An update of the classical and novel methods used for measuring fast neurotransmitters during normal and brain altered function.

To understand better the cerebral functions, several methods have been developed to study the brain activity, they could be related with morphological, electrophysiological, molecular and neurochemical techniques. Monitoring neurotransmitter concentration is a key role to know better how the brain works during normal or pathological conditions, as well as for studying the changes in neurotransmitter concentration with the use of several drugs that could affect or reestablish the normal brain activity. Immediate response of the brain to environmental conditions is related with the release of the fast acting neurotransmission by glutamate (Glu), γ-aminobutyric acid (GABA) and acetylcholine (ACh) through the opening of ligand-operated ion channels. Neurotransmitter release is mainly determined by the classical microdialysis technique, this is generally coupled to high performance liquid chromatography (HPLC). Detection of neurotransmitters can be done by fluorescence, optical density, electrochemistry or other detection systems more sophisticated. Although the microdialysis method is the golden technique to monitor the brain neurotransmitters, it has a poor temporal resolution. Recently, with the use of biosensor the drawback of temporal resolution has been improved considerably, however other inconveniences have merged, such as stability, reproducibility and the lack of reliable biosensors mainly for GABA. The aim of this review is to show the important advances in the different ways to measure neurotransmitter concentrations; both with the use of classic techniques as well as with the novel methods and alternant approaches to improve the temporal resolution.

INTRODUCTION

Several methods have been used to study the brain functions, these consider several aspects: like behavior, morphology, as well as electrophysiological and biochemical activity. It is important to study the brain with reliable methods and techniques, to understand better the brain activity during normal and pathological conditions. The results of these studies are important to design new strategies, drug treatments and to know the basic mechanisms of drug action. Some techniques to study the brain can be carried out in real time, and with precise spatial resolution, like electroencephalography (EEG), magnetic resonance imaging (MRI), proton nuclear magnetic resonance and biosensors, which could be used to study behavioral and electro-physiological activity. However, the biochemical studies possess low temporal resolution due to the time needed for the sample collection from brain tissue, cerebral spinal fluid or microdyalisates.

The brain microdialysis technique is the preferred method to monitor several neurotransmitters and changes in these during brain activity, which can be related to a particular behavior or brain-altered function. This procedure has gotten a big impact in biochemical studies since Delgado et al., introduced it in 1972 [1]. This technique is usually coupled to HPLC methods. The type of neurotransmitter to be studied determines the separation method and the detection system that could include: optical density, fluorescence, electrochemical, luminescence, and mass spectroscopy (MS). Despite the fact microdialysis procedure can be considered as a golden technique to monitor neurotransmitters in brain, it has some disadvantages, like the poor temporal resolution which depends on the speed of sample collection (generally, it is carried out at 2 μl/min) and the minimum volume of sample required for the HPLC analysis is about 5-15 min of time collection. Another disadvantage is the insertion of the probe itself that produces a local injury. With long periods of monitoring, a glial scar could emerge as a consequence of damage, resulting in a poor recovery or underestimation of the neurotransmitter to analyze. With recent advances in HPLC systems, bigger sensitivity can be reached, the volume needed has been reduced considerably and the temporal resolution has been improved using variants of a chromato-graphic method like capillary electrophoresis (CE) [2]. However, handling small volumes could be an important issue, since a great number of samples need to be processed individually before the separation procedure [3]. Another disadvantage is the time needed for the separation and the analysis process, besides it is important to determine the dead time from the tip of a microdialysis probe to the collection zone if a good correlation between behavior and EEG activity is attempted, since these events occur in real time [4]. The use of biosensors has provided a useful alternative to avoid the separation process and study the rapid events that can be correlated with a particular neuro-physiological activity. These are mainly electrochemical, using oxidases which produce hydrogen peroxide (H2O2). Biosensors have allowed the monitoring of changes in Glu and ACh with good temporal and spatial resolution, allowing behavior and EEG activity to be studied simultaneously [5-7]. The use of biosensors opens the possibility to obtain sufficient temporal resolution to correlate changes in a particular neurotransmitter with a specific behavior or EEG activity. At the beginning they could only monitor a single neurotransmitter at once and in the case of ACh they do not distinguish choline (Ch) from ACh. Although the use of multiplexed electrodes can detect more than one neuro-transmitter, using different enzymatic coatings [8]. Moreover, additional biosensors for other important neurotransmitters, like GABA are being developed. Nowadays, biosensors are available in a very small size and they have incorporated more advanced technologies. An example for a nanometrical scale biosensors are the carbon fiber (CF) biosensors, CF is a 7 µm graphite monofilament. CF has several mechanical and electrical properties and is an excellent electrode for electrophysiological and electrochemical measurements and has been used since 1979. It has been used for electro-chemical detection of catecholamines, ACh, Ch, glucose and Glu [9]. A recent alternative to monitor neurotransmitters with high temporal resolution involves biosensors to design enzymatic reactions to generate a “derivate” from a neurotransmitter as a substrate that could be measured by luminesce [10, 11], electrochemistry [5] and fluorescence [12]. The use of enzymatic reactors avoids the drawback of fixed enzymes in a biosensor, giving a more active enzyme and a better control of the enzymatic reactions as well as the amount of substrate, cofactors and other conditions like pH and temperature. In the light of the above, the objective of this review is to show the recent advances in the classical and new methods to measure neurotransmitter during normal or pathological conditions and their relationship with the use of different pharmacological tools.

Glu Measurement

Glu is the main excitatory neurotransmitter in the central nervous system (CNS) and an important energy and nitrogen source in eukaryotic and mammalian cells [13]. It also plays an important role in several brain functions like learning and memory [14]. It has been found that long lasting increases in extracellular Glu concentrations have excitotoxic properties [15]. Glu alterations have been related with essentially all forms of behavior, including consciousness, sensory perception, motor control, mood [16], seizures [17] and processes like cognitive, formation of memory, learning, biomaterial sensitivity, and synaptic plasticity of the CNS [13, 14, 18]. Furthermore, alterations in Glu levels have been shown to be linked to several neurodegenerative disorders such as Alzheimer's [19], Parkinson's [20], Huntington's disease [21], epilepsy [22], amyotrophic lateral sclerosis [23], mood disorders [24], ischemic stroke [25] and schizophrenia [26]. Studies about the role of Glu and other neurotransmitters could explain the onset and progression of several neurological disorders [8]. Thus measuring this neurotransmitter in vitro and in vivo is fundamental to understand in a better way its role in normal as well as in pathological conditions. The methods used more frequently are chromatographic, coupled to the following detection systems; spectrophotometric [27], fluorometric [28, 29] and electrochemical [30]. Despite the fact of the existence of several techniques there is a necessity for a reliable method to monitor this neurotransmitter with real time resolution, which could help to design better-personalized medicine, biomedical research and its biotechnological applications [31].

