CN1173161C - A micro-sampling device for an on-line detection system of a biological reaction process - Google Patents

Abstract

The invention relates to a sampling dilution device of an online detection system of a biological reaction process, in particular to a micro sampling device and a cascade dilution device. Sampling dilution adopts the method of continuous flow and intermittent measurement. The micro-sampling device mainly places a ceramic membrane filter at the end of the sampling probe. The reaction liquid is filtered in situ in the membrane in the tank, cross-flow infiltrates, and the permeate is led out by the diversion jacket thread outside the membrane, so as to realize the transfer from the outside of the tank to the tank. Internal automatic micro sampling. The cascade dilution device is mainly composed of vertical three-stage cascade dilution plates with overflow chambers. The fluid flow depends on the gravity of the fluid itself, and the micro-full flow path structure of organic materials keeps the flow of samples and diluents stable. The device takes very little sample, does not affect the optimal control of the biological reaction process, is easy to control the dilution ratio, is convenient and flexible to operate, simple to manufacture, and low in price. The main components can be made as standard parts, which are easy to clean and replace. The sample is well represented and is suitable for the application of on-line detection of biological reaction process and micro-total analysis (μ-TAS).

Description

A micro-sampling device for an on-line detection system of a biological reaction process

technical field

The invention relates to a sampling and dilution device for an online detection system of a biological reaction process, which is matched with a biosensor and a microcomputer to realize the on-line detection of substance concentration in the biological reaction process, and specifically relates to a micro sampling device and a cascade dilution device.

current technology

In addition to traditional brewing, today's fermentation industry has developed towards the fermentation production of antibiotics, amino acids, nucleic acids, etc. In recent years, the use of technologies such as microbial recombination and cell culture is being studied to produce biologically active substances. Along with the above-mentioned development process, the control and optimization of the fermentation process has also developed from experience management to computer automation management.

Various physical and chemical sensors are widely used in the control of processes. Sensors for detecting parameters such as temperature, pH, dissolved oxygen, carbon monoxide, pressure, flow, and stirring functions are connected with a microcomputer to form a control system for biochemical and fermentation processes. But it can only indirectly control biochemical reactions, and cannot directly understand the changes of substances in complex biochemical processes. The biosensor solves many substances that were difficult to analyze in the past, and can measure the concentration of raw materials and fermentation products that directly represent the state of the fermentation process. After more than 20 years of research and development, biosensors have formed a unique category, juxtaposed with physical sensors and chemical sensors, and are regarded as the third category of sensors.

Biosensors have the characteristics of good specificity, enzymes can be reused many times, fast analysis speed, high accuracy, and relatively simple operating system, which prepares feasible sensor conditions for direct control of biological reaction processes by microcomputers. However, no analysis system has been reported that can realize on-line detection of biological and chemical quantities in the general biological reaction process. Biological and chemical quantities cannot be detected online. The root cause is that the detection interface technology between biosensors and complex biological reaction processes cannot pass the test. This has become the "bottleneck" technology for biosensors to achieve online detection.

The schematic diagram of the online detection system for the biological reaction process is shown in Figure 1, including a micro sampler, a diluter, a micro biosensor group, bioengineering data processing and analysis software and a microcomputer. And the micro sampler and the diluter are the technical problems to be solved in the present invention.

In scientific research work, experimenters use fully automatic small fermenters to optimize the fermentation process conditions. Small tanks with a volume of 2-30L are generally used, and the sampling volume is as high as 15ml. Multiple samplings cause the volume of the fermentation broth to decrease, which brings great harm to the fermentation process. A larger impact will lead to an increase in the error of process optimization. Therefore, it is urgent to design a micro-sampling device to reduce the sampling volume and minimize the interference of sampling to detection.

Currently existing dilution devices, such as robotic arms and flow injection analysis online dilution technology, provide one of the effective methods for online determination of high-concentration samples; however, time controllers, computer-controlled valves and high-precision peristaltic pumps are required. Therefore, the requirements for mechanical processing are high, the price is expensive, and the control of the dilution ratio is inflexible, which seriously affects the online detection application of biosensors in the biological reaction process.

Contents of the invention

(1) Technical problems to be solved

Provide a micro-sampling device for the on-line detection system of the biological reaction process, which requires solving the technical problem of micro-sampling the fermentation tank from the outside of the tank, which not only overcomes the heat-resistant characteristics of the biosensor, but also overcomes the impact of oxygen concentration and temperature on the bioelectrode. The impact of the sample volume is reduced, the interference of sampling to the detection is reduced as much as possible, and the need for continuous online sampling and intermittent measurement is maintained.

