Non-invasive micro-test technology and applications

Abstract

Non-invasive micro-test technology (NMT) reveals dynamic ionic/molecular concentration gradients by measuring fluxes of ions and small molecules in liquid media in 1D, 2D or 3D fashions with sensitivity up to pico- (10⁻¹²) or femto- (10⁻¹⁵) moles per cm² per second. NMT has been applied to study metabolism, signal transduction, genes and/or proteins physiological functions related to transmembrane ionic/molecular activities with live samples under normal conditions or stress. Data on ion and/or molecule homeostasis (IMH) by NMT in biomedical sciences, plant and crop sciences, environmental sciences, marine and space biology as well as traditional Chinese medicine are reviewed.

Graphical abstract

INTRODUCTION

Ion and/or molecule homeostasis (IMH) is a fundamental biological phenomenon essential for all living organisms (Ahmadi et al. 2023; Huang et al. 2022; Song et al. 2024; Sun et al. 2009a, 2009b, 2010; Xu 2023; Xu et al. 2022; Yang et al. 2010). Pathology of humans, animals, crops, insects and microbes is accompanied by abnormal IMHs. Excessive H⁺ accumulations, for instance, are the result of lactate formation even in the presence of O₂ in tumor cells (Martinez-Outschoorn et al. 2011; Semenza 2007; Seyfried and Shelton 2010; Warburg 1956; Warburg et al. 1927). It is also well known that a plant will not grow properly if the supply of NH₄⁺, NO₃⁻, PO₄³⁻, K⁺ are out of balance (Guan et al. 2024; Martínez-Ballesta et al. 2020; Tyler 2017; Zhou et al. 2019).

However, it has been a challenge for biologists to fully comprehend IMHs due to: (1) ionic and molecular information related to IMH have to be collected with live samples; (2) multiple ions/molecules are preferably measured simultaneously in order to construct the network among relevant ion(s)/molecule(s); (3) ionic/molecular data in different levels, i.e. cell, tissue to organ, has to be collected ; and (4) since biological materials are 3-dimensional per se, 1D or 2D form of data is no longer accurate to reflect the 3D world (Kunkel et al. 2001, 2006; Wan et al. 2011; Xu et al. 2006; Yang et al. 2012).

Non-invasive micro-test technology (NMT) is a collection of techniques. Initial NMT hardware was developed based on the Vibration Probe (VP) Technique invented by Dr. Lionel Jaffe at Marine Biological Laboratory in the early 1990s (Degenhardt et al. 1998; Kochian et al. 1992; Kuhtreiber and Jaffe 1990; Smith 1995). VP was primarily focused on solving the problems of voltage signal drifts and instrumental background noise (Kunkel et al. 2006; McLamore and Porterfield 2011), while NMT went further emphasizing: (1) developing more ionic and molecular micro-sensors; (2) simultaneous multiple ion/ion and/or ion/molecule measurements; (3) 3D-flux data collection and visualization and (4) automation of NMT to be more efficient to collect flux data (Kunkel et al. 2006; Xu et al. 2006; Yin et al. 2006). The term Non-invasive Micro-test Technology, initially proposed by Yue Jeff Xu, is now widely accepted (Xu 2023).

This review will describe NMT about: (1) working principles based on the 2-point measurement scheme (2PMS); (2) spatial relationships among samples, NMT sensors and ionic/molecular gradients from physical and chemical perspectives; and (3) ionic/molecular fluxes in relation to their gradients.

Comparisons will be made between NMT with other ion/molecule detection techniques, such as patch clamp and ion-selective microelectrodes.

NMT is now widely used in plant and crop sciences (Liu et al. 2023), animal science, microbiology, environmental science, and traditional Chinese medicine, as outlined below.

NON-INVASIVE MICRO-TEST TECHNOLOGY (NMT)

Principles

Two-points measurement scheme

Ionic/molecular fluxes are detectable when ions/molecules move from high concentrations to low concentrations. The concentration differences then result in ionic/molecular gradients (Fig. 1). In live biological samples, transmembrane activities of ions/molecules result in ionic/molecular gradients via their effluxes and/or influxes (Kunkel et al. 2001). In order to measure these ions/molecules selectively or specifically in a quantitative way, various sensors made from glass, optical fiber, and platinum-iridium alloy have been developed. These sensors’ fabrications are based on different theories/mechanisms, such as ionophores, enzymatic reactions, electrochemistry, and fluorescent dyes (McLamore and Porterfield 2011).

