Introduction to research status and application analysis of biosensors

I. Introduction

From 1962, Clark and Lyons first proposed the idea of ​​biosensors for 40 years. Biosensors have been deeply valued and widely used in fermentation processes, environmental monitoring, food engineering, clinical medicine, military and military medicine. In the first 15 years, biosensors were mainly based on biosensors made with enzyme electrodes. However, because enzymes are expensive and not stable enough, the use of enzymes as sensitive materials has limited applications.

In recent years, the continuous development of microbial immobilization technology has produced microbial electrodes. Microbial electrodes use microbial organisms as molecular recognition elements, which are unique compared to enzyme electrodes. It can overcome weaknesses such as high price, difficult extraction and instability. In addition, coenzymes in microorganisms can also be used to process complex reactions. At present, the application of optical fiber biosensors is more and more extensive. And with the development of polymerase chain reaction technology (PCR), there are more and more DNA biosensors using PCR.

2. Research status and main application areas

1. Fermentation industry

Among various biosensors, microbial sensors are suitable for the determination of the fermentation industry. Because there are often interfering substances to the enzyme in the fermentation process, and the fermentation broth is often not clear and transparent, so it is not suitable for determination by spectroscopy and other methods. The application of microbial sensors is very likely to eliminate interference and is not limited by the degree of turbidity of the fermentation broth. At the same time, because the fermentation industry is a large-scale production, the low cost of the microbial sensor and its simple equipment make it a great advantage.

(1). Determination of raw materials and metabolites

Microbial sensors can be used for the determination of raw materials such as molasses, acetic acid, etc., and the determination of metabolic products such as cephalosporin, glutamic acid, formic acid, 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, and the amount of oxygen reduction is measured by measuring the change of 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. Psoudomonas fluorescens (Psoudomonas fluorescens) metabolizes the role of glucose consumption, and the oxygen electrode is used to detect the concentration of glucose. Compared with the electrode type of glucose enzyme, the measurement results of this kind of microbial electrode are similar, and the microbial electrode has high sensitivity, good repeatability, and does not need to use expensive glucose enzyme.

When acetic acid is used as a carbon source for the cultivation of microorganisms, the concentration of acetic acid above a certain concentration will inhibit the growth of microorganisms, so online measurement is required. The microbial sensor composed of immobilized yeast (Trichosporon brassicae), gas-permeable membrane and oxygen electrode can determine the concentration of acetic acid.

In addition, there is a combination of E.coli (E.coli) carbon dioxide gas sensor, which can constitute a microbial sensor for measuring glutamic acid, which immobilizes the whole cell of Citrobacter in the collagen membrane. The microbial sensor composed of glass electrodes can be applied to the determination of cephalosporin in fermentation broth and so on.

(2). Determination of the total number of microbial cells

In terms of fermentation control, there has been a need for a simple and continuous method for 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, and the result is the same as that of the traditional plaque counting method [1].

(3). Identification of metabolic tests

The identification of traditional microbial metabolism types is based on the growth of microorganisms on a certain medium. These experimental methods require long cultivation time and specialized techniques. The assimilation of microorganisms to substrates can be measured by their respiratory activity. The oxygen electrode can 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 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 objects, selection of microorganism preservation methods, etc. [2].

2. Environmental monitoring

(1). Determination of biochemical oxygen demand

The determination of biochemical oxygen dem (BOD) is a common indicator for monitoring the pollution of water bodies by organic matter. The routine BOD measurement requires a 5-day incubation period. The operation is complex, poor repeatability, time-consuming, labor-intensive, and disturbing. It is not suitable for on-site monitoring, so a new method that is simple, fast, accurate, highly automated, and widely applicable is urgently needed To determine. At present, researchers have isolated two new yeast species SPT1 and SPT2, and fixed it on the glassy carbon pole to form a microbial sensor for measuring BOD, and its repeatability is within ± 10. The sensor is used to measure the BOD in the wastewater of the pulp mill, and its measurement value can reach 2 mg / l, and the time used is 5min [3]. There is also a new microbial sensor that uses yeast strains resistant to high osmotic pressure As a sensitive material, it can work normally under high osmotic pressure. And its strains can be stored dry for a long time, and the activity will be restored after soaking, which provides 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 15min, the suitable working condition is 30 ° C, pH = 7. This sensor system is almost not affected by chloride ion (in the range of 1000mg / l), 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 kind of BOD biosensor after light treatment (that is, using TiO2 as a semiconductor, irradiated with 6 W lamp for about 4min), the sensitivity is greatly improved, and it is suitable for the measurement of lower BOD in river water [5]. At the same time, a compact The optical biosensor has been developed to measure the BOD value of multiple samples simultaneously. It uses three pairs of light-emitting diodes and silicon photodiodes. Pseudomonas fluorescens is fixed on the bottom of the reactor with photo-crosslinked resin. This measurement method is fast and simple. It can be used at 4 ℃ for six weeks. It has been used in the process of factory wastewater treatment [5].