Microdialysis is a useful method to measure Glu in the extracellular space in vivo conditions. Samples collected are mainly evaluated by HPLC [32, 33]. This method offers some advantages, like the sample collection that can be carried out in freely moving or anesthetized animals and, information of the compound of interest and its metabolites can be obtained simultaneously. Also the microdialysis probe can be used to administer testing drugs locally to explore their basic mechanisms of action related with a particular neurotransmitter. Nevertheless, there are some important disadvantages, such as the speed at which the information is obtained from microdialysis procedure, as it is considerably slower in relation to other dynamic processes in brain and is carried out between 5-15 min [15]; although some attempts have been done to improve the time resolution [3, 34]. Also the size of the probe can produce an important tissue insult and glial scar; which can be seen 1.4 mm apart from the implanted zone and the data collected could represent pathological conditions instead physiological [35]. Despite of these disadvantages, Glu measurement with this technique has been widely used, and Glu concentrations have been determined in the range of 1.0 to 5.0 µM in different brain regions without considering probe recovery [36-38]. Chemical stimulation by some drugs administrated with reverse microdialysis induces an increase in extracellular Glu, like 4-aminopyridine (4-AP), which blocks potassium channels, increasing its time aperture [17]. Infusion of the inhibitor of the high affinity Glu transporter, DL-treo-β-benzyloxyaspartate (DL-TBOA), produces an increase of 300% in Glu in nucleus accumbens of young rats [39]. The dialysates were analyzed by HPLC with fluorimetric detection. Also, the local administration of the NMDA and GABA antagonist in the frontal cerebral cortex induced an increase in Glu concentration by 278% with respect to baseline concentrations detected by HPLC in neostriatum [40]. Increases in Glu by electrical stimulation, have also been detected in dialysates, these activate glutamatergic pathways when the nerve endings are stimulated in bone marrow, sciatic nerve and raphe magnus nucleus [41, 42]. Also, the application of electric pulses for 2 minutes increased Glu concentrations from 200 to 300% in samples for 30 second in the nucleus accumbens, returning to baseline within 60 second by the administration of metabotropic agonist trans-1-aminocyclopentane-1,3 dicarboxylate (ACPD) [43]. Certain types of stimulations may affect behavioral changes that reflect Glu increases. In a study of moderate stress a sustained increase in Glu concentrations of 200 to 450% above baseline in 10 to 20 min measured in prefrontal cortex, hippocampus, nucleus accumbens and striatum was reported [44].

The use of biosensors has attracted the attention of many researchers to measure several compounds and now is a popular technique widely used; this due to their simplicity and the relative high sensitivity. Electrochemical biosensors are built with high technology in order to reduce their size and to avoid tissue damage. Several strategies for enzyme immobilization over the electrodes have appeared and used [45]. Although, there are still several obstacles to be solved like the stability, reproducibility, response and a high potential demanded over the working electrode. Most of the biosensors designed to measure Glu in vivo are based on the use of glutamate oxidase (GluOx) to generate H2O2, which is electrochemically detectable [46]. GluOx is an enzyme that catalyzes the oxidative deamination of Glu in the presence of molecular oxygen (O2) to produce α ketoglutarate, ammonia (NH3) and H2O2 [47]. Other electroactive species that could interfere with these sensors can be excluded by the use of polymers with selective permeability, giving a more specific signal [48]. Different approaches related with the use of biosensors based on electrochemical sensing of Glu have been developed, like immobilization of GluOx with carbon nanotubes (CNTs) and polymers over platinum (Pt). These have been used to build biosensors with improved response (7 second) and limit of detection ~0.3 µM [49]. Electrochemical microsensors were used to evaluate the extracellular Glu concentration in vivo after blocking the Glu transporters EAAT-3 and EAAC1, these studies showed that this method is able to detect discrete changes in this neurotransmitter [31].

Electrochemical methods using microelectrodes allow direct real-time assessment of physiological levels of Glu in vitro and in vivo with high spatial resolution [50]. These have led to the development of different biosensors that couple microelectrodes with cerium oxide and titanium nanoparticles deposited on a surface of platinum microelectrodes and GluOx, which catalyzes Glu, generating α-ketoglutarate and H2O2, which is electrochemically active. The LOD was 0.493 µM with a sensitivity of 793 pA / mM, with 2 and 5 s of time resolution [46]. Also with a flexible microsensor, based on a polymer substrate, having an array of microelectrodes allows online monitoring in specific regions. Pt microelectrodes with electrochemical biosensor for Glu allow detection with high sensitivity of 2.16 nA/mm2/μM [31]. The advantage of ultra microelectrodes, nanoelectrodes or smaller electrodes is the benefit obtained from the enhanced mass transport which takes place. As electrodes decrease in size, radial (3-dimensional) diffusion becomes dominant and results in faster mass transport. This high rate of mass transport (diffusion) at small electrodes enables measurement of kinetics by steady-state experiments rather than by transient techniques [51].

Recently Glu electrochemical detection was improved by the development of nano graphene plates. Graphene exhibits excellent electron transfer promoting ability for some enzymes and excellent catalytic behavior toward small biomolecules such as H2O2 and NADH, which makes graphene extremely attractive for enzyme-based biosensors [52]. The incorporation of Pt nanoparticles catalyzes the electrooxidation of H2O2 to oxidize Glu by GluOx on a layer of poly-phenylenediamine and showed a greater selectivity, displaying a competitive performance [53]. Graphene quantum dots have been successfully used as a fluorescence chemical sensor, concentrations of Glu at a nanoscale have been efficiently detected in Tris-HCl (pH = 9) buffer solution at a wavelength of 430 nm. The fluorescence intensity of the quantum dot gradually improves with increase in the concentration of Glu and any change in fluorescence intensity is directly proportional to the concentration of Glu. Under optimal conditions, the linear range for detection of glutamate was 0.16 µM to 10 µM with a detection limit of 52 nM. The sensor showed high selectivity for Glu compared with other amino acids [54].

