What is happening at the electrode interface when the electrical field is modified due to the change of ion concentration after an AP?

What is happening at the electrode interface when the electrical field is modified due to the change of ion concentration after an AP?

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I have been working for quite a while now on electrophysiology and electrode fabrication.

I studied what is happening at the neuron level during an action potential (polarization/depolarization, change in ion concentration inside/outside the cell membrane) as well as the interface electrode/electrolyte (double layer, electrical model of an electrode, etc… ), but I am still struggling to tie all of those principle together. My question is, what is actually happening when an electrode is recording an AP (extracellular recording). To my knowledge, this is what is happening:

  1. AP triggered, change of ion concentration outside the cell membrane.
  2. As the ion concentration is modified, electrical field is modified around the cell

My question is now, how does the electrode record this change of electrical field? In almost all of the literature, a simple, but unexplained, shortcut is usually done: the authors usually speak about the change in ion concentration and then directly link it to a "change of potential at the electrode", but how can we explain this change of potential? What is happening at the electrode interface when the electrical field is modified due to the change of ion concentration after the AP? Is there any current flowing inside the electrode?

Thank you in advance

There is an art to expressing the nature of any kind of uncertainty in writing, and especially in experimental literature, to stating no more than has been empirically shown. An electrode interface only measures a one dimensional trace of what actually happens during an action potential cycle or spike, and it could be that in most of the literature, the precise mechanism by which the spike is measurable takes lower priority than the existence (or non-existence) of a particular signal.

According to Hodgkin-Huxley theory, the action potential is controlled through changes to the ionic permeability of certain membranes. The precise mechanisms by which this happens depend on the molecular structure of channels through which sodium and potassium ions flow. However, it is not hard to explain how a change in the permeability of a membrane can cause changes to an electric potential gradient (over 'large' length scales); the "deep concept" at work here is the second law of thermodynamics.

Let's suppose that you have two ions, one positive and one negative, confined to one side of a box as shown:

You might notice that there are more negative ions than there are positive ions, so there would be a nonzero electric potential gradient between the left and right hand sides of the container.

Now let's suppose that the confining membrane is replaced with one that allows only negative ions to pass (through the purple or tapered channels.) You might expect a fraction of the negative charge to flow over to the other side of container where the potential is slightly higher (as shown):

The result is that the original potential gradient (from left to right) is dampened slightly.

The OP is probably aware of this, but it turns out that replacing the membrane with one that allows positive charge to pass through results in a fraction of the total positive ions moving to the left side of the chamber (even though it would be attracted to the right hand side by the net negative charge)*. The reason for this is that the entropy gained by the first positive charge to move into the (log-macroscopically large) empty chamber vastly outweighs the (microscopic) entropy cost of leaving the occupied chamber with fewer positive ions to offset the negative ones.

This results in a slightly larger potential gradient than in the first (impermeable) case, as there are now positive charges in the region of higher electric potential.

The electrode can pick up on changes to the potential gradient by "grounding" the circuit at several points. In particular, this requires a circuit that is soft or sensitive enough that it does not disturb the ambient potential significantly (a tape measure or height rod adapts to your height, not vice versa.) That is, whatever current flows between the electrodes should be weak enough that it does not completely drain or equilibrate whatever local potential difference might exist, and other circuit elements should be weak enough that they do not cause flooding or, in a sense, drought of the natural electrodynamic state. Beyond this, the complex and densely packed environment of neurons would make it a challenge to place electrodes to isolate a single action potential, or even a potentially useful signal across several action potentials. Also, there is inevitably a certain amount of excessive unnatural current flow below a certain length scale (the scale at which the electrode starts to look like a bulk conductor.) Because of this, neuroscientists probably care more about the presence or absence of noise at this point than about the precise physiological origin of the signal, except insofar as the disturbance from the electrode is correctable and within tolerance.

*If the experiment is performed at nonzero temperature, which seems to be unavoidable.

The diffusion of generated H + ions in glucose sensing process is critical for the sensitivity of sensor. Although many glucose sensors assembled by two dimensional (2D) materials (MXene, graphene, MoS2 et al.) have been extensively studied, the detailed analyses about the diffusion of H + ions are still rarely reported. Herein, hybrid Ti3C2-rGO film is fabricated by electrostatic self-assembly utilizing the 2D Ti3C2 and graphene sheets to focus on the H + ion’s diffusion dynamic properties related to the sensing performance. Through controlling the graphene content, this hybrid Ti3C2-rGO film provide the effective H + ion diffusion channels. Based on the comprehensive analyses of cyclic voltammogram (CV) and electrochemical impedance spectroscopy (EIS), the corresponding localized conduction and enzymatic reaction at interfaces between the Ti3C2-rGO electrodes and glucose solution were investigated. According to the Warburg impedance in Nyquist plot from EIS, it is certified that the H + ion diffusion in low frequency range may play a significant role in the sensing mechanism of the glucose sensor. Moreover, the relationship derived from the interaction including interlayer spacing, H + ion diffusion and the sensing performance further reveal the H + ion diffusion as the dominant origin of the enhanced glucose sensitivity. This analysis will provide fundamental insight into the electrode structures assembled with 2D materials to optimize the sensor design in terms of ion transport dynamics.

Changying Cao is currently a master student at Shanghai Normal University in China. Her research interest focuses on the 2D materials and their applications in smart sensors.

Quanhong Chang received his Ph.D. degree from Harbin Institute of Technology, China in 2015. His research interests focus on the printing electronics, including the synthesis of 2D materials and electronic ink, and their applications in flexible printed energy storage and healthy sensors.

Huijie Qiao is currently a master student at Shanghai Normal University in China. His current research interests include the preparation and functionalization of the Fe-based magnetic nanostructured materials for printed sensors and memory decices.

Runze Shao is currently a master student at Shanghai Normal University in China. His current research interest comprises the synthesis of MXene-based 2D materials for future valuable memory devices.


With the increasing energy demand together with the deteriorating environment and decreasing fossil fuel resources, the development of highly efficient energy conversion and storage devices is one of the key challenges of both fundamental and applied research in energy technology. Melamine sponges (MS) with low density, high nitrogen content, and high porosity have been used to design and obtain three-dimensional porous carbon electrode materials. More importantly, they are inexpensive, environment-friendly, and easy to synthesize. There have been many reports on the modification of carbonized MS and MS-based composites for supercapacitor and lithium battery electrode materials. In this paper, recent studies on the fabrication of electrode materials using MS as raw materials have been mainly reviewed, including carbonation, doping activation, and composite modification of MS, and expectations for the development of porous carbon materials for energy storage as a reference with excellent performance, environment-friendliness, and long life.

Author summary

"Swine flu" illustrated that the spread of influenza pandemics in the modern era is rapid, making antiviral drugs the best way of limiting disease. One proven influenza drug target is the M2 proton channel, which plays an essential role during virus entry. However, resistance against licensed drugs targeting this protein is now ubiquitous, largely due to an S31N change in the M2 sequence. Understandably, considerable effort has focused on developing M2-N31 inhibitors, yet this has been hampered by controversy surrounding two potential drug binding sites. Here, we show that both sites can in fact be targeted by new M2-N31 inhibitors, generating synergistic antiviral effects. Developing such drug combinations should improve patient outcomes and minimise the emergence of future drug resistance.

Citation: Scott C, Kankanala J, Foster TL, Goldhill DH, Bao P, Simmons K, et al. (2020) Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza. PLoS Pathog 16(8): e1008716.

Editor: Anice C. Lowen, Emory University School of Medicine, UNITED STATES

Received: September 18, 2019 Accepted: June 19, 2020 Published: August 11, 2020

Copyright: © 2020 Scott et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by a Medical Research Council studentship awarded to CS (SG, RF), Medical Research Council grants G0700124 (S.G.) and L018578 (J.R.S), a Yorkshire Cancer Research Pump-Priming Award (S.G., LPP025), and a Pfizer Pump-Priming Award (SG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Dr. Paul Targett-Adams is a former employee of Pfizer Ltd. He was involved collaboratively with this project during inception and early stages and acted as a contact and advocate during the application for the Pfizer pump-priming award. He is presently an employee of Sygnature Discovery, who are not involved with this work. C. Scott is a previous employee of ReViral Ltd. and is now an employee of Covance. However, neither body supplied funding to, nor influenced this manuscript.

Tumor markers

Synthesize nanomaterials within cage-like protein templates has been demonstrated to be a suitable approach to produce uniform [35]. Ferritin nanocages provide surface modification and specific targeting abilities for synthesizing ferritin-based nanozymes [36]. Biomineralization synthesis of cobalt nanozyme in SP94-ferritin nanocage was reported for prognostic diagnosis of hepatocellular carcinoma (HCC) [37]. In this report, ferritin-based cobalt nanozyme (HccFn(Co3O4)) was designed for HCC diagnosis and therapy. SP94 peptide was modified onto the exterior surface of ferritin nanocage (HccFn) for specifically binding to HCC cells. HccFn(Co3O4) nanozymes specifically bound to HCC tissues and catalyze the oxidation of peroxidase substrate diaminobenzidine (DAB) to produce deep brown colorimetric reaction. In comparison with Fn(Co3O4) control group, HccFn(Co3O4) nanozymes specifically recognized and visualized HCC tissues and could distinguish tumor cells from normal tissues (Fig. 1).

