Application progress of Non-invasive Micro-test Technology in environmental bioremediation and stress physiology research

ABSTRACT Non-invasive Micro-test Technology (NMT) measures the ions/molecules in and out of the samples in a real-time, three-dimensional and dynamic manner without damaging the biological samples to obtain ions/molecules concentration, flow rate and movement direction, providing evidence for the study of the transport mechanism of biological and external environmental substances. This paper introduces the structure, operation system and technical advantages of NMT, and compares it with other ion testing technologies. Additionally, we conclude that it possesses the following attributes: no damage to biological activity, large range of measurable samples and targets, high sensitivity, and rich data content. Furthermore, its application progress in recent years has been reviewed and prospected from the perspectives of environmental bioremediation and stress physiology, which provides a reference for sustainable biological and environmental research.


Introduction
Organisms are often affected by adverse environmental factors during their growth.Plants are subjected to heavy metals (Asfa and Mohd Saghir 2018;Sorrentino et al. 2018), salinity (Fan et al. 2021), drought (Chandra et al. 2021) and other stresses.Microbial growth activities are affected by such environmental factors as temperature (Lu et al. 2022a) and pH (Muskus et al. 2022).In order to discover the transport process of organisms and external substances, the repair mechanism of pollutants and the response of organisms to adversity in a more intuitive manner, many approaches have been adopted to study the principle of ions and molecules entering and leaving organisms.Traditional ions/molecules testing technologies are usually applied to pretreat samples, indicating that subsequent tests cannot be performed in a near-real physiological environment, and such treatment may cause irreversible damage to organisms (Lemtiri et al. 2016;Vymazal 2016).
Non-invasive Micro-test Technology (NMT) is a tool that can measure various ions/molecules flows in and out of biological samples in a three-dimensional, dynamic and realtime manner to obtain such information as ions/molecules flow concentration and flow rate (Ole et al. 2020).NMT was first proposed by American scientist Lionel F. Jaffe in 1974. In 1990, it was successfully used to determine the Ca 2+ flow rate and direction of movement in and out of cells, creating a milestone from static measurement to dynamic measurement in the physiological function research (Jaffe 2010).After long-term development and improvement, a variety of non-invasive biological testing technologies have been derived from a calcium-specific vibrating electrode.In particular, scanning ion-selective electrode technique (SIET) (Yen et al. 2019;Zhou et al. 2020;Wang et al. 2021) and microelectrode ion flux estimation (MIFE) (Shabala et al. 2001) are its main derivative technologies, which provide a good experimental system platform for studying the physiological characteristics of organisms.
NMT has been widely applied in medicine, botany, environmental science, zoology, and microbiology, etc., with many high-level scientific and technological achievements gained in various fields (Wang et al. 2022).It has become a powerful tool for studying physiological and molecular mechanisms (Jia et al. 2021a).In recent years, more and more scholars have applied this technology to sustainable research.The test object has been expanded from ion measurement of plant root tissue to measurement of microbial cell entry and exit substances (Zhang et al. 2020;Chen et al. 2021).The research field has been developed from the application of early plant stress resistance to the application of microorganisms and plant-microorganism assisted remediation of environmental pollution (Han et al. 2021).The application fields have become increasingly extensive, accompanied by more and more diversified types of samples and measured ions.In view of this, we summarized the applications of NMT in various tests in recent years, analyzed the value of NMT, and prospected its research potentials, hoping to attract more attentions and use it more effectively.

