Advances in Microfluidic Biosensors Based on Luminescent Bacteria

Advances in Microfluidic Biosensors Based on Luminescent Bacteria

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 2, February 2019 Online English edition of the Chinese language journal Cite this article as...

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CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 2, February 2019 Online English edition of the Chinese language journal

Cite this article as: Chinese J. Anal. Chem., 2019, 47(2): 181–190

REVIEW

Advances in Microfluidic Biosensors Based on Luminescent Bacteria JIN Xiao-Wei1, LI Zhe-Yu1, XU Pian-Pian1, ZHANG Xiao-Yan1, REN Nan-Qi1, Vitaliy V. Kurilenko2, SUN Kai1,* 1 2

State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China Institute of Earth Sciences, Saint-Petersburg State University, Saint-Petersburg 199034, Russian Federation

Abstract: Luminescent bacteria can emit visible light at 450–490 nm, and its luminous intensity decreases with the increase of the concentration of toxic substances in test solution. The method using luminescent bacteria is widely used in acute toxicity analysis of water quality because it is a simple, rapid and low-cost way. In recent years, biosensors based on luminescent bacteria have attracted more and more attention, and the reports of bioluminescence biosensors based on microfluidic systems are increasing day by day. Based on the characteristics and mechanism of luminescent bacteria, this paper introduces their applications in the environmental monitoring, and summarizes several new bioluminescence sensors. Key Words:

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Luminescent bacteria; Acute toxicity; Environmental pollutant; Microfluidic; Biosensor; Review

Introduction

With the increasingly serious environmental pollution, the demand for monitoring environmental pollutants is increasing. There are many kinds of toxic substances in the environment. The chemical, physical and biological methods have been gradually established to detect bacteria. Among them, the chemical methods are established earlier and the detection systems are relatively perfect, which can realize the qualitative and quantitative detection of most pollutants. However, those methods can only obtain the composition and concentration of the pollutant, but cannot directly reflect the toxic effect of the pollutant on organisms. The physical methods are complex in analysis and expensive in equipment, and are widely used in remote sensing monitoring of atmospheric and large-area water pollution. Bioanalytical methods use organisms such as microorganisms, plants, invertebrates and fish to evaluate the changes in toxic substances in environment. Microbial-based bioanalytical methods are widely used because of their simplicity, rapidity, and reproducibility in water quality

toxicity test. Luminescent bacteria are a kind of bacteria that can emit visible blue-green light to the naked eye in darkroom. The luminescent bacteria are often used to detect water toxicity. Biotoxicity detection of luminescent bacteria depends on its response to environmental pollutants. It can reflect the effects of pollutants on living individuals[1] and toxicity effects [2]. It has many advantages such as simplicity, rapidity, simple operation and good reproducibility. Therefore, the biotoxicity detection methods of luminescent bacteria are applied to the toxicity analysis of various environmental pollutants. Westlund et al[3] studied the acute and chronic toxicity of more than ten pesticides towards Vibrio fischei. The results suggested that the EC50 values of some pesticides at 15 min and 30 min were dozens of times different. It showed that only considering the EC50 value at 15 min was easy to underestimate the toxicity of some pollutants. Liu et al[4] studied the single and binary combined toxicity of 6 pesticides by using Vibrio qinghaiensis as target organisms. It was found that whether the toxicity of binary combination was higher

________________________ Received 12 May 2018; accepted 7 November 2018 *Corresponding author. Email: ksun@ hit.edu.cn This work was supported by the National Natural Science Foundation of China (No. 61674046). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61139-4

