I. INTRODUCTION From 1962, Clark and Lyons first proposed the idea of ​​a biosensor 40 years ago. Biosensors have received great attention and extensive applications in fermentation processes, environmental monitoring, food engineering, clinical medicine, military and military medicine. In the first fifteen years, biosensors were mainly based on biosensors fabricated with enzyme electrodes. However, due to the high cost and instability of enzymes, sensors that use enzymes as sensitive materials have limited application.
In recent years, the continuous development of microbial immobilization technology has produced microbial electrodes. Microbial electrodes use microbiological organisms as molecular recognition elements, which are unique compared to enzyme electrodes. It can overcome the weaknesses of high prices, difficulties in extraction and instability. In addition, it is also possible to use a coenzyme in vivo to treat complex reactions. At present, the application of optical fiber biosensors has become more and more widespread. Moreover, with the development of polymerase chain reaction (PCR), more and more DNA biosensors have been used for PCR.
Second, research status and the main application areas 1, a variety of biosensor fermentation industry, microbial sensors are most suitable for the determination of the fermentation industry. Because there are often interfering substances in the fermentation process, and the fermentation broth is often not clear and transparent, it is not suitable for the determination of spectra and Other methods. The use of microbial sensors is highly likely to eliminate interference and is not limited by the degree of turbidity of the fermentation broth. At the same time, due to the large-scale production of the fermentation industry, microbial sensors have great advantages due to their low cost and simple equipment.
(1) Determination of raw materials and metabolites The microbial sensor can be used for the determination of raw materials such as molasses, acetic acid, and the like, and metabolites such as cephalosporin, glutamic acid, formic acid, methane, alcohols, penicillin, lactic acid, and the like. The principle of measurement is basically composed of a suitable microbial electrode and an oxygen electrode, which utilizes the assimilation of microorganisms to consume oxygen, and measures the reduction amount of oxygen by measuring the change amount of the current of the oxygen electrode 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-depleting effect of Pseudomonas fluorescens metabolism can be estimated through the oxygen electrode and glucose concentration can be estimated. This microbial electrode is similar to the glucoenzyme electrode type in that the assay results are similar, and the microbial electrode has high sensitivity, good repeatability, and does not require the use of expensive glucoenzymes.
When acetic acid is used as a carbon source for the cultivation of microorganisms, an acetic acid content higher than a certain concentration inhibits the growth of microorganisms, and therefore it requires on-line measurement. The concentration of acetic acid can be determined using a microbe sensor consisting of an immobilized yeast (Trichosporon brassicae), a gas permeable membrane, and an oxygen electrode.
In addition, there is a combination of carbon dioxide gas-sensing electrode with E. coli that can constitute a microbial sensor for measuring glutamic acid, immobilizing Citrobacter bacterium intact cells in the collagen membrane, and using a bacteria-collagen membrane reactor and A combination of glass electrode microsensors can be applied to the determination of cephalosporin in fermentation broth.
(2) Determination of the total number of microbial cells In the aspect of fermentation control, there has 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 can be directly oxidized and generate electricity. This electrochemical system has been applied to the determination of the number of cells, the results of which are the same as the conventional plaque counting method [1].
(3) Identification of metabolic tests The identification of traditional microbial metabolic types is based on the growth of microorganisms on a certain medium. These experimental methods require longer incubation times and specialized techniques. The assimilation of a substrate by a microorganism can be determined by its respiratory activity. The oxygen electrode can be used to directly measure the respiratory activity of microorganisms. Therefore, microbial sensors can be used to determine the metabolic characteristics of microorganisms. This system has been used for simple identification of microorganisms, selection of microbial media, determination of microbial enzyme activity, estimation of biodegradable substances in wastewater, selection of microorganisms for wastewater treatment, assimilation test of activated sludge, biodegradation The determination of the substance, the choice of methods for the preservation of microorganisms, etc. [2].