Device structure
Non-invasive Micro-test Technology contains both the hardware system and the software system, the former of which is installed in a vibration isolation platform, including microelectrode sensor, motion control system, image capture system, microscope and signal amplifier.And the software system applies the IF-Luxes system that processes the electrical signals generated by the sensor and the real-time video data generated by the imaging system (Figure 1).
The living sample is placed on a slide, and the distance between the microelectrode and the sample is adjusted under the microscope.The computer controls the movement of the microelectrode so that a phase-correct waveform is induced on the output signal.The amplitude of this signal should be proportional to the concentration of the analyte.This calibration operation is necessitated.The microelectrode repeatedly measures the two points of the sample at a known distance to obtain a three-dimensional signal.The chemical signal is converted into an electrical signal.The amplifier amplifies the differential waveform.The signal processing system digitally analyzes the differential signal, and then presents it in IF-Luxes.IF-Luxes records, displays and processes the data in real time.Meanwhile, it is connected with the image capture system and the electrode motion controller.During the test, the detection screen can be observed in real time to precisely control the microelectrode (Mclamore and Porterfield 2011).

Electrochemical sensors
The sensitivity, selectivity and persistence of sensors are common schemes for comparing ion detection techniques.Currently, self-reference microsensors function mainly based on electrochemical and optical theories.Electrochemical sensors are divided into two categories in terms of ampere and potential.The ion-selective electrode of NMT is a potential microsensor that detects ion activity by measuring the electromotive force between the two electrochemical sites.
A liquid ion exchange membrane (LIX) is injected at the tip of the microelectrode, and an electrolyte is injected at the rear end (Xie et al. 2021).The Ag/AgCl wire is stably immersed in the electrolyte (Liu et al. 2019a).Figure 2 takes the measurement of Ca 2+ selective microelectrode into and out of the root tip as an example to demonstrate the recognition-transduction mechanism of NMT.Ca 2+ is recognized by the ion carriers that are immobilized in porous membranes.Ca 2+ in the liquid near the tip of the microelectrode react with valinemycin in LIX, causing changes in electromotive force.The electron exchange reaction on the surface of the electroplated Ag/AgCl wire in the electrolyte immerses this electromotive force into a voltage signal (Mclamore and Porterfield 2011).

Governing equations
The concentration and flow rate of ions/molecules of the sample are calculated by the Fick's first law and the Nernst equation (Mclamore and Porterfield 2011).The concentration depends on the voltage of two different sites and Nernst slope.The flow rate of ions/molecules is obtained by combining the concentration and the distance between the near pole and the far pole. where

Technical features
Firstly, the sample analyzed by NMT can be a complete seedling, a tissue or even a cell.No complex pretreatment of the sample will be required, and the sample will not be contacted in the testing process.It has not only reduced the workload but also guaranteed the physiological activity and integrity of biological samples (Hawkins and Kranabetter 2017).Secondly, NMT can measure a wide range of concentrations and flow rates, and it can also measure many kinds of ions and molecules (Ma et al. 2020;Zheng et al. 2020).The concentration, flow rate and motion direction of the target ions/ molecules can be detected simultaneously by controlling the IF-Luxes software.In addition, NMT has high spatial and temporal resolution, and measurement time and response time are short.Therefore, it is often used for long-term and real-time measurement.Its technical parameters and characteristics are shown in Table 1.NMT is frequently employed to measure the influx and efflux of Ca 2+ , Cd 2+ , H + and Pb 2+ in plant root tissues and mesophyll cells in environmental bioremediation and stress biophysiological studies (Wang et al. 2020;Deng et al. 2022).It provides a possibility for long-term and real-time monitor the dynamic changes of ions/molecules in and out of the living tissues and cells.