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than that of single substance mainly depended on the interaction between the two substances, such as antagonistic effect that made joint toxicity decrease, synergistic effect that enhanced joint toxicity and so on. Ding et al[5] studied the toxicity of six phthalate esters and Cadmium (Cd2+) to Vibrio Qinghaiensis and pointed out that the binary toxicity of these six phthalate esters and Cd2+ had additive effect. Luminescent bacteria are also commonly used to evaluate the bio-toxicity of new materials. Rossetto et al[6] tested the acute and chronic toxicity of nano-CuO particles and micro-CuO particles by Vibrio fischei. The results presented that the toxicity of nanoCuO particles was higher than that of micro-CuO particles. This work provided a method and a convenient tool for toxicology research. Costa et al[7] discussed the toxic effects of seven ionic liquids on Vibrio fischei through a fully automated sequential injection analysis system. It was demonstrated that the toxic effects of ionic liquids were related to their structure and composition. The toxicity of cations containing benzene ring was greater than that of structures without benzene ring, while the toxicity of fluorine-containing anions was greater than that of structures without fluorine ring. In recent years, with the combination of microfluidics, biological toxicity detection with luminescent bacteria has been developing in the direction of portability, integration and intelligence. Microfluidic system is a kind of analytical device which can complete the process of sampling, reaction and detection on a smaller chip. It has many advantages such as fast reaction, high efficiency and low energy consumption. Researchers have constructed a new bioluminescent microanalysis system based on the combination of toxicity detection with luminescent bacteria and microfluidics. The system consists of three parts. (1) Microfluidic chip, which contains microchamber arrays and microchannels; (2) living cells in the microchamber, which are used to sense the target material and feedback the changes of bioluminescent signals; (3) converter, which converts bioluminescent signals into electrical signals. Based on the luminescent principle of luminescent bacteria and microfluidic technology, this paper summarizes the principles and applications of several microfluidic biosensors.

There is an enzyme system consisting of NAD(P)H:FMN oxidoreductase and luciferase in the luminescent bacteria. When flavin acid (FMN), coenzyme II [NAD(P)H], long chain aliphatic aldehyde (RCHO) and molecular oxygen (O2) exist, the enzyme system emits about 490 nm light. The reaction equation is as follows: (1) (2) Bacterial luciferase is a monooxygenase. It can transfer one oxygen atom of molecular oxygen (O2) to reduced flavin mononucleotide (FMNH2), which is oxidized to a flavin mononucleotide (FMN). And another oxygen atom is added to long chain aliphatic aldehyde (RCHO) to form corresponding long chain fatty acids. Luciferase cannot catalyze the reduction of flavin, so it is necessary to complete the bioluminescence process with the help of coenzyme II [NAD(P)H]. FMNH2/FMN is also an important component of bacterial respiratory metabolism, thus bacterial luminescence reaction can be regarded as a metabolic bypass of electron transfer to oxygen molecules in respiratory action, which can regulate the level of reduced flavin mononucleotides. In the dehydrogenation reaction of energy metabolism or catabolism, coenzyme I (NAD) must transfer H to FMN after accepting H and reduce FMN to FMNH2 before it enter the stage of respiratory productivity. At this time, bacterial luciferase, like a flavin monooxygenase or a balanced oxidase, can reduce the production of reduced flavin mononucleotides and increase the reduction function of electronic metabolic pathways. Therefore, it is favorable for cell metabolism[11]. Molecular oxygen and reduced flavin mononucleotides produce luciferase-flavin peroxide. The energy generated by peroxide degradation generates singlet excites molecules and emits photons during deexcitation. Usually the peroxide bond breaks and is replaced by two stronger chemical bonds, and accompanied by energy generation, part of which is used for bioluminescence.

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Luminescent bacteria and their luminous mechanism

Luminescent bacteria are widely distributed, mainly in the marine environment. They emit weak bluish green light, of which the maximum wavelength is between 450 and 490 nm[8]. It is a gram-negative, facultative aerobic bacterium[9]. Luminescent bacteria are divided into 4 genera: Vibrio, Photobacterium, Shewanella and Photorhabdus[10]. Among them, the typical bacteria such as Photobacterium phosphoreum, Vibrio fischei and Vibrio qinghaiensis, are widely used in toxicity analysis.