2. Environmental Monitoring (1) Determination of Biochemical Oxygen Demand (BOD) Biochemical oxygen demand (BOD) is the most commonly used indicator for monitoring the status of water pollution by organic matter. The conventional BOD measurement requires a five-day incubation period, which requires complicated operation, poor repeatability, time-consuming and labor-intensive, and large interference. It is not suitable for on-site monitoring. Therefore, there is an urgent need for a new method that is simple, rapid, accurate, highly automated, and widely applicable. To determine. Currently, researchers have isolated two new yeast strains, SPT1 and SPT2, and immobilized them on a glass carbon electrode to constitute a microbial sensor for measuring the BOD with repeatability within ±10%. The sensor was used to measure the BOD in pulp mill sewage. The minimum value of the measurement was 2 mg/l, and the time was 5 min [3]. There is also a new microbial sensor that uses high osmotic pressure yeast strains as sensitive materials and can work normally under high osmotic pressure. And its strains can be stored dry for a long period of time, and then reactivated after soaking, providing a quick and easy method for the determination of BOD in sea water [4].
In addition to microbial sensors, there is also an optical fiber biosensor that has been developed to measure lower BOD values ​​in river waters. The reaction time of the sensor is 15 min, the optimum working condition is 30°C, pH=7. This sensor system is virtually immune to chloride (in the 1000 mg/l range) and is not affected by heavy metals (Fe3+, Cu2+, Mn2+, Cr3+, Zn2+). The sensor has been applied to the determination of BOD in river water and good results have been obtained [4].
Now there is a way to measure the sensitivity of BOD biosensor after it has been treated with light (that is, TiO2 is used as a semiconductor and irradiated with 6 W lamp for about 4 minutes), which is very suitable for the measurement of lower BOD in river water [5]. At the same time, a compact optical biosensor has been developed for simultaneous measurement of multiple sample BOD values. It uses three pairs of light-emitting diodes and silicon photodiodes, and Pseudomonas fluorescens is immobilized on the bottom of the reactor with photo-crosslinked resin. The measurement method is quick and easy. It can be used at 4°C for six weeks. It has been used in the process of factory wastewater treatment [5].
(2) Concentrations of ammonia, nitrites, sulfides, phosphates, carcinogens, mutagens, heavy metal ions, phenolic compounds, surfactants, and other important pollutants commonly used in the determination of various pollutants. A variety of biosensors for measuring various types of pollutants have been developed and put into practical use.
The microbial sensors that measure ammonia and nitrates are mostly made up of a combination of nitrifying bacteria and oxygen electrodes that are separated from wastewater treatment plants. There is currently a microbial sensor that can measure nitrate and nitrite (NOx-) in dark and light conditions, and its measurement in a saline environment makes it immune to the effects of other types of nitrogen oxides. Using it to measure NOx in the estuary, the effect is better [6].
The sulphide is a microbial sensor made from an obligate, autotrophic, and aerobic oxidizing thiobacilli isolated and screened from acidic soil near pyrite. Measurements were performed 200 times a week at pH=2.5 and 31°C, and the activity remained unchanged, and the activity decreased by 20% after two weeks. The sensor life is 7 days, its equipment is simple, the cost is low, and the operation is convenient. There is also a photomicrobial electrode for measuring sulfide content. The bacteria used is Chromatium. SP, connected with hydrogen electrodes constitutes [7].
Scientists have recently isolated a bacterium that can fluoresce in contaminated areas. The bacteria contain fluorescent genes that, when stimulated by a source of contamination, produce fluorescent proteins that fluoresce. This gene can be introduced into suitable bacteria by genetic engineering methods to make microbial sensors for environmental monitoring. Luciferase has been introduced into E. coli to detect arsenic toxic compounds [8].