GABA Measurements

GABA has been established as the main inhibitory neurotransmitter in the CNS [55]. GABA is homogenous and widely distributed across the CNS and it has an important role in brain physiology. Precise and accurate measurement of GABA in biological fluids is important to the study of neuropharmacology, since changes in its concentration are of relevance in many neurological pathologies such as; epilepsy, seizures, schizophrenia, anxiety and bipolar disorders [56-61]. Also the GABAergic system is one of the principle targets of general anesthetics [62]. Several methods of analysis have been developed based on the available instruments, the sensitivity required and the species or tissues to be analyzed. The first methods used were paper [63] and column chromatography [64-67]. The presence of GABA in vivo can be determined by three ways. The first requires a sample collection carried out through microdialysis procedure [68], push-pull [69] or low-flow push-pull [70]. The second option consists of in vivo and in situ quantification via microsensor or microelectrodes implantation; and the third option are non-invasive imaging techniques in vivo, which allows the detection of metabolites in human [71, 72], like the functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) [73].

The direct measurement of GABA in the brain, faces several significant challenges since is neither fluorescent nor electroactive and it is difficult to detect by enzymatic reactions because not an oxidase nor hydrogenase have been found. GABA detection via fluorescence and electrochemical methods requires derivatization using different reagents with some fluorogenic compounds [65, 74-76]. However, some derivatizant reagent can cause interference in the chromato-graphy. Another challenge for the direct measurement of GABA is the few reports on GABA biosensors. This is probably due to poor sensitivity and an insufficient detection limit for determining GABA in physiological fluids [77].

The most common technique to measure GABA in microdialysates is HPLC coupled with fluorescence 65, 78-81, electrochemical detection [77], MS [82] and liquid chromatography/tandem MS [83, 84]. Nonetheless, it is necessary a prolonged collection time (20 min) to obtain enough sample to be measured, resulting in poor temporal resolution [76, 85]. GABA measurements in dialysates require ultrasensitive methods, since it is present in very low concentration (5-50 nM) in normal physiological conditions [78]. Since GABA lacks of fluorescent or electroactive properties, it has to be derivatized to generate a compound that has these characteristics. Several reagents have been tested with this purpose, including: 1-fluoro-2,4-dinitrophenyl-5-alaninamide (FDA) [86], o-phthalaldehyde (OPA) [64, 65, 74, 87], naphthalene-2 3-dicarboxaldehyde (NDA) [67], phenylisothiocyanate (PITC) [88], 9-fluorenylmethyl chloroformate (FMOC), 5-dimethylamino-1 naphthalene-sulfonyl-chloride (DANSYL-Cl) and 4-(Dimethylamino)azobenzene-4´-sulfonyl chloride (DABSYL-Cl) [89]. These processes have a good limit of detection, in the range of 0.03 pmol for electrochemical detection [90] and 0.5 to 1 pmol for fluorescence and have been improved to 100 fmol with microbore columns [78], besides it has excellent selectivity and recovery [91]. Nevertheless electrochemical detection with OPA affects the working electrode and reduces considerably its lifespan and increases the background noise [78]. However, the sensitivity of these methods would be insufficient to quantify GABA in vivo microdialysates. After obtaining samples, a prepurification process is not required; for this reason there is no risk of enzymatic degradation. Disadvantages of microdialysis technique should be considered for its applicability, since a stressing stereotaxic surgery is necessary and, as mentioned before, glial scar could result as a consequence of damage. Besides, GABA release is not sensitive to tetrodotoxin (TTX) and calcium [15] although it responds to several pharmacological and behavior stimulation. In this way, the GABA content in microdialysates could give information about the neurotransmission mediated by GABA [92]. In different way, the GABA contained in microdialysates is not related with neuronal activity and has been considered as a non-synaptic signaling molecule [93].

The different HPLC techniques previously mentioned have been used to measure the changes in the concentration of GABA on diverse physical and drug stimuli: the fluctuations in GABA concentration due to high potassium infusion has been measured in microdialysates samples through a HPLC system coupled with fluorescence [94] or MS detection [95]. Also, HPLC-fluorescence detection has been used to examine the effect of lithium, valproic acid [96], sevoflurane [80], lamotrigine [97], and kinurenic acid [98] on GABA concentration, while the effect of menin protein on extracellular GABA concentration was analyzed by electrochemical detection [99].

Capillary Electrophoresis

GABA and other amino acid neurotransmitters are generally analyzed by HPLC and fluorescence or electrochemical detection and, as it was mentioned above, these methods require large volume of sample. CE does not require such volumes [100], instead, low volumes are necessary and the temporal resolution is highly improved [2], especially when the fluorescence is induced with a laser beam. The technique is named capillary electrophoresis-laser induced fluorescence detection (CE-LIFD). For precolumn derivatization, NDA [101] and 4-cloro-7-nitro-2,1,3-benzoxadiazol (NBD-Cl) are commonly used [102]. Additionally, several derivatizing compounds have been tried to get reliable signal detection of GABA and other amino acids, such as fluorescein isotiocyanate (FITC), 5-carboxy- fluorescein succinimidil ester, 6-oxy-(N-succinimidil acetate)-9-(2-methoxycarbonyl) fluorescein, 3-(2-furoyl)- quinoline-2-carboxaldehyde, and 3-(4-carboxy benzoyl)-2- quinoline carboxaldehyde [103]. Some advantages of CE is the low volume required, also other neurotransmitters besides GABA can be measured simultaneously, like Glu, aspartic acid and some drugs like vigabatrin [101] and low limit of detection (LOD) (0.016 μM) has been achieved. A particular disadvantage with respect to GABA analysis by CE is the temperature used for derivatization; in some cases 50oC is necessary; which implies and additional treatment process for samples, leading to a poor temporal resolution [102]. CE-LIFD now is a reliable technique, absolutely validated bringing new data to the interaction of GABA with several drugs [101].