Also provided is a cascade dilution device of an online detection system for a biological reaction process, which requires solving the technical problem that online serial dilution can be performed. The buffer solution is used to dilute the sample tens to hundreds of times, and the dilution rate is flexibly controlled to achieve the linear range of biosensor detection. Another function of dilution is to adjust the pH value of the sample to match the working pH value of the sensor and reduce interference.

(2) Technical solution

micro sampling device

Fig. 2 is a schematic structural diagram of a micro-sampling device, which consists of a sampling probe and a sample flow control part. The end of the probe is the membrane filter part, and the other end is the permeate outlet pipe and the sealing part connected with the tank. The sampling probe is composed of a probe cylinder, a membrane filter, a diversion jacket outside the membrane, a diversion connector, a lead-out tube, and a casing. The flow control part is jointly controlled by the needle valve and the metering valve. The sampling probe, the needle valve and the metering valve are connected by an outlet pipe.

The membrane filter adopts a ceramic membrane, and the membrane filter is tubular, with a membrane layer on the inner wall and a support layer on the outside, with an inner diameter of 6-10mm and an outer diameter of 10-14mm. The length of the membrane filter is 8-12mm, and the membrane pore diameter is 0.1-0.2μm.

The outer membrane diversion jacket is horizontally drilled on the end of the vertically placed probe cylinder, which is the outer membrane diversion jacket. The inner wall of the diversion jacket outside the membrane is threaded for diversion, and the difference between the major diameter and the minor diameter of the thread is less than 2mm. The membrane filter is placed in the drill hole of the outer membrane diversion jacket of the probe cylinder. The inner thread of the outer membrane diversion jacket is in contact with the outer wall of the membrane filter. The outer membrane diversion jacket and the membrane filter adopt an interference fit. Both ends are sealed with silicone. The probe cylinder is made of polyvinylidene fluoride or polytetrafluoroethylene.

A small cylindrical hole with a conical head is opened in the middle of the circular surface at the end where the probe cylinder and the casing are connected. The cylindrical part of the small hole has internal threads. The small hole communicates with the middle part of the diversion jacket, and a diversion connector is built in the small hole. . The diversion connector is a small cylinder with a tapered head, the cylindrical part of the small cylinder is provided with external threads, the diversion connector and the probe cylinder are threaded, and the conical head of the diversion connector is close to the diversion jacket outside the membrane to reduce dead volume. Open a hole in the middle of the cone of the diversion connector, and insert the outlet tube. The diversion connector is made of polytetrafluoroethylene.

The lead-out tube is a plastic elastic tube, and a polysilicone elastic tube or a polyvinylidene fluoride elastic tube is selected.

One end of the bushing is provided with an internal thread and the external thread at one end of the probe cylinder is threadedly matched. The casing is made of stainless steel to protect the outlet pipe.

Sampling adopts cross-flow filtration and osmosis technology. The in-situ filter is used to allow the reaction liquid to flow through the membrane and filter directly in the tank. The membrane is used to separate the fermentation liquid from the solid. The outside of the membrane is guided by threads to lead out the filtrate. After processing directly, continuous filtration. The permeate flow is controlled by a valve, and the diffusion amount of the membrane is equal to the permeate flow in a steady state, so that the permeate is continuously drawn out. The permeate is taken as a measurement sample at regular intervals, that is, intermittent measurement, and the permeate that is not used as a measurement sample is directly discharged.

The design parameters are: permeate flow rate 0.4ml/min, lag time less than 1min, sampler pipeline volume ≤0.7ml, sampling volume 10-1000μl/min.