The determination of the concentration of phenols and surfactants in water has been greatly advanced. At present, there are nine gram-negative bacteria isolated from the soil of the West Siberian oil basin, with phenol as the sole carbon source and energy source. These strains can increase the sensitivity of the sensor portion of the biosensor. Its monitoring limit for phenol is 5'10-9 mol. The optimal conditions for this sensor to work are: pH = 7.4, 35 °C, continuous working time 30h [9]. There is also an amperometric biosensor made from Pseudomonas rathonis that measures surfactant concentration, immobilizing microbial cells on gels (agar, agarose, and calcium alginate) and polyethyl alcohol membranes. The cross-linking of microbial cells in the gel by chromatography paper GF/A, or glutamate, can be maintained over long distances to maintain their activity and growth in high-concentration surfactant assays. The sensor can quickly recover the activity of the sensor after the measurement is completed [10].
There is also an amperometric biosensor for the determination of organophosphorus pesticides using artificial enzymes. Using the organophosphorus insecticide hydrolase, the measurement limit of p-nitrophenol and diethylphenol is 100′10-9 mol, and it takes only 4 minutes at 40° C. [11]. There is also a newly developed phosphate biosensor, using pyruvate oxidase G, in combination with an automated system CL-FIA desktop computer, which can detect (32-96) 10-9 mol of phosphate and can be used at 25°C. More than two weeks of use, high reproducibility [12].
Recently, there is a novel microbial sensor that uses bacterial cells as a biological component to determine the content of nonyl-phenol etoxylate (NP-80E) in surface water. Using an amperometric oxygen electrode as a sensor, the microbial cells were immobilized on a dialysis membrane on an oxygen electrode. The measuring principle was to measure the respiratory activity of Trichosporum grablata cells. The biosensor has a reaction time of 15 to 20 minutes and a life span of 7 to 10 days (for continuous measurement). In the concentration range of 0.5 to 6.0 mg/l, the electrical signal is linearly related to the concentration of NP-80E, which is very suitable for the detection of molecular surfactants in contaminated surface water [13].
In addition, the determination of the concentration of heavy metal ions in sewage can not be ignored. A complete monitoring and analysis system for the determination of the bioavailability of heavy metal ions based on immobilized microorganisms and bioluminescence measurement technology has been successfully designed. An operon from Vibrio fischeri was introduced into Alcaligenes eutrophus (AE1239) under the control of a copper-inducible promoter. The bacteria luminesced under the induction of copper ions. The ion concentration is proportional to. By embedding microorganisms and optical fibers in a polymer matrix, a biosensor having high sensitivity, good selectivity, wide measurement range, and strong storage stability can be obtained. At present, this microbial sensor can reach the minimum measured concentration of 1'10-9 mol [14].
There is also a current-based microbial sensor that measures copper ions. It uses a recombinant strain of Saccharomyces cerevisiae as a biological element. These strains carry a fusion of the copper ion-inducible promoter on the CUP1 gene of E. vinifera and the E. coli lacZ gene. Its working principle is that the CUP1 promoter is first induced by Cu2+, and then lactose is used as a substrate for measurement. If Cu2+ is present in solution, these recombinant bacteria can use lactose as a carbon source, which will lead to changes in the oxygen demand of these aerobic cells. The biosensor can measure CuSO4 solution within the range of concentration (0.5-2)' 10-3 mol. Currently, various types of metal ion-inducible promoters have been transformed into E. coli, so that E. coli luminescence reactions occur in solutions containing various metal ions. Based on the intensity of its luminescence, the concentration of heavy metal ions can be determined. The measurement range can be from nanomolar to micromolar, and the time required is 60-100 min [15] [16].
Biosensors for the measurement of zinc concentration in sewage have also been developed, using the basophilic bacteria Alcaligenes cutrophus and used to measure the concentration and bioavailability of zinc in the sewage. The results are satisfactory [17].