HccFn(Co3O4) nanozymes specifically recognize and visualize clinical HCC tissues. a HccFn(Co3O4) nanozymes showed peroxidase-like activity and catalyzed the oxidation of peroxidase substrate diaminobenzidine (DAB) to produce colorimetric reaction. b Schematic diagram of HccFn(Co3O4)-based immunohistochemical approach. c HccFn(Co3O4)-based immunohistochemical staining (top row) and Fn(Co3O4)-based immunohistochemical staining (bottom row) of HCC tissues and non-tumor liver tissues

The nanomaterial-mediated colorimetric sensor is an attractive system for advance instrument-free bioanalysis due to its unique advantages of simplicity in operating analysis via camera or smartphone [38, 39]. Several colorimetric assays based on the 3,3,5,5 tetramethylbenzidine (TMB)-H2O2 system catalyzed by enzyme mimic nanomaterials have been extensively developed for immunoassay [40,41,42]. For instance, Alizadeh et al. present a paper-based microfluidic colorimetric immunosensor for the detection of carcinoembryonic antigen (CEA), using Co2(OH)2CO3-CeO2 nanocomposite with extraordinary intrinsic peroxidase like activity [43]. The proposed immunosensor facilely prepared by modifying mixture of ionic liquid and chitosan functionalized primary antibodies (Ab1) on the surface of paper. Co2(OH)2CO3-CeO2 peroxidase mimicking enzyme was functionalized secondary antibodies (Ab2) and used as a signal tag. Co2(OH)2CO3–CeO2 nanocomposite catalyzed the oxidation of 3,3′,5,5′-tetramethyl benzidine in the presence of H2O2, resulting in a color change, which acquired as the immunosensor response. The color change was distinct by the naked eye and analyzed by an installed application on the smartphone (Fig. 2).

Schematic illustration and assay procedure of CEA detection on the paper-based chip

In colorimetric assays, color changes and photothermal effect of TMB-H2O2 colorimetric system have been prospected [44]. In this regard, nanoparticle (NPs)-mediated photothermal immunoassay platform was developed for detection of prostate-specific antigen (PSA) using a common thermometer as the quantitative signal reader [45]. The iron oxide NPs-labeled antibody was applied as the detection probe, on basis of sandwich-type proof-of-concept immunoassay. In the immunoassay, iron oxide artificial enzyme demonstrated color changes and also a strong NIR laser-driven photothermal effect, simultaneously. The oxidized TMB acted as a highly sensitive photothermal probe to convert the immunoassay signal into heat via its photothermal effect (Fig. 3).

Schematic illustration of the photothermal immunoassay platform based on the photothermal effect of the iron oxide NPs mediated TMB-H2O2 colorimetric system

Aptamers are artificial synthetic single-stranded DNA or RNA oligonucleotides, which can bind with various targets such as protein, peptide, organic/inorganic molecule, and cell with high affinity and specificity. Aptamer with superiority to antibodies, including high stability, ease of synthesis, low cost and easy chemical modification, have attracted a lot of attention in biomedical and bioanalysis research [46,47,48]. Zhao et al. selected three hairpin anti-MUC1 DNA aptamers for construction of a sensitive electrochemical aptasensor based on catalytic hairpin assembly coupled with PtPdNPs peroxidase-like activity [49]. After binding with target protein, Apt-HP1 containing aptamer sequence was opened and MUC1-aptamer binding complex formed (Fig. 4). Next, the exposed segment of HP1 would attack HP2 immobilized on the electrode to form a double strand structure. Then, the new exposed segment of HP2 hybridized with the toehold of PtPdNPs modified HP3. Finally, MUC1-A was released via the strand displacement process, and the released MUC1-A could participate in the subsequent reaction cycles. The carried PtPdNPs, as a mimic peroxidase probe, catalyzed the TMB by H2O2, leading to the electrochemical signal generation.

Schematic representation of the aptasensor for the detection of MUC1


Understanding ion relaxation dynamics in overlapping electric double layers (EDLs) is critical for the development of efficient nanotechnology-based electrochemical energy storage, electrochemomechanical energy conversion, and bioelectrochemical sensing devices as well as the controlled synthesis of nanostructured materials. Here, a lattice Boltzmann (LB) method is employed to simulate an electrolytic nanocapacitor subjected to a step potential at t = 0 for various degrees of EDL overlap, solvent viscosities, ratios of cation-to-anion diffusivity, and electrode separations. The use of a novel continuously varying and Galilean-invariant molecular-speed-dependent relaxation time (MSDRT) with the LB equation recovers a correct microscopic description of the molecular-collision phenomena and enhances the stability of the LB algorithm. Results for large EDL overlaps indicated oscillatory behavior for the ionic current density, in contrast to monotonic relaxation to equilibrium for low EDL overlaps. Further, at low solvent viscosities and large EDL overlaps, anomalous plasmalike spatial oscillations of the electric field were observed that appeared to be purely an effect of nanoscale confinement. Employing MSDRT in our simulations enabled modeling of the fundamental physics of the transient charge relaxation dynamics in electrochemical systems operating away from equilibrium wherein Nernst–Einstein relation is known to be violated.

The skin of adult frogs is well known as a transporting epithelium that sustains a TEP of ∼100 mV, inside positive, across the multilayered epithelium (102). The ion transport properties of the apical and basolateral cell membranes differ and are polarized (Fig. 1D). The apical cell membrane contains specialized amiloride-sensitive sodium channels allowing Na + to enter the cells, while the basolateral membranes contain the well-known “sodium pump,” the ouabain-sensitive Na + -K + -ATPase that electrogenically pumps three Na + from the cytoplasm out into the extracellular fluid in exchange for two K + entering the cells. Net ion transport therefore occurs across the epithelium, with Na + being transported from pond water into the animal. A high-resistance electrical “seal” exists between neighboring cells in most epithelial sheets including amphibian skin. This is formed by tight junctions, and these greatly reduce the electrical conductivity of the paracellular space (5). Viewed end-on, the apical surface of each epithelial cell appears to be encircled by strands of specialized tight junctional proteins. Strands on neighboring cells abut each other to form a “seal” that restricts the paracellular passage of solutes and water. The whole structure may be pictured as similar to a series of interconnecting hoops around the end of a barrel. The same basic elements of polarized channels, pumps, and tight junctions are found in embryonic frog skin, and these also establish a TEP from very early stages of development (131, 168).

1. Voltage gradients exist within the extracellular spaces underneath the skin

Crucially, the potential difference across the skin is different in different parts of the developing embryo (180). With the use of standard glass microelectrodes, stable gradients of voltage have been measured around the neural plate area in axolotl embryos during the period of neurulation. Skin potentials are higher rostrally than caudally (Fig. 4, A–C). This is particularly marked at the head end of the embryo, where EFs of 75–100 mV/mm have been measured in the extracellular space below the epithelium (180), whilst EFs of 30 mV/mm are present in the region rostral to the developing blastopore (173). In both cases the orientation of the steady voltage gradient is rostrocaudal, that is, along the long axis of the embryo (head to tail). The skin potential also is high at the midline of the neural plate as it begins to fold over to become the neural tube, decreases at the neural folds, and increases again further laterally on the flank of the embryo. This pattern of skin potential gives rise to standing voltage gradients on either side of the dorsal midline, with a mediolateral orientation (Fig. 4, A–C).

FIG. 4.Spatial differences in the transepithelial potential difference (TEP) generate electric fields within intact embryos. A: TEP measurements made using glass voltage-sensing electrodes. The TEP of axolotl embryos was measured relative to a bath ground electrode at the time of early neural tube formation (stage 16) in three positions on the same embryo. Measurement sites are shown in B: a, at the rostral end of the neural groove b, at the lateral edge of the neural fold c, at the lateral epithelium d, halfway along the neural groove e, at the caudal end of the neural groove near the blastopore. The TEP at each site was positive on the inside of the embryo. [Data from Shi and Borgens (180).] B: TEP measurements made at sites a, b, and c in 8 embryos demonstrating that the TEP is highest in the center of the neural groove (a) and lowest at the lateral edge of the neural ridge (b). Measurements from 20 embryos at sites a, d, and e indicate a rostral to caudal TEP gradient. [Data from Shi and Borgens (180) embryo in B and D modified from Borgens and Shi (26).] C: an artist’s impression of the spatial differences of TEP in a stage 16 axolotl embryo. Colors represent the magnitude of the TEP. Yellow is highest, and purple is lowest. The slope of the line indicates the magnitude of the resulting local electric field in the subepidermal tissues. [Modified from Shi and Borgens (180).] D: current loops detected using a noninvasive vibrating electrode. The electrode vibrates rapidly near the embryo in an electrically conductive medium (e.g., pond water). The stainless steel electrode has a small voltage-sensing platinum ball at its tip, which is vibrated rapidly over a distance of ∼20 μm. The electrode (red) is shown at the extremes of its vibration. The voltage is determined at each point, and the current density at the measurement site is calculated using known values for distance from the embryo and the resistivity of the bathing medium. As would be predicted from the spatial variation of TEP illustrated in A and B, there is outward current at the lateral edges of the neural ridges, inward current at the center of the neural groove, inward current at the lateral skin, and a large outward current at the blastopore.