(2). Determination of various pollutants

Commonly used important pollution indicators are the concentration of ammonia, hypochlorite, sulfide, phosphate, carcinogens and mutagenic substances, heavy metal ions, phenolic compounds, surfactants and other substances. At present, a variety of biosensors for measuring various types of pollutants have been developed and put into practical use.

Microbial sensors for measuring ammonia and salt are mostly composed of a combination of nitrifying bacteria and oxygen electrodes separated from wastewater treatment equipment. There is currently a microbial sensor that can measure salt and sub-salt (NOx-) under dark and light conditions. Its measurement in a salt environment makes it immune to other types of nitrogen oxides. Using it to measure NOx- in the estuary, the effect is better [6].

The measurement of sulfide is a microbial sensor made of specific, autotrophic, aerobic thiooxidans that are isolated and screened from acid soil near pyrite. When measured at pH = 2.5 and 31 ℃ for more than 200 times a week, the activity remains unchanged, and the activity decreases after two weeks 20. The sensor life is 7 days, its equipment is simple, the cost is low, and the operation is convenient. At present, a photomicrobial electrode is used to measure the sulfide content. The bacteria used is Chromatium.SP, which is connected to the hydrogen electrode [7].

Nearly scientists have isolated a bacterium that can fluoresce in the polluted area. This bacterium contains fluorescent genes, which can produce fluorescent proteins under the stimulation of the pollution source, 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 E. coli to detect toxic compounds of arsenic [8].

The concentration of phenols and surfactants in water has been greatly developed. Currently, nine species of Gram-negative bacteria have been isolated from the soil of the Western Siberian oil basin, using phenol as the carbon source and energy source. These strains can increase the sensitivity of the sensor part of the biosensor. Its monitoring limit for phenol is 5? 10-9mol. The suitable conditions for the sensor to work are: pH = 7.4, 35 ℃, continuous working time is 30h [9]. There is also a current made of Pseudomonas rathonis to measure the concentration of surfactant -Type biosensor, fix the microbial cells on the gel (agar, agarose and calcium alginate) and polyethanol membrane, you can use chromatographic test paper GF / A, or microbial cells caused by glutamic acid in the gel In the cross-linking, they can maintain their activity and growth in the detection of high concentration surfactant for a long distance. The sensor can quickly restore the activity of the sensitive element after the measurement [10].

There is also a galvanic biosensor for the determination of organophosphorus pesticides, using artificial enzymes. Using organophosphorus insecticide hydrolase, the measurement limit of p-nitrophenol and diethylphenol is 100? 10-9mol, only 4min at 40 ℃ [11]. There is also a newly developed phosphate biosensor that Pyruvate oxidase G, combined with the automatic system CL-FIA desktop computer, can be detected (32 ~ 96)? 10-9mol phosphate can be used for more than two weeks at 25 ° C, with high repeatability [12].

Recently, there is a new type of microbial sensor that uses bacterial cells as biological components to determine the content of nonyl-phenol etoxylate (NP-80E) in surface water. A current-type oxygen electrode is used as the sensor, and the 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 response time of the biosensor is 15 ~ 20min, and the life span is 7 ~ 10 days (for continuous measurement). 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 contaminated surface water [13].

In addition, the determination of the concentration of heavy metal ions in sewage cannot be ignored. At present, we have successfully designed a complete monitoring and analysis system for the determination of the bioavailability of heavy metal ions based on immobilized microorganisms and bioluminescence measurement technology. An operon in Vibrio fischeri was introduced into Alcaligenes bacteria (Alcaligenes eutrophus (AE1239)) under the control of a copper-inducible promoter. The ion concentration is proportional to. By embedding microorganisms and optical fibers together in a polymer matrix, a biosensor with high sensitivity, good selectivity, wide measurement range, and strong storage stability can be obtained. At present, this microbial sensor can reach a low measurement concentration of 1? 10-9mol [14].

There is also a current-type microbial sensor that specifically measures copper ions. It uses recombinant strains of Saccharomyces cerevisiae as biological elements. These strains carry a fusion of a copper ion-inducible promoter on the CUP1 gene of S. cerevisiae and the lacZ gene of E. coli. 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 the solution, these recombinant bacteria can use lactose as a carbon source, which will cause changes in the oxygen demand of these aerobic cells. Can the biosensor be in the concentration range (0.5 ~ 2)? The CuSO4 solution was measured in the range of 10-3 mol. At present, various metal ion-inducible promoters have been transferred into E. coli, so that E. coli will have a luminescent reaction in the solution containing various metal ions. According to the intensity of its luminescence, the concentration of heavy metal ions can be determined, and the measurement range can be from nanomolar to micromolar, and the time required is 60 ~ 100min [15] [16].

