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Abstract: This article briefly describes the research and application of biosensors, especially microbial sensors, in the fermentation industry and environmental monitoring fields in recent years. Its development prospects and marketization are predicted and forecasted. Bioelectrodes are sensitive materials that use immobilized biological components as molecular recognition elements. They form biosensors with oxygen electrodes, membrane electrodes, and fuel electrodes. They are widely used in fermentation industry, environmental monitoring, food monitoring, clinical medicine, etc. Biosensors have good specificity, are easy to operate, have simple equipment, fast and accurate measurement, and have a wide range of applications. With the development of immobilization technology, biosensors are extremely competitive in the market.
Keywords: biosensor; fermentation industry; environmental monitoring.
1. Introduction
It has been 40 years since Clark and Lyons first proposed the idea of ??biosensors in 1962. Biosensors have been deeply valued and widely used in fermentation technology, environmental monitoring, food engineering, clinical medicine, military and military medicine, etc. In the first 15 years, biosensors were mainly based on the development of enzyme electrodes. However, because enzymes are expensive and not stable enough, the applications of sensors using enzymes as sensitive materials are subject to certain limitations.
In recent years, the continuous development of microbial immobilization technology has produced microbial electrodes. Microbial electrodes use living microorganisms as molecular recognition elements, which are unique compared with enzyme electrodes. It can overcome the disadvantages of high price, difficulty in extraction and instability. In addition, coenzymes in microorganisms can also be used to handle complex reactions at the same time. At present, the application of optical fiber biosensors is becoming more and more widespread. Moreover, with the development of polymerase chain reaction technology (PCR), more and more DNA biosensors using PCR are being used.
2. Research status and main application fields
1. Fermentation industry
Among various biosensors, microbial sensors are most suitable for determination in the fermentation industry. Because substances that interfere with enzymes often exist during the fermentation process, and the fermentation broth is often not clear and transparent, it is not suitable for measurement by spectroscopy and other methods. The application of microbial sensors has a high possibility of eliminating interference and is not limited by the turbidity of the fermentation broth. At the same time, since the fermentation industry is a large-scale production, microbial sensors have great advantages due to their low cost and simple equipment.
(1). Determination of raw materials and metabolites
Microbiological sensors can be used for the determination of raw materials such as molasses, acetic acid, etc., and metabolites such as cephalosporin, glutamic acid, formic acid, Determination of methane, alcohols, penicillin, lactic acid, etc. The principle of measurement is basically composed of a suitable microbial electrode and an oxygen electrode. The assimilation of microorganisms is used to consume oxygen. The reduction of oxygen is measured by measuring the change in the oxygen electrode current, so as to achieve the purpose of measuring the substrate concentration.
The determination of glucose in various raw materials is particularly important for process control. The glucose metabolism and consumption of glucose by Psoudomonas fluorescens is detected through an oxygen electrode, and the concentration of glucose can be estimated. Compared with the glucose enzyme electrode type, the measurement results of this microbial electrode are similar, and the microbial electrode has high sensitivity, good repeatability and no need to use expensive glucose enzyme.
When acetic acid is used as a carbon source for microbial culture, acetic acid content higher than a certain concentration will inhibit the growth of microorganisms, so online measurement is required. The concentration of acetic acid can be measured using a microbial sensor composed of immobilized yeast (Trichosporon brassicae), a gas-permeable membrane, and an oxygen electrode.
In addition, Escherichia coli (E.coli) combined with a carbon dioxide gas-sensitive electrode can be used to form a microbial sensor for measuring glutamate. Complete Citrobacter cells are immobilized in a collagen membrane, and the bacteria are —The microbial sensor composed of a collagen membrane reactor and a combined glass electrode can be applied to the determination of cephalosporins in fermentation broth, etc.
(2). Determination of the total number of microbial cells
In fermentation control, there has always been a need for a simple and continuous method of directly measuring the number of cells. It was found that on the surface of the anode, bacteria could be oxidized directly and generate an electric current. This electrochemical system has been applied to the determination of cell number, and the results are the same as the traditional plaque counting method [1].
(3). Identification of metabolic tests
Traditional identification of microbial metabolic types is based on the growth of microorganisms on a certain culture medium. These experimental methods require long incubation times and specialized techniques. The assimilation of substrates by microorganisms can be measured by their respiratory activity. The respiratory activity of microorganisms can be directly measured using an oxygen electrode. Therefore, microbial sensors can be used to measure the metabolic characteristics of microorganisms. This system has been used for simple identification of microorganisms, selection of microbial culture media, determination of microbial enzyme activity, estimation of biodegradable substances in wastewater, selection of microorganisms for wastewater treatment, assimilation test of activated sludge, biodegradation Determination of organisms, selection of preservation methods for microorganisms, etc. [2].