Microfluidic system based on luminescent bacteria

When the luminescent bacteria contact with pollutants, the destruction of the enzyme system and cell death will result in the decrease of their luminous intensity. Some pollutants can bind to luciferase of luminescent bacteria, thus interfering with the bioluminescent process of bacteria. Some pollutants disrupt the function of cell membrane by destroying the receptors on the cell surface. Even chemical reactions with cellular components cause cell inactivation, and thus reduce the intensity of luminescence. Therefore, the luminescent bacteria can be used to evaluate the toxic effects of pollutants

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according to the different inhibitory effects of pollutants on the luminescent intensity of luminescent bacteria[12]. Since Karube et al[13] put forward the concept of biosensor, biosensors have made great progress in the detection of environmental pollutants, especially microfluidic biosensors. In the microfluidic biosensor based on luminescent bacteria, the state of bacteria is in various forms[14], including suspended state in liquid (Fig.1A), freeze-dried state made into freeze-dried powder (Fig.1B) and stationary state fixed on disposable sample pool, optical fiber or biochip (Fig.1C). Among them, the suspended state and the fixed state are commonly used. 3.1

Biosensor with immobilized bacteria

Bacteria-fixed microfluidic biosensors can be divided into calcium alginate immobilization, agar immobilization and sodium alginate sheet immobilization according to the different ways of bacterial immobilization. In this paper, several fixed biosensors are introduced in detail. Eltzov et al[18] developed an on-line optical fiber monitoring system for detecting toxic pollutants in water. Two bacterial suspensions carrying recA (DNA damage) and grpE (heat-shock) initiation factors were mixed with sodium alginate solution (2%) in a volume ratio of 1:1, and then coated on the head of optical fiber. Subsequently, calcium chloride solution (0.5 M) was coated to form calcium alginate

which could fix the bacteria on the optical fiber tip. The author first optimized the conditions required for the continuous operation of the system for 24 h. The pollutants in tap water and surface water were detected in the flow state. Preliminary experiments proved that adding 7.5% LB medium was a necessary condition to ensure the long-term operation of the equipment in flowing water (> 1 h). In the water quality monitoring of tap water, the system could run steadily for 24 h. However, in the water quality monitoring of surface rivers, it was difficult for the system to respond to pollutants after 20 h of operation, as the gradual reduction of fixed luminescent bacteria on the optical fiber tip due to fluid erosion. Subsequently, the author optimized the system in depth[20]. The composition and concentration of the medium were adjusted firstly. Then the influence of different flow rates on the test results was studied by adding peristaltic pumps at the outlet of the fluid. A flow buffer shield was added to the optical fiber probe to reduce the fluid shear force. The optimized device is shown in Fig.2. The optimized monitoring system can not only detect the changes of water quality quickly, but also increase the maximum flow rate from 3 L h–1 to 10 L h–1. A larger volume of water sample detection was completed at the same time. Simultaneously, the equipment realized the real-time monitoring of pollutant toxicity in flowing water. It could provide early warning function for water quality safety of important nodes such as water intake in water source area or drinking water pipe network.

Fig.1 Three kinds of biosensors based on luminescent bacteria: (A) biosensor with bacteria in suspension[15], (B) biosensor with freeze-dried bacteria[16] and (C) biosensor with immobilized bacteria: (1) Immobilization of bacteria in a disposable card[17]; (2) Immobilization of bacteria onto optical fiber tips[18]; (3) Immobilization of bacteria in the biochips[19]. (a) The photograph of the fabricated micro-well-chip, (b) the microscopic view of the channel and the region where the bacteria is immobilized

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Fig.2 Biosensor for detecting water toxicity after optimization[20]: (A) schematic of the optimized biosensor, (B) photo of Perspex flow unit, and (C) calcium (2%, V/V)-polymerized alginate with six layers forming the fiber optic probe