The seaweed sensor that estimates the pollution of the outflow from the estuary is composed of a cyanobacterium Spirlina subsalsa and a gas-sensing electrode. By monitoring the extent to which photosynthesis is inhibited, the change in the toxicity of water due to the presence of environmental contaminants is estimated. Using standard natural water as the medium, different concentrations of the three major pollutants (heavy metals, herbicides, carbamate insecticides) were measured and their toxic reactions were monitored. Repeatability and reproducibility were very high. Gao [18].
Recently, due to the rapid development of polymerase chain reaction (PCR) technology 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 determine a specific bacterial toxin. Biotinylated probes were immobilized on quartz crystals with a platinum-gold surface of the enzyme antibiotic, and circulating measurements were performed on the same crystal surface with 1'10-6 mol of hydrochloric acid. The same hybridization reaction was performed with DNA extracted from bacteria and amplified by PCR. The product was a special gene fragment of Aeromonas hydrophila. The piezoelectric biosensor can identify whether the gene is contained in the sample, which provides the possibility to detect from the water sample whether there are various Aeromonas with this pathogen [19].
There is also a channel biosensor that can detect toxins such as neurotoxins produced by organisms such as phytoplankton and jellyfish, and has now been able to measure trace amounts of PSP toxins contained in a planktonic cell [20]. DNA sensors are also being rapidly applied. There is currently a miniaturized DNA biosensor that converts DNA recognition signals into electrical signals for measuring cryptospores and other waterborne pathogens in water samples. The sensor focuses on improving the recognition of nucleic acids and enhancing the specificity and sensitivity of the sensor, and seeks new methods for translating hybridization signals into useful signals. The current research work is the integration of recognition devices and conversion devices [21].
Microalgae is a bacterial hepatotoxin produced from blooms caused by cyanobacteria. A biosensor with a surface cell plasmid genome immobilized on it has been prepared for the measurement of microalgae in water. Its direct measurement range is 50～1000′10-6g/l[22].
The idea of ​​multiple biosensors based on enzyme-based inhibition analysis for measuring toxic substances has also been proposed. In this multiple biosensor, two types of transducers are used—pH-sensitive electronic transistors and heat-sensitive thin-film electrodes, and three enzymes—urease, acetylcholinesterase, and butyrylcholinesterase. The performance of this biosensor has been tested with good results [23].
In addition to the fermentation industry and environmental monitoring, biosensors are also used in food engineering, clinical medicine, military and military medicine, and are mainly used to measure glucose, acetic acid, lactic acid, lactose, uric acid, urea, antibiotics, and glutamic acid. Amino acids, as well as various carcinogenic and mutagenic substances.
Third, discuss and look forward to the United States Harold H. Weetal pointed out that the commercialization of biosensors must meet the following conditions:
Sufficient sensitivity and accuracy, easy operation, low cost, easy mass production, and quality monitoring during production. Among them, the cheap price determines the sensor's competitiveness in the market. Among various biosensors, the biggest advantages of microbiological sensors are low cost, simple operation, and simple equipment, so their market prospects are very large and attractive. In comparison, enzyme biosensors, etc., are relatively expensive. However, microbial sensors also have their own shortcomings. The main drawback is that the selectivity is not good enough. This is caused by the presence of multiple enzymes in the microbial cells. It has been reported that adding special inhibitors to solve the problem of microbial electrode selectivity. In addition, the method of immobilizing microorganisms needs to be further improved. First of all, it is necessary to ensure the activity of the cells as much as possible. Secondly, the cells must be tightly bound to the basic membrane to avoid the loss of cells. In addition, the long-term preservation of microbial membranes needs to be further improved, otherwise it is difficult to achieve large-scale commercialization.
In conclusion, commonly used microbial electrodes and enzyme electrodes have their advantages in various applications. If a stable, highly active, low cost free enzyme is readily available, the enzyme electrode is ideal for the user. Conversely, if biocatalysis needs to go through complex pathways, requires coenzymes, or if the desired enzymes are not suitable for separation or instability, microbial electrodes are a better choice. While various other forms of biosensors are also booming, their applications have become more widespread. With the further improvement of the immobilization technology, with the constant deepening of people's understanding of living organisms, biosensors will open up a new world in the market.