Due to the special demands of temporal and spatial resolutions, conventional ionic/molecular sensors usually have to be modified to fit NMT hardware configuration. Therefore, NMT ionic/molecular micro-sensors are designated to differentiate themselves from those traditional micro-sensors.

1D, 2D, 3D and 6D measurements

NMT flux data in 1D, 2D and 3D are essential to (1) validate results from molecular biology, biochemistry, microscopy, and omics experiments; (2) provide clues for research on new genes, and new mechanisms; (3) for network map connecting among genes, proteins, metabolites.

An online interactive 3D animation application has been developed to not only visualize 3D flux data but also for additional features, such as zooming, spinning, panning, movie making and coloring (Fig. 2) (Wang et al. 2023b).

A recent NMT hardware design allows both NMT micro-sensor and the tested sample to move in their own 3D system independently, which is even more powerful with a versatile 6D NMT system (Xu 2023).

Limitations of NMT

The types of NMT sensors and their temporal and spatial resolutions are limiting factors of NMT.

NMT Micro-sensors

Knowing exactly which ions/molecules are measured is very straightforward, but adding new members to the current NMT sensor’s list is not an easy task. For example, some breakthroughs have been made to the most desired Fe²⁺/³⁺ and PO₄³⁻ sensors recently after years of endeavors and efforts. The reasons that this progress has been slow are (1) only a few scientists are working in the field; (2) due to the inter-disciplinary nature of NMT sensor’s development; (3) unwillingness to develop new NMT sensors for the lack of commercial incentives.

Temporal resolution

NMT’s temporal resolution is mainly determined by its responding time. When obtaining reliable and stable readings, the NMT sensor requires responding time in a few seconds range versus dozens of seconds or even minutes of conventional microsensors.

Although temporal resolution may be a limiting factor for NMT to go below seconds, biologists are taking advantage of its relative long-term detection capability of up to hours or even days (Fig. 3).

Comparative advantages over other techniques

NMT vs. Micro-electrodes

NMT is different from traditional Ion Selective and/or molecular specific Micro-electrodes. On one hand, it is true to a certain extent because they both inform about the ions and/or molecules, on the other hand, much difference exists upon how much information each technique can reveal (Fig. 4).

NMT vs. Patch Clamp

E. Neher and B. Sakmann were awarded the Nobel Prize in 1991, after they invented the patch clamp technique to measure ionic currents in the plasma membrane of living cells (Xu and Qiu 1993).

One of the major breakthroughs with patch clamp is the successful GigaΩ seal between the polished glass micro-pipette and the plasma membrane. Yet, it also brings in significant mechanical impacts or even damages to the cells, which makes the mechanical stimulations unavoidable throughout every patch clamp experiment (Yang et al. 2012).

Therefore, a non-invasive way of detecting ions across cell membranes is very attractive. Especially for plant biologists to study salt or water processes, there is no need to enzymatically peel away the cell walls of plant cells which perturb the properties of cell membranes significantly (Xu 2023).

NMT can detect ion(s) movement(s) even under electrical neutral conditions (Fig. 5).

A valuable by-product of no-touching measurements of NMT is that it can measure a wide range of live samples in different sizes, from small intact organisms, isolated organs, tissues, cell layers, and single cells to aggregated organelles or microbes.

Detection of multi-ions/molecules simultaneously is another unique feature of NMT, which allows scientists to study the inter-relationships not only among ions but also among ions and molecules potentially paving the way to understand vital physiological functions of life (Kunkel et al. 2006; Xu et al. 2006).

However, patch clamps have good temporal and spatial resolutions (Fig. 3), which allow scientists to take advantage of both techniques to tackle tough scientific questions (Chen et al. 2007; Shabala et al. 2006; Smith et al. 1999).