Jouanneau et al[17] designed two kinds of biosensors (Lumisens III and Lumisens IV) based on different storage modes of luminescent bacteria (Fig.3A), which was used for on-line detection of heavy metals in environmental samples. The bacterial suspension was fixed in a micro-well driven by continuous flow with 4% agarose solution in Lumisens III system which had a higher biological activity. The bacteria used in Lumisens IV system were freeze-dried in 96-well microplates, resulting in the relatively low biological activity. Both systems were enclosed in the dark chamber, and the light signals emitted by bacteria were captured and recorded by CCD camera. In 10 days, the two biosensors were used to continuously detect mercury (Hg) in distilled water or environmental samples. The luminescent bacteria in the system were exposed to Hg samples (500 nM) for 100 min every day, and in the rest of the time, the bacterial culture medium was continuously fed into. The results indicated that Lumisens III system generated response signal after 6 hours of starting up (Fig.3B). The level bioluminescence in Lumisens III system was unstable within 10 days, with a change rate of 40%. The immobilized bacteria were in a stable growth stage on the 3–6 day, and the reproducibility and repeatability of bioluminescence signals were close to 5%. The response signal of Lumisens IV system could be obtained after 1.5 h of start-up. The reproducibility of bioluminescent signals was 3% in Lumisens IV system, which could reach the stable detection of Hg within 10 days. In Lumisens III system, bacteria were susceptible to exposure to pollutants, leading to the decrease of bacterial activity in subsequent experiments and affecting the consistency of experimental results. In Lumisens IV system, bacteria were confined to each micropore in the microporous plate to perform independent

single analysis, but the operation was relatively cumbersome and the repeatability was low. Elad et al[21] developed a flow-through biosensor for on-line continuous monitoring of water quality toxicity. The modular biochip of recombinant luminescent bacteria immobilized on agar was the core part of the device. The single photon avalanche diode detector (SPD) was a detection device to respond to the signals (Fig.4A). The authors prepared two strains, inducible and constitutive, and kept them in a continuous water flow for 10 days. During this period, naphthoic acid, paraquat, trivalent arsenicals and the mixture of the above three substances and a sample of industrial wastewater were continuously pumped into the biosensor for 2 h. By comparing the response intensity of fixed or suspended bacteria to the same concentration of pollutants, it was found that suspended bacteria were more sensitive to pollutants. However, their stability was slightly lower than that of the fixed bacteria, and their repeatability was close to 20%. The sensor had a good recognition effect on As(III) of 6 mg L–1 and 0.01 mg L–1, and Sb(III) of 0.02 mg L–1 and 0.005 mg L–1 (Fig.4B). And the detection concentration met the detection limit requirements of drinking water quality safety standards of the United States and the European Union. The biosensor could detect all simulated pollution events within 0.5–2.5 h and fed back specific response signals according to the nature of pollution. It could be used to prevent accidental leakage of toxic chemicals or deliberate release of toxic substances from water sources. It is complementary to the relevant physical and chemical methods to form a water quality safety guarantee network. In recent years, biosensors based on luminescent bacteria were widely used for water quality toxicity detection, and few

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Fig.3 Biosensor for online detection of metals[17]: (A) fludic chart of the two systems of Lumisens(III) and Lumisens(IV) and (B) real-time monitoring data of Lumisens(III) when exposed to Hg solution (500 nM) daily. (a) Comparison of the results between twice experiments; (b) Growth curves of the cells (CFU, colony forming unit); (c) CCD camera imaging of the immobilized bacteria in multi-well card

Fig.4 Schematic of flow-through biosensor[21]: (A) Top view of the flow-through chambers, including the single photon advanced diode (SPAD) detectors, the stepper axis, and a cross-section of a flow-through chamber. Each flow-through chamber is constructed of three layers (glass, PDMS, and PMMA), which forms a flow channel with 12 wells in its path; (B) Response curves of As and Sb

reports were reported on gas toxicity detection. The air toxicity sensor designed by Eltzov et al[22] had excellent performance in indoor air toxicity monitoring. The core parts of the system mainly included (1) photomultiplier tube for detecting bacterial response, (2) liquid light guide for optical signal transmission and (3) calcium alginate pads for bacterial immobilization. The schematic diagram of the equipment is shown in Fig.5. The E. coli modified by grpE was mixed with a filter-sterilized 2% (w/V) sodium alginate solution at a ratio of 1:1. A fixed sheet was prepared by dropping 30 mL of CaCl2 solution (0.5 mM) into 50 mL of mixture of sodium alginate and luminescent bacteria in a cylinder with a diameter of 0.6 mm. The bacterial fixed slice was firstly optimized. The