Comparison of NMT and traditional ion detection techniques
Radioactive tracer technique (RTT), patch clamp technique (PCT) and fluorescent probe technique (FPT) are used to detect ion transmembrane transport in cells and tissues.
RTT is a traditional technology for detecting the absorption and transport of elements by organisms.Its applications have been frequently reported in the field of environmental protection (Belyaev et al. 2009;Cusnir et al. 2022).For example, radioactive tracers can be added to the plant culture soil to detect heavy metals in leaves, stems, roots and pods, and observe their enrichment in different tissues.PCT is a technique used for measuring ion channels in cell membranes (Yu et al. 2019;Jovanovic 2021).It is often used to study the kinetic characteristics of ion entry and exit in animal cell membranes.For plant samples, it is destructive and cannot be measured in real physiological conditions.Moreover, the operation of this technique is complicated and requires accurate operation by the experimenter, which limits the application range of the patch clamp.FPT is used to qualitatively analyze ions/molecules based on the characteristic changes of fluorescence spectra as well as ions/molecules based on the relationship between fluorescence properties and ion concentration (Mistri et al. 2018;Mondal et al. 2020).Like NMT, FPT is also non-invasive and has high sensitivity (You et al. 2022), but its measurable ion/molecule types are limited.Many fluorescent probes are affected by the test environment, resulting in low reliability of the results (Okamoto et al. 2022).In Table 2, these four ion measurement techniques are compared from several performance parameters.
Compared with other ion measurement techniques, the most prominent advantage of NMT is that the measurement is conducted without damaging plant and animal samples, the measurable sample and ion ranges are relatively wide, and the results are more accurate and comprehensive (Ye J et al. 2021).

Application of NMT in plant heavy metal remediation
With the development of industry and urbanization, heavy metal contaminated sites have become increasingly prominent (Song et al. 2022).There are many kinds of heavy metal remediation technologies, including physio-chemical  remediation, electrochemical remediation and phytoremediation, etc. (Lin et al. 2022).Physio-chemical remediation based on the adsorptive capabilities of certain materials for heavy metals and the modification of these materials to extract heavy metals (Krishnani et al. 2009;Kong et al. 2021).Krishnani (2016) developed partially delignified Ca 2 + -and-Mg 2+ -ion-exchanged product from lignocellulosic wheat straw, which can be used to remove eight different heavy metals.Electrochemical remediation transfers heavy metal ions to the electrode by introducing low direct current into the soil.Based on this method, Zhao et al. (2023) developed a new electrokinetic adsorption remediation method and the average Cd(II) removal rate was approximately 85%.Phytoremediation is widely used due to its cost-effectiveness.Plants adsorb exogenous metals through physiological reactions to effectively eliminate pollution in the environment (Liu et al. 2023).Throughout the remediation process, plants may promote the immobilization and translocation of free metal ions within the organism by changing the metal speciation (Alves et al. 2011).Kińska et al. (2022) identified dihydroxynicotianamine as the predominant ligand for Cu and Zn in Hypericum laricifolium.Cao et al. (2020) speculated that malic acid may be an important metal speciation for Cu in the phloem of willow (Salix integra Thunb.).However, there are two main problems in phytoremediation of heavy metals: One is how to improve the enrichment effect of plants on heavy metals, and the other is how to improve the tolerance of plants to high concentrations of heavy metal pollutants.In order to promote the enrichment and tolerance of hyperaccumulators, many studies have been conducted to demonstrate the mechanism of material transformation between the plants and the outside world by using modern biotechnology tools (Dai et al. 2022;He et al. 2022).The outstanding advantages of NMT in the studies of plant heavy metal remediation are as follows: (1) NMT can not only measure the entry and exit of heavy metal ions such as Cd 2+ , Cu 2+ and Pb 2+ in plants to observe the absorption and enrichment of plants, but also measure the flow of Ca 2+ , K + and H + ions to speculate the effect of heavy metal stress on plant physiological state.(2) Heavy metal hyperaccumulators usually have rich roots to adsorb and fix external substances.A complete root can be directly used as a sample for measurement, so the results are closer to the normal physiological state of the plants and more authentic.In addition, NMT can be used to determine the transport of ions in xylem and the absorption of root cells, and meet the test requirements of relevant scientific research from multiple perspectives.In order to clarify the effect of oxalic acid on cadmium transport in Sedum alfredii Hance under cadmium stress, NMT was used to measure the Cd 2+ inflow in the 0-10 mm area of root tip.Combined with the fluorescence imaging technology, it was found that the Cd 2+ inflow increased with the increase of oxalic acid secretion in the root (Tao et al. 2016).Li et al. (2019)   In addition, more and more researchers have carried out in-depth exploration from the perspective of molecular science.In particular, NMT is one of the important means used by such researchers.Zhang et al. (2021b) adopted molecular methods and NMT to study the effect of the transcription of Populus euphratica cell gene into Arabidopsis thaliana homologous gene on the efficiency of remediation of cadmium contaminated soil.It was found that the Cd 2+ inflow of transgenic Arabidopsis thaliana root tip was significantly higher than that of the control group, which was helpful for Arabidopsis thaliana to absorb and enrich cadmium.Liao et al. (2019) used NMT to measure H + , Cd 2+ and NO 3 -in plant mature vacuoles in real time, and found that Brassica napus with two different Arabidopsis thaliana genes responded differently to vacuoles and NO 3 -in vacuoles under cadmium stress.The balance of Cd 2+ and NO 3 -can be changed by regulating the activity of transporters, which can effectively enhance the tolerance of Brassica napus to Cd 2+ and improve the potential of cadmium repair.In Table 3, there are some other cases of NMT application in phytoremediation.