The point was made above that voltage gradients and current flow are linked inextricably, and these findings of voltage gradients using glass microelectrodes impaling the skin have been paralleled using a second, less familiar technique that measures current flow noninvasively around the outside of the embryonic skin. This technique uses a vibrating microelectrode (90, 91) that is driven at 300 Hz between two extreme positions roughly 20 μm apart. With the vibrating microelectrode placed close to an embryo within an electrically conducting medium and all signals filtered out other than those at 300 Hz, the probe records the voltage at the two extremes of its excursion, close to and more distant from the skin surface (Fig. 4D). With the use of the resistivity of the medium bathing the embryo, the differential voltage signals between the extremes of vibration are converted to current flowing in or out of the embryo at one point. Moving the location of the probe allows current flow to be mapped spatially around an embryo. Currents of 100 μA/cm 2 have been measured exiting the blastopore in Xenopus embryos at developmental stages 15–20 (a period spanning the formation of the neural tube Fig. 4D) (84). The size of these measured currents is consistent with the rostrocaudal and mediolateral TEP gradients described in Figure 4. Since the resistivity of soft tissues is ∼1,000 Ω · cm, current densities of 100 μA/cm 2 give rise to voltage gradients of ∼10 mV/mm. Importantly, the anterior neural folds also are sites of current exit, and here current densities of 2 μA/cm 2 have been measured. The sites of current leaks are regions of major tissue movements (173). Because tissue movements disrupt tight junctional seals transiently (47) and therefore reduce tissue resistivity locally (but not in distal areas), current flows parallel to the tightly sealed epithelium in areas of high resistivity (intact tight junctions) and exits the embryo in regions of low resistivity (where tight junctions have broken down) (Fig. 4D).

Although these two techniques demonstrate clearly that electrical signals exist during neurulation in amphibians, the signals at the blastopore and at the neural folds may have different functions. This is because the blastopore current persists after closure of the neural tube, but the rostrocaudal and mediolateral voltage gradients at the buckling neural plate stage disappear as the neural folds fuse at the end of neurulation (Fig. 5). The functional significance of switching spatially localized DC EFs on and off during gastrulation and neurulation, which are major developmental milestones, has been tested.

FIG. 5.Neural tube development. A: neural tube formation begins with a thickening of the ectoderm at the dorsal midline to form a neural groove flanked by raised neural ridges. B: the neural ridges fold upward gradually. C: eventually they fuse at the dorsal midline. D: after fusion the neural tube is a distinct structure, covered by a continuous epithelium (green). The resulting neural tube is therefore derived from the ectoderm in such a way that the lumen of the neural tube is historically the equivalent of the apical side of the ion-transporting embryonic epidermis. Note that the walls of the neural tube are thicker laterally and thinner ventrally at the floor plate (yellow), nearest to the notochord.

2. Disrupting the natural EFs in amphibians disrupts development

If the EFs in embryos play a significant role in development, disrupting the normal electrical milieu of embryos would cause developmental defects. This has been tested directly in amphibians using two experimental approaches. The first involved placing whole axolotl embryos in an externally applied EF of physiological magnitude for a period spanning either gastrulation or neurulation (∼18–22 h). The applied EF was designed to disrupt the pattern and magnitude of the endogenous EFs by altering the TEP in a predictable way (139).

With no EF exposure (normal TEP), abnormalities occurred in no more than 17% of embryos. In embryos with no specific orientation to the imposed EF, 62% developed abnormally in an EF of 75 mV/mm and 43% at 50 mV/mm. Because the main axes of the measured endogenous voltage gradients run rostrocaudally and mediolaterally within the neural plate (Fig. 4), experiments were made using embryos with these body axes aligned with the EF vector. When the EF was imposed during neurulation with a cathode at the rostral end of the embryos, 95% (19/20) developed abnormally. When the cathode was caudal, 93% were abnormal, and of those with the long (rostrocaudal) embryonic axis perpendicular to the applied EF, 75% (12/16) were abnormal. The types of defects seen were profound and included absence of the cranium, loss of one or both eyes, a misshapen head, abnormal or absent brachial development, and incomplete closure of the neural folds in focused areas. In some cases, cells from the neural plate migrated out from the embryo onto the dish and continued to develop autonomously. In another experiment an EF of identical duration and magnitude was applied to embryos undergoing gastrulation. The EF was switched off at the end of gastrulation, and the embryos were allowed to develop to stage 36. Importantly, these embryos developed completely normally.

Interestingly, the polarity of the applied EF predicted the polarity of the developmental abnormalities that were induced. The most striking disruptions were seen in areas facing a cathode, where the skin TEP had become hyperpolarized. When the rostral (head) end of the embryo faced the cathode, head defects predominated, and when the caudal (tail) end of the embryo faced the cathode, lower abdominal and tail defects predominated (139).

The disruption to the embryonic skin TEP by applying an EF externally was marked and was polarized in a predictable manner. The TEP, which is normally internally positive, depolarized and switched polarity to become increasingly negative internally in regions of skin at the anode in an EF of 25–100 mV/mm. At the other end of the embryo, the TEP of skin under the cathode hyperpolarized and became increasingly more positive internally. Because the TEP was altered in this striking manner by the externally applied EF, the endogenous gradients of extracellular voltage under the skin will be scrambled by this imposed applied EF.

These experiments allow three conclusions. 1) Endogenous EFs with normal polarity and magnitude are essential for normal development of the nervous system and other tissues. 2) There was no generalized harmful effect of an applied EF since embryos exposed at gastrulation developed normally. 3) Embryos responded to scrambling of their endogenous EF only during the period when cells were undergoing neurulation. This suggests that neuronal cells gain and then lose the ability to respond to an applied EF during a specific window of developmental time.

The second approach to modifying endogenous EFs involved impaling Xenopus embryos with glass microelectrodes, similar to those used to measure the TEP. Current was passed through the electrodes so that the endogenous current leaving the blastopore was reduced or reversed (84). The effect of injecting current on the magnitude of the blastopore currents was measured directly using the vibrating microprobe. Injected current at 100 nA nulled the blastopore current, and 500 nA approximately reversed it. Eighty-seven percent of embryos (20/23) injected with this level of current for 9–11 h at stage 14–16 showed gross external developmental abnormalities. These included the formation of ventral pigmented bulges, failure of the anterior neural tube to close, reduced head development, retarded eye formation, the extrusion of cells from the blastopore into the dish, and failure of embryos to form functional cilia. Control embryos with long-term impalement of an electrode in the same region but no current, those with low current (10 nA, which did not affect the magnitude of the natural blastopore current), or those with reversed current that augmented the blastopore current, showed a much lower rate of abnormality.

Therefore, although two markedly different methods have been used to disrupt the endogenous EFs of amphibian embryos, they nevertheless induced a surprisingly uniform and striking array of developmental defects. Brain and tail structures were especially vulnerable and, significantly, these are regions of endogenous outward currents and of measurable steady DC voltage gradients in the embryo. The high incidence of neural tube defects also may be significant. Mutations in a number of genes such as sonic hedgehog cause neural tube defects (41), so it would be instructive to determine the expression levels of these genes in normal embryos and in embryos where the endogenous EFs have been disrupted. Conversely, it would be useful to determine whether disruption of endogenous EF affects expression patterns of key developmental genes.

One interpretation of the experiments outlined above is that steady voltage gradients within embryos provide a gross template for the development of pattern during ontogeny and that developmental abnormalities resulted from experimentally scrambling electrical cues necessary for patterning and cell migration within the embryo.