A biosensor for measuring the concentration of zinc in sewage has also been successfully developed, using the alkaliphilic bacteria Alcaligenes cutrophus, and used to measure the concentration and biological effectiveness of zinc in sewage, and the results are satisfactory [17].

The algae sensor that estimates the pollution of the estuary effluent is composed of a cyanobacterium Spirlina subsalsa and a gas-sensitive electrode. By monitoring the extent to which photosynthesis is inhibited, we can estimate changes in water toxicity due to the presence of environmental pollutants. Using standard natural water as the medium, the different concentrations of the three main pollutants (heavy metals, herbicides, carbamate insecticides) were measured, and their toxic reactions could be monitored, with both 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, which can detect a special bacterial toxin. The biotinylated probe was fixed on the quartz crystal equipped with platinum enzyme streptomycin on the surface, using 1? 10-6mol hydrochloric acid can make the circulating measurement on the same crystal surface. DNA samples extracted from bacteria were subjected to the same hybridization reaction and amplified by PCR. The product was a special gene fragment of Aeromonas hydrophila. This piezoelectric biosensor can identify whether the sample contains this gene, which provides the possibility of detecting the presence of various Aeromonas with this pathogen from water samples [19].

There is also a channel biosensor that can detect toxic substances such as lumbar flagellate neurotoxin produced by organisms such as phytoplankton and jellyfish. It has been able to measure the extremely small amount of PSP toxin contained in a plankton cell [20]. DNA sensor also In rapid application, there is currently a miniaturized DNA biosensor that can convert DNA recognition signals into electrical signals for measuring cryptosporidium and other infectious agents in water samples. The sensor focuses on improving the recognition of nucleic acids and enhancing the specificity and sensitivity of the sensor, and seeks a new method to convert the hybridization signal into a useful signal. The current research work is the integration of recognition devices and conversion devices [21].

Microalgae is a bacterial liver toxin produced from water blooms caused by cyanobacteria. A biosensor immobilized on the surface cell plasmid genome has been prepared to measure the content of microalgae in water. Its direct measurement range is 50 ~ 1000? 10-6g / l [22].

A multi-biosensor based on enzyme inhibition analysis has also been proposed for measuring toxic substances. In this multiple biosensor, two types of electronic transistors sensitive to pH and thermally sensitive thin-film electrodes are used, as well as three enzymes urease, acetylcholinesterase and butyrylcholinesterase. The performance of the biosensor has been tested and the effect is better [23].

In addition to fermentation industry and environmental monitoring, biosensors are also deeply 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, glutamic acid, etc. An amino acid, as well as various carcinogenic and mutagenic substances.

3. Discussion and Outlook

Harold H. Weetal of the United States pointed out that the commercialization of biosensors must have the following conditions: sufficient sensitivity and accuracy, easy operation, low price, easy mass production, and quality monitoring during the production process. Among them, the cheap price determines whether the sensor is competitive in the market. Among various biosensors, the big advantages of microbial sensors are low cost, easy operation, and simple equipment. Therefore, its prospects in the market are very huge and attractive. In comparison, enzyme biosensors and the like are more expensive. But microbial sensors also have their own shortcomings. The main shortcoming is that the selectivity is not good enough, which is caused by the multiple enzymes contained in the microbial cells. It has been reported to add special inhibitors to solve the selectivity problem of microbial electrodes. In addition, the method of microbial immobilization also needs to be further improved. First of all, it is necessary to ensure the activity of the cells as much as possible, and secondly, the combination of the cells and the basal membrane should be firm to avoid the loss of cells. In addition, the problem of long-term preservation of microbial membranes needs further improvement, otherwise it is difficult to achieve large-scale commercialization.

In short, the commonly used microbial electrodes and enzyme electrodes have their own advantages in various applications. If it is easy to obtain free enzyme with stable, high activity and low cost, the enzyme electrode is ideal for users. Conversely, if biocatalysis needs to go through complex pathways, coenzymes are needed, or the desired enzymes are not suitable for separation or instability, microbial electrodes are a more ideal choice. And other forms of biosensors are also booming, and their applications are becoming more and more widespread. With the further improvement of immobilization technology and the continuous deepening of people's understanding of organisms, biosensors will surely open up a new world in the market.

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