Application of NMT in microbial remediation
Microorganisms have been widely used in the field of environmental remediation because of their high metabolism, good environmental adaptability and strong ability to degrade pollutants (Xiao et al. 2021).Microorganisms can not only act as natural sensors for monitoring pollutants, but also degrade pollutants through life activities (Shing et al. 2013;Techtmann and Hazen 2016).Microorganisms convert toxic substances into non-toxic or low-toxic metabolites through a series of enzymatic reactions.For example, cyanobacteria produce oxygen and release organic matter through photosynthesis.This fosters the biosorption and absorption by microbial communities and the biomineralization of Fe/ Mn oxides, ultimately facilitating the oxidation of As(III) to As(V) and promoting the immobilization of arsenic (Chen et al. 2023a).Through the analysis of the metabolic pathway and transformation products of Green alga Chlamydomonas reinhardtii, Liakh et al. (2023) discovered its remarkable effective in removing diclofenac from aquatic environments.The research results unveil a novel dimension in the field of mediation and contribute to the advancement of microalgae-based bioremediation techniques.As an important technique of environmental remediation, it is of great significance for understanding its toxic response to pollutants and its adsorption mechanism to promote the development of microbial remediation technique.NMT has been applied in the field of microbial remediation because of its high sensitivity and the ability to test cells.It is mainly used to measure heavy metal ions and small molecular substances transported across the membranes of single microbial cells.
Cyanobacteria can indicate water ecosystem degradation.In order to explore the reaction of Microcystis and Nostoc under mass transfer and temperature shock, Xiao et al. (2020) used NMT to test the flux of NH 4 + and O 2 in and out of cells.The result showed that the outer mass transfer and instantaneous temperature shock produced different effects on the flow of NH 4 + and O 2 , which is of great significance for controlling the external conditions to promote the physiological activities of cyanobacteria and play its biological monitoring role.In addition to observing the physiological state of microorganisms by detecting NH 4 + and O 2 , NMT was also used to detect the transmembrane transport of heavy metal ions in microorganisms to improve the environmental remediation effect of microorganisms.It was previously reported that marine diatoms degraded heavy metal cadmium in the ocean (Lane et al. 2008), but there are few in-depth studies on the effect of silicate imbalance on cadmium accumulation in marine diatoms.Therefore, Ma et al. (2018) measured the Cd 2+ flux into the cell wall of living diatoms by NMT and clarified the positive effect of silicon on the adsorption of cadmium by diatoms, which strongly proved the important role of diatoms in marine heavy metal remediation.White-rot fungi can remove over 95% of Cr(VI) at an initial concentration of 10 mg/L, which is a promising way to remove heavy metals.Feng et al. (2017) observed the effect of Cr(VI) on the life activities of white-rot fungi by using NMT to monitor the K + flux in real time.The results indicated that Cr(VI) treatment could promote the K + efflux, confirming such a view that Cr(VI) induced apoptosis.