GABA Sensor

Neurotransmitter sensors have been used to pursuit a better spatial and temporal resolution, which in some experiments is 0.1 second with spatial resolution of some microns [104-107].

The sensing of GABA by acoustic means [108] and immune [109], amperometric [110], and fluorescent [111] assays has been used in recent years.

Immunosensing technique for acoustic wave based on mass changes caused by surface binding reactions, is a tool for real-time measurement. Compared to other similar standard methods, like ELISA and radioimmunoassay, mass-sensitive sensor does not require labeling of antigens or antibodies. Another technique of free sensing is surface plasmon resonance (SPR) [112]. It is sensitive to variations in the refractive index at the sensing surface [113, 114]. However, a disadvantage of using this method is that sensors based on optical fibers and SPR require relatively expensive optical analysis system. Furthermore acoustic sensors have several attractive advantages, such as simplicity, convenience, real-time responses and the ability to provide multiple interface information between the medium and the sensor. The sensitivity achieved with this methodology allows sensing GABA concentrations up to 38 μM [108], which is a value above the physiological range and this is of interest for many pharmacological aspects.

The amperometric sensing is based on the catalytic activity of the enzymes GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSDH) known as GABAse; and horseradish peroxidase (HRP) [115, 116]. The product of the GABAse (reaction 1 and 2) is nicotinamide adenine dinucleotide phosphate (NADPH), which is then oxidized by the HRP (3) [117, 118]. Enzymatic reactions can be used separately or together. Therefore, the analytic signal is always given by the measurement of oxygen consumption in reaction 3; it allows the measurement of NADPH formed (in reaction 2), and then, the concentration of GABA in reaction 1.

The steady state conditions for the concentration of GABA in the CNS are determined by a continuous balance between GABA, formed by decarboxylation of glutamic acid, catalyzed by the enzyme glutamate decarboxylase (GAD) and GABA interaction with α -ketoglutarate to form glutamic acid together with succinic semialdehyde again. Therefore, the simultaneous measurement of both GABA and glutamic acid could be a valuable tool for both assessment of various pathologies that occur in the CNS, and new active pharmaceutical agents in GABAse and on GABA receptors [110].

The immunosensing known as quartz crystal microbalance (QCM) involves sensitive quartz chemical mass transducers widely used as detection devices for studying the affinity of antibodies and antigen interactions fluid-solid interface [119]. Such measurements are simple and easy to use; and require no potentially hazardous materials [120]. These also provide a direct measurement of the reactions of the antigen-antibody interaction at the sensor surface; which is represented by a change in the mass of glass due to the formation of an immune complex on the surface. This produces a proportional change in the resonant frequency according to the Sauerbrey equation [121]. When operating in a liquid environment, the response of the quartz resonator and the viscosity also depends on the density of the liquid. The purpose of this sensor is to detect GABA without the need for an enzyme or a fluorescent marker. For sensing low molecular weight antigens as GABA with a mass sensitive approach direct binding does not produce a sufficient frequency to be detected. Therefore GABA is fixed on a QCM gold surface via a layer of the polysaccharide Dextran. A predetermined amount of anti-GABA is mixed with free GABA and GABA immobilized on the sensor surface. GABA molecules bound and free, compete for binding sites on the antibodies. The molecular interactions of GABA molecules immobilized on the surface with anti-GABA antibody can be detected by the change in the resonance frequency corresponding to the amount of antibody bound to the immobilized GABA sensor surface. The decrease in resonant frequency of the sensor is then inversely related to free GABA concentration in the sample [109].

The fluorescent sensing is another alternative available to quantify GABA. For this purpose the GABAse reactions (1) and (2) are used, which produces NADPH. The NADPH is a fluorescent molecule when it is excited upon it with a laser at a wavelength of 340 nm emits fluorescence at 450 nm [122], this fact allows for indirect quantification of the concentration of GABA as production of NADPH, it is stoichiometric and reflects GABA levels in the sample [123]. The sensitivity of this method has allowed the quantification of GABA in the range 500 μM to 10 mM [122-124].

Additionally, NADPH can be used as a donor molecule and can be oxidized through a dehydrogenase like a diaphorase, also resazurin is reduced to resorufin. In this case, the excitation is produced at 544 nm and the fluorescence detected at 590 nm, the increased sensitivity of detection is about 8 times compared with the GABAse assay [122].

Other techniques that are being developed are GABAse luminescent techniques, based on the Photobacterium luciferase enzyme. Because NADPH is the product of the GABAse reaction, it is possible to shoot a luminescent reaction with Photobacterium luciferase [125]. The reaction involves the oxidoreductase enzyme (4) and the luciferase itself (5). The light emission produced is proportional to the concentration of GABA [126]. The sensitivity of this technique depends on the purity of the enzymes involved, and can detect at micromolar levels of GABA. Using a purified enzyme a nanomolar range of NADPH can be detected as referred by NOVOCIB SAS (Lyon, France), nevertheless there is not published data quantifying GABA at this level.

Magnetic Resonance Spectroscopy

Another method widely used for GABA determination is magnetic resonance spectroscopy (MRS), which allows the detection of metabolites in humans. The principle is based on identifying radiofrequency signals emitted by hydrogen nuclear spins that have chemically specific frequencies, determined by the chemical environment of the hydrogen spins. There are three approaches to resolve GABA signals by MRS: (1) using an editing method, which includes J-difference editing, like the MEGA-PRESS (J-difference edited point-resolved spectroscopy) method [127]. This method is based in two experiments; in the first a frequency selective pulse which affects an specific GABA signal is added; the second experiment lacks of this pulse [72]. The result is reported as the difference of these experiments. (2) Through two-dimensional MRS, this consists in plotting the signals in two frequency axes by doing a series of experiments that differ by one parameter (spin echo or spin delay), in this category are included the dimensional J-editing and correlation spectroscopy-based methods [128]. (3) The final approach is to move the edited methods field strength, from a common 3 Teslas (T), to a higher value (usually 7T) [72, 129]. The GABA detection by these “edited methods” has been validated by correlation with chromatographic measurements [130]. The first unambiguous detection of GABA in vivo was made using J-edited technique [131].