3. Endogenous currents and voltage gradients are present in chick embryo: disrupting these disrupts development

Importantly, currents and voltage gradients that are analogous to the blastopore and neural fold currents in amphibians have been measured in chick embryos (81). In stage 15–22 chick embryos, ionic currents greater than 100 μA/cm 2 leave the embryo via the posterior intestinal portal (PIP). This is the period of tail gut reduction when there is extensive cell death at the caudal end of the embryo. The PIP is the opening into the hindgut from the yolk sac. These currents enter through the ectoderm of the embryo and upon flowing through the embryo generate a caudally negative voltage gradient of ∼20 mV/mm. If these voltage gradients play a necessary role in embryogenesis, then disrupting them should alter development. This has been tested by creating an alternative path for current flow out of the embryo. Hollow capillaries that formed an ectopic region of low resistance were implanted to create, in effect, a permanent, nonhealing wound (83). This procedure reduced the magnitude and altered the internal pattern of the natural EF. Conductive implants designed to shunt currents out of the embryo were placed under the dorsal skin at the midtrunk level (stage 11–15). These hollow capillary shunts were ∼100 μm outside diameter and 1 mm long and were filled with saline, in some cases gelled with 2% agarose to control against bulk fluid transfer between the embryo and its surroundings. They were placed perpendicular to the neural tube in a slit ∼250 μm long and inserted around 500 μm under the ectodermal epithelium parallel to the neural axis. Currents of 18 μA/cm 2 left the conductive shunts. The net effect was to reduce the current leaving the PIP of these embryos by 30%. Ninety-two percent (25/27) of these embryos developed with gross abnormalities. The most common defect was in tail development, with the neural tube, notochord, and somites all either missing or truncated in the tail region. There were defects also in limb and head/brain development, but the frequency of defects increased in a rostrocaudal direction. Forty-four percent of embryos showed multiple developmental defects. Nonconductive, solid rod implants of the same dimensions were used in control embryos. No currents were measured escaping from these implants, nor did the implants influence the magnitude of the currents leaving the PIP. Only 11% (2/18) of control embryos with solid implants showed any developmental abnormalities all the others developed completely normally, despite the continuous presence of the nonconducting implant. The abnormalities seen in experimental embryos were very similar to those produced in rumpless chicks, a naturally occurring mutation which can result in complete absence of all caudal structures (228). Vibrating probe measurements from rumpless chicks showed that currents leaving the PIP were ∼41% of the PIP current in normal embryos (83), suggesting that this electrical deficit contributes, at least in part, to the tail structure deletions.

Several aspects of these experiments are important. 1) They confirm that currents and endogenous voltage gradients are present during major episodes of chick development and are greatest in the tail region. 2) Reducing the PIP currents by shunting current out at an ectopic dorsal location has the greatest developmentally disruptive effect in the tail region. 3) The shunt placement is several millimeters away from the site of the main defects, indicating that current shunting did not have nonspecific and deleterious local effects. 4) Solid shunts had no effect. 5) A naturally occurring chick mutation may cause tail deformities because of aberrant electrical signals.

A further point of interest and one which requires further study is that the primitive streak of the chick embryo, which is analogous to the amphibian blastopore, also is a site of large outward currents of ∼100 μA/cm 2 (94). A physiological role for these currents has not been explored.

4. A voltage gradient exists across the neural tube and neuroblasts differentiate in this gradient

The vertebrate neural tube forms when the lateral edges of the neural plate thicken, rise up, and fold over to fuse with each other at the dorsal midline (Fig. 5). A hollow tube called the neural tube that develops to become the brain and spinal cord forms from the folded ectoderm and then detaches to lie below the skin. The luminal surface of the neural tube therefore is equivalent to the outer surface of embryonic skin. Because spatial and temporal differences in the electrical properties of embryonic skin generate steady endogenous electrical signals (see above), the neural tube has been investigated to determine whether similar electrical signals are generated across its wall. Amphibian neural tube does establish a potential difference across its wall known as the transneural tube potential (TNTP Fig. 6) (82, 179). In axolotl this may be as large as 90 mV, with the lumen negative with respect to the extracellular space at stage 28. Because the wall of the neural tube is roughly 50 μm wide, this large potential difference would create a steady voltage gradient across cells in the neural tube wall of a remarkable 1,800 mV/mm [90 mV/50 μm = 180 mV/100 μm = 1,800 mV/mm]. The neuroblasts (neuronal precursors) within the wall must migrate, differentiate, and sprout directed axonal projections whilst exposed to this high, continuous extracellular EF. The TNTP is largely the result of transporting Na + out of the lumen, and this can be prevented pharmacologically by injecting benzamil or amiloride into the lumen. When this was done in axolotl embryos at stage 21–23 and the embryos were allowed to develop for 36–52 h, by which time uninjected embryos had developed to stage 34–36, the TNTP collapsed for several hours. In all of 28 embryos tested, collapse of the TNTP caused major abnormalities in cranial and central nervous system (CNS) development. The defects were characterized by a disaggregation of cells from structures that had already begun to form (26). The cells that had comprised the optic and otic primordia, brain, neural tube, and notochord disaggregated, but did not die, whilst new internal structures failed to form. In effect, the internal structure of most embryos had been reduced to a formless mass of apparently dedifferentiated cells, simply by collapsing the TNTP. Remarkably, the external form of some embryos with collapsed TNTP continued to develop, despite the complete absence of concomitant internal histogenesis (26). Making similar injections of a vehicle solution into the neural tube, or of the active agents amiloride, or benzamil beneath the embryonic skin immediately adjacent to the neural tube had no effect on the TNTP and did not disturb development. This shows that neither the injection, nor the drugs, had a generalized toxic effect on the embryos and that the disaggregation of the neural tube and other internal structures was a consequence of collapse of the TNTP.

FIG. 6.Measurement of the trans-neural tube potential (TNTP) in an axolotl embryo by steady advance of a glass voltage-sensing electrode. A: initially the electrode penetrates the ectoderm (1) and records the TEP. It then advances through the wall of the neural tube, resting in the lumen (2) to record the neural tube potential (NTP). Then the electrode penetrates the far side of the neural tube to again record the TEP (3). The diagram shows the recording position of the tip of a single electrode as it advances through the tissue layers. B: a sample recording from the experiment described in A. Penetration of the ectoderm (1) indicates a TEP (blue bar) of ∼20 mV, inside positive (relative to a reference electrode in the bath). At 2, the electrode penetrates into the lumen of the neural tube, recording the NTP. The sharp downward deflection indicates that the lumen is negative (−30 mV) relative to the bath (pink bar). The sum of the TEP and NTP represents the TNTP (green bar). In this example the TNTP is about −50 mV (lumen negative relative to the outside of the neural tube). When the electrode tip is advanced out of the lumen through the far wall of the neural tube (3), there is a sharp upward deflection, in which the TEP of −20 mV is recorded again (second blue bar). At 4, the electrode is withdrawn from the embryo. There is a sharp return to the reference baseline, which has remained stable throughout the experiment. C: a cross-section through a stage 23 axolotl embryo. To confirm that the electrode was positioned in the neural tube lumen, a fluorescent label (TRITC-con A) was iontophoresed into the neural tube from the same electrode used to measure the NTP at point 2 above. [B and C redrawn from Shi and Borgens (179).]

The presence of a strong electrical gradient across the wall of the neural tube and its role in maintaining the development of the neural tube itself (and other internal organ systems) are surprising findings with profound implications. These include 1) the voltage drop across the wall of the neural tube will not be uniform, but will be steepest across the cells lining the lumen of the neural tube, because this region of tight junctional sealing is the area with the highest electrical resistance. Division and differentiation of presumptive CNS neurons begins at the lumen, and intriguingly, we have shown that the axis of cell division can be determined by applied and endogenous EFs an order of magnitude less than those across the neural tube (220, 184 see below). The axis of presumptive neuroblast cell division is regulated developmentally by segregating and polarizing a variety of proteins (e.g., numb, miranda, prospero) within neuroblasts as they prepare to divide. It would be worth testing whether the polar distribution of these molecules, which determines the axis of neuroblast division, is determined by the polarity of the TNTP. 2) Because the neural tube varies in thickness, the largest EF (given a spatially uniform TNTP) will be across the thinnest region of the wall, which is the floor plate. This is an area of key importance in CNS patterning and neuronal differentiation. 3) Finally, the number of neuroblasts that are stimulated to develop in culture increases markedly when a small DC EF is applied across these cells. Borgens suggests that this could be because culturing developing neurons without a weak polarized gradient of voltage imposed across them does not adequately mimic their in vivo environment (26).

In short, vertebrate embryos possess steady voltage gradients, particularly in areas where major developmental events related to cell movement and cell division are occurring. Disrupting these electrical fields disrupts normal development.

What is a Biosensor?

Biosensors can be defined as analytical devices which include a combination of biological detecting elements like a sensor system and a transducer. When we compare with any other presently existing diagnostic device, these sensors are advanced in the conditions of selectivity as well as sensitivity. The applications of these Biosensors mainly include checking ecological pollution control, in the agriculture field as well as food industries. The main features of biosensors are stability, cost, sensitivity, and reproducibility.

The short form of the biological sensor is known as a biosensor. In this sensor, a biological element is maybe an enzyme, a nucleic acid otherwise an antibody. The bio-element communicates through the analyte being checked & the biological reply can be changed into an electrical signal using the transducer. Based on the application, biosensors are classified into different types like resonant mirrors, immune, chemical canaries, optrodes, bio-computers, glucometers & biochips.

