入并从 样品出口 111输出, 并且将清洗液从试剂入口 112输入并从试剂出口 113输出, 从而清洗该装置 100, 以便于下次测量使用。 The method may further include: step S19, inputting the cleaning liquid from the sample inlet 110 and outputting from the sample outlet 111, and inputting the cleaning liquid from the reagent inlet 112 and outputting from the reagent outlet 113, thereby cleaning the device 100 for the next Used for secondary measurements.
该方法也可以包括校准流程: 以定标溶液代替发酵液执行上述步 骤 S 11至步骤 S 18 , 从而测量定标溶液中被分析物的浓度; 利用定标溶 液中被分析物浓度的标准值和测量得到的被分析物浓度, 来校准测量 得到的发酵液样品中的被分析浓度。 The method may also include a calibration process: performing the above steps S11 to S18 with the calibration solution instead of the fermentation liquid, thereby measuring the concentration of the analyte in the calibration solution; using the standard value of the analyte concentration in the calibration solution and The resulting analyte concentration is measured to calibrate the measured concentration in the measured fermentation broth sample.
从公式 (3 ) 可以看出, 当 L越小时, 测量的敏度度越高。 因此, 对于不同的发酵液, 上述方法还可以调节探测室 114中的光路长度 L, 来改变测量的灵敏度。 例如, 增大光路长度 L, 降低测量的灵敏度; 减 小光路长度 L, 提高测量的灵敏度。 It can be seen from the formula (3) that the smaller the L, the higher the measured sensitivity. Therefore, for different fermentation broths, the above method can also adjust the optical path length L in the detection chamber 114 to change the sensitivity of the measurement. For example, increasing the length L of the optical path reduces the sensitivity of the measurement; reduces the length L of the optical path to increase the sensitivity of the measurement.
图 3是本发明中用于在线测量发酵过程组分浓度的装置的另一实 施例。 图 3所示的装置在与图 1和图 2所示装置 100的基础上, 在上 部部件 101的样品流路 104中增加了一个用来控制样品流路 104中发 酵液样品的温度的温度控制元件, 例如珀耳帖 ( Peltier ) 线 201。 利用 对该珀耳帖线 201施加不同方向的电流, 可以控制样品流路 104中发 酵液样品的温度。 例如, 在一个方向方向施加电流时, 珀耳帖线 201 吸收热量, 从而降低发酵液样品的温度, 而在另一个方向即相反方向 施加电流时, 珀耳帖线 201放出热量, 从而提高发酵液样品的温度。 Fig. 3 is another embodiment of the apparatus for online measurement of the concentration of components in a fermentation process in the present invention. The apparatus shown in FIG. 3 adds a temperature control for controlling the temperature of the fermentation broth sample in the sample flow path 104 in the sample flow path 104 of the upper member 101 on the basis of the apparatus 100 shown in FIGS. 1 and 2. A component, such as a Peltier line 201. By applying a current in a different direction to the Peltier line 201, the temperature of the fermentation broth sample in the sample flow path 104 can be controlled. For example, when a current is applied in one direction, the Peltier line 201 absorbs heat, thereby lowering the temperature of the fermentation liquid sample, and when the current is applied in the other direction, that is, in the opposite direction, the Peltier line 201 emits heat, thereby increasing the fermentation liquid. The temperature of the sample.
图 3所示的装置还可以进一步在上部部件 101的样品流路 104中 设置一个用来测量中样品流路 104 中发酵液样品的温度的温度测量元 件, 例如温度传感器 202、 温度计等。
当上述装置 100 同时包括温度控制元件和温度测量元件时, 可以 实现对样品流路 104中发酵液温度的闭环控制。 以珀耳帖线 201和温 度传感器 102为例, 利用温度传感器 202测量发酵液样品的温度, 当 温度过高时, 通过对珀耳帖线 201施加电流来降低发酵液样品的温度 , 当温度过低时, 通过对珀耳帖线 201 施加电流来提高发酵液样品的温 度。 这样, 可以将发酵液样品的温度控制在一个合适的范围内。 The apparatus shown in Fig. 3 can further provide a temperature measuring element for measuring the temperature of the fermentation liquid sample in the medium sample flow path 104 in the sample flow path 104 of the upper member 101, such as a temperature sensor 202, a thermometer, or the like. When the above apparatus 100 includes both the temperature control element and the temperature measuring element, closed loop control of the temperature of the fermentation liquid in the sample flow path 104 can be achieved. Taking the Peltier line 201 and the temperature sensor 102 as an example, the temperature of the fermentation broth sample is measured by the temperature sensor 202. When the temperature is too high, the temperature of the fermentation broth sample is lowered by applying a current to the Peltier line 201. When low, the temperature of the fermentation broth sample is increased by applying a current to the Peltier line 201. In this way, the temperature of the fermentation broth sample can be controlled within a suitable range.