cascade dilution device

Figure 3 is a schematic diagram of the interface flow of the three-stage cascade device in the online detection system. The cascade dilution device consists of a buffer overflow section and a vertical three-stage cascade dilution plate with an overflow chamber. The buffer overflow part includes a buffer storage tank, a constant flow pump, and three buffer overflow tanks, and the buffer flow is controlled by a buffer flow regulating valve on the buffer flow path. Fig. 4 is a schematic structural diagram of a vertical three-stage cascaded dilution plate with an overflow chamber. The structure is: the sample flow path with an overflow chamber and the buffer flow path are merged into a dilution mixing flow path, as a primary diluter, three primary diluters are vertically connected in series to form a three-stage cascade dilution plate, and the first The first-stage dilution mixing flow path is connected to the second-stage overflow chamber, and the second-stage dilution mixing flow path is connected to the third-stage overflow chamber. The first-level diluted mixed solution is used as the second-level sample to continue diluting, the second-level diluted mixed solution is used as the third-level sample for further dilution, and the third-level diluted mixed solution is used as the test sample for detection. A drain valve is arranged at the bottom of the overflow chamber, and a vent hole is arranged at the top of the overflow chamber. The sample flow is controlled by the sample flow regulating valve on the sample flow path, and the cascade dilution plate is made of organic materials, such as polyvinylidene fluoride or polytetrafluoroethylene. The fluid flow path adopts a micro total flow structure compatible with the micro total analysis system (μ-TAS), which fully shortens the residence time of the sample, but does not form droplets. The fluid flow depends on the gravity of the fluid itself, which not only fully maintains the static state of the fluid Pressure, generate as little dynamic pressure as possible, all operations must be continuous and stable flow, continuous sampling, continuous dilution, and intermittent measurement.

The diameter of the fluid flow path is 0.1-1mm, the length of the dilution and mixing flow path is 5-10cm, and the depth of the overflow cavity is 3-6mm. The single-stage dilution ratio is controlled at 4-10, and the dilution error is not greater than 1%, and the third-stage dilution ratio is up to 1000, and the dilution error is not greater than 2.5%.

(3) Beneficial effect

The biggest feature of the micro-sampling device and three-stage cascade dilution device designed in the present invention is micro-sampling and micro-dilution for micro-detection of micro-biosensors. The sample consumption is 10-100 μL/min, fermented for 100 hours, and continuously sampled The sample loss is only 60-600ml, which can meet the requirements of sampling and testing of small fermenters.

The micro-sampling device replaces the manual sampling method with an automatic sampling method, adopts cross-flow filtration technology, and uses a membrane to separate the fermentation liquid from the solid. The filter membrane layer permeates into the jacket thread, forming a cross-flow permeation, and realizes the first-in-first-out liquid, that is, the reaction liquid flows into the membrane tube, and the permeate is drawn out.

The in-situ filter is used to filter directly in the tank, and the filtrate is taken out as a sample. The advantages are: the complexity of the tank is not increased, and the filter membrane is installed at the probe of the tank, which will not increase the chance of bacterial contamination. The membrane and The pipelines are easy to clean, and all are standard parts, which can be replaced in time.

The cascade dilution device is designed to operate relying on the gravity of the fluid itself, that is, overflow to control the stability of the diluted sample and buffer flow, without the need for a time controller, computer-controlled valve and high-precision peristaltic pump. It is easy to operate and flexible, and is suitable for biological Sample dilution requirements for online detection during the reaction process. Its three-stage dilution and the micro-full-flow structure of the fluid flow path make the sample form a stable continuous flow, and the dilution rate does not change or changes little with the change of the sample or buffer flow.

The online detection system adopts the methods of continuous automatic sampling, dilution and intermittent measurement. The process of continuous sampling and dilution is to let the sample flow through the sampling pipeline and the dilution pipeline in this way. The sample is in a continuous flow, and its flow rate is easy to control. There is no need to empty the dead sample, and the memory effect is small, which has little influence on the representativeness of the sample. The continuous sampling and dilution system is simple to manufacture, cheap, and has good sample representation, and is very suitable for on-line analysis and detection. The cost of biological reaction control can be further reduced, and the detection accuracy of the biosensor can be improved.

Description of drawings

Fig. 1 Schematic diagram of online detection system for biological reaction process.

Figure 2 Schematic diagram of the structure of the micro-sampling device. (a) Schematic diagram of the front view of the micro-sampling device; (b) Schematic diagram of the side view of the sampling probe.

Figure 3 is a schematic diagram of the interface flow of the three-stage cascade device in the online detection system.

Fig. 4 Schematic diagram of the structure of a vertical three-stage cascaded dilution plate with an overflow chamber.

In the accompanying drawings, 1 probe cylinder, 2 membrane filter, 3 membrane outer diversion jacket, 4 diversion connector, 5 outlet pipe, 6 set of valves, 7 needle valve, 8 metering valve, 9 overflow chamber, 10 Sample flow path, 11 sample flow regulating valve, 12 drain valve, 13 air vent, 14 buffer flow regulating valve, 15 buffer flow path, 16 dilution mixing flow path.

