The research and application of biosensors, especially microbial sensors, in the fields of fermentation industry and environmental monitoring in recent years are briefly described, and its development prospect and marketization are predicted and prospected. Bio-electrode is a sensitive material with immobilized organisms as molecular recognition elements, which forms a biosensor with oxygen electrode, membrane electrode and fuel electrode. It has been widely used in fermentation industry, environmental monitoring, food monitoring, clinical medicine and other fields. The biosensor has the advantages of good specificity, simple operation, simple equipment, rapid and accurate measurement and wide application range. With the development of immobilization technology, biosensors are very competitive in the market.
Keywords: biosensor; Fermentation industry; Environmental monitoring.
I. Introduction
It has been 40 years since Clark and Lyons first put forward the idea of biosensor from 1962. Biosensors have been widely used in fermentation technology, environmental monitoring, food engineering, clinical medicine, military and military medicine. Before 15 years, biosensor was mainly based on the development of enzyme electrode. However, due to its high price and instability, its application as a sensitive material is limited.
In recent years, with the continuous development of microbial immobilization technology, microbial electrodes have been produced. Compared with enzyme electrode, microbial electrode is unique in that it takes living microorganisms as molecular recognition elements. It can overcome the shortcomings of high price, difficult extraction and instability. In addition, we can also use coenzyme in microorganisms to deal with complex reactions. At present, the application of optical fiber biosensor is more and more extensive. With the development of polymerase chain reaction, we should
There are more and more DNA biosensors using PCR.
Second, the research status and main application fields
1, fermentation industry
Among all kinds of biosensors, microbial sensors are most suitable for the detection of fermentation industry. Because there are often interfering substances in the fermentation process, the fermentation broth is often not clear and transparent, which is not suitable for spectral determination. The application of microbial sensor is likely to eliminate interference and is not limited by the turbidity of fermentation broth. At the same time, due to the large-scale production of fermentation industry, microbial sensors have great advantages because of their low cost and simple equipment.
(1). Determination of raw materials and metabolites
Microbial sensors can be used to determine raw materials such as molasses and acetic acid, as well as metabolites such as cephalosporin, glutamic acid, formic acid, methane, alcohol, penicillin and lactic acid. The measuring principle is basically composed of appropriate microbial electrodes and oxygen electrodes. Oxygen is consumed by the assimilation of microorganisms, and the reduction of oxygen is measured by measuring the change of oxygen electrode current, thus measuring the substrate concentration.
Determination of glucose in various raw materials is particularly important for process control. Using the metabolism and consumption of glucose by Pseudomonas fluorescens, the concentration of glucose can be estimated by oxygen electrode detection. Compared with the glucose enzyme electrode type, this microbial electrode has similar measurement results, while the microbial electrode has high sensitivity, good repeatability and practicability, and does not need to use expensive glucose enzyme.
When acetic acid is used as carbon source for microbial culture, the content of acetic acid above a certain concentration will inhibit the growth of microorganisms, so it is necessary to measure it online. The concentration of acetic acid can be determined by a microbial sensor consisting of immobilized yeast, gas permeable membrane and oxygen electrode.
In addition, the combination of Escherichia coli and carbon dioxide gas sensor can form a microbial sensor for glutamic acid determination, and the whole cell of Citrobacter is fixed in collagen membrane. The microbial sensor composed of bacteria-collagen membrane reactor and combined glass electrode can be used for the determination of cephalosporins in fermentation broth.
(2) Determination of total microbial cells
In fermentation control, a simple, continuous and direct method for measuring cell number is always needed. It is found that bacteria can be directly oxidized to generate current on the anode surface. The electrochemical system has been applied to the determination of cell number, and the results are the same as those of the traditional plaque counting method [1].
(3) Identification of metabolic test
The traditional identification of microbial metabolic types is based on the growth of microorganisms on a certain culture medium. These experimental methods require a long culture time and special techniques. The assimilation of substrates by microorganisms can be measured by their respiratory activity. Oxygen electrode can directly measure the respiratory activity of microorganisms. Therefore, microbial sensors can be used to measure the metabolic characteristics of microorganisms. The system has been used for simple identification of microorganisms, selection of microbial culture medium, determination of microbial enzyme activity, estimation of biodegradable substances in wastewater, selection of microorganisms for wastewater treatment, assimilation test of activated sludge, determination of biodegradable substances and selection of microbial preservation methods [2].