Using some of the MRS variants mentioned before, it has been possible to estimate the GABA concentrations in healthy and sick people, as well as in animal models. Thus, the concentrations of brain GABA under the effects of topiramate; a GABAA receptor agonist, were determined in patients with partial epileptic seizures [132]. Also the effects of vigabatrin and gabapentin (irreversible inhibitor of GABA-T and GABA analogue, respectively), were studied in the occipital lobe of patients using J-edited techniques, and has been shown to raise the levels of GABA in healthy and epileptic subjects [133-139]. A comparative study of gabapentin, pregabalin and vigabatrin in brain GABA on healthy Long Evans rats was reported, only vigabatrin showed a significant increase on the levels of the neurotransmitter [140]. Similar results were obtained in another research, were the effects of vigabatrin and gabapentin; on rat and human neocortical slices were compared, vigabatrin showed a significant increase in GABA levels in both neocortical slices; nonetheless gabapentin increased the levels of this neurotransmitter only in human neocortical slices [141]. Finally the influence of 5’-pyridoxal phosphate and vigabatrin was measured in healthy patients via MRS methods, where only vigabatrin showed an increase in brain GABA levels [142]. As shown, a variety of drugs has been tested with NMR methodologies, allowing a valuable information about the GABA dynamics in real time.

Catecholamines

Catecholamines (CAs) are a group of neurotransmitters derived from tyrosine, throughout enzymatic action of tyrosine hydroxylase to form Dopamine (DA), and later aromatic acid decarboxylase to form noradrenaline. In the brain, DA is considered the more abundant catecholaminergic neurotransmitter, with an important role in attention modulation, voluntary motor control, and a series of aspect related with motivated behavior and reinforcement learning [143]. Also, alterations in its extracellular concentrations have been related with schizophrenia [144] and Parkinson's disease [145]. Interest in brain CAs is focused on DA, which, like others electroactives neurotransmitters, has been measured in dialysate fractions or homogenates by HPLC coupled with electrochemical (amperometric or voltammetry) detection [146, 147]. However, these methodologies have some disadvantages: CAs require relatively high potentials to oxidize, producing interferences by others molecules with similar potentials, importantly ascorbic acid and uric acid [148]; as a result, there are deposition of polymeric films onto the electrode, and decay of electrochemical response [149]. To overcome this effect, different electrode materials and electrode modifications have been tested with good results [150, 151].

A relevant methodology to measurement DA and others CAs in vivo is the fast-scan cyclic voltammetry associated with carbon fibers microelectrodes, located directly in a brain nucleus of rodents [152-154] and awake monkeys [155]. In this approach, electrodes show changes in the voltage that are proportional to the amount of neurotransmitter present in the site of sensor, with high temporal resolution [156, 157]. Some modifications have been introduced in the microelectrode construction designs to improve the electrical signal, like zeolites-graphite mixtures [158] and diamond nanostructures [159].

Acetylcholine

ACh was identified first as a neurotransmitter in the neuromuscular junction [160], and later accepted as a putative neurotransmitter in the CNS. The biological importance of ACh lies in its association with certain pathologies such as Alzheimer's, schizophrenia and Parkinson's disease, as well as its relationship with seizures and neuropathology associated with organophosphorus compounds, especially those related to the development of biological weapons [161].

The quantification of ACh is complicated, since it is neither electroactive nor chromogenic. In addition, its cationic nature complicates its retention in the conventional reverse-phase columns for chromatographic separation. In the past decade a variety of methods have been developed for ACh quantification, such as quimioluminiscent [162], electrochemical [163], fluorescent [164], and MS [165], but the search for methodological improvements that enable to increase the LOD and limit of quantification (LOQ) still continues.

HPLC-Enzymatic Reactor-Electrochemical Detection (HPLC-ER-ED)

The most common way to determine the content of ACh in samples of brain dialysates or in tissue homogenates, is a combination of HPLC-ER-ED, in which the separation of ACh is carried out in columns for ion pair chromatography or ion exchange retention mechanisms. After the analytes are separated, these are passed through a post-column arrangement with enzymes immobilized in small "cartridges" available in the market, in which the ACh is hydrolyzed by acetylcholinesterase (AChE) in acetate and Ch. Ch is subsequently oxidized by Choline-Oxidase (ChOx), in betaine and H2O2. H2O2 is detected electrochemically on a carbon or Pt electrode at a determined applied potential in a potentiostat [166, 167]. Finally the arrangement is completed with a reference (Ag/AgCl) and counter electrodes. This method has been used for the determination of extracellular ACh content in multiple regions of the CNS under different paradigms [168-172] with a LOD of 10 fmoles [171, 172] in dialysate fractions between 12 and 30 µL, representing collection time of 6 to 15 min [170, 171]. With these methodologies it has been possible to establish the involvement of cholinergic neurotransmission in several cerebral functions, including different types of memory [171, 173], learning [174], attention [175], and homeostatic aspects; such as body temperature and blood pressure [176], as well as the association of this neurotransmitter with Alzheimer's, Parkinson's, Hungtinton disease and schizophrenia [177]. Although the method was introduced almost 30 years ago [178], it is still valid. However, it has some major drawbacks. A relatively large amount of sample is required to obtain a reliable quantification (> 10 µl). Which means large amount of time for the sample collection to reach the LOD described in the methodology (in the nM range), thus preventing a direct relationship between the neurochemical event of release and its quantification. In addition, the quantification of ACh presents two aspects to solve. The first is the rapid inactivation of ACh by the enzymatic action of AChE, maintaining the basal extracellular concentrations of ACh at very low level. This requires the use of inhibitors to enrich the concentration of ACh in the samples of dialysate, among which the most used, is neostigmine [179-181]. The second problem is the presence of electroactive elements with similar oxidative potential to ACh, mainly the ascorbic acid (AA). These molecules could interfere with the electro-chemical determination of ACh and other neurotransmitters (DA, serotonin) [182, 183].

HPLC Coupled with MS

With the idea of increasing the sensitivity for the analysis of ACh, it was developed a methodology that couples the HPLC with MS [184], in which the ACh molecules, previously separated in a chromatographic system, are introduced in a generating "electrospray interface” at high voltage and temperature; the resulting electrospray (ES) contains ions generated by the breakdown of the original molecule (daughter ions), and enters to the mass spectrometer. This approach provides a sensitive and selective analysis that is unique for individual analytes [185]. In the case of ACh, this has a quaternary amine that carries a positive charge in acidic conditions. This prevents the ionization of the molecule improving its sensitivity. In this way, ACh molecule becomes fragmented, producing the so-called molecular ion m/z 87, which is quantified in the systems of MS. This methodology uses the retention time and the molecular mass to establish the identity of compounds.