bacterial fixed sheet was placed in a 20-mL tube and 1 L of chloroform was added around it. By studying the effects of different orientation positions, ambient temperature and exposure time on the sensitivity of the sensor, it was presented that front orientation could increase the diffusion rate of chemical substances into the matrix. The result indicated that increasing ambient temperature or prolonging exposure time could enhance the response intensity of cells. Afterward, an integrated device was put in an office where several common pollutants in air, such as cigarette smoke, acetone and paint (containing trichloromethane) were released. The responses of the biosensor to various pollutants were recorded. This study suggested that the response of biosensors to several pollutants

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Fig.5 Biosensor for detecting air toxicity[22]: (A) scheme of the biosensor; (B) response curves of biosensor exposed to acetone and chloroform. (a) Response of biosensor to the “spilling accidents” of 2 mL of acetone in office room; (b) Response of biosensor to “spilling accidents” of 5 and 10 mL of chloroform in office room

showed their responsiveness to toxic substances in indoor environment. The response to acetone was the strongest, followed by trichloromethane. Biosensors and traditional analytical methods are capable of qualitatively analyzing the type of contaminants in air samples. However, the traditional methods still have some problems, such as high price, special laboratory equipment and unsuitable for real-time monitoring. Moreover, strict technical requirements and high salaries of operators are the biggest issues to prevent widespread application of traditional methods in toxicity monitoring. In contrast, the biosensor is simple, sensitive and convenient, which has important guiding significance for the development of new air quality monitoring devices in the future. Immobilization of bacteria can limit the spread of bacteria to the surrounding environment. Some biological elements can be reused after removal of pollutants to improve the service life of biosensors. However, there are some problems in the in-situ application of this method. For example, fixed bacteria will change with time and affect the response to pollutants. Owing to the limitation of bacterial diffusion and swimming, the contact between bacteria and contaminants is insufficient, which leads to low detection signal in practical application. The disposable chips used to immobilize bacteria need to be replaced frequently, thus they consume a large amount of pollutants in rapid detection. Above all, the suspension type biosensor has more advantages for continuous rapid detection in a short time. 3.2

Biosensor with bacteria in suspension

As early as 1996, Gu et al[23] first developed a

continuous-flow micro-bioreactor for testing the response of cell to toxic substances. The microreactor was similar as a simplified bioreactor without probe control. The recombinant bioluminescent Escherichia coli maintained the optimal physiological state in continuous flow culture. With a volume of approximately 58 mL, this micro bioreactor could be operated continuously for long periods of time with high stability and reliability. The authors studied the response of recombinant bioluminescent E. coli to ethanol under continuous flow. It was found that the level of luminescence of bacteria in continuous culture depended on the final dilution times of the bacteria solution. Luminescent bacteria under high dilution ratio were in a very active metabolic state and respond quickly to environmental stimuli, and the higher the dilution ratio of the bacterial solution induced the higher sensitive reactor and the quicker response. In 1999, a multi-channel micro-reactor mainly consisted of two reactors was developed and fabricated. One reactor was used to maintain the bacterial cells in a stable state and continuously supply fresh cells to another reactor. The other was used for mixing the luminescent bacteria with the substances to be tested to complete the detection and analysis[24]. However, since the bacteria in the system were not fixed, the stability of the detection could not be guaranteed. In 2003, Thouand et al[25] developed a biosensor with genetically engineered luminescent bacteria based on trimethyltin-induced luminescence. Bacteria were cultured in a dedicated bioreactor with a volume of approximately 100 mL. An entrance and an exit for the culture medium and the substance were provided on top of the reactor, and the sensor was inserted to measure the pH, temperature, dissolved oxygen and cell density in the reactor in real time to monitor