NMT APPLICATIONS

Medicine and healthy

Disease treatments

Glioma is a common brain tumor with high morbidity and mortality. Recent studies have shown that photodynamic therapy (PDT) can be used to kill the glioma cells due to increased extracellular glutamate concentrations in C6 gliomas and upregulated glutamate receptor AMPA expression. However, how PDT affects the related transportations of Ca²⁺ and K⁺ across the cell membranes remains elusive. Researchers have demonstrated with NMT that significant Ca²⁺ uptake and K⁺ loss occurred after PDT treatment resulting in the death of C6 glioma cells (Hu et al. 2014). Antagonist CNQX eliminated the changes of Ca²⁺ and K⁺ therefore direct evidence was provided for the involvement of changes in Ca²⁺ and K⁺ transportations in PDT interventions (Hu et al. 2014). To date, NMT has been successfully used to correlate both O₂ and Ca²⁺ fluxes to the early signals of PDT-induced apoptosis as well as the intracellular ROS (Song et al. 2015). Intracellular pH (pHi) and extracellular pH (pHe) are important parameters for maintaining various physiological functions. The drug resistance study method (DRSM) was established using non-invasive micro-test technology (NMT), which can be used to study the correlations among organs, tissues, extracellular ions, molecular activities and anti-cancer drug resistance (Ma et al. 2012). The study suggested the correlation between extracellular H⁺ activity and anti-cancer drug resistance by using ADR (doxorubicin) with both sensitive and resistant strains of MCF-7 cells (Song et al. 2008).

Ca²⁺ has been demonstrated to be a key player in pancreatic (Ren et al. 2022) functions (Cui and Kanno 1997). Abnormal insulin secretion of pancreas β cells is a major defect in type 2 diabetes. Impaired mitochondrial function and abnormal Ca²⁺ absorption have been previously found in studies in human and animal diabetic models. NMT experiments found that the change in Ca²⁺ influx caused by the abnormal mitochondria of pancreatic beta cells rather than Ca²⁺ channel activity is the main reason for abnormal insulin secretion in diabetic mice (Li et al. 2012a). Islet β cell transplantation is an ideal treatment for type-I diabetes and generating β cells from induced pluripotent stem cells (iPSCs) from patients is a promising strategy. Increased glucose levels lead to membrane depolarization resulting in an influx of Ca²⁺ which triggers insulin exocytosis. Therefore, researchers used Ca²⁺ influxes as one of the benchmarks to evaluate whether the pancreas β cells derived from iPSCs are healthy (Bai et al. 2021).

NMT also was widely applied in pharmacological research. Bumetanide is a potent loop diuretic increasing the urine flow by blocking NKCC2, which may relate to its side effects in the digestive system, such as loss of appetite, nausea or/and vomiting due to abnormal H⁺ secretion. H⁺ fluxes in the gastric mucosa of mice revealed by NMT demonstrate that bumetanide (10 μmo/L) significantly inhibited histamine-induced H⁺ fluxes, but had no effect on basal H⁺ fluxes, suggesting NKCC plays a role in acid secretion (Zheng et al. 2020). Entacapone, an inhibitor of Catechol-O-methyltransferase (COMT), is widely used in the treatment of Parkinson's disease (PD). However, about 10% of PD patients with entacapone treatment clinically experience diarrhea. NMT results indicate that the cause of the gastrointestinal side effects of entacapone is primarily the profound secretion of Cl⁻ (Li et al. 2015). Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a well-known ingredient of herb-based laxatives, but the working mechanism is still unclear. Cl⁻ secretion was found to be the main player in the emodin-induced colonic physiological process accompanied by mast cell degranulation and activation of cholinergic and non-cholinergic submucosal neurons (Xu et al. 2012).

NMT has also been used to detect apoptosis, which often occurs in inflammatory responses. Similar to Ca²⁺, which has been demonstrated to be involved in ROS production and mitochondria depolarization (Hu et al. 2008), K⁺ has been proven to be a player in apoptosis. Programmed cell death (apoptosis) occurs in almost all cells, including oocytes and embryos. Understanding the mechanism of apoptosis can provide cues for preterm birth, cancer and aging. When cells initiate programmed death by H₂O₂, cells shrink, potassium channels are activated, and K⁺ effluxes are detected outside the embryos (Trimarchi et al. 2000a, b).