2. Environmental monitoring
(1). Measurement of biochemical oxygen demand
The measurement of biochemical oxygen demand (BOD) is monitoring The most commonly used indicator of water body contamination by organic matter. Conventional BOD determination requires a 5-day incubation period. The operation is complex, has poor repeatability, is time-consuming and labor-intensive, and is highly disruptive. It is not suitable for on-site monitoring. Therefore, there is an urgent need for a new method that is simple to operate, fast, accurate, highly automated, and widely applicable. to measure. Currently, researchers have isolated two new yeast strains, SPT1 and SPT2, and fixed them on glass carbon poles to form microbial sensors for measuring BOD, with repeatability within ±10. This sensor is used to measure BOD in pulp mill sewage, and the minimum measurement value can reach 2 mg/l, and the time taken is 5 minutes [3]. There is also a new microbial sensor that uses high osmotic pressure-resistant yeast strains as sensitive materials and can work normally under high osmotic pressure. Moreover, its strains can be stored dry for a long time and recover their activity after soaking, providing a quick and easy method for the determination of BOD in seawater [4].
In addition to microbial sensors, an optical fiber biosensor has been developed to measure lower BOD values ??in river water. The reaction time of the sensor is 15 minutes, and the optimal working conditions are 30°C and pH=7. This sensor system is almost unaffected by chloride ions (in the range of 1000mg/l) and is not affected by heavy metals (Fe3, Cu2, Mn2, Cr3, Zn2). This sensor has been applied to the determination of river water BOD and achieved good results [4].
There is now a BOD biosensor that undergoes light treatment (that is, using TiO2 as a semiconductor and irradiating it with a 6 W lamp for about 4 minutes). The sensitivity is greatly improved and is very suitable for measuring lower BOD in river water [5 ]. Meanwhile, a compact optical biosensor has been developed to measure the BOD values ??of multiple samples simultaneously. It uses three pairs of light-emitting diodes and silicon photodiodes. Pseudomonas fluorescens is immobilized on the bottom of the reactor with photo-cross-linked resin. The measurement method is fast and simple and can be used for six weeks at 4°C. It has been used in the process of factory wastewater treatment [5].
(2). Determination of various pollutants
Commonly used important pollution indicators include ammonia, nitrite, sulfide, phosphate, carcinogens and mutagens, and heavy metals Concentrations of ions, phenolic compounds, surfactants, etc. A variety of biosensors for measuring various pollutants have been developed and put into practical applications.
Microbiological sensors that measure ammonia and nitrate are mostly composed of nitrifying bacteria isolated from wastewater treatment devices and an oxygen electrode.
There is currently a microbial sensor that can measure nitrate and nitrite (NOx-) in dark and light conditions. Its measurement in a salt environment makes it unaffected by other types of nitrogen oxides. It has been used to measure NOx- in the estuary, and the effect is good [6].
The determination of sulfide is a microbial sensor made of obligate, autotrophic, aerobic Thiobacillus thiooxidans isolated and screened from acidic soil near pyrite. At pH=2.5 and 31°C, the activity was measured more than 200 times a week and the activity remained unchanged. After two weeks, the activity decreased by 20%. The sensor life is 7 days, and its equipment is simple, low cost and easy to operate. Currently, a photomicrobial electrode is used to measure sulfide content. The bacteria used are Chromatium.SP, which is connected to a hydrogen electrode [7].
Recently, scientists have isolated a fluorescent bacterium in a polluted area. This bacterium contains fluorescent genes and can produce fluorescent proteins when stimulated by pollution sources, thereby emitting fluorescence. This gene can be introduced into suitable bacteria through genetic engineering methods to make microbial sensors for environmental monitoring. Luciferase has now been introduced into Escherichia coli (E.coli) to detect toxic compounds of arsenic [8].
The concentration determination of phenols and surfactants in water has made great progress. Currently, nine species of Gram-negative bacteria have been isolated from soils in the West Siberian Petroleum Basin and use phenol as the sole source of carbon and energy. These bacterial species can improve the sensitivity of the sensor part of the biosensor. Its monitoring limit for phenol is 5?10-9mol. The optimal working conditions of this sensor are: pH=7.4, 35℃, and the continuous working time is 30h[9]. There is also an amperometric biosensor made of Pseudomonas rathonis that measures surfactant concentration. Microbial cells are fixed on gels (agar, agarose and alginate calcium salts) and polyethyl alcohol membranes. You can use chromatographic test paper GF/A or glutamic acid aldehyde to cause cross-linking of microbial cells in the gel to maintain their activity and growth over a long period of time in high-concentration surfactant detection. This sensor can quickly restore the activity of the sensitive element after the measurement is completed [10].