Application of NMT in microbial-plant collaborative remediation
Plants play a pivotal role in assisting microbial communities transform, remove and contain contaminants within soil in plant-assisted bioremediation (Giaccio et al. 2023).Inoculation of exogenous fungi in plant roots is one of the effective ways to improve the ability of plants to resist external stresses, and it is also one of the important means H + , K + , Ca 2+ , Mg 2+ in the root system were changed with the increase of aluminum concentration in the environment.(Ma et al. 2016) to improve the effects of plant environmental remediation.
More and more evidences have proved that the collaboration between symbiotic bacteria and plants is crucial for effective transformation of foreign substances (Chane et al. 2023;Narayanan and Ma 2023).The implementation of a plant-assisted bioremediation approach demonstrated significant reductions in PCBs and heavy metals within the vicinity where poplar trees (Monviso) were cultivated.Concurrently, rhizosphere microbial activity exhibited a general increase, leading to an enhancement in soil quality (Ancona et al. 2017).
NMT can be used to detect plant roots, which provides strong technical support for exploring the transmembrane transport before and after the inoculation of symbiotic bacteria and clarifying the detoxification mechanism of the two.Arbuscular mycorrhizal fungi (AMF) are used to assist phytoremediation and are usually found in metal contaminated sites (Rufyikiri et al. 2000;Zhang et al. 2019aZhang et al. , 2019b)).In order to explore how AMF mediated cadmium accumulation in plants, Han et al. (2021) inoculated two different AMF mycorrhiza to perennial ryegrass (Lolium perenne), and compared the effects of the two on cadmium absorption and tolerance.NMT measurements showed that the inoculation of mycorrhizal increased the net inflow of Cd 2+ in roots, and the accumulation of cadmium in roots was higher than that in the control group.Leguminous plant-rhizobium symbiosis can possibly remediate the soil contaminated by chlorinated organic compounds.Wang et al. (2018) symbiotically cultured Medicago sativa and Sinorhizobium meliloti, used NMT to measure the Cl -and K + fluxes of 3-5 cm long nodules, and recorded the realtime flux for 10 min.The results proved for the first time that symbiotic nitrogen fixation is a driving force for the dechlorination of tetrachlorobiphenyl, which provides a new method for using rhizobia to strengthen the phytoremediation of halogenated organic compounds.

Application of NMT in the physiological study on plant resistance to soil salinization
Soil salinization is a key environmental problem that limits the productivity in many regions (Parihar et al. 2015).Because the salinity can affect the photosynthesis, respiration, nutrient assimilation and hormone imbalance of soil crops (Hussin et al. 2013), which can seriously limit the yield of crops.To find the key components of plant salt tolerance network and make improvement in this regard, NMT has become an important method to solve this problem.With the development of genetics, transcriptology, proteomics and metabolomics, researchers have improved the salt tolerance of plants from the perspectives of genes (Shu et al. 2022), transcription factors, proteins (Mahmoud et al. 2022) and metabolites.In recent years, more and more researchers have focused on the dynamic changes of ions in organisms such as Na + and K + and used NMT to analyze the salt tolerance mechanism of plants.
In order to clarify the salt tolerance mechanism of sugar beet, Dong et al. (2021) combined NMT and gas chromatography to determine the Cl -influx and Cl -efflux rate of sugar beet seedling roots under 750 μmol/L boron and 300 mM NaCl treatment.The results revealed that the Cl -influx rate and efflux rate of seedling roots under 750 μmol/L boron and 300 μmol/L NaCl treatment were significantly lower than those of the control group.The results demonstrated the effectiveness of the interaction between boron and NaCl for sugar beet resistance to salt stress, and provided a new method for improving sugar beet planting in the saline-alkali land.Nitrogen is an indispensable element for plant growth.Different plants have diversified nutritional demands for ammonium nitrogen and nitrate nitrogen (Ren et al. 2021).Dai et al. (2015) used NMT to measure the net flux of sodium (Na + ), ammonium (NH 4 + ) and nitrate (NO 3 -) in plant roots.It was found that the sensitivity of plants applying nitrate nitrogen under salt stress was lower than that of ammonium nitrogen plants, and the inhibition of root activity was relatively low.This provides a theoretical basis for fertilizer selection for cotton cultivation in the saltstressed soil.Feng et al. (2020) used NMT to compare the Na + and K + fluxes of two different types of willow roots.It was found that the Na + and K + flow rates of willows became faster under NaCl treatment, the Na + and K + fluxes of frame willows were significantly higher than those of willows, and the salt tolerance was strong.This species may become a promising species for greening the saline-alkali land.Table 4 lists some other cases of salt stress applications.These cases are the application of NMT in different tissues such as seeds and genetically engineered cells, which is mainly used to measure the effects of salt stress on Na + and K + fluxes rate.