图 4显示了将用于在线测量发酵过程组分浓度的装置与一个 SIA 流动系统 300相结合的实施例。 图 4中的在线测量发酵过程组分浓度 的装置可以采用图 1、 图 2或图 3所示的装置 100, 这里不再赘述。 Figure 4 shows an embodiment in which a device for measuring the concentration of components in a fermentation process is combined with an SIA flow system 300. The apparatus for measuring the concentration of components in the fermentation process on-line in Fig. 4 may employ the apparatus 100 shown in Fig. 1, Fig. 2 or Fig. 3, and will not be described again.
图 4中所示 SIA流动系统 300包括一个泵 301、 "-个多通道阀 302、 一个储液管 303以及连接它们的管路。 泵 301与储液管 303的一端相 连接。 储液管 303的另一端、 样品流路 104的入口 110、 试剂流路 105 的入口 112、 发酵液容器 (例如发酵罐, 图中未示出)、 试剂容器 304 分别与多通道阀 302的一个端口相连接。 泵 301和储液管 303相连接 的一端还与一个清洗液容器 305相连接。 并且, 多通道阀 302还具有 一个用于排放废液的排放端口。利用 SIA流动系统 300,可以完成上述 测量发酵过程组分浓度的过程。 The SIA flow system 300 shown in Figure 4 includes a pump 301, "- a multi-channel valve 302, a reservoir 303, and a line connecting them. The pump 301 is coupled to one end of the reservoir 303. The reservoir 303 The other end, the inlet 110 of the sample flow path 104, the inlet 112 of the reagent flow path 105, the fermentation liquid container (e.g., fermentor, not shown), and the reagent container 304 are respectively connected to one port of the multi-channel valve 302. The end to which the pump 301 and the liquid storage tube 303 are connected is also connected to a cleaning liquid container 305. Also, the multi-channel valve 302 has a discharge port for discharging waste liquid. The above measurement fermentation can be completed by the SIA flow system 300. The process of process component concentration.
在测量过程中 , 将泵 301通过储液管 303、 多通道阀 302连接至发 酵液容器, 利用泵 301 将发酵液容器中的发酵液样品抽到储液管 303 中, 然后将泵 301通过储液管 303、 多通道阀 302连接至样品流路 104 的样品入口 110,利用泵 301将储液管 303中的发酵液样品推送至样品 入口 110, 从而将发酵液样品输入样品流路 104。 利用泵 301抽取清洗 液容器 305中的清洗液来清洗储液管 303, 并将清洗液从多通道阀 302 的排放端口排出。 将泵 301通过储液管 303、 多通道阀 302连接至试剂 容器 304, 利用泵 301将试剂容器 304中的试剂抽到储液管 303中, 然 后将泵 301通过储液管 303、多通道阀 302连接至试剂流路 104的试剂 入口 112,利用泵 301将储液管 303中的试剂推送至试剂流路 105的试 剂入口 112, 从而将试剂输入试剂流路 105。 通过上述操作, 就可以将 发酵液样品和试剂分别输入装置 100的样品流路 104和试剂流路 105 中。
此时, 样品流路 104 当中发酵液样品中的葡萄糖等被分析物经过 透析膜 103扩散到试剂流路 105的试剂中, 并且与试剂发生反应。 如 果需要, 可以等待一段时间, 以便反应能够更充分。 通过对试剂流路 105下游的监测室 114中的反应后试剂进行测量, 得到光吸收率 A, 然 后根据公式 (3 ) 可以计算得到葡 -萄糖浓度, 从而可以得知发酵液中 的葡萄糖浓度。 During the measurement, the pump 301 is connected to the fermentation liquid container through the liquid storage tube 303, the multi-channel valve 302, and the sample of the fermentation liquid in the fermentation liquid container is pumped into the liquid storage tube 303 by the pump 301, and then the pump 301 is passed through the storage. The liquid tube 303, the multi-channel valve 302 is connected to the sample inlet 110 of the sample flow path 104, and the fermentation liquid sample in the liquid storage tube 303 is pushed to the sample inlet 110 by the pump 301, thereby inputting the fermentation liquid sample into the sample flow path 104. The reservoir 303 is purged by the pump 301 to purge the reservoir 303, and the purge is discharged from the discharge port of the multi-channel valve 302. The pump 301 is connected to the reagent container 304 through the reservoir 303, the multi-channel valve 302, the reagent in the reagent container 304 is pumped into the reservoir 303 by the pump 301, and then the pump 301 is passed through the reservoir 303, the multi-channel valve 302 is connected to the reagent inlet 112 of the reagent flow path 104, and the reagent in the liquid storage tube 303 is pushed by the pump 301 to the reagent inlet 112 of the reagent flow path 105, thereby inputting the reagent into the reagent flow path 105. By the above operation, the fermentation broth sample and the reagent can be separately input into the sample flow path 104 and the reagent flow path 105 of the apparatus 100. At this time, in the sample flow path 104, the analyte such as glucose in the fermentation liquid sample is diffused into the reagent of the reagent flow path 105 through the dialysis membrane 103, and reacts with the reagent. If you need to, you can wait a while for the reaction to be more adequate. By measuring the post-reaction reagent in the monitoring chamber 114 downstream of the reagent flow path 105, the light absorption rate A is obtained, and then the glucose-glucose concentration can be calculated according to the formula (3), so that the glucose concentration in the fermentation liquid can be known. .