Alzheimer's disease (AD) is a common, irreversible central nervous system degenerative disorder characterized by progressive cognitive dysfunction, memory loss, and psychobehavioral changes. Its pathology is characterized by massive extracellular deposition of amyloid-β (Aβ) that forms senile plaques and the intracellular accumulation of abnormally modified tau protein that forms neurofibrillary tangles (NFTs). The main pathogenesis hypotheses include: Aβ tangles, Tau hyperphosphorylation, energy metabolism disorders, oxidative stress, excitatory neurotoxicity, metal ion homeostasis, etc. (Chung et al. 2010; Ray et al. 2011; Shabala et al. 2010). Among them, the changes of Ca²⁺, K⁺, H⁺, O₂, and H₂O₂ plasma dynamic balance are closely related to the development of AD, which could be detected using NMT. Evidence suggest that transmembrane Ca²⁺ transportation regulation is crucial to understand the mechanism of many human diseases (Li et al. 2011). Especially, the intracellular Ca²⁺ overload is an important mechanism for the cytotoxic effects of Aβ. Therefore, rapid and accurate detection of dynamic changes in neuronal transmembrane Ca²⁺ flux can not only help to understand the mechanism of cell maintenance of Ca²⁺ homeostasis and normal functional activities, but also help to reveal the pathogenesis of AD and other diseases related to Ca²⁺ signaling. Researchers used NMT to study the effect of a novel anti-T2DM drug GLP-1/GIP/Gcg tri-receptor agonist (Triagonist) on the transmembrane fluxes of Ca²⁺ in neurons in ex vivo brain slices. The results showed that Triagonist maintained the Ca²⁺ homeostasis of neurons and avoided intracellular Ca²⁺ overload by regulating the transmembrane Ca²⁺ fluxes of neurons in the CA1 region of the hippocampus of 3xTg-AD mice (Li et al. 2020). Moreover, both K⁺ and Ca²⁺ have been found to be key players in the immune system and the brain (Shu et al. 2007). It is imperative to correlate the deposition of amyloid-β (Aβ) that forms senile plaques and the homeostasis of K⁺ and Ca²⁺ to tackle the mechanism of how AD develops. K⁺/Ca²⁺ fluxes data also suggest that long term exposure to Aβ is detrimental since it diminishes the ability of cortical neurons to maintain K⁺/Ca²⁺ homeostasis, which in turn may provide a new insight into the early indicators of AD (Ray et al. 2011; Shabala et al. 2010). One well-known feature of AD is a relatively higher level of reactive oxygen species (ROS) that overcomes the anti-oxidative stress mechanism of the brain. Meanwhile, the Cu (II)–Aβ complex has been demonstrated to be highly toxic to neurons, and Zn7MT-2A can alleviate the toxicity. In a previous study, a significant K⁺ efflux was detected with the presence of Cu(II)-Aβ1-40 suggesting that Cu(II)–Aβ can lead to disruption of K⁺ homeostasis in cultured cortical neurons, while the addition of Zn7MT-2A can inhibit the disruption (Chung et al. 2010; Howells et al. 2012).

Toxicology

Zebrafish embryos were used as a model to investigate the toxic effects of CuNPs. The results show that CuNP exposure can impair two subtypes of ionocytes and their associated functions, including Na⁺/Ca²⁺ uptake and H⁺/NH₄⁺ excretion in zebrafish embryos (Lee et al. 2020; Shabala et al. 2012).

Clinical medicine

Cell volume regulation is the basis of various cellular functions, such as cell division, cell proliferation, apoptosis, cell migration, and cell regulation, all of which require changes in cell volume and the involvement of transmembrane ions and molecules (Chen et al. 2011). Previously, K⁺ and Cl⁻ were found to be tightly coupled in regulatory volume reduction (RVD), it has been proved, however, not the case by using NMT. Not only for the first time that the transport of K⁺ and Cl⁻ is not coupled during hypotonic-induced RVD, but also H⁺ efflux has been found to play an important role in cell volume regulation, which may be a target for the treatment of nasopharyngeal carcinoma (Yang et al. 2012).

Myopia has been a public health issue because of its increasingly high prevalence worldwide. The occurrence of myopia correlates with both the disturbance of ionic homeostasis and the change in the local microenvironment of the ciliary muscle. K⁺ fluxes in ciliary muscles from guinea pigs demonstrated myopia-triggered K⁺ influx, which leads to the disturbance of the microenvironment within ciliary muscles, providing a possible approach to treating myopia in clinical practice (Wu et al. 2020). Using live catfish retinal level cells, H⁺ fluxes under the stimulation of a variety of substances suggested that the neurotransmitter Glu induced alkalinization at the outer surface of the cell membrane, and the size of the effect of Glu was related to the buffer, especially extracellular Ca²⁺ (Kreitzer et al. 2007).

Understanding the ionic homeostasis of wound healing at the single cell level is crucial to decipher the tissue, organ and or disease physiology (Zhang et al. 2022a).

Ca²⁺ influxes are required for single cell wound healing while tested with Xenopus laevis oocytes (Luxardi et al. 2014).