There is also an amperometric biosensor for measuring organophosphorus pesticides that uses artificial enzymes. Using organophosphorus pesticide hydrolase, the measurement limit of p-nitrophenol and diethylphenol is 100?10-9mol, which only takes 4 minutes at 40℃[11]. There is also a newly developed phosphate biosensor that uses pyruvate oxidase G and is combined with the automatic system CL-FIA desktop computer to detect (32~96)?10-9mol phosphate at 25°C. It is used for more than two weeks and has high repeatability [12].
Recently, a new type of microbial sensor has been developed that uses bacterial cells as biological components to measure the content of nonyl-phenol etoxylate - NP-80E in surface water. A current-type oxygen electrode is used as a sensor, and microbial cells are fixed on the dialysis membrane on the oxygen electrode. The measurement principle is to measure the respiratory activity of Trichosporum grablata cells. The biosensor has a reaction time of 15~20min and a lifespan of 7~10 days (when used for continuous measurement). Within 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 concentrations in sewage cannot be ignored. A complete monitoring and analysis system for the bioavailability determination of heavy metal ions based on immobilized microorganisms and bioluminescence measurement technology has been successfully designed. An operon from Vibrio fischeri is introduced into Alcaligenes eutrophus (AE1239) under the control of a copper-inducible promoter. The bacteria emit light under the induction of copper ions, and the degree of luminescence is similar to that of Alcaligenes eutrophus (AE1239). proportional to ion concentration.
By embedding microorganisms and optical fibers in a polymer matrix, a biosensor with high sensitivity, good selectivity, wide measurement range, and strong storage stability can be obtained. Currently, this microbial sensor can reach the lowest measurement concentration of 1?10-9mol [14].
There is also an amperometric microbial sensor that specifically measures copper ions. It uses recombinant strains of Saccharomyces cerevisiae as biological elements. These strains carry a fusion of the copper ion-inducible promoter on the Saccharomyces CUP1 gene and the E. coli lacZ gene. How it works, first the CUP1 promoter is induced by Cu2, and then lactose is used as a substrate for measurement. If Cu2 is present in solution, these recombinant bacteria can utilize lactose as a carbon source, which will lead to changes in the oxygen demand of these aerobic cells. This biosensor can measure CuSO4 solution in the concentration range (0.5~2)?10-3mol. At present, various metal ion-induced promoters have been transferred into E. coli, so that E. coli will produce luminescent reactions in solutions containing various metal ions. The concentration of heavy metal ions can be measured according to its luminous intensity. The measurement range can be from nanomolar to micromolar, and the required time is 60~100min[15][16].
A biosensor for measuring zinc concentration in sewage has also been successfully developed, using the alkaline bacterium Alcaligenes cutrophus, and used to measure the concentration and bioavailability of zinc in sewage, with satisfactory results. [17].
The algae sensor that estimates pollution in estuary effluents is composed of a cyanobacterium Spirlina subsalsa and a gas-sensitive electrode. Estimating changes in water toxicity due to the presence of environmental pollutants by monitoring the extent to which photosynthesis is inhibited. Using standard natural water as the medium, different concentrations of three major pollutants (heavy metals, herbicides, and carbamate pesticides) were measured, and their toxic reactions could be monitored, with excellent repeatability and reproducibility. High[18].
Recently, due to the rapid development of polymerase chain reaction technology (PCR) and its wide application in environmental monitoring, many scientists have begun to combine it with biosensor technology. There is a DNA piezoelectric biosensor using PCR technology that can detect a special bacterial toxin. The biotinylated probe is fixed on a quartz crystal equipped with a streptozotocin platinum surface, and cyclic measurements can be performed on the same crystal surface using 1 to 10-6 mol of hydrochloric acid. The same hybridization reaction was performed using DNA samples extracted from bacteria and amplified by PCR. The product was a special gene fragment of Aeromonas hydrophila. This piezoelectric biosensor can identify whether a sample contains this gene, which provides the possibility to detect whether various Aeromonas bacteria carrying this pathogen are contained in water samples [19].
There is also a channel biosensor that can detect toxic substances such as dinoflagellate neurotoxins produced by organisms such as phytoplankton and jellyfish. It is currently able to measure very small amounts of PSP toxins contained in a plankton cell. [20]. DNA sensors are also rapidly being applied. Currently, there is a miniaturized DNA biosensor that can convert DNA recognition signals into electrical signals and is used to measure Cryptosporidium and other waterborne infectious agents in water samples. This sensor focuses on improving the recognition of nucleic acids and enhancing the specificity and sensitivity of the sensor, and seeks new methods to convert hybridization signals into useful signals. The current research work is the integration of recognition devices and conversion devices [21].