In recent years, the continuous development of microbial immobilization technology has produced microbial electrodes. Microbial electrodes use microbiological organisms as molecular recognition elements, which are unique compared to enzyme electrodes. It can overcome the weaknesses of high prices, difficulties in extraction and instability. In addition, it is also possible to use a coenzyme in vivo to treat complex reactions. At present, the application of optical fiber biosensors has become more and more widespread. Moreover, with the development of polymerase chain reaction (PCR), more and more DNA biosensors have been used for PCR.
Second, research status and the main application areas 1, a variety of biosensor fermentation industry, microbial sensors are most suitable for the determination of the fermentation industry. Because there are often interfering substances in the fermentation process, and the fermentation broth is often not clear and transparent, it is not suitable for the determination of spectra and Other methods. The use of microbial sensors is highly likely to eliminate interference and is not limited by the degree of turbidity of the fermentation broth. At the same time, due to the large-scale production of the fermentation industry, microbial sensors have great advantages due to their low cost and simple equipment.
(1) Determination of raw materials and metabolites The microbial sensor can be used for the determination of raw materials such as molasses, acetic acid, and the like, and metabolites such as cephalosporin, glutamic acid, formic acid, methane, alcohols, penicillin, lactic acid, and the like. The principle of measurement is basically composed of a suitable microbial electrode and an oxygen electrode, which utilizes the assimilation of microorganisms to consume oxygen, and measures the reduction amount of oxygen by measuring the change amount of the current of the oxygen electrode 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-depleting effect of Pseudomonas fluorescens metabolism can be estimated through the oxygen electrode and glucose concentration can be estimated. This microbial electrode is similar to the glucoenzyme electrode type in that the assay results are similar, and the microbial electrode has high sensitivity, good repeatability, and does not require the use of expensive glucoenzymes.
When acetic acid is used as a carbon source for the cultivation of microorganisms, an acetic acid content higher than a certain concentration inhibits the growth of microorganisms, and therefore it requires on-line measurement. The concentration of acetic acid can be determined using a microbe sensor consisting of an immobilized yeast (Trichosporon brassicae), a gas permeable membrane, and an oxygen electrode.
In addition, there is a combination of carbon dioxide gas-sensing electrode with E. coli that can constitute a microbial sensor for measuring glutamic acid, immobilizing Citrobacter bacterium intact cells in the collagen membrane, and using a bacteria-collagen membrane reactor and A combination of glass electrode microsensors can be applied to the determination of cephalosporin in fermentation broth.
(2) Determination of the total number of microbial cells In the aspect of fermentation control, there has 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 can be directly oxidized and generate electricity. This electrochemical system has been applied to the determination of the number of cells, the results of which are the same as the conventional plaque counting method [1].
(3) Identification of metabolic tests The identification of traditional microbial metabolic types is based on the growth of microorganisms on a certain medium. These experimental methods require longer incubation times and specialized techniques. The assimilation of a substrate by a microorganism can be determined by its respiratory activity. The oxygen electrode can be used to directly measure the respiratory activity of microorganisms. Therefore, microbial sensors can be used to determine the metabolic characteristics of microorganisms. This system has been used for simple identification of microorganisms, selection of microbial media, determination of microbial enzyme activity, estimation of biodegradable substances in wastewater, selection of microorganisms for wastewater treatment, assimilation test of activated sludge, biodegradation The determination of the substance, the choice of methods for the preservation of microorganisms, etc. [2].