Application of NMT in temperature stress
With the intensification of global climate change, the frequency and intensity of extreme weather events such as high temperatures, droughts and frosts have gradually increased, posing a major threat to biodiversity and ecosystem-related functions (Zhan et al. 2023).High temperature is the most important abiotic stressor, which is the key factor that cause crop yield reduction and even lead to species extinction (Zhu et al. 2022;Chen et al. 2023b).Abiotic stress induce alteration in the uptake of external substances by organisms, the intracellular movement of ions and molecules, and the morphological transformation of pollutants in organisms (Strano et al. 2022;Chen et al. 2023c).It was reported that the efficiency of converting external macronutrients into biomass will be reduced at higher temperatures.Compared with 25°C, the uptake of nitrate and phosphate by T.seucica at 31°C changed significantly (Lo et al. 2022).In order to elucidate the effects of combined abiotic stresses of heavy metals and high temperature on Glycine max L., Bilal et al. (2020) co-inoculated the Synergistic association, which evaluated the degree of stress damage to organisms by detecting the absorption and transport of heavy metals (Ni, Cd, and Al) and macronutrients (Ca, K, and Mg).In addition, temperature changes can affect energy metabolism by stimulating oxidative stress in organisms, which is manifested in a series of intracellular reactions of H 2 O 2 , O 2 and H 2 O in the presence of antioxidant enzymes (Zhao et al. 2022).In these studies, ion and molecular tests serve as pivotal proof factors, and NMT can obtain more comprehensive data.It emerges as an effective instrument for future application in this domain.
Seed germination is the basis of seedling establishment and the most basic development stage in the life cycle.Temperature is the most critical environmental factors (Xu and Du 2023).In order to investigate the roles of plant hormone abscisic acid (ABA) and reactive oxygen species (ROS) in rice seed germination under high temperature.Liu et al. (2019b) adopted NMT to detect H 2 O 2 flux at the site of coleoptile emergence.High temperature stress can lead to H 2 O 2 efflux.Tang et al. (2016) studied the morphological and physiological responses of roots and leaves in autumn by warming the night growth environment of two coniferous species, Picea asperata and Abies faxoniana.The fluxes of NH 4 + , NO 3 -, H + and O 2 in the roots were measured by NMT.The results showed that nighttime warming led to a significant increase in the net influxes of NH 4 + and NO 3 -, corresponding well with net H + efflux and O 2 net influx.In addition, NMT also has applications in low temperature stress and suboptimal root zone temperature (Table 4), providing evidence for elucidating the effects of temperature on plant physiology.