Furthermore, NMT has also been widely used in the mechanism study of traditional Chinese Medicine. Traditional Chinese medicine and leucous, astragalus sugar complex have been proposed to facilitate the rapid repair process of intestinal mucosa via activation signaling pathway of potassium channel mediated by polyamine. K⁺ effluxes were observed with leucocus and astragalus sugar complex treatments promoting the repair of gastrointestinal mucosal injury (Wang et al. 2018).

NMT was adopted to study the acupuncture mechanism. The results indicated that dynamic changes of Ca²⁺, Na⁺, and H₂O₂ fluxes had been observed. (1) In the early phase of skeletal muscle regeneration, Ca²⁺ efflux decreased, while Na⁺ influx increased, accompanied by increased H₂O₂ efflux; (2) Acupuncture intervention increased Ca²⁺ efflux in the early phase of skeletal muscle regeneration and advanced the Na⁺ influx phase, with the decrease of H₂O₂ efflux, and the effect was related to the interaction of TRP with NOX2 (Liu et al. 2018).

Membrane depolarization has been found to be accompanied by the transmural pressure, the typical myogenic response of renal and cerebral arteries of the rat. A Cl⁻ efflux from rat cerebral arteries with a temperature dependence correlated with myogenic contraction suggesting that Cl⁻ efflux through Cl⁻ channels contributes to the depolarization associated with myogenic contraction (Doughty and Langton 2001).

Oseltamivir phosphate (OP) is an antiviral drug that is used in the treatment and prophylaxis of both influenza A and influenza B. It is effective against all known influenza viruses that can infect humans, including pandemic influenza viruses and may be the most appropriate antiviral option against avian influenza caused by H5N1. An OP sensor based on ion association complexes and electrochemical reactions using a polyvinyl chloride (PVC) membrane has been successfully developed. The sensor can be used to determine (OP) in tablets. Compared to the reported HPLC method, the developed method is simple, accurate and precise (Hamza et al. 2017).

Acupuncture has a good therapeutic effect on skeletal muscle injury, and according to recent research trends, the mechanism is related to the TRP channel-mediated reactive oxygen species signaling pathway to restore early cytoplasmic Ca²⁺ homeostasis. The changes of Ca²⁺, Na⁺, and H₂O₂ dynamic flow rates in the early phase after skeletal muscle injury were detected by acupuncture intervention, aiming to explore the role of the interaction between TRP channel and NADPH oxidase 2 in the mechanism of acupuncture effect (Liu and Zhang 2018).

Uncoupling protein (UCP) is an inner mitochondrial membrane protein that eliminates transmembrane proton gradients on both sides of the inner mitochondrial membrane, slowing down the oxidative phosphorylation process driven by the proton gradient and hindering the normal production of ATP. Reduced UCP3 in prediabetes and diabetes is associated with insulin resistance, but the function of UCP3 is unclear. The study of the O₂ fluxes caused by UCP3 demonstrates no significant change in oxygen consumption in the UCP3 super expression group, and the chemical decoupling agent significantly increased oxygen consumption (MacLellan et al. 2005).

Animal study

Entomology

In insects, heterodimeric glycoprotein hormones play an important role in myelinization and cuticle hardening during development. With the recent discovery of two novel subunits forming an additional heterodimeric glycoprotein hormone in mammals, similar glycoprotein hormone forming subunits have been identified in insects. Research done by O’Donnell et al. suggests that AedaeGAP2/GPB5 can be used to regulate the physiological phenomenon of low Na⁺ and high K⁺ in the process of digestion and absorption of red blood cells by female mosquitoes, and direct evidence of physiological function has been obtained (Nguyen and Donini 2010; Paluzzi et al. 2014).

Animal physiology and behavior

E-C coupling contraction of the muscle relies on the movement of Ca²⁺ from different sources, such as the intracellular stores and/or from the extracellular media. The Ca²⁺ flux data suggested that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na-Ca exchanger as demonstrated by Li⁺ substitution experiments. NMT has provided new insights into the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling (Devlin and Smith 1996).