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the growth of bacteria in the reactor at all times. The authors used broth medium and glucose medium to culture bacteria and compared their differences to select the optimum medium for the sensor. In the exponential phase of bacteria, different concentrations of trimethyltin (0.001–10.0 μM) were added to induce the growth. The results exhibited that the bacteria had the strongest luminescence when the concentration of trimethyltin reached 1.5 μM in glucose medium. The luminescence intensity decreased with the increase of sample concentration and toxicity. In broth medium, the highest luminescence intensity occurred at the concentration of trimethyltin reached 10 μM. Its luminous intensity was only a quarter of the former. The main features of the system were as follows: (1) the optical fibers were fixed on the cover of the reactor without contact with bacteria and samples, thereby avoiding the loss of light output caused by biofilm formation; (2) several microprobes were introduced to evaluate the activity of bacteria during continuous growth; and (3) the equipment had the advantages of automation, integration and portability. However, the limitation of the biosensor lies in its long detection time and low flux. Zhao et al[26] developed a microfluidic device for detecting toxic substances in drinking water based on luminescent bacteria (Fig.6). Vibrio fischei was used as the biological monitor for the potential cytotoxic substances such as heavy metal ions and phenol in water, and an independent continuous bacterial culture system was equipped. The chip consisted of two countercurrent mixers, a T-type droplet generator and six spiral microchannels. This design could realize the continuous detection of the system. Cell suspension and water sample were mixed fully in the micro mixer, and then dispersed into droplets in the air flow to ensure that the

luminescent bacteria in the sensor could obtain adequate nutrient supply. Copper (Cu2+), zinc (Zn2+), potassium dichromate and 3,5-dichlorophenol were selected as typical toxicants to test the sensitivity of the system. There was an obvious non-linear relationship between the concentration of Zn2+ and the relative luminescence units. Preliminary tests suggested that this system could only roughly estimate the concentration of simple toxic chemicals in water, and could not identify or quantify specific contaminants. However, it could quickly screen toxic substances in a large number of samples. As an effective tool for screening acute toxic substances, it has good application prospects.

4 Gene manipulation for performance improvement of luminescent bacterial biosensors Recombinant luminescent bacteria are constructed by inserting or transfecting plasmids carrying promoters and target genes (lux genes) into host cells. The promoter is responsible for specific recognition, and the target gene directs the synthesis of luciferase, which makes the recombinant bacteria have luminescent properties[27]. The promoter is a non-coding DNA sequence located at the front of the coding gene, which is initiated after the transcription of its downstream genes. Therefore, researchers usually perform molecular operations on the promoter region to enhance the overall analytical performance of luminescent bacterial biosensors. Researchers constructed a large number of genetic engineering strains containing different promoters based on the substances to be detected. The specific characteristics of bioluminescent genes from different sources must be considered in the process of introducing them into bacteria.

Fig.6 Schematic of water quality detection system[26]: (A) droplet flow is generated by cell-based LOC and moved into observation chamber, the PMT is located on top of the observation chamber; (B) dynamic diagram of the observation chamber, the droplets flow from the cell-based LOC through the observation chamber continuously, the buffer will be replaced with the mixture of bacteria cells and contaminants within a short period