Application of NMT in drought stress
Climate change poses a significant threat to biodiversity and ecosystem-related functions (Trenberth et al. 2014).According to the report of the fifteenth session of the Conference of the Parties to the United Nations Convention to Combat Desertification, the duration and severity of drought are increasing.Since 2000, the number and duration have increased by 29%, accounting for 15% of natural disasters.The frequent occurrence of drought disasters has caused serious threats to agricultural crops, urban gardens, and biodiversity (Ji et al. 2023;Zhang et al. 2023).Under drought stress, in order to maintain the homeostasis of organisms, plants regulate a series of physiological responses, such as photosynthesis (Wang et al. 2019), water potential balance (Walthert et al. 2021), metabolites and so on (Tong et al. 2020).In this process, it is also accompanied by abundant ion transport within organisms and material exchange with the outside world.NMT can identify drought-related responses by testing various ions (K + , H + ) and small molecule substances (IAA) in organisms, and provide help for studying the effects of drought stress on plants (Liu et al. 2018;Yang et al. 2018;Qi et al. 2019).Liu et al. (2018) used NMT to determine the auxin (IAA) flux along the root surface in the root hair area (3 cm from the root tip) and the lateral roots in different orders during the exploration of the factors affecting the growth of root hair and the level of lateral root hormones under drought stress.Li et al. (2021) measured in situ the net K + , H + and Ca 2+ fluxes of guard cells and mesophyll cells by NMT.It was confirmed that the increase of [ABA] leaf coincided with the increase of K + efflux and Ca 2+ influx and the decrease of stomatal conductance in WT under short-term drought stress.Zhang et al. (2019c) cut the tea leaves into small pieces (5 mm × 8 mm) and exposed the mesophyll cells with a sharp blade at 30°.The NMT measurement results indicated that the K + efflux of tea mesophyll cells under drought stress showed a strong negative correlation with the overall drought tolerance.In Table 4, there are some examples of the application of NMT in cells and tissues under drought stress.