2. Environmental Monitoring (1) Determination of Biochemical Oxygen Demand (BOD) Biochemical oxygen demand (BOD) is the most commonly used indicator for monitoring the status of water pollution by organic matter. The conventional BOD measurement requires a five-day incubation period, which requires complicated operation, poor repeatability, time-consuming and labor-intensive, and large interference. It is not suitable for on-site monitoring. Therefore, there is an urgent need for a new method that is simple, rapid, accurate, highly automated, and widely applicable. To determine. Currently, researchers have isolated two new yeast strains, SPT1 and SPT2, and immobilized them on a glass carbon electrode to constitute a microbial sensor for measuring the BOD with repeatability within ±10%. The sensor was used to measure the BOD in pulp mill sewage. The minimum value of the measurement was 2 mg/l, and the time was 5 min [3]. There is also a new microbial sensor that uses high osmotic pressure yeast strains as sensitive materials and can work normally under high osmotic pressure. And its strains can be stored dry for a long period of time, and then reactivated after soaking, providing a quick and easy method for the determination of BOD in sea water [4].
In addition to microbial sensors, there is also an optical fiber biosensor that has been developed to measure lower BOD values ​​in river waters. The reaction time of the sensor is 15 min, the optimum working condition is 30°C, pH=7. This sensor system is virtually immune to chloride (in the 1000 mg/l range) and is not affected by heavy metals (Fe3+, Cu2+, Mn2+, Cr3+, Zn2+). The sensor has been applied to the determination of BOD in river water and good results have been obtained [4].
Now there is a way to measure the sensitivity of BOD biosensor after it has been treated with light (that is, TiO2 is used as a semiconductor and irradiated with 6 W lamp for about 4 minutes), which is very suitable for the measurement of lower BOD in river water [5]. At the same time, a compact optical biosensor has been developed for simultaneous measurement of multiple sample BOD values. It uses three pairs of light-emitting diodes and silicon photodiodes, and Pseudomonas fluorescens is immobilized on the bottom of the reactor with photo-crosslinked resin. The measurement method is quick and easy. It can be used at 4°C for six weeks. It has been used in the process of factory wastewater treatment [5].
(2) Concentrations of ammonia, nitrites, sulfides, phosphates, carcinogens, mutagens, heavy metal ions, phenolic compounds, surfactants, and other important pollutants commonly used in the determination of various pollutants. A variety of biosensors for measuring various types of pollutants have been developed and put into practical use.
The microbial sensors that measure ammonia and nitrates are mostly made up of a combination of nitrifying bacteria and oxygen electrodes that are separated from wastewater treatment plants. There is currently a microbial sensor that can measure nitrate and nitrite (NOx-) in dark and light conditions, and its measurement in a saline environment makes it immune to the effects of other types of nitrogen oxides. Using it to measure NOx in the estuary, the effect is better [6].
The sulphide is a microbial sensor made from an obligate, autotrophic, and aerobic oxidizing thiobacilli isolated and screened from acidic soil near pyrite. Measurements were performed 200 times a week at pH=2.5 and 31°C, and the activity remained unchanged, and the activity decreased by 20% after two weeks. The sensor life is 7 days, its equipment is simple, the cost is low, and the operation is convenient. There is also a photomicrobial electrode for measuring sulfide content. The bacteria used is Chromatium. SP, connected with hydrogen electrodes constitutes [7].
Scientists have recently isolated a bacterium that can fluoresce in contaminated areas. The bacteria contain fluorescent genes that, when stimulated by a source of contamination, produce fluorescent proteins that fluoresce. This gene can be introduced into suitable bacteria by genetic engineering methods to make microbial sensors for environmental monitoring. Luciferase has been introduced into E. coli to detect arsenic toxic compounds [8].