2. Environmental monitoring
(1). Determination of biochemical oxygen demand
Determination of biochemical oxygen demand (BOD) is the most commonly used index to monitor water pollution by organic matter. Conventional BOD determination needs a five-day incubation period, which is complex, repetitive, time-consuming and laborious, and is not suitable for on-site monitoring. Therefore, there is an urgent need for a new method with simple operation, high accuracy, high degree of automation and wide application. At present, researchers have isolated two new yeast species SPT 1 and SPT2, and fixed them on a glass carbon electrode to form a microbial sensor for measuring BOD, and the repeatability is within 10%. The sensor is used to measure BOD in pulp mill wastewater, and the minimum measurement value is 2 mg/l, and the time is 5min[3]. There is also a new type of microbial sensor, which uses high osmotic pressure-resistant yeast species as sensitive materials and can work normally under high osmotic pressure. Moreover, the strain can be dried and preserved for a long time, and its activity will recover after soaking, which provides a rapid and simple method for the determination of BOD in seawater [4].
In addition to microbial sensors, optical fiber biosensors have been developed to determine the lower BOD value in river water. The reaction time of the sensor is 65438 05 minutes, and the optimal working conditions are 30°C and pH=7. The sensor system is almost unaffected by chloride ions (in the range of 1000mg/l) and heavy metals (Fe3+, Cu2+, Mn2+, Cr3+, Zn2+). The sensor has been applied to the determination of BOD in river water and achieved good results [4].
At present, there is a BOD biosensor, which is very suitable for the determination of low BOD in river water after illumination treatment (that is, using TiO2 as semiconductor and irradiating with 6 W lamp for about 4min). At the same time, a compact optical biosensor is developed, which can measure BOD values of multiple samples at the same time. It uses three pairs of light emitting diodes and silicon photodiodes, and Pseudomonas is fixed at the bottom of the reactor with photocrosslinking resin. This measurement method is fast and simple, and can be used for six weeks at 4℃, and has been used in the process of wastewater treatment in factories [5].
(2) Determination of various pollutants
Commonly used important pollution indicators include the concentrations of ammonia, nitrite, sulfide, phosphate, carcinogens and mutagens, heavy metal ions, phenolic compounds, surfactants and other substances. At present, various biosensors for measuring various pollutants have been developed and put into practical application.
Microbial sensors for measuring ammonia nitrogen and nitrate are mostly composed of nitrifying bacteria separated from wastewater treatment equipment and oxygen electrode. At present, there is a microbial sensor that can measure nitrate and nitrite (nitrogen oxides-) in dark and light conditions, and it is not affected by other kinds of nitrogen oxides when it is measured in salt environment. It is used to measure nitrogen oxides in estuaries, and the effect is good [6].
The determination of sulfide is a microbial sensor made of specialized, autotrophic and aerobic thiobacillus thiooxidans separated and screened from acid soil near pyrite mine. When pH=2.5, 3 1℃ was measured more than 200 times a week, the activity remained unchanged and decreased by 20% after two weeks. The service life of the sensor is 7 days, with simple equipment, low cost and convenient operation. At present, microbial electrodes are used to measure sulfide content, and the bacteria used are chromatin. SP is connected with the hydrogen electrode [7].
Recently, scientists isolated a bacterium that can emit fluorescence in polluted areas. This bacterium contains fluorescent genes, which can produce fluorescent proteins under the stimulation of pollution sources, thus emitting fluorescence. This gene can be introduced into suitable bacteria through genetic engineering to make microbial sensors for environmental monitoring. At present, luciferase has been introduced into Escherichia coli to detect toxic compounds of arsenic [8].
Great progress has been made in the determination of phenols and surfactants in water. At present, nine kinds of Gram-negative bacteria have been isolated from the soil in the oil basin of Western Siberia, and phenol is the only carbon source and energy source. These strains can improve the sensitivity of the receptor part of biosensor. Its monitoring limit for phenol is 5? 10-9 mol. The optimum working conditions of the sensor are: pH=7.4, 35℃, and continuous working time is 30h[9]. There is also an amperometric biosensor for measuring the concentration of surfactants manufactured by Pseudomonas. Microbial cells are immobilized on gel (agar, agarose and calcium alginate) and polyethanol membrane. The cross-linking of microbial cells in gel caused by chromatographic test paper GF/A or glutamic aldehyde can keep its activity and growth in the long-distance detection of high concentration surfactants. The sensor can quickly restore the activity of the sensitive element after measurement [10].