The advantage of MS is the possibility to get a direct measure of ACh, without using inhibitors of AChE, and without derivatizing the compound of interest through an enzymatic reactor. However, in this methodology the criteria of selection of HPLC columns and mobile phases are important issues for the detection. Nevertheless, due to poor retention of polar molecules in reversed phase columns, it is necessary to use the ion pair chromatography agents, which significantly interfere with the process of generation of ES. In addition, the relatively high concentrations of organic solvent decrease the efficiency of molecular ions production. For this reason several works have been published proposing various modifications on the column material and the buffers used. From the first attempts to match the typical methodology for ACh detection by HPLC through microbore C18 columns, using octane-sulfonic acid as ion-pairing agent by the production of ES [186], a lot of proposals were quickly developed, which improved the LOD and LOQ for ACh. Hows et al. [187] present a HPLC-MS analytical system, in which microdialysis samples (fractions of 24 µL) are separated by cation-exchange chromatography, with mobile phase of ammonium acetate, ammonium formate and acetonitrile. The reported LOD was 1 fmol on column, and allows the quantification of ACh without AChE inhibitors. The same methodology was used for the quantification of ACh, iso-ACh and Ch in the hippocampus of animals in free movement, where the formation of molecular ions from ACh was optimized by reducing the voltage of both production of ES (from 3.5 to 0.9 kV) and the detection of molecular ions (from 30 to 18 kV). Under these conditions, the intensity of ACh-derived ions was not altered with the change of organic composition in the mobile phase [185]. In addition, the use of β-methyl-ACh as internal standard in samples with artificial cerebral spinal fluid (aCSF) significantly improved the quantification, achieving a LOD of 0.01 nM (0.2 fmol on column) and a LOQ in solution of aCSF of 0.05 nM (1 fmol on column) to ACh. Carrozo et al., [188] optimized the separation at low voltages, with the regulation of the acetonitrile concentration entering the ES system, by use a mixing tee post-column system, located after the six-valve port and before ES inlet, in order to improve sensitivity preserving the optimized chromatographic conditions. By this way, the sensitivity of the method increases since acetonitrile improves ionization of ACh with no effect on chromatographic separation. Since the mixing tee is inserted after six-valve port, the inorganic salts of the Ringer’s solution were wasted before getting in contact with acetonitrile preventing precipitation in ES. LOD was 0.01 nM (0.05 fmol on colum) for ACh measured in Ringer’s solution and the LOQ at signal-to-noise ratio of 10, were 0.05 mM (0.25 fmol on colum) to ACh.

Hydrophilic interaction chromatography (HILIC) has been used since the hydrophilic/polar nature of the ACh. Using polar stationary phases (also known as "aqueous normal phase columns") in combination with organic aqueous mobile phases, it is possible to generate an aqueous layer over the polar stationary phase, which allows the distribution of the analyte between the two phases, according to their polarity. Stationary phases of silica [189], amide [190], polyhydroxyethyl aspartamide [191, 192], or diol [193] have been used for ACh. HILIC systems have become popular for the quantification of ACh, since they represent a good strategy to avoid interference due to ion pair chromatography agents and mobile phases with high concentrations of salts.

Using HILIC mobile phase of 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid as ion-pairing agent, combined with the atmospheric pressure photoionization (APPI) method rather than ES for the production of derivatives of ion m/z 146, a LOD of 0.15 nM (1.5 fmol on-column) was obtained [194]. Even, miniaturization has been achieved using a microchip "nebulizer" [195], which has been used to quantify ACh in some paradigms of behavior [196]. However, the method of detection by APPI so far has not been validated by other groups.

The use of ACh analogues as internal standards in HILIC system has been reported, showing a significant improvement in the sensitivity of detection of ACh. The incorporation of ACh-d9 bromide as isotope-labeled internal standard and collision-induced dissociation to determine the resulting daughter ions, allowed to obtain LOQ of 0.1 nM (0.2 fmol on-colum) for ACh in dialysates of rat frontal cortex [197]. ACh tetradeuterated (ACh-d4), used as a calibration standard and internal standard, has been used under the “analyte replacement" strategy (introduced by Li and Cohen, in 2003) [198] to eliminate the interference of iso-ACh, the main ACh isomer, which is also an endogenous substance with the same ionic transition to ACh, and it is difficult analyte to separate in HPLC-MS/MS systems. This approach was validated to quantify ACh in SAMP8 mice, used as a model for Alzheimer's disease [199].

An alternative for chromatographic separation is the separation by CE also coupled to MS. This allows a quick and efficient separation of charged compounds in small volumes of sample. ACh has been quantified by CE coupled with MS at used cell extracts, with a LOD of 5 nM/6 nL, introduced into the capillary via hydrodynamic injections [200] in microdialysate fractions, with a LOD of 0.15 nM in 15 µL of dialysate [201], and even there are reports of detections of ACh online using CE, where a LOD of 40 pM was reported [202]. The separation is based on differences in mobility of ions within a capillary tube of silica, with a length between 30-100 cm and an inner diameter less than 100 µm. One end of the capillary tube is placed at the anode and the outlet is located in the cathode. The capillary is subject to procedure of deprotonation of the silanol groups (which gives a negative charge to the inside of the tube) followed by perfusion with a buffer electrolyte to form a double layer in which hydrated positive ions migrate towards the cathode under the influence of an electric field. This creates a natural pumping mechanism inside the capillary, called electro-osmotic flow, which increases the pH. The electrophoretic mobility, which depends on the charge-size relationship, is faster for positive ions, followed by the neutral and finally for negative [166].

The coupling between HPLC and MS has mean a breakthrough for the LOD and LOQ, though it does not resolve the issue of time quantification of ACh, despite the high sensitivities reported, the decrease in the volume of sample and the effectiveness in the separation of the analytes, is still requiring the collection of samples and their subsequent analysis.