Conclusion and prospect
It will be of great significance for the study of biological mechanism whether it can be measured in a physiological state without damaging biological samples.NMT based on the measurement of ions/molecules concentration and flow rate of transmembrane transport in various tissues and cells of organisms, providing data support for the study on the transport mechanism of various plants and microorganisms.Faced with the problems of plants, microorganisms and plant-microorganism collaborative remediation of environmental heavy metals, NMT cannot only directly reflect the absorption and transport of external heavy metal ions by measuring the target ions, but also analyze the absorption and transport mechanism from the perspective of ion channels by measuring other ions (Yang et al. 2020;Zhang et al. 2021c).In the process of the resistance of plants and microorganisms to environmental stresses such as salinization, drought and high temperature, dynamic measurement of related ions can more accurately find the key physiological positions and related factors of biological response to environmental stresses, regulate, control and improve the biological tolerance (Dai et al. 2019;Lu et al. 2022b).
It can be seen that in the field of plant heavy metal remediation, NMT, as an ions/molecules test technique, provides direct evidence for studying the removal effect of heavy metals in the environment by plants as well as systematic evidence for studying the mechanism of plant absorption, fixation and transport of pollutants.More importantly, it is accompanied by the development of other emerging technologies, providing strong evidences for the application of many advanced technologies in the field of phytoremediation (Ma et al. 2016).Compared with phytoremediation, there are relatively few studies on the mechanism of microbial remediation from the perspective of ions/molecules transmembrane transport, and the measurement of microbial ions/molecules requires stronger technical support.NMT takes single cells as the test object so that a can be conducted to explore the mechanism of microbial transmembrane transport from a more microscopic perspective, and promote the in-depth development of microbial remediation technology in the field of environmental science (Feng et al. 2017).Plant-assisted bioremediation has been used in heavy metal pollution remediation, organic pollution remediation and other fields.Their root symbiotic bacteria are the key to this method, and NMT can measure plant root parameters in this field and help the development of the Plant-assisted bioremediation (Han et al. 2021).The physiological changes of plants under salt stress are mainly manifested in Na + , K + and H + .By monitoring these parameters, NMT can find out the reasons for plant salt tolerance, improve the survival ability of plants in the process of soil salinization, and screen out excellent environmental functional plants.The high temperature and drought caused by climate change have a significant impact on organisms.NMT can judge the ecological impact of climate change by measuring the absorption of external environmental nutrients and the exchange of internal ions with the outside world (Li et al. 2021).
As an emerging technology, NMT still has some problems that need to be improved: (1) As selective glass microelectrodes are extremely fine glass products, in the filling process of LIX solution and ion exchange solution, tip damage and LIX leakage occur easily, which will not only increase consumables but also reduce the accuracy of measurement results.It is difficult to calibrate the selective electrode and the polarographic electrode before testing.The measurement process fluctuates due to some external factors, and the test process is unstable (Mclamore and Porterfield 2011;Yang et al. 2019).( 2) The combination of NMT in plant physiological research and other technologies needs to be strengthened due to limited types of ions/molecules that can be measured.Although NMT has many application examples combined with other technologies in the study of plant stress resistance mechanism, the application range of NMT still has some limitations due to the high precision requirements of the system for selective microelectrodes and operation environment.How to solve its limitations and combine it with other technologies effectively and systematically and achieve more comprehensive measurable ions/molecules, more simple measurement method, more diverse measurement data, and more extensive measurement range have become an important development direction of NMT in plant resistance to environmental stress and repair of heavy metals (Ye et al. 2021).( 3) Compared with phytoremediation, there are fewer NMT studies in the field of microbial ecological restoration.The stability of cells and sensors and the physical/chemical interference between them are the key issues to be weighed and solved.The testing of singlecell microorganisms not only requires a high-precision requirement for the instrument but also requests the experimenters to improve their abilities to meet the measurement requirements of standardized technology.(4) Researchers should make full use of the advantages of NMT, expand its application scope in bioremediation, and strengthen its research depth of biological growth mechanism under environmental stress.This will promote more and more hyperaccumulation plants to be screened and modified to improve their stress tolerance and repair effect, which is of great value for future development.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Notes on contributors
Wenxuan Jiang is a master's student at the School of Environmental Science and Engineering in the Guilin University of Technology.She received her bachelor degree in 2021, major in Environmental Engineering from the Guilin University of Electronic Technology.Her research interests include heavy metal soil remediation and plant toxicology.Yucui Shi is a PhD student at the School of Environmental Science and Engineering in the Guilin University of Technology.She received her bachelor degree in 2017, major in water and wastewater science and engineering from the Guilin University of Technology.She received her master's degree in 2019, major in municipal engineering from the Guilin University of Technology.Her research interests include heavy metal soil remediation and water pollution control project.

Shaohong
Pingping Jiang is a laboratory at the College of Earth Science in the Guilin University of Technology.She received her bachelor degree in 2013, master's degree in 2016, and PhD degree in 2020, major in Environmental Science and Engineering from the Guilin University of Technology.Her research interests include heavy metal soil remediation and water pollution control project.
Nernst slope, b = yintercept of Nernst calibration plot [mV], E (x) = electrode potential at the near pole [mV],and E (x + Dx) = electrode potential at the far pole [mV].

Figure 1 .
Figure 1.Composition system and workflow of NMT.
This work was supported by National Natural Science Foundation of China [grant number 52170154]; Natural Science Foundation of Guangxi Province [grant number 2021GXNSFBA196023].
You is a PhD candidate at the School of Environmental Science and Engineering in the Guilin University of Technology.He received his bachelor degree in 2001 and his master's degree in 2007, major in Environmental Engineering from the Guilin University of Technology.His research interests include water treatment technology, heavy metal pollution phytoremediation, and environmental impact assessment.

Table 1 .
Technical characteristic parameters of NMT.

Table 2 .
Comparison of NMT with other ion/molecule detection techniques.

Table 3 .
Application of NMT in ecological restoration of heavy metals in plants.

Table 4 .
Application of NMT in the study of stress physiological response.