The determination of the concentration of phenols and surfactants in water has been greatly advanced. At present, there are nine gram-negative bacteria isolated from the soil of the West Siberian oil basin, with phenol as the sole carbon source and energy source. These strains can increase the sensitivity of the sensor portion of the biosensor. Its monitoring limit for phenol is 5'10-9 mol. The optimal conditions for this sensor to work are: pH = 7.4, 35 °C, continuous working time 30h [9]. There is also an amperometric biosensor made from Pseudomonas rathonis that measures surfactant concentration, immobilizing microbial cells on gels (agar, agarose, and calcium alginate) and polyethyl alcohol membranes. The cross-linking of microbial cells in the gel by chromatography paper GF/A, or glutamate, can be maintained over long distances to maintain their activity and growth in high-concentration surfactant assays. The sensor can quickly recover the activity of the sensor after the measurement is completed [10].
There is also an amperometric biosensor for the determination of organophosphorus pesticides using artificial enzymes. Using the organophosphorus insecticide hydrolase, the measurement limit of p-nitrophenol and diethylphenol is 100′10-9 mol, and it takes only 4 minutes at 40° C. [11]. There is also a newly developed phosphate biosensor, using pyruvate oxidase G, in combination with an automated system CL-FIA desktop computer, which can detect (32-96) 10-9 mol of phosphate and can be used at 25°C. More than two weeks of use, high reproducibility [12].
Recently, there is a novel microbial sensor that uses bacterial cells as a biological component to determine the content of nonyl-phenol etoxylate (NP-80E) in surface water. Using an amperometric oxygen electrode as a sensor, the microbial cells were immobilized on a dialysis membrane on an oxygen electrode. The measuring principle was to measure the respiratory activity of Trichosporum grablata cells. The biosensor has a reaction time of 15 to 20 minutes and a life span of 7 to 10 days (for continuous measurement). In the concentration range of 0.5 to 6.0 mg/l, the electrical signal is linearly related to the concentration of NP-80E, which is very suitable for the detection of molecular surfactants in contaminated surface water [13].
In addition, the determination of the concentration of heavy metal ions in sewage can not be ignored. A complete monitoring and analysis system for the determination of the bioavailability of heavy metal ions based on immobilized microorganisms and bioluminescence measurement technology has been successfully designed. An operon from Vibrio fischeri was introduced into Alcaligenes eutrophus (AE1239) under the control of a copper-inducible promoter. The bacteria luminesced under the induction of copper ions. The ion concentration is proportional to. By embedding microorganisms and optical fibers in a polymer matrix, a biosensor having high sensitivity, good selectivity, wide measurement range, and strong storage stability can be obtained. At present, this microbial sensor can reach the minimum measured concentration of 1'10-9 mol [14].
There is also a current-based microbial sensor that measures copper ions. It uses a recombinant strain of Saccharomyces cerevisiae as a biological element. These strains carry a fusion of the copper ion-inducible promoter on the CUP1 gene of E. vinifera and the E. coli lacZ gene. Its working principle is that the CUP1 promoter is first induced by Cu2+, and then lactose is used as a substrate for measurement. If Cu2+ is present in solution, these recombinant bacteria can use lactose as a carbon source, which will lead to changes in the oxygen demand of these aerobic cells. The biosensor can measure CuSO4 solution within the range of concentration (0.5-2)' 10-3 mol. Currently, various types of metal ion-inducible promoters have been transformed into E. coli, so that E. coli luminescence reactions occur in solutions containing various metal ions. Based on the intensity of its luminescence, the concentration of heavy metal ions can be determined. The measurement range can be from nanomolar to micromolar, and the time required is 60-100 min [15] [16].
Biosensors for the measurement of zinc concentration in sewage have also been developed, using the basophilic bacteria Alcaligenes cutrophus and used to measure the concentration and bioavailability of zinc in the sewage. The results are satisfactory [17].
The seaweed sensor that estimates the pollution of the outflow from the estuary is composed of a cyanobacterium Spirlina subsalsa and a gas-sensing electrode. By monitoring the extent to which photosynthesis is inhibited, the change in the toxicity of water due to the presence of environmental contaminants is estimated. Using standard natural water as the medium, different concentrations of the three major pollutants (heavy metals, herbicides, carbamate insecticides) were measured and their toxic reactions were monitored. Repeatability and reproducibility were very high. Gao [18].