Micro-Biosensors

Here we have grouped various types and designs, which involve the inclusion of sensors directly in the brain tissue, in the areas where the determination of neurotransmitters is tried. In general, biosensors contain a biological component (an enzyme, antibody, nucleic acid, membrane receptors, cells) to identify a molecule of interest, producing in the process a chemical signal, which will be captured by a transducer component, whose expression will be optical or electrochemical [203, 204]. Both the biological component and the transducer are a single unit of small size (commonly a Pt wire or carbon fiber), allowing insert it in vivo in a discrete region of the brain with minimal injury compared with microdialysis probe [205]. For the quantification of ACh and other neurotransmitters, biosensors are designed with oxidases to oxidize the primary analyte, with the concomitant production of H2O2, which is detected amperometrically at potential between 500-700 mV, in relation to a reference of Ag/AgCl electrode [206]. It is important to mention that biosensors are constructed in a multienzyme format; with ACh and ChOx, given the need to produce consecutive reactions to generate H2O2. Because the presence of electroactive molecules, such as AA or CAs, generates nonspecific signals, the main challenge for miniaturization of biosensors is to achieve the immobilization of enzymes on a matrix suitable to maintain the activity and biological stability, and also to form a part of a barrier for molecules that are not of interest. With this purpose, systems for enzyme immobilization with crosslinkable polymers as styryl pyrydinium-polivynil alcohol or tetrathiafulvalene-tetracyanoquinodimethane crystals have been developed, reaching LOD between 0.1-0.3 μM to ACh in vitro [207, 208]. Another biosensor reported was constructed with poly m-(1,3)-phenylenediamine electro-polymerized on Pt-iridium (Pt-Ir) wire as a template for immobilization of multienzyme layer containing ChOx, AChE, and ascorbic acid oxidase (AAO) immobilized with bovine serum albumin (BSA) and cross-linked with glutaraldeyhyde [209] or base on N-acetylaniline (used as to selective film to reject interferences) electropolymerized onto the Pt electrode with a second layer of zinc oxide sol-gel as a matrix to immobilize a ChOx/AChE/BSA solution [210]. Typically, these micro-biosensors have a LOD of 0.5-0.7 μM for ACh, but there is no evidence of its usage for in vivo experiments.

Given the limited real use of micro-biosensors in vivo, a biosensor for intracerebral measurement of ACh in awake animals was implemented [211] in which the method reported by Hu et al., [212] was used with several modifications. These biosensors showed a linear response at 20 µM - 80 µM of ACh when tested in vitro with a time resolution of 20 second. Also, it was insensitive to AA and norepinephrine. The extracellular concentration of ACh was monitored during induced seizure activity, making evident a significant increase in ACh related with strong seizure behavior activity. The biosensor retains its activity beyond one month when these were store at 4°C [211].

Others micro-biosensors have been developed with CNTs as transducer element. CNTs are 1-2 nm tubules of graphite carbon, that form aggregates among them, providing a large edge/basal plane ratio, enhanced electronic properties, and rapid electrode kinetics [213-215], which improve the electrontransfer reactions of enzymatically generated species, such as H2O2 [216]. The CNTs can form a negatively charged surface over which can be deposited an electrostatically and self-layer-by-layer assembly of AChE/polymer, and have become useful elements to estimate the presence of organophosphorus pesticides used in agriculture, medicine, industry and chemical warfare agents [217, 218]. Although the use of microsensors to measure ACh directly into the brain is not a reality, as reflected by the lack of publications about in vivo applications, new combinations of CNTs with biological materials are emerging: in a recent paper, Kim et al., [219] reported the construction of a lipid membrane sensor for ACh based on CNTs containing M1 type ACh receptors, on a silicon chip. In this device, the binding of ACh onto the receptor was detected by measuring the electrical current change in the underlying CNTs, enabling the real-time detection of 100 pM of ACh concentration. According to this and other reports, this kind of device has a strong potential to be used in a small-size needle-like configurations for in vivo applications [219]. Also, the combination of CNTs/sol-gel surface with self-assembly gold nanoparticles-AChE matrices on Pt wire has been proved to the ACh amperometric detection, reaching a LOD of 1 μM in vitro [214].

Others Biosensors

In the last 5-6 years, a number of novel biosensors have been developed, which eliminates the requirement of enzyme confinement to a surface and takes some characteristics from biological molecules and processes. Although the sensitivity of most of them have low range, they are not adequate for monitoring the ACh concentration in the brain. In the future it is possible that some of these ideas could be developed with greater sensitivity to achieve the necessary nanomolar range. In this sense, Schena and Johnsson [220] introduced the semisynthetic ACh-SNIFIT protein, as a fluorescent probe for the direct quantification of ACh within the extracellular matrix. The sensor is constructed with ACh binding to CLIP/SNAP tags: CLIP is labeled with a fluorescence resonance energy transfer (FRET) donor and SNAP with a FRET acceptor. The molecular assembly has the capacity of anchoring to the outer cell membrane through a growth factor receptor transmembrane domain in a cell line derived from embryonic kidney cells. The interaction of ACh with the AChE causes an opening of the assembly, to decrease in the FRET and the emission of a fluorescent signal. The response of this probe was tested by perfusing cells with concentrations of ACh between 1-10 mM, with good response [220].

In a recent paper, the capacity of cysteine to form aggregates with gold nanoparticles was exploited to implement the detection of H2O2 generated by the AChE/ChOx biocatalytic cascade. In this case, the aggregation process leading to a color changes from the red to violet corresponding to the aggregated nanoparticles. The H2O2 produced in the catalysis of ACh inhibits these aggregations, and the change in color can be visualized with a spectrophotometer. Results of this paper show linear response between 20 and 200 µM of ACh, the probe was tested only in calibration mode [221]. Also, immobilization of TiO2 nanoparticles and BiOI nanoflakes onto Indium tin oxide micro-slices has been used as surface to attach anti-AChE antibody/A-protein complex. AChE was after fixed by its immunoreaction with its antibody. Theoretically, the enzymatic reaction between bound AChE and free ACh will produce thiocholine, which acts as electron donor [222]. This array produces a photoelectrochemical signal proportional to the amount of ACh added.