Main Components of a Biosensor

The block diagram of the biosensor includes three segments namely, sensor, transducer, and associated electrons. In the first segment, the sensor is a responsive biological part, the second segment is the detector part that changes the resulting signal from the contact of the analyte, and for the results, it displays in an accessible way. The final section comprises an amplifier which is known as a signal conditioning circuit, a display unit as well as the processor.

Image Source

Working Principle of Biosensor

Usually, a specific enzyme or preferred biological material is deactivated by some of the usual methods, and the deactivated biological material is in near contact with the transducer. The analyte connects to the biological object to shape a clear analyte which in turn gives the electronic reaction that can be calculated. In some examples, the analyte is changed to a device that may be connected to the discharge of gas, heat, electron ions, or hydrogen ions. In this, the transducer can alter the device linked convert it into electrical signals which can be changed and calculated.

Working of Biosensors

The electrical signal of the transducer is frequently low and overlays upon a fairly high baseline. Generally, the signal processing includes deducting a position baseline signal, obtained from a related transducer without any biocatalyst covering.

The comparatively slow character of the biosensor reaction significantly eases the electrical noise filtration issue. In this stage, the direct output will be an analog signal however it is altered into digital form and accepted to a microprocessor phase where the information is progressed, influenced to preferred units, and o/p to a data store.

Before discussing the different types of biosensors and their uses, we have to discuss the simple example of this biosensor like Glucometer. This is most frequently used in different medical applications. We know that diabetes is one of the dangerous diseases that characterize the glucose levels within the blood of human bodies. So for diabetes patients, checking glucose levels within the blood is essential. For that, a glucometer is used as a biosensor to measure the glucose concentration within the human blood.
Generally, a glucometer includes a strip for testing.

This strip collects the blood sample and checks the glucose level within the blood. This strip includes a trigger as well as a reference-type electrode. Once a blood sample is poured on the strip, then a chemical reaction takes place to generate an electrical current that is directly proportional to the glucose concentration. The processor used in the glucometer is Cortex-M3 otherwise Cortex-M4 through the flow of current toward filter, amplifier, voltage converter, a display unit.

Evolution of Biosensor

The classification of Biosensors can be done into 3 generations based on the amount of incorporation of the separate component like the technique of connection of the bioreceptor molecule otherwise biorecognition toward the element of the base transducer.

In the 1st generation, the molecule of the bioreceptor is entrapped physically within the area of the base sensor after a discriminating membrane like a dialysis membrane. In the next generations, the achievement of immobilization can be done through covalent bonds on a properly customized transducer interface otherwise by inclusion into a polymer matrix on the surface of transduction.
In the 2nd generation, the individual components stay separate like control electronics, bio-molecule & electrode.

In the 3rd generation, the molecule-like bio-receptor turns into an essential element of the base sensing element whereas these definitions were possibly planned for enzyme electrode systems, related classifications are suitable to biosensors usually can be made. It is within the 2nd & 3rd generations of families that the main development attempt can currently be observed.


A biosensor includes two main distinct components like Biological component such as cell, enzyme and a physical component like an amplifier and transducer.

The biological component identifies as well as communicates through the analyte for generating a signal that can be sensed through the transducer. The biological material is properly immobilized over the transducer & these can be frequently used numerous times for a long period.


In 1886, Eugen Goldstein observed rays in gas discharges under low pressure that traveled away from the anode and through channels in a perforated cathode, opposite to the direction of negatively charged cathode rays (which travel from cathode to anode). Goldstein called these positively charged anode rays "Kanalstrahlen" the standard translation of this term into English is "canal rays". Wilhelm Wien found that strong electric or magnetic fields deflected the canal rays and, in 1899, constructed a device with perpendicular electric and magnetic fields that separated the positive rays according to their charge-to-mass ratio (Q/m). Wien found that the charge-to-mass ratio depended on the nature of the gas in the discharge tube. English scientist J. J. Thomson later improved on the work of Wien by reducing the pressure to create the mass spectrograph.

The word spectrograph had become part of the international scientific vocabulary by 1884. [2] [3] Early spectrometry devices that measured the mass-to-charge ratio of ions were called mass spectrographs which consisted of instruments that recorded a spectrum of mass values on a photographic plate. [4] [5] A mass spectroscope is similar to a mass spectrograph except that the beam of ions is directed onto a phosphor screen. [6] A mass spectroscope configuration was used in early instruments when it was desired that the effects of adjustments be quickly observed. Once the instrument was properly adjusted, a photographic plate was inserted and exposed. The term mass spectroscope continued to be used even though the direct illumination of a phosphor screen was replaced by indirect measurements with an oscilloscope. [7] The use of the term mass spectroscopy is now discouraged due to the possibility of confusion with light spectroscopy. [1] [8] Mass spectrometry is often abbreviated as mass-spec or simply as MS. [1]

Modern techniques of mass spectrometry were devised by Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919 respectively.

Sector mass spectrometers known as calutrons were developed by Ernest O. Lawrence and used for separating the isotopes of uranium during the Manhattan Project. [9] Calutron mass spectrometers were used for uranium enrichment at the Oak Ridge, Tennessee Y-12 plant established during World War II.

In 1989, half of the Nobel Prize in Physics was awarded to Hans Dehmelt and Wolfgang Paul for the development of the ion trap technique in the 1950s and 1960s.

In 2002, the Nobel Prize in Chemistry was awarded to John Bennett Fenn for the development of electrospray ionization (ESI) and Koichi Tanaka for the development of soft laser desorption (SLD) and their application to the ionization of biological macromolecules, especially proteins. [10]

A mass spectrometer consists of three components: an ion source, a mass analyzer, and a detector. The ionizer converts a portion of the sample into ions. There is a wide variety of ionization techniques, depending on the phase (solid, liquid, gas) of the sample and the efficiency of various ionization mechanisms for the unknown species. An extraction system removes ions from the sample, which are then targeted through the mass analyzer and into the detector. The differences in masses of the fragments allows the mass analyzer to sort the ions by their mass-to-charge ratio. The detector measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. Some detectors also give spatial information, e.g., a multichannel plate.

Theoretical example Edit

The following example describes the operation of a spectrometer mass analyzer, which is of the sector type. (Other analyzer types are treated below.) Consider a sample of sodium chloride (table salt). In the ion source, the sample is vaporized (turned into gas) and ionized (transformed into electrically charged particles) into sodium (Na + ) and chloride (Cl − ) ions. Sodium atoms and ions are monoisotopic, with a mass of about 23 u. Chloride atoms and ions come in two isotopes with masses of approximately 35 u (at a natural abundance of about 75 percent) and approximately 37 u (at a natural abundance of about 25 percent). The analyzer part of the spectrometer contains electric and magnetic fields, which exert forces on ions traveling through these fields. The speed of a charged particle may be increased or decreased while passing through the electric field, and its direction may be altered by the magnetic field. The magnitude of the deflection of the moving ion's trajectory depends on its mass-to-charge ratio. Lighter ions get deflected by the magnetic force more than heavier ions (based on Newton's second law of motion, F = ma). The streams of sorted ions pass from the analyzer to the detector, which records the relative abundance of each ion type. This information is used to determine the chemical element composition of the original sample (i.e. that both sodium and chlorine are present in the sample) and the isotopic composition of its constituents (the ratio of 35 Cl to 37 Cl).

The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte). The ions are then transported by magnetic or electric fields to the mass analyzer.

Techniques for ionization have been key to determining what types of samples can be analyzed by mass spectrometry. Electron ionization and chemical ionization are used for gases and vapors. In chemical ionization sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source. Two techniques often used with liquid and solid biological samples include electrospray ionization (invented by John Fenn [11] ) and matrix-assisted laser desorption/ionization (MALDI, initially developed as a similar technique "Soft Laser Desorption (SLD)" by K. Tanaka [12] for which a Nobel Prize was awarded and as MALDI by M. Karas and F. Hillenkamp [13] ).

Hard ionization and soft ionization Edit

In mass spectrometry, ionization refers to the production of gas phase ions suitable for resolution in the mass analyser or mass filter. Ionization occurs in the ion source. There are several ion sources available each has advantages and disadvantages for particular applications. For example, electron ionization (EI) gives a high degree of fragmentation, yielding highly detailed mass spectra which when skilfully analysed can provide important information for structural elucidation/characterisation and facilitate identification of unknown compounds by comparison to mass spectral libraries obtained under identical operating conditions. However, EI is not suitable for coupling to HPLC, i.e. LC-MS, since at atmospheric pressure, the filaments used to generate electrons burn out rapidly. Thus EI is coupled predominantly with GC, i.e. GC-MS, where the entire system is under high vacuum.

Hard ionization techniques are processes which impart high quantities of residual energy in the subject molecule invoking large degrees of fragmentation (i.e. the systematic rupturing of bonds acts to remove the excess energy, restoring stability to the resulting ion). Resultant ions tend to have m/z lower than the molecular mass (other than in the case of proton transfer and not including isotope peaks). The most common example of hard ionization is electron ionization (EI).