Detailed ways

Example 1

Micro-sampling device: The membrane filter adopts the ceramic membrane of a certain company. The membrane is tubular, the membrane layer is on the inner wall, the support layer is outside, the inner diameter d1=8mm, and the outer diameter d2=12mm. The pressure drop during filtration is about 10KPa, the membrane pore size is 0.2μm, the filtrate flow rate is required to be 0.4ml/min, and the length of the membrane piece is 10mm. According to the calculation according to the relevant formula, it can meet the permeate flow requirement.

Example 2

The membrane pore diameter of the membrane filter element is 0.1 μm, and other conditions are the same as those in Example 1, which can also meet the requirement of the flow rate of the permeate.

Example 3

The total length of the three-stage cascaded dilution plate fluid flow path is 90mm, and the flow path diameter is 1mm, so the total volume is 0.09ml. Considering the flow rate of 0.1ml/min, the residence time is only 0.9min, and the sampling rate is 10-1000L/min. The level dilution ratio is controlled at 4-10, the single-level dilution error is not greater than 1%, the third-level dilution ratio is up to 1000, and the dilution error is not greater than 2.5%. Glucose was used for the sample, and its original concentration was 278.54g/l, and a three-stage dilution test was carried out, and the results of the dilution precision test were:

        Table 1 Test results of three-stage cascaded dilution accuracy

    Primary Dilution Secondary Dilution Tertiary Dilution Total Dilution Ratio Error Rate %

Average concentration 52.23 10.89 2.45

Dilution factor 5.33 4.80 4.44 113.69 0.6

Average concentration 47.42 6.65 0.94

Dilution factor 5.87 7.13 7.07 296.32 1.2

Average concentration 28.83 3.30 0.45

Dilution factor 9.66 8.74 7.33 618.98 1.8

Average concentration 28.83 2.88 0.28

Dilution factor 9.66 10.00 10.35 1000 2.5

Claims (2)

1. A micro-sampling device for online detection of biological reaction process, which is characterized by a sampling probe and a sample flow control part. The end of the probe is a membrane filter part, and the other end is a permeate outlet tube and a sealing part connected to the tank , the sampling probe is composed of a probe cylinder (1), a membrane filter (2), a diversion jacket outside the membrane (3), a diversion connector (4), an outlet tube (5), and a sleeve (6). The control part is jointly controlled by the needle valve (7) and the metering valve (8), and the sampling probe, the needle valve and the metering valve are connected by an outlet pipe; The membrane filter (2) adopts a ceramic membrane, the membrane filter is tubular, the membrane layer is on the inner wall, the support layer is outside, the inner diameter is 6-10mm, the outer diameter is 10-14mm, the length of the membrane filter is 8-12mm, and the membrane aperture is 0.1 -0.2μm, The outer membrane diversion jacket (3) is horizontally drilled on the end of the vertically placed probe cylinder (1), which is the outer membrane diversion jacket (3), and the inner wall of the outer membrane diversion jacket (3) Made into a thread shape for diversion, the membrane filter (2) is placed in the drill hole of the outer membrane diversion jacket of the probe cylinder (1), the inner thread of the outer membrane diversion jacket and the membrane filter (2) The outer wall is in contact, the diversion jacket (3) outside the membrane and the membrane filter (2) adopt an interference fit, and the two ends of the fit are sealed with silica gel. A small cylindrical hole with a conical head is opened in the middle of the circular surface at the end where the probe cylinder and the casing are connected. The cylindrical part of the small hole has internal threads. The built-in diversion connector (4), the diversion connector (4) is a small cylinder with a tapered head, the cylindrical part of the small cylinder is provided with external threads, the diversion connector (4) and the probe cylinder (1) are threaded Cooperate, the conical head of the diversion connector (4) is close to the outer diversion jacket (3) of the membrane, perforates in the middle of the diversion connector (4) column cone, inserts the outlet pipe (5), and the sleeve pipe ( 6) One end is provided with an internal thread and the external thread at one end of the probe cylinder (1) is threadedly matched. 2. The micro-sampling device of the online detection system according to claim 1, characterized in that the probe cylinder (1) is made of polyvinylidene fluoride or polytetrafluoroethylene, and the diversion connector (4) is made of polytetrafluoroethylene As for the material, the outlet pipe (5) is made of polysilicone elastic pipe or polyvinylidene fluoride elastic pipe, and the sleeve pipe (6) is made of stainless steel.

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