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For example, in the process of implanting the luminescent gene of Vibrio fischei into E. coli, the temperature required for luciferase activity to remain in Vibrio fischei was lower than that of E. coli (37 °C). The study found that the temperature required for Photobacterium phosphoreum to maintain its activity was the same as that for E. coli, thus their light-emitting genes was transfected into E. coli and a shorter response time was obtained[28,29]. Yagur-Kroll et al[30] proposed four methods to enhance the performance of luminescent bacterial biosensors by manipulating promoters: (1) modifying the length of DNA fragments containing promoter regions; (2) introducing a random gene mutant through directed evolution; (3) introducing more specific site mutants into promoter sequences; (4) replicating promoter sequences to increase binding sites of RNA polymerase. Through these four methods, the sensitivity, response time and emission intensity of biosensors would be significantly improved. LuxCDABEG, a lux operator, makes the bacteria be luminous. LuxA and luxB genes are responsible for coding the alpha and beta subunits of luciferase in bacteria, respectively. LuxC, luxD, and luxE genes individually encode r, s, and t peptides in the fatty acid reductase. LuxG is responsible for coding flavin reductase[31]. The number and types of lux genes isolated from different luminescent bacteria are different. Apart from luxG, which isn’t found in the genus photobacterium, the other five genes mentioned above are present in all luminescent bacteria that have been found. Thus far, lux gene of luminescent bacteria is often used to produce recombinant bacteria for biological testing, which can improve the bioluminescent intensity, reduce detection limit and shorten response time. The lux gene in bacteria is regulated by the upstream promoter sequence. Therefore, in addition to molecular regulation of promoter region, the enhancement of bioluminescence performance can also be achieved by splitting lux gene[32]. LuxCDABE can be divided into two smaller gene fragments: luxAB which codes luciferase and luxCDE which directs the synthesis of long-chain fatty aldehydes required for bioluminescence reactions. The two gene fragments were modified to be controlled by the inducible stress response promoter or the synthetic constitutive promoter. After multiple combinations of luxAB and luxCDE, researchers found that the optimal combination of luxAB of the inducible type and luxCDE of the constitutive type was able to enhance the luminescence intensity of the bacteria and accelerate the reaction speed. With the gradual maturity of genetic engineering technology, the method of transfecting lux gene plasmid into non-luminescent bacteria has been widely used. Researchers can select suitable bacteria for genetic modification according to specific pollutants to obtain specific recombinant luminescent bacteria. This operation can obtain the biosensor

for luminescent bacteria with higher sensitivity and wider response range. Nonetheless, whether the growth environment, conditions and laws of genetic engineered bacteria will change, whether the sensitivity and stability of pollutant toxicity detection will be improved, and what conditions will be needed to induce pollutants should be further addressed. Hence, the gradient generated in the micro environments and high throughput by the characteristics of micro chambers, micro mixers and in-situ detection technologies will greatly accelerate the determination of optimal screening conditions.

5

Conclusions

The biological toxicity detection with luminescent bacteria plays an important role in the detection of acute toxicity of water quality because of its high sensitivity, fast reaction speed, simple operation and easy to achieve high-throughput detection. By combination with optical fiber technology, sensor technology and microfluidic technology, the method based on luminescent bacteria can flexibly deal with various complex detection environments. With the increasing prominence of environmental issues and the need for toxicity assessment of pollutants, the toxicity analysis method of luminescent bacteria is suitable for rapid screening of a large number of samples, and it can be combined with other analysis to conduct in-depth detection of samples to provide comprehensive risk assessments. Multiple biosensors based on luminescent bacteria will play an important role in preventing the deliberate or accidental infiltration of toxic chemicals into surface water or municipal water network systems, and protect the environmental safety of the people. The development of molecular biology is expected to make luminescent bacteria respond specifically to specific pollutants and expand the application of luminescent bacteria. Combining luminescent bacteria with advanced materials, equipment or technology (such as micro/nanofluids, advanced optical materials, etc.) to improve the efficiency of the entire testing system will also become an important direction for the development of luminescent bacteria. Additionally, the effects of high pressure and low dissolved oxygen on the luminescent intensity of luminescent bacteria in microchannels still exist. Nevertheless, the generation and manipulation of micro bubbles in microfluidics will be the key to solve this problem.

References [1]

Ma X Y, Wang X C, Liu Y J. J. Hazard. Mater., 2011, 190(1): 100–105

[2]

Cairns J, Pratt J R. Hydrobiologia, 1989, 188(1): 5–20

[3]

Westlund P, Nasuhoglu D, Isazadeh S, Yargeau V. Arch. Environ. Contam. Toxicol., 2017: 1–11

[4]

Liu S S, Wang C L, Zhang J, Zhu X W, Li W Y. Ecotoxicol. Environ. Saf., 2013, 95: 98–103

JIN Xiao-Wei et al. / Chinese Journal of Analytical Chemistry, 2019, 47(2): 181–190

[5]

Ding K K, Lu L P, Wang J Y, Wang J P, Zhou M Q, Zheng C W, Liu J S, Zhang C L, Zhuang S L. Sci. Total Environ., 2017, 580: 1078–1084