Plants biology

Abiotic stress

Quantitative K⁺/H⁺ fluxes under salt stress detected by NMT suggested a K⁺ preservation anti-salt stress mechanism via repolarization of the plasma membrane and silence of Guard cell outward-rectifying K⁺ (GORK) channel (Gong et al. 2023; Wu et al. 2024; Zeng et al. 2024). The other way to control K⁺ fluxes is to go through the key signal ROS that activates the Non-selective cation channel (NSCC) and the pathways generated extracellular of ROS (Pang et al. 2006; Shabala and Hariadi 2005; Sun et al. 2009c; Tang et al. 2018). Taking advantage of direct physiological evidences from NMT, a number of attempts have been made to find out a way to maintain IMH, Na⁺/K⁺ homeostasis specifically, to combat with plant salt stress (Chinnusamy et al. 2004; Guo et al. 2009; Lou et al. 2020; Qu et al. 2021; Shabala et al. 2005; Shahzad et al. 2022; Zhang et al. 2022b).

Extreme temperature is a key factor limiting global crop plant distribution and yield. NMT was able to demonstrate that there was a significant difference in Ca²⁺ influxes after cold treatment between COLD1/transgenic lines and corresponding wild types (Ma et al. 2015). Supported by Ca²⁺, H⁺, K⁺, and Na⁺ fluxes data, Lsi1 and OsCNGC9 genes have been found to enhance the cold tolerance of crops by regulating low-temperature-induced calcium influx and cytosolic calcium elevation (Liu et al. 2024; Wang et al. 2021).

Cd²⁺/Cu²⁺/Pb²⁺ influxes have been detected in live samples, such as roots, leaves, algae, biofilms, bacteria, and fungi, as well as with different live parts/organs, such as root xylem, stem, xylem duct, mesophyll cells, intact vacuoles, etc. (Cheng et al. 2023; Cuin and Shabala 2007; Jiang et al. 2022; Li et al. 2016, 2017a, 2024; Lv et al. 2018; Papoyan et al. 2007; Yang et al. 2022b; Zhang et al. 2020). The changes in the absorption of Mg²⁺, K⁺, and Ca²⁺ were studied with plant tissues and cells in the presence of heavy metals (Li et al. 2017a, 2017b, 2012b; Lin et al. 2013; Ma et al. 2016; Wang et al. 2022, 2023a). NMT data of Cu²⁺ and Cd²⁺ fluxes help to identify OsHIPP9/BcHIPP16 membrane proteins to absorb nutrient metal Cu and heavy metal Cd in plants (Niu et al. 2021; Xiong et al. 2023).

Drought is a great threat to crop yields and quality worldwide. lows of H⁺, K⁺, Ca²⁺ and H₂O₂ have been monitored with live plant samples to correlate energy, nutrient metabolisms, K⁺ homeostasis, signal transduction, ROS response to water control in plants (Chen et al. 2005; Li et al. 2019; Mak et al. 2014; Sun et al. 2024). NMT study provides important evidence for the dual role of plasma membrane H⁺-ATPases in light-induced stomatal opening and ABA-induced stomatal closure, highlighting the importance of H⁺ in plasma membrane cation and anion influx and efflux (Pei et al. 2022).

Plant immunity

NMT results found that herbivory pretreatment altered Ca²⁺ flux via different Ca²⁺ channels or transporter which help Ammopiptanthus nanus to prevent K⁺ leakage (Chen et al. 2020). Ca²⁺ flux data also indicated that the wheat susceptibility factor TaBln1, which interacts with TaCaM3 to impair Ca²⁺ influx could inhibit plant defenses (Guo et al. 2022). Ca²⁺ influx and H⁺ efflux data indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein, ROS, cytosolic Ca²⁺, and plasma membrane H⁺-ATPase (Gong et al. 2023; Hao et al. 2012; Sun et al. 2012; Zhao et al. 2016). NMT results indicated that melatonin, H₂O₂ and Ca²⁺ attenuated ABA‐induced K⁺ efflux and subsequent cell death (Guo et al. 2023).

Plant hormones signaling

After Indole acetic acid(IAA) treatment, the H⁺, K⁺, and Ca²⁺ fluxes of Arabidopsis thaliana roots were detected (Yang et al. 2022a), which provided key evidences for IAA-induced H⁺ alkalinization and then H⁺-induced exosomes in roots (Li et al. 2021). NMT experiments have indicated that the Ca²⁺ uptake of the mutant is impaired resulting in the pollen tube tip not moving towards the ovule. Meanwhile, the fluxes of Cl⁻ and K⁺ did not differ significantly between mutant and wild type (Meng et al. 2020).