There is also an amperometric biosensor for the determination of organophosphorus pesticides, which uses artificial enzymes. The determination limit of p-nitrophenol and diethyl phenol is 100 by using organophosphorus pesticide hydrolase. 10-9mol, only 4 min at 40℃ [11]. There is also a newly developed phosphate biosensor, which can detect (32~96) by using pyruvate oxidase G combined with CL-FIA desktop computer. 10-9mol phosphate can be used at 25°C for more than two weeks with high repeatability [12].
Recently, there is a new microbial sensor, which uses bacterial cells as biological components to determine the content of nonylphenol -NP-80e in surface water. Using amperometric oxygen electrode as a sensor, microbial cells are fixed on the dialysis membrane on the oxygen electrode, and its measuring principle is to measure the respiratory activity of Trichosporon cells. The reaction time of the biosensor is 15~20min, and its life span is 7~ 10 days (when used for continuous determination). In the concentration range of 0.5~6.0mg/l, the electrical signal has a linear relationship with the concentration of NP-80E, which is very suitable for the detection of molecular surfactants in polluted surface water [13].
In addition, the determination of heavy metal ion concentration in sewage can not be ignored. At present, based on immobilized microorganisms and bioluminescence measurement technology, a complete monitoring and analysis system for determining the bioavailability of heavy metal ions has been successfully designed. An operon of Vibrio fischeri was introduced into Alcaligenes eutrophica (AE 1239) controlled by a copper-induced promoter, and the bacteria were induced to emit light by copper ions, and the degree of luminescence was proportional to the ion concentration. A biosensor with high sensitivity, good selectivity, wide measurement range and strong storage stability can be obtained by embedding microorganisms and optical fibers in polymer matrix. At present, the minimum measurement concentration of this microbial sensor can reach 1 10-9 mol.
There is also an amperometric microbial sensor for measuring copper ions. It takes the recombinant strains of Saccharomyces cerevisiae as biological elements, and these strains fuse the promoter induced by copper ion on the CUP 1 gene of Saccharomyces cerevisiae and the lacZ gene of Escherichia coli. Its working principle is that Cu2+ is used to induce the promoter of CUP 1, and then lactose is used as the substrate for determination. If Cu2+ exists in the solution, these recombinant bacteria can use lactose as carbon source, which will lead to the change of oxygen demand of these aerobic cells. Can the biosensor be used in the concentration range (0.5~2)? Determination of copper sulfate solution in the range of 10-3mol. At present, various metal ion inducible promoters have been transferred into Escherichia coli, which makes Escherichia coli glow in solutions containing various metal ions. According to its luminous intensity, the concentration of heavy metal ions can be determined, and the measurement range can be from nanomole to micromole, and the required time is 60 ~100 min [15] [16].
A biosensor for measuring zinc concentration in sewage has also been successfully developed. The concentration and bioavailability of zinc in sewage were determined by alkaline bacteria Alcaligenes, and the results were satisfactory [17].
The algae sensor used to estimate the pollution of estuary effluent consists of cyanobacteria Spirlina subsalsa and gas sensor. By monitoring the inhibition degree of photosynthesis, we can estimate the toxicity change of water caused by the existence of environmental pollutants. Using standard natural water as medium, the different concentrations of three main pollutants (heavy metals, herbicides and carbamate pesticides) can be determined and their toxic reactions can be monitored, which has high repeatability and reproducibility [18].
In recent years, due to the rapid development of polymerase chain reaction (PCR) and its wide application in environmental monitoring, many scientists began to combine it with biosensor technology. There is a DNA piezoelectric biosensor using PCR technology, which can determine a special bacterial toxin. The biotinylated probe was immobilized on the crystal loaded with streptomycin on the platinum surface, and the probe was immobilized with 1? 10-6mol hydrochloric acid can be measured circularly on the same crystal plane. The DNA samples extracted from bacteria were subjected to the same hybridization reaction, and the products were specific gene fragments of Aeromonas hydrophila after PCR amplification. This piezoelectric biosensor can identify whether the sample contains this gene, which makes it possible to detect whether the water sample contains various Aeromonas with this pathogen [19].
There is also a channel biosensor, which can detect toxic substances such as dinoflagellate neurotoxin produced by phytoplankton and jellyfish, and it has been able to measure a very small amount of PSP toxin contained in plankton cells [20]. DNA sensors are also rapidly applied. At present, there is a miniaturized DNA biosensor, which can convert DNA recognition signals into electrical signals for measuring cryptosporidium and other water-borne infectious bodies in water samples. The sensor focuses on improving the recognition function of nucleic acid, strengthening the specificity and sensitivity of the sensor, and seeking a new method to convert hybridization signals into useful signals. The current research work is the integration of identification devices and conversion devices [2 1].