[6]

De O F, Rossetto A L, Melegari S P, Ouriques L C, Matials W G. Sci. Total Environ., 2014, 490: 807–814

[7]

Costa S P F, Pinto P C A G, Lapa R A S, Saraiva M F S. J. Hazard. Mater., 2015, 284: 136‒142

[8]

Tu S C, Mager H I X. Photochem. Photobiol., 1995, 62(4): 615-624

[9]

Kaeding A J, Ast J C, Pearce M M, Urbanczyk H, Kimura S, Endo H, Nakamura M, Dunlap P V. Appl. Environ. Microbiol., 2007, 73(10): 3173–3182

[10] Zhu W J, Zheng T L, Li W M. Luminescent Bacteria and Environmental Toxicity Detection. China Light Industry Press, 2009: 21 [11] Dunlap P. Bioluminescence: Fundamentals and Applications in Biotechnology-Volume 1. Springer Berlin Heidelberg, 2014: 37–64 [12] Yousef S E A, Brendan J M, George D D. Ecotoxico. Environ. Saf., 2002, 51(1): 12–21 [13]

Karube I, Suganuma T, Suzuki S. Biotechnol. Bioeng., 1977, 19(3): 301–309

[14] Woutersen M, Gaag B, Abrafi Boakye A, Mink J, Marks R S, Wagenvoort A J, Ketelaars H A M, Brouwer B, Heringa M B. Sensors, 2017, 17(11): s17112682 [15] Charrier T, Chapeau C, Bendria L, Picart P, Daniel P, Thouand G. Anal. Bioanal. Chem., 2011, 400(4): 1061–1070 [16] Choi S H, Man B G. Biosens. Bioelectron., 2002, 17(5): 433–440 [17] Jouanneau S, Durand M J, Thouand G. Environ. Sci. Technol., 2012, 46(21): 11979‒11987 [18] Eltzov E, Marks R S, Voost S, Wullings B A, Heringa M B.

Sens. Actuators B, 2009, 142(1): 11–18 [19] Yoo S, Lee J, Yun S, Man B G, Lee J H. Biosens. Bioelectron., 2007, 22(8): 1586–1592 [20] Eltzov E, Slobodnik V, Ionescu R E, Marks R S. Talanta, 2015, 132: 583–590 [21] Elad T, Almog R, Yagurkroll S, Levkov K, Melamed S, Diamand Y S, Belkin S. Environ. Sci. Technol., 2011, 45(19): 8536–8544 [22] Eltzov E, Cohen A, Marks R S. Anal. Chem., 2015, 87(7): 3655–3661 [23] Gu M B, Dhurjati P S, Van Dyk T K, La Rossa R A. Biotechnol. Prog., 1996, 12(3): 393–397 [24] Gu M B, Gil G C, Kim J H. Biosens. Bioelectron., 1999, 14(4): 355–361 [25] Thouand G, Horry H, Durand M J, Picart P, Bendriaa L, Daniel P, Du Bow M S. Appl. Microbiol. Biotechnol., 2003, 62(2-3): 218–225 [26] Zhao X Y, Dong T. Int. J. Environ. Res. Public Health, 2013, 10(12): 6748–6763 [27] Yagur-Kroll S, Belkin S. Bioluminescence: Fundamentals and Applications in Biotechnology-Volume 2. Springer Berlin Heidelberg, 2014: 137‒149 [28] Szittner R, Meighen E. J. Biol. Chem., 1990, 265(27): 16581‒16587 [29] Davidov Y, Rozen R, Smulski D R, Van Dyk T K, Vollmer A C, Elsemore D A, La Rossa R A, Belkin S. Mut. Res.-Gen. Tox. En., 2000, 466(1): 97–107 [30] Yagur-Kroll S, Bilic B, Belkin S. Microb. Biotechnol., 2010, 3(3): 300–310 [31] Meighen E A. Microbiol. Rev., 1991, 55(1): 123–142 [32] Yagurkroll S, Belkin S. Anal. Bioanal. Chem., 2011, 400(4): 1071–1082