最后, 利用泵 301抽取清洗液容器 305 中的清洗液, 并通过储液 管 303、 多通道阀 302将清洗液分别推送至样品流路 104及试剂流路 105, 清洗这两个流路。 清洗后的液体可以直接从样品流路 104的样品 出口 111、 试剂流路 105的试剂出口 113 4非出。 Finally, the cleaning liquid in the cleaning liquid container 305 is pumped by the pump 301, and the cleaning liquid is pushed to the sample flow path 104 and the reagent flow path 105 through the liquid storage tube 303 and the multi-channel valve 302, respectively, to clean the two flow paths. The cleaned liquid can be directly discharged from the sample outlet 111 of the sample flow path 104 and the reagent outlet 113 4 of the reagent flow path 105.
如图 4所示, SIA流动系统 300中的多通道阀 302还可以具有一个 与一个盛有定标溶液的定标溶液容器 306相连接的端口。 通过上述流 程测量定标溶液中的被分析物浓度, 利用所测量的被分析物浓度和定 标溶液实际的被分析物浓度, 可以校准所测量得到的发酵液样品的被 分析物浓度。 As shown in Figure 4, the multi-channel valve 302 in the SIA flow system 300 can also have a port that is coupled to a calibration solution container 306 containing a calibration solution. The concentration of the analyte in the calibration solution is measured by the above process, and the analyte concentration of the measured fermentation broth sample can be calibrated using the measured analyte concentration and the actual analyte concentration of the calibration solution.
以上所述仅为本发明的较佳实施例而已, 并不用以限制本发明, 凡在本发明的精神和原则之内, 所作的任何修改、 等同替换、 改进等, 均应包含在本发明的保护范围之内。
The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalents, improvements, etc., which are included in the spirit and scope of the present invention, should be included in the present invention. Within the scope of protection.
Claims
权 利 要 求 书 Claims
1、 一种用于测量发酵过程组分浓度的装置 ( 100), 该装置 ( 100) 包括: 1. A device (100) for measuring the concentration of a component of a fermentation process, the device (100) comprising:
一个样品流路( 104 ), 用来使受驱动的发酵液样品在其中流过; 一个试剂流路( 105), 用来使受驱动的试剂在其中流过; 所述的 试剂流路( 105) 的形状这样设计, 使得所述样品流路 ( 104) 与所述 试剂流路( 105) 至少部分彼此重叠沿所述样品流路 ( 105) 形成一条 腔体; a sample flow path (104) for flowing a sample of the driven fermentation broth therein; a reagent flow path (105) for flowing a driven reagent therein; said reagent flow path (105) The shape is such that the sample flow path (104) and the reagent flow path (105) at least partially overlap each other to form a cavity along the sample flow path (105);
一个透析膜( 103 ), 位于所述样品流路 ( 104 )和试剂流路( 105 ) 之间, 允许所述发酵液样品中的被分析物从所述样品流路( 104)扩散 到所述试剂流路( 105) 中, 以便所述被分析物与所述试剂发生反应; 一个监测室 ( 114), 设置在所述试剂流路上, 用于测量反应后试 剂的光吸收度。 a dialysis membrane (103) located between the sample flow path (104) and the reagent flow path (105), allowing analytes in the fermentation broth sample to diffuse from the sample flow path (104) to the a reagent flow path (105) for reacting the analyte with the reagent; a monitoring chamber (114) disposed on the reagent flow path for measuring the light absorbance of the reagent after the reaction.