Soft ionization refers to the processes which impart little residual energy onto the subject molecule and as such result in little fragmentation. Examples include fast atom bombardment (FAB), chemical ionization (CI), atmospheric-pressure chemical ionization (APCI), atmospheric-pressure photoionization (APPI), electrospray ionization (ESI), desorption electrospray ionization (DESI), and matrix-assisted laser desorption/ionization (MALDI).

Inductively coupled plasma Edit

Inductively coupled plasma (ICP) sources are used primarily for cation analysis of a wide array of sample types. In this source, a plasma that is electrically neutral overall, but that has had a substantial fraction of its atoms ionized by high temperature, is used to atomize introduced sample molecules and to further strip the outer electrons from those atoms. The plasma is usually generated from argon gas, since the first ionization energy of argon atoms is higher than the first of any other elements except He, F and Ne, but lower than the second ionization energy of all except the most electropositive metals. The heating is achieved by a radio-frequency current passed through a coil surrounding the plasma.

Photoionization mass spectrometry Edit

Photoionization can be used in experiments which seek to use mass spectrometry as a means of resolving chemical kinetics mechanisms and isomeric product branching. [14] In such instances a high energy photon, either X-ray or uv, is used to dissociate stable gaseous molecules in a carrier gas of He or Ar. In instances where a synchrotron light source is utilized, a tuneable photon energy can be utilized to acquire a photoionization efficiency curve which can be used in conjunction with the charge ratio m/z to fingerprint molecular and ionic species. More recently atmospheric-pressure photoionization (APPI) has been developed to ionize molecules mostly as effluents of LC-MS systems.

Ambient ionization Edit

Some applications for ambient ionization include environmental applications as well as clinical applications. In these techniques, ions form in an ion source outside the mass spectrometer. Sampling becomes easy as the samples don't need previous separation nor preparation. Some examples of ambient ionization techniques are DESI, SESI, LAESI, and Desorption atmospheric-pressure chemical ionization (DAPCI), among others.

Other ionization techniques Edit

Mass analyzers separate the ions according to their mass-to-charge ratio. The following two laws govern the dynamics of charged particles in electric and magnetic fields in vacuum:

F = Q ( E + v × B ) =Q(mathbf +mathbf imes mathbf )> (Lorentz force law) F = m a =mmathbf > (Newton's second law of motion in the non-relativistic case, i.e. valid only at ion velocity much lower than the speed of light).

Here F is the force applied to the ion, m is the mass of the ion, a is the acceleration, Q is the ion charge, E is the electric field, and v × B is the vector cross product of the ion velocity and the magnetic field

Equating the above expressions for the force applied to the ion yields:

This differential equation is the classic equation of motion for charged particles. Together with the particle's initial conditions, it completely determines the particle's motion in space and time in terms of m/Q. Thus mass spectrometers could be thought of as "mass-to-charge spectrometers". When presenting data, it is common to use the (officially) dimensionless m/z, where z is the number of elementary charges (e) on the ion (z=Q/e). This quantity, although it is informally called the mass-to-charge ratio, more accurately speaking represents the ratio of the mass number and the charge number, z.

There are many types of mass analyzers, using either static or dynamic fields, and magnetic or electric fields, but all operate according to the above differential equation. Each analyzer type has its strengths and weaknesses. Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS/MS). In addition to the more common mass analyzers listed below, there are others designed for special situations.

There are several important analyzer characteristics. The mass resolving power is the measure of the ability to distinguish two peaks of slightly different m/z. The mass accuracy is the ratio of the m/z measurement error to the true m/z. Mass accuracy is usually measured in ppm or milli mass units. The mass range is the range of m/z amenable to analysis by a given analyzer. The linear dynamic range is the range over which ion signal is linear with analyte concentration. Speed refers to the time frame of the experiment and ultimately is used to determine the number of spectra per unit time that can be generated.

Sector instruments Edit

A sector field mass analyzer uses a static electric and/or magnetic field to affect the path and/or velocity of the charged particles in some way. As shown above, sector instruments bend the trajectories of the ions as they pass through the mass analyzer, according to their mass-to-charge ratios, deflecting the more charged and faster-moving, lighter ions more. The analyzer can be used to select a narrow range of m/z or to scan through a range of m/z to catalog the ions present. [16]

Time-of-flight Edit

The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through the same potential, and then measures the time they take to reach the detector. If the particles all have the same charge, their kinetic energies will be identical, and their velocities will depend only on their masses. Ions with a lower mass will reach the detector first. [17] However, in reality, even particles with the same m/z can arrive at different times at the detector, because they have different initial velocities. The initial velocity is often not dependent on the mass of the ion TOF-MS, and will turn into a difference in the final velocity. Because of this, ions with the same m/z ratio will reach the detector at a variety of times, which broadens the peaks shown on the count vs m/z plot, but will generally not change the central location of the peaks, since the average starting velocity of ions relative to the other analyzed ions is generally centered at zero. To fix this problem, time-lag focusing/delayed extraction has been coupled with TOF-MS. [18]

Quadrupole mass filter Edit

Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or destabilize the paths of ions passing through a radio frequency (RF) quadrupole field created between 4 parallel rods. Only the ions in a certain range of mass/charge ratio are passed through the system at any time, but changes to the potentials on the rods allow a wide range of m/z values to be swept rapidly, either continuously or in a succession of discrete hops. A quadrupole mass analyzer acts as a mass-selective filter and is closely related to the quadrupole ion trap, particularly the linear quadrupole ion trap except that it is designed to pass the untrapped ions rather than collect the trapped ones, and is for that reason referred to as a transmission quadrupole. A magnetically enhanced quadrupole mass analyzer includes the addition of a magnetic field, either applied axially or transversely. This novel type of instrument leads to an additional performance enhancement in terms of resolution and/or sensitivity depending upon the magnitude and orientation of the applied magnetic field. [19] [20] A common variation of the transmission quadrupole is the triple quadrupole mass spectrometer. The “triple quad” has three consecutive quadrupole stages, the first acting as a mass filter to transmit a particular incoming ion to the second quadrupole, a collision chamber, wherein that ion can be broken into fragments. The third quadrupole also acts as a mass filter, to transmit a particular fragment ion to the detector. If a quadrupole is made to rapidly and repetitively cycle through a range of mass filter settings, full spectra can be reported. Likewise, a triple quad can be made to perform various scan types characteristic of tandem mass spectrometry.

Ion traps Edit

Three-dimensional quadrupole ion trap Edit

The quadrupole ion trap works on the same physical principles as the quadrupole mass analyzer, but the ions are trapped and sequentially ejected. Ions are trapped in a mainly quadrupole RF field, in a space defined by a ring electrode (usually connected to the main RF potential) between two endcap electrodes (typically connected to DC or auxiliary AC potentials). The sample is ionized either internally (e.g. with an electron or laser beam), or externally, in which case the ions are often introduced through an aperture in an endcap electrode.

There are many mass/charge separation and isolation methods but the most commonly used is the mass instability mode in which the RF potential is ramped so that the orbit of ions with a mass a > b are stable while ions with mass b become unstable and are ejected on the z-axis onto a detector. There are also non-destructive analysis methods.

Ions may also be ejected by the resonance excitation method, whereby a supplemental oscillatory excitation voltage is applied to the endcap electrodes, and the trapping voltage amplitude and/or excitation voltage frequency is varied to bring ions into a resonance condition in order of their mass/charge ratio. [21] [22]

Cylindrical ion trap Edit

The cylindrical ion trap mass spectrometer (CIT) is a derivative of the quadrupole ion trap where the electrodes are formed from flat rings rather than hyperbolic shaped electrodes. The architecture lends itself well to miniaturization because as the size of a trap is reduced, the shape of the electric field near the center of the trap, the region where the ions are trapped, forms a shape similar to that of a hyperbolic trap.

Linear quadrupole ion trap Edit

A linear quadrupole ion trap is similar to a quadrupole ion trap, but it traps ions in a two dimensional quadrupole field, instead of a three-dimensional quadrupole field as in a 3D quadrupole ion trap. Thermo Fisher's LTQ ("linear trap quadrupole") is an example of the linear ion trap. [23]

A toroidal ion trap can be visualized as a linear quadrupole curved around and connected at the ends or as a cross-section of a 3D ion trap rotated on edge to form the toroid, donut-shaped trap. The trap can store large volumes of ions by distributing them throughout the ring-like trap structure. This toroidal shaped trap is a configuration that allows the increased miniaturization of an ion trap mass analyzer. Additionally, all ions are stored in the same trapping field and ejected together simplifying detection that can be complicated with array configurations due to variations in detector alignment and machining of the arrays. [24]

As with the toroidal trap, linear traps and 3D quadrupole ion traps are the most commonly miniaturized mass analyzers due to their high sensitivity, tolerance for mTorr pressure, and capabilities for single analyzer tandem mass spectrometry (e.g. product ion scans). [25]

Orbitrap Edit

Orbitrap instruments are similar to Fourier transform ion cyclotron resonance mass spectrometers (see text below). Ions are electrostatically trapped in an orbit around a central, spindle shaped electrode. The electrode confines the ions so that they both orbit around the central electrode and oscillate back and forth along the central electrode's long axis. This oscillation generates an image current in the detector plates which is recorded by the instrument. The frequencies of these image currents depend on the mass-to-charge ratios of the ions. Mass spectra are obtained by Fourier transformation of the recorded image currents.