Recently, due to the rapid development of polymerase chain reaction (PCR) technology 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 determine a specific bacterial toxin. Biotinylated probes were immobilized on quartz crystals with a platinum-gold surface of the enzyme antibiotic, and circulating measurements were performed on the same crystal surface with 1'10-6 mol of hydrochloric acid. The same hybridization reaction was performed with DNA extracted from bacteria and amplified by PCR. The product was a special gene fragment of Aeromonas hydrophila. The piezoelectric biosensor can identify whether the gene is contained in the sample, which provides the possibility to detect from the water sample whether there are various Aeromonas with this pathogen [19].
There is also a channel biosensor that can detect toxins such as neurotoxins produced by organisms such as phytoplankton and jellyfish, and has now been able to measure trace amounts of PSP toxins contained in a planktonic cell [20]. DNA sensors are also being rapidly applied. There is currently a miniaturized DNA biosensor that converts DNA recognition signals into electrical signals for measuring cryptospores and other waterborne pathogens in water samples. The sensor focuses on improving the recognition of nucleic acids and enhancing the specificity and sensitivity of the sensor, and seeks new methods for translating hybridization signals into useful signals. The current research work is the integration of recognition devices and conversion devices [21].
Microalgae is a bacterial hepatotoxin produced from blooms caused by cyanobacteria. A biosensor with a surface cell plasmid genome immobilized on it has been prepared for the measurement of microalgae in water. Its direct measurement range is 50～1000′10-6g/l[22].
The idea of ​​multiple biosensors based on enzyme-based inhibition analysis for measuring toxic substances has also been proposed. In this multiple biosensor, two types of transducers are used—pH-sensitive electronic transistors and heat-sensitive thin-film electrodes, and three enzymes—urease, acetylcholinesterase, and butyrylcholinesterase. The performance of this biosensor has been tested with good results [23].
In addition to the fermentation industry and environmental monitoring, biosensors are also used in food engineering, clinical medicine, military and military medicine, and are mainly used to measure glucose, acetic acid, lactic acid, lactose, uric acid, urea, antibiotics, and glutamic acid. Amino acids, as well as various carcinogenic and mutagenic substances.
Third, discuss and look forward to the United States Harold H. Weetal pointed out that the commercialization of biosensors must meet the following conditions:
Sufficient sensitivity and accuracy, easy operation, low cost, easy mass production, and quality monitoring during production. Among them, the cheap price determines the sensor's competitiveness in the market. Among various biosensors, the biggest advantages of microbiological sensors are low cost, simple operation, and simple equipment, so their market prospects are very large and attractive. In comparison, enzyme biosensors, etc., are relatively expensive. However, microbial sensors also have their own shortcomings. The main drawback is that the selectivity is not good enough. This is caused by the presence of multiple enzymes in the microbial cells. It has been reported that adding special inhibitors to solve the problem of microbial electrode selectivity. In addition, the method of immobilizing microorganisms needs to be further improved. First of all, it is necessary to ensure the activity of the cells as much as possible. Secondly, the cells must be tightly bound to the basic membrane to avoid the loss of cells. In addition, the long-term preservation of microbial membranes needs to be further improved, otherwise it is difficult to achieve large-scale commercialization.
In conclusion, commonly used microbial electrodes and enzyme electrodes have their advantages in various applications. If a stable, highly active, low cost free enzyme is readily available, the enzyme electrode is ideal for the user. Conversely, if biocatalysis needs to go through complex pathways, requires coenzymes, or if the desired enzymes are not suitable for separation or instability, microbial electrodes are a better choice. While various other forms of biosensors are also booming, their applications have become more widespread. With the further improvement of the immobilization technology, with the constant deepening of people's understanding of living organisms, biosensors will open up a new world in the market.
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