2、根据权利要求 1所述的装置( 100 ), 其中, 所述样品流路( 104 ) 是成型在一个上部部件 ( 101 ) 上的槽, 所述试剂流路 ( 105) 是成型 在一个下部部件 (102) 上的槽, 所述上部部件 ( 101 ) 和 /或所述下部 部件 (102) 由聚甲基丙烯酸曱酯 (PMMA)、 玻璃或者硅材料制成。 2. Apparatus (100) according to claim 1, wherein said sample flow path (104) is a groove formed in an upper part (101), said reagent flow path (105) being formed in a lower portion A groove on the component (102), the upper component (101) and/or the lower component (102) is made of polymethyl methacrylate (PMMA), glass or silicon material.
3、根据权利要求 1所述的装置( 100 ), 其中, 所述样品流路 ( 104 ) 和所述试剂流路( 105 )分别是一条沿纵向剖开的半管。 The apparatus (100) according to claim 1, wherein the sample flow path (104) and the reagent flow path (105) are each a half tube cut longitudinally.
4、 根据权利要求 1至 3之一所述的装置 ( 100), 其中, 所述样品 流路( 104) 和所述试剂流路 ( 105) 迂回曲折布置, 在有限的空间内 延长流路。 The apparatus (100) according to any one of claims 1 to 3, wherein the sample flow path (104) and the reagent flow path (105) are arranged in a meandering manner to extend the flow path in a limited space.
5、 根据权利要求 1至 3之一所述的装置 ( 100), 其中, 所述样品 流路( 104 ) 的下游端设有一个发酵液出口, 所述试剂流路( 105 ) 的 下游端设有一个试剂出口。 The device (100) according to any one of claims 1 to 3, wherein a downstream end of the sample flow path (104) is provided with a fermentation liquid outlet, and a downstream end of the reagent flow path (105) is provided. There is a reagent outlet.
6、根据权利要求 2所述的装置( 100),其中, 所述上部部件( 101 ) 和所述下部部件( 102)上分别设置有彼此相配合的定位机构, 在所述 上部部件与下部部件合拢时, 使所述样品流路( 104)和所述试剂流路 ( 105 )彼此对准形成一条腔体。
7、 根据权利要求 1至 3之一所述的装置 (100), 其中, 所述样品 流路( 104) 中设置有一个温度测量元件 (202) 和一个温度控制元件 (201 ), 所述温度控制元件 (201 ) 用来调节所述样品流路 ( 104) 中 发酵液样品的温度。 The device (100) according to claim 2, wherein the upper member (101) and the lower member (102) are respectively provided with positioning mechanisms that cooperate with each other, the upper member and the lower member When closed, the sample flow path (104) and the reagent flow path (105) are aligned with each other to form a cavity. The device (100) according to any one of claims 1 to 3, wherein a temperature measuring element (202) and a temperature control element (201) are disposed in the sample flow path (104), the temperature A control element (201) is used to adjust the temperature of the fermentation broth sample in the sample flow path (104).
8、 根据权利要求 1所述的装置 ( 100), 其中, 所述透析膜( 103 ) 可布满所述样品流路 ( 104) 所在区域。 8. Apparatus (100) according to claim 1, wherein said dialysis membrane (103) can fill the area of said sample flow path (104).
9、 根据权利要求 1所述的装置 (100), 其中, 所述监测室 ( 114) 位于所述试剂流路( 105) 的下游, 并位于所述试剂流路( 105) 与所 述样品流路( 104) 重叠部分之外。 9. The apparatus (100) according to claim 1, wherein the monitoring chamber (114) is located downstream of the reagent flow path (105) and is located in the reagent flow path (105) and the sample flow Road (104) is outside the overlap.
10、 根据权利要求 1或 9所述的装置 (100), 其中, 所述监测室 10. Apparatus (100) according to claim 1 or 9, wherein said monitoring room
( 114) 的一侧连接有一条输入光纤 ( 106), 另一侧连接有一条输出光 纤 ( 107), 所述输入光纤 ( 106) 和所述输出光纤 ( 107)彼此对准; 所述输入光纤 (106) 与一个光源 ( 108)相连, 所述输出光纤 ( 107) 与一个探测器 ( 109)相连。 An input fiber (106) is connected to one side of (114), and an output fiber (107) is connected to the other side, and the input fiber (106) and the output fiber (107) are aligned with each other; (106) is coupled to a light source (108) that is coupled to a detector (109).