Orbitraps have a high mass accuracy, high sensitivity and a good dynamic range. [26]

Fourier transform ion cyclotron resonance Edit

Fourier transform mass spectrometry (FTMS), or more precisely Fourier transform ion cyclotron resonance MS, measures mass by detecting the image current produced by ions cyclotroning in the presence of a magnetic field. Instead of measuring the deflection of ions with a detector such as an electron multiplier, the ions are injected into a Penning trap (a static electric/magnetic ion trap) where they effectively form part of a circuit. Detectors at fixed positions in space measure the electrical signal of ions which pass near them over time, producing a periodic signal. Since the frequency of an ion's cycling is determined by its mass-to-charge ratio, this can be deconvoluted by performing a Fourier transform on the signal. FTMS has the advantage of high sensitivity (since each ion is "counted" more than once) and much higher resolution and thus precision. [27] [28]

Ion cyclotron resonance (ICR) is an older mass analysis technique similar to FTMS except that ions are detected with a traditional detector. Ions trapped in a Penning trap are excited by an RF electric field until they impact the wall of the trap, where the detector is located. Ions of different mass are resolved according to impact time.

The final element of the mass spectrometer is the detector. The detector records either the charge induced or the current produced when an ion passes by or hits a surface. In a scanning instrument, the signal produced in the detector during the course of the scan versus where the instrument is in the scan (at what m/Q) will produce a mass spectrum, a record of ions as a function of m/Q.

Typically, some type of electron multiplier is used, though other detectors including Faraday cups and ion-to-photon detectors are also used. Because the number of ions leaving the mass analyzer at a particular instant is typically quite small, considerable amplification is often necessary to get a signal. Microchannel plate detectors are commonly used in modern commercial instruments. [29] In FTMS and Orbitraps, the detector consists of a pair of metal surfaces within the mass analyzer/ion trap region which the ions only pass near as they oscillate. No direct current is produced, only a weak AC image current is produced in a circuit between the electrodes. Other inductive detectors have also been used. [30]

A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation. For example, one mass analyzer can isolate one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), electron-detachment dissociation (EDD) and surface-induced dissociation (SID). An important application using tandem mass spectrometry is in protein identification. [31]

Tandem mass spectrometry enables a variety of experimental sequences. Many commercial mass spectrometers are designed to expedite the execution of such routine sequences as selected reaction monitoring (SRM) and precursor ion scanning. In SRM, the first analyzer allows only a single mass through and the second analyzer monitors for multiple user-defined fragment ions. SRM is most often used with scanning instruments where the second mass analysis event is duty cycle limited. These experiments are used to increase specificity of detection of known molecules, notably in pharmacokinetic studies. Precursor ion scanning refers to monitoring for a specific loss from the precursor ion. The first and second mass analyzers scan across the spectrum as partitioned by a user-defined m/z value. This experiment is used to detect specific motifs within unknown molecules.

Another type of tandem mass spectrometry used for radiocarbon dating is accelerator mass spectrometry (AMS), which uses very high voltages, usually in the mega-volt range, to accelerate negative ions into a type of tandem mass spectrometer.

The METLIN Metabolite and Chemical Entity Database [32] [33] [34] is the largest repository of experimental tandem mass spectrometry data acquired from standards. The tandem mass spectrometry data on over 850,000 molecular standards (as of 24 August 2020) [32] is provided to facilitate the identification of chemical entities from tandem mass spectrometry experiments. In addition to the identification of known molecules it is also useful for identifying unknowns using its similarity searching/analysis. [35] All tandem mass spectrometry data comes from the experimental analysis of standards at multiple collision energies and in both positive and negative ionization modes. [32]

When a specific combination of source, analyzer, and detector becomes conventional in practice, a compound acronym may arise to designate it succinctly. One example is MALDI-TOF, which refers to a combination of a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer. Other examples include inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS) and spark source mass spectrometry (SSMS).

Certain applications of mass spectrometry have developed monikers that although strictly speaking would seem to refer to a broad application, in practice have come instead to connote a specific or a limited number of instrument configurations. An example of this is isotope ratio mass spectrometry (IRMS), which refers in practice to the use of a limited number of sector based mass analyzers this name is used to refer to both the application and the instrument used for the application.

An important enhancement to the mass resolving and mass determining capabilities of mass spectrometry is using it in tandem with chromatographic and other separation techniques.

Gas chromatography Edit

A common combination is gas chromatography-mass spectrometry (GC/MS or GC-MS). In this technique, a gas chromatograph is used to separate different compounds. This stream of separated compounds is fed online into the ion source, a metallic filament to which voltage is applied. This filament emits electrons which ionize the compounds. The ions can then further fragment, yielding predictable patterns. Intact ions and fragments pass into the mass spectrometer's analyzer and are eventually detected. [36] However, the high temperatures (300°C) used in the GC-MS injection port (and oven) can result in thermal degradation of injected molecules, thus resulting in the measurement of degradation products instead of the actual molecule(s) of interest. [37]

Liquid chromatography Edit

Similar to gas chromatography MS (GC-MS), liquid chromatography-mass spectrometry (LC/MS or LC-MS) separates compounds chromatographically before they are introduced to the ion source and mass spectrometer. It differs from GC-MS in that the mobile phase is liquid, usually a mixture of water and organic solvents, instead of gas. Most commonly, an electrospray ionization source is used in LC-MS. Other popular and commercially available LC-MS ion sources are atmospheric pressure chemical ionization and atmospheric pressure photoionization. There are also some newly developed ionization techniques like laser spray.

Capillary electrophoresis–mass spectrometry Edit

Capillary electrophoresis–mass spectrometry (CE-MS) is a technique that combines the liquid separation process of capillary electrophoresis with mass spectrometry. [38] CE-MS is typically coupled to electrospray ionization. [39]

Ion mobility Edit

Ion mobility spectrometry-mass spectrometry (IMS/MS or IMMS) is a technique where ions are first separated by drift time through some neutral gas under an applied electrical potential gradient before being introduced into a mass spectrometer. [40] Drift time is a measure of the radius relative to the charge of the ion. The duty cycle of IMS (the time over which the experiment takes place) is longer than most mass spectrometric techniques, such that the mass spectrometer can sample along the course of the IMS separation. This produces data about the IMS separation and the mass-to-charge ratio of the ions in a manner similar to LC-MS. [41]

The duty cycle of IMS is short relative to liquid chromatography or gas chromatography separations and can thus be coupled to such techniques, producing triple modalities such as LC/IMS/MS. [42]

Data representations Edit

Mass spectrometry produces various types of data. The most common data representation is the mass spectrum.

Certain types of mass spectrometry data are best represented as a mass chromatogram. Types of chromatograms include selected ion monitoring (SIM), total ion current (TIC), and selected reaction monitoring (SRM), among many others.

Other types of mass spectrometry data are well represented as a three-dimensional contour map. In this form, the mass-to-charge, m/z is on the x-axis, intensity the y-axis, and an additional experimental parameter, such as time, is recorded on the z-axis.

Data analysis Edit

Mass spectrometry data analysis is specific to the type of experiment producing the data. General subdivisions of data are fundamental to understanding any data.

Many mass spectrometers work in either negative ion mode or positive ion mode. It is very important to know whether the observed ions are negatively or positively charged. This is often important in determining the neutral mass but it also indicates something about the nature of the molecules.

Different types of ion source result in different arrays of fragments produced from the original molecules. An electron ionization source produces many fragments and mostly single-charged (1-) radicals (odd number of electrons), whereas an electrospray source usually produces non-radical quasimolecular ions that are frequently multiply charged. Tandem mass spectrometry purposely produces fragment ions post-source and can drastically change the sort of data achieved by an experiment.

Knowledge of the origin of a sample can provide insight into the component molecules of the sample and their fragmentations. A sample from a synthesis/manufacturing process will probably contain impurities chemically related to the target component. A crudely prepared biological sample will probably contain a certain amount of salt, which may form adducts with the analyte molecules in certain analyses.

Results can also depend heavily on sample preparation and how it was run/introduced. An important example is the issue of which matrix is used for MALDI spotting, since much of the energetics of the desorption/ionization event is controlled by the matrix rather than the laser power. Sometimes samples are spiked with sodium or another ion-carrying species to produce adducts rather than a protonated species.