Plant nutrition

Sa et al. (2019) found that root-associated ectomycorrhizal fungi (EMF) are involved in promoting root NO₃⁻ uptake of poplar by detecting NO₃⁻ fluxes around the root surface. Similarly, Peng et al. (2021) used the NMT to monitor and compare K⁺ flux profiles across the meristematic zone, root elongation and mature zones of Liquidambar styraciflua and observed that more K⁺ is absorbed by the roots colonized by a common soil saprotroph, Clitopilus hobsonii. Sun et al. (2022) demonstrated that fungus-regulated NO₃⁻/NH₄⁺ dynamics alters plant response to NO₃⁻/NH₄⁺ nutrition during symbiotic fungus Phomopsis liquidambaris-Arabidopsis interaction by examining N fluxes at the plant-fungal interface (Peng et al. 2021; Sa et al. 2019; Sun et al. 2022).

The acidity and alkalinity of the surrounding environment also affect ion exchange. In soybeans and coniferous trees, proton efflux at lower pH is beneficial for maintaining sustained absorption of NH₄⁺, for plants to adapt to acidic soil (Hawkins and Robbins 2010). Glomus mosseae (GM, fungus) and Bradyrhizobium japonicum (BJ, fungus) promoted the outflow of H⁺ in soybean roots, and the increase of phosphorus content was positively correlated with the outflow of H⁺ (Ding et al. 2012).

Environmental science

Bacteria biofilms use potassium channel-mediated electrical signals for cell-to-cell communication although it is unknown whether these signals play a role in Geobacter sp. when surrounded by an intense electric field.

Steady and reliable K⁺ fluxes detected by NMT provide preliminary evidence to reveal the role of potassium channels in electroactive biofilms in Agrobacterium reducing ensembles (Jing et al. 2020).

Seagrass is the only higher angiosperm that submerges in seawater. The seagrass ecosystem is, nevertheless, subject to numerous stressors brought on by human activity, including hypoxia, hyper salinity, eutrophication, temperature, and others. The findings from NH₄⁺ and H⁺ flux data demonstrated that an increase in NH₄⁺ content in seawater encouraged absorption, which might reprogram the nitrogen metabolism (Chen et al. 2019; Fang et al. 2020).

The safety of freshwater ecosystems is critical to humans, so real-time monitoring of levels of harmful chemicals and biological agents in freshwater environments is essential. Using O₂ fluxes as a Biosafety Indicator, a real-time effect of atrazine, cadmium chloride, pentachlorophenol, malathion, and potassium cyanide on respiratory oxygen consumption in fathead minnow embryos two days after fertilization was detected (Sanchez et al. 2008).

Plants use environmental signals to guide root growth and response so that they can adapt to the environment. For instance, roots can direct their growth and response in relation to gravity, light, gradients of temperature, humidity, ions, chemicals and oxygen. These environmental signals will be translated into physiological responses via ionic/molecular transportation. In root gravitropism, H⁺ efflux or Ca²⁺ influx in root apexes was weaker in IPG-grown roots than those in TPG-grown roots (Xu et al. 2013).

A list of live samples tested by NMT is provided in Table 1, and a list of categorized references by NMT applications fields is also made available as a quick reference with NMT (supplementary Table S1).

CONCLUSION

Abnormal IMHs can lead to pathology, while the activities of ions and small molecules are relatively difficult to study.

NMT, an integration of multi-techniques and multi-disciplinary knowledge, is evolving. It is constantly being improved functions to better fit their specific studies (Chi et al. 2021; Li et al. 2014; Lv et al. 2013; Song et al. 2017; Zhu et al. 2007).

The involvements of H⁺ and O₂ in tumor physiology have been studied (Hsu and Sabatini 2008; Parks et al. 2013; Seyfried and Shelton 2010), as well as H⁺ and/or O₂ manipulations in tumor micro-environments (Bailey et al. 2012; Parks et al. 2013; Yang and Li 2019; Zhang et al. 2021).

A few decades have passed since the first Ca²⁺ flux data was collected by biologists from a single cell (Jaffe and Nuccitelli 1974; Kuhtreiber and Jaffe 1990). NMT-based ions and molecules Omics (imOmics) is the study of IMH pertinent to a specific physiological process via collections and integral analysis of ions and/or molecules activities (Xu et al. 2023).

In this multi-dimensional-omics age, scientists are trying to make a functional network map, it has been demonstrated that the study of IMHs by NMT could play a significant role in not only connecting but verifying data from other techniques for a better understanding of a specific physiological function, more importantly from live samples.

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