11、 根据权利要求 1所述的装置( 100), 其中, 所述监测室( 114) 位于所述输入光纤( 106)和输出光纤( 107)之间的光路长度可调节。 11. Apparatus (100) according to claim 1, wherein the length of the optical path between the input fiber (106) and the output fiber (107) of the monitoring chamber (114) is adjustable.
12、 居权利要求 10所述的装置( 100 ),其中,所述下部部件( 102 ) 中设置有两个槽,分别用于固定所述输入光纤( 106)和输出光纤( 107 )。 12. Apparatus (100) according to claim 10, wherein said lower part (102) is provided with two slots for securing said input fiber (106) and output fiber (107), respectively.
13、 根据权利要求 1所述的装置 ( 100), 其中, 该装置 ( 100) 与 一个顺序注射分析流动系统 ( 300 ) 相连接, 所述顺序注射流动系统 13. Apparatus (100) according to claim 1, wherein the apparatus (100) is coupled to a sequential injection analysis flow system (300), the sequential injection flow system
( 300 )包括:一个泵( 301 )、一个多通道阀( 302 )和一个储液管( 303 ), 其中, 所述泵(301 )与储液管(303 )的一端连接, 所述多通道阀(302) 具有复数个端口,分别连接所述储液管( 303 )的另一端、样品流路( 104 ) 的入口、 试剂流路( 105) 的入口、 试剂容器 (304 ) 的出口、 以及待 测量发酵液的容器的一个出口。 (300) comprising: a pump (301), a multi-channel valve (302) and a liquid storage tube (303), wherein the pump (301) is connected to one end of the liquid storage tube (303), the multi-channel The valve (302) has a plurality of ports respectively connected to the other end of the liquid storage tube (303), the inlet of the sample flow path (104), the inlet of the reagent flow path (105), the outlet of the reagent container (304), and An outlet of the container of the fermentation broth to be measured.
14、根据权利要求 13所述的装置( 100),其中,所述多通道阀( 302 ) 还包括: 一个与定标溶液的容器相连接的端口和 /或一个用于排放废液 的端口。 14. Apparatus (100) according to claim 13, wherein said multi-channel valve (302) further comprises: a port connected to the container of the calibration solution and/or a port for discharging the waste liquid.
15、 一种测量发酵过程组分浓度的方法, 包括: 15. A method of measuring the concentration of a component of a fermentation process, comprising:
将发酵液样品输入一个样品流路( 104), 将试剂输入一个试剂流
路( 105 ); Enter the fermentation broth sample into a sample flow path ( 104) and enter the reagent into a reagent stream Road (105);
利用位于所述样品流路和试剂流路之间的透析膜 ( 103 ), 使发酵 液样品中的被分析物透过透析膜( 103 )进入所述试剂流路 ( 105 ), 并 与所述试剂发生反应; Using the dialysis membrane (103) between the sample flow path and the reagent flow path, the analyte in the fermentation broth sample is passed through the dialysis membrane (103) into the reagent flow path (105), and Reagent reaction;
将光穿过所述试剂流路( 105 ) 中的反应后试剂, 并测量所述反应 后试剂的光吸收度; Passing light through the post-reaction reagent in the reagent flow path (105), and measuring the light absorbance of the reagent after the reaction;
根据所述光吸收度计算所述发酵液样品中的被分析物浓度。 The analyte concentration in the fermentation broth sample is calculated based on the light absorbance.
16、 根据权利要求 15所述的方法, 其中, 该方法在所述测量反应 后试剂的光吸收度之前进一步包括: 调节反应后试剂中的光路长度。 16. The method according to claim 15, wherein the method further comprises: adjusting the optical path length in the reagent after the reaction before the measuring the light absorbance of the reagent after the reaction.
17、 根据权利要求 15所述的方法, 其中, 该方法进一步包括: 利用定标溶液代替所述发酵液样品, 并测量所述定标溶液中的被 分析物浓度; 17. The method according to claim 15, wherein the method further comprises: replacing the fermentation broth sample with a calibration solution, and measuring an analyte concentration in the calibration solution;
根据所述定标溶液中被分析物浓度的标准值和测量得到的被分析 物浓度, 校准所述发酵液样品中的被分析浓度。
The analyzed concentration in the fermentation broth sample is calibrated based on the standard value of the analyte concentration in the calibration solution and the measured analyte concentration.
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