Mass spectrometry can measure molar mass, molecular structure, and sample purity. Each of these questions requires a different experimental procedure therefore, adequate definition of the experimental goal is a prerequisite for collecting the proper data and successfully interpreting it.

Interpretation of mass spectra Edit

Since the precise structure or peptide sequence of a molecule is deciphered through the set of fragment masses, the interpretation of mass spectra requires combined use of various techniques. Usually the first strategy for identifying an unknown compound is to compare its experimental mass spectrum against a library of mass spectra. If no matches result from the search, then manual interpretation [43] or software assisted interpretation of mass spectra must be performed. Computer simulation of ionization and fragmentation processes occurring in mass spectrometer is the primary tool for assigning structure or peptide sequence to a molecule. An a priori structural information is fragmented in silico and the resulting pattern is compared with observed spectrum. Such simulation is often supported by a fragmentation library [44] that contains published patterns of known decomposition reactions. Software taking advantage of this idea has been developed for both small molecules and proteins.

Analysis of mass spectra can also be spectra with accurate mass. A mass-to-charge ratio value (m/z) with only integer precision can represent an immense number of theoretically possible ion structures however, more precise mass figures significantly reduce the number of candidate molecular formulas. A computer algorithm called formula generator calculates all molecular formulas that theoretically fit a given mass with specified tolerance.

A recent technique for structure elucidation in mass spectrometry, called precursor ion fingerprinting, identifies individual pieces of structural information by conducting a search of the tandem spectra of the molecule under investigation against a library of the product-ion spectra of structurally characterized precursor ions. [45]

Mass spectrometry has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is now commonly used in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.

As an analytical technique it possesses distinct advantages such as: Increased sensitivity over most other analytical techniques because the analyzer, as a mass-charge filter, reduces background interference, Excellent specificity from characteristic fragmentation patterns to identify unknowns or confirm the presence of suspected compounds, Information about molecular weight, Information about the isotopic abundance of elements, Temporally resolved chemical data.

A few of the disadvantages of the method is that it often fails to distinguish between optical and geometrical isomers and the positions of substituent in o-, m- and p- positions in an aromatic ring. Also, its scope is limited in identifying hydrocarbons that produce similar fragmented ions.

Isotope ratio MS: isotope dating and tracing Edit

Mass spectrometry is also used to determine the isotopic composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an element are typically very rare, so a very sensitive instrument is required. These instruments, sometimes referred to as isotope ratio mass spectrometers (IR-MS), usually use a single magnet to bend a beam of ionized particles towards a series of Faraday cups which convert particle impacts to electric current. A fast on-line analysis of deuterium content of water can be done using flowing afterglow mass spectrometry, FA-MS. Probably the most sensitive and accurate mass spectrometer for this purpose is the accelerator mass spectrometer (AMS). This is because it provides ultimate sensitivity, capable of measuring individual atoms and measuring nuclides with a dynamic range of

10 15 relative to the major stable isotope. [46] Isotope ratios are important markers of a variety of processes. Some isotope ratios are used to determine the age of materials for example as in carbon dating. Labeling with stable isotopes is also used for protein quantification. (see protein characterization below)

Membrane-introduction mass spectrometry: measuring gases in solution Edit

Membrane-introduction mass spectrometry combines the isotope ratio MS with a reaction chamber/cell separated by a gas-permeable membrane. This method allows the study of gases as they evolve in solution. This method has been extensively used for the study of the production of oxygen by Photosystem II. [47]

Trace gas analysis Edit

Several techniques use ions created in a dedicated ion source injected into a flow tube or a drift tube: selected ion flow tube (SIFT-MS), and proton transfer reaction (PTR-MS), are variants of chemical ionization dedicated for trace gas analysis of air, breath or liquid headspace using well defined reaction time allowing calculations of analyte concentrations from the known reaction kinetics without the need for internal standard or calibration.

Another technique with applications in trace gas analysis field is secondary electrospray ionization (SESI-MS), which is a variant of electrospray ionization. SESI consist of an electrospray plume of pure acidified solvent that interacts with neutral vapors. Vapor molecules get ionized at atmospheric pressure when charge is transferred from the ions formed in the electrospray to the molecules. One advantage of this approach is that it is compatible with most ESI-MS systems. [48] [49]

Atom probe Edit

An atom probe is an instrument that combines time-of-flight mass spectrometry and field-evaporation microscopy to map the location of individual atoms.

Pharmacokinetics Edit

Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time point data. The most common instrumentation used in this application is LC-MS with a triple quadrupole mass spectrometer. Tandem mass spectrometry is usually employed for added specificity. Standard curves and internal standards are used for quantitation of usually a single pharmaceutical in the samples. The samples represent different time points as a pharmaceutical is administered and then metabolized or cleared from the body. Blank or t=0 samples taken before administration are important in determining background and ensuring data integrity with such complex sample matrices. Much attention is paid to the linearity of the standard curve however it is not uncommon to use curve fitting with more complex functions such as quadratics since the response of most mass spectrometers is less than linear across large concentration ranges. [50] [51] [52]

There is currently considerable interest in the use of very high sensitivity mass spectrometry for microdosing studies, which are seen as a promising alternative to animal experimentation.

Recent studies show that secondary electrospray ionization (SESI) is a powerful technique to monitor drug kinetics via breath analysis. [53] [54] Because breath is naturally produced, several datapoints can be readily collected. This allows for the number of collected data-points to be greatly increased. [55] In animal studies, this approach SESI can reduce animal sacrifice. [54] In humans, SESI-MS non-invasive analysis of breath can help study the kinetics of drugs at a personalized level. [53] [56] [57]

Protein characterization Edit

Mass spectrometry is an important method for the characterization and sequencing of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as "top-down" strategy of protein analysis. The top-down approach however is largely limited to low-throughput single-protein studies. In the second, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in solution or in gel after electrophoretic separation. Other proteolytic agents are also used. The collection of peptide products are often separated by chromatography prior to introduction to the mass analyzer. When the characteristic pattern of peptides is used for the identification of the protein the method is called peptide mass fingerprinting (PMF), if the identification is performed using the sequence data determined in tandem MS analysis it is called de novo peptide sequencing. These procedures of protein analysis are also referred to as the "bottom-up" approach, and have also been used to analyse the distribution and position of post-translational modifications such as phosphorylation on proteins. [58] A third approach is also beginning to be used, this intermediate "middle-down" approach involves analyzing proteolytic peptides that are larger than the typical tryptic peptide. [59]

Space exploration Edit

As a standard method for analysis, mass spectrometers have reached other planets and moons. Two were taken to Mars by the Viking program. In early 2005 the Cassini–Huygens mission delivered a specialized GC-MS instrument aboard the Huygens probe through the atmosphere of Titan, the largest moon of the planet Saturn. This instrument analyzed atmospheric samples along its descent trajectory and was able to vaporize and analyze samples of Titan's frozen, hydrocarbon covered surface once the probe had landed. These measurements compare the abundance of isotope(s) of each particle comparatively to earth's natural abundance. [60] Also on board the Cassini–Huygens spacecraft was an ion and neutral mass spectrometer which had been taking measurements of Titan's atmospheric composition as well as the composition of Enceladus' plumes. A Thermal and Evolved Gas Analyzer mass spectrometer was carried by the Mars Phoenix Lander launched in 2007. [61]

Mass spectrometers are also widely used in space missions to measure the composition of plasmas. For example, the Cassini spacecraft carried the Cassini Plasma Spectrometer (CAPS), [62] which measured the mass of ions in Saturn's magnetosphere.

Respired gas monitor Edit

Mass spectrometers were used in hospitals for respiratory gas analysis beginning around 1975 through the end of the century. Some are probably still in use but none are currently being manufactured. [63]

Found mostly in the operating room, they were a part of a complex system, in which respired gas samples from patients undergoing anesthesia were drawn into the instrument through a valve mechanism designed to sequentially connect up to 32 rooms to the mass spectrometer. A computer directed all operations of the system. The data collected from the mass spectrometer was delivered to the individual rooms for the anesthesiologist to use.

The uniqueness of this magnetic sector mass spectrometer may have been the fact that a plane of detectors, each purposely positioned to collect all of the ion species expected to be in the samples, allowed the instrument to simultaneously report all of the gases respired by the patient. Although the mass range was limited to slightly over 120 u, fragmentation of some of the heavier molecules negated the need for a higher detection limit. [64]

Preparative mass spectrometry Edit

The primary function of mass spectrometry is as a tool for chemical analyses based on detection and quantification of ions according to their mass-to-charge ratio. However, mass spectrometry also shows promise for material synthesis. [46] Ion soft landing is characterized by deposition of intact species on surfaces at low kinetic energies which precludes the fragmentation of the incident species. [65] The soft landing technique was first reported in 1977 for the reaction of low energy sulfur containing ions on a lead surface. [66]


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