Buiculescu et al., [223] developed quantum dots (QD) semiconductors, these have several characteristics of interest, such as small size, high photoluminescent capacity and; narrow, symmetric and size-tunable emission of the spectra covering the blue-red region. Also, the capacity of QD for being conjugated with antibodies, proteins or receptors is important. The construct QD/AChE is conjugated on poly-L- lysine-templated silica matrix. The product analyzed is AA, which is generated in the enzymatic reaction and lowers the pH of the surrounding environment and as a result, a decrease in the photoluminescence is observed. This decrease in photoluminescence is proportional to the AA generated. The LOD obtained in this work was 1 µM [223].

Another method using QD was proposed by Wei et al., [224], based on the fact that the fluorescence of QD has linear relation with the production of H2O2 in the concentration range of 0.1-40 μM and in presence of Fe2+, by Fenton reaction. Once the enzymatic reaction produces H2O2; QD and Fe2+ can be introduced into the solution, and the turn-off in fluorescence intensity can be determined. The LOD reported in this work was 0.1 µM.

Finally, interesting attempts have been made to construct molecular reporters by molecular engineering, in a methodology named cell-based neurotransmitter fluorescence engineered reporters (CNiFERs) that allows monitoring in situ neurotransmitter receptor activation, using a modified cell line to express the fluorescent protein TN-XXL combined with the quantification of the FRET [225]. Nguyen et al., [226] developed a system based on the Ca2+ overload by activation of muscarinic M1 intracellular receptors, via the Gq/inositol triphosphate second messenger pathway. Intracellular Ca2+ bind to TN-XXL and induces a conformational change that enhances the FRET between two separated protein domains, and produces a fluorescent signal. The CNiFERs was stereotaxically implanted in the brain neocortex of adult rats, and its functionality was assessed by direct deposition of ACh near the implantation site. Besides, to determine if CNiFERs can sense ACh physiological burst, the nucleus basalis magnocellularis was stimulated, which is known to send cholinergic projections into the neocortex, obtaining a positive response in the fluorescence associated to CNiFERs.

CONCLUSION

Measuring the fast neurotransmitters in CNS has a relevant interest in neuroscience field; this is due to the role of these compounds during normal of pathophysiological activity. Alterations in their homeostasis could mean the appearance of a neurological disease. Although, these neurotransmitters are not commonly measured for clinical purpose, they are normally determined in laboratories research to evaluate the effects on many possible therapeutic drugs in order to determine their mechanisms of action, as well as the alteration due to a pathological state. Since many years ago, microdialysis procedure has been a very popular technique despite of its poor temporal resolution; thousands of papers have been published around this method, which means that this technique is still valid. The use of biosensors is positioning as another alternative method and although several advantages come with them, like the improved spatial and temporal resolution and a lesser damage induced in the brain, they can only determine the concentration of one neurotransmitter at a time and perhaps there will not be biosensors for all the neurotransmitters. NMRS methods have a great temporal resolution and are non-invasive, which permits the study in humans. Other method is the immunosensing by acoustic wave, this does not require the use of hazardous materials or an enzymatic reaction, and gives a good temporal resolution. With the appearance of optical methods for measuring compounds in a non-invasive way like: functional near infrared spectroscopy (fNIRS) which determine the difference in the absorption spectra of des-oxy-hemoglobin and oxy-hemoglobin allowing to quantify the relative change in hemoglobin concentration and also, the Raman spectroscopy has been used to try to measure glucose concentration in human patients without the necessity of a catheter to get a sample. Perhaps in the future, the methods reviewed here could be adapted to measure not only the fast neurotransmitters but also several compounds of biological interest in real time and with enough sensitivity to associate their changes with behavior, normal of pathophysiological states. Although a lot of effort must be put to reach this goal.

Table 1

Limits of detection and temporal resolution for techniques used for the quantification of GABA, AChand Glu.

Analyte	Technique	Sensibility	Limit of Detection	Temporal Resolution	References
GABA	HPLC		9.7 nM	minutes	[227]
	HPLC- ED	0.03 pmol		minutes	[228]
	HPLC-FLD, in a boron columns	0.5-1 pmol		minutes	[78]
	HPLC-FLD, in a microboron column	100 fmol		minutes	[78]
	CE	0.016 µM		minutes	[102]
	CE-LIFD		5.1 nM	minutes	[229]
	LC-FLD		10 nM	minutes	[230]
	LC/MS/MS	5 nM	0.5 nM	minutes	[231]
	HPLC-FLD		0.97 µM	minutes	[232]
	LC-MS		48 nM	minutes	[82]
	Peroxidase based amperometric biosensor	50 µM – 1.2mM	20 µM	minutes	[105]
	Acoustic biosensor	38 µM		seconds	[108]
	GABAse Fluorescence-Coupled Assay	0.78 µM	0.41 µM	minutes	[122]
	Immunosensor using QCM		42 µM	seconds	[109]
	Whole-cell sniffer		100 nM	miliseconds	[233]
	Bioluminescent assay		5 pmol/µL	minutes	[126]
	MRS J-difference edited		1mM	real time	[129]
	MRS MEGA-editing		0.75 µmol/g	real time	[129]
ACh	GC-FID detector		0.02 nmol	minutes	[234]
	HPLC-ED		20 fmol	minutes	[235]
	CF electrode		1 µM	minutes	[236]
	HPLC-ER-ED		10 fmol	minutes	[171]
	HPLC-MS		1 fmol	minutes	[187]
	HPLC-MS using Ringer solution	0.05 mM	0.01 nM	minutes	[188]
	HILIC		0.15 nM	minutes	[199]
	CE-MS		5 nM	minutes	[200]
	Pt-electrode based biosensor		0.5 µM	seconds	[210]
	CNTs/sol-gel gold nanoparticles with AChE		1 µM	seconds	[214]
	CLIP/SNAP ACh sensor	1-10 mM		seconds	[220]
	QD/AChE-Poly-L-Lysine-templated silica matrix		1 µM	seconds	[223]
Glu	CE-LIFD		0.070±0.71 nM	seconds	[237]
	CE-LED-IF		1.2 nM	seconds	[238]
	LC-FLD		3-30 nM	minutes	[230]
	LC-ESI-MS		0.5-5 nM		[231]
	LC-MS		0.65 µM	minutes	[239]
	HPLC-MS		0.1 µM	minutes	[83]
	HPLC-FLD		0.1-20 µM	minutes	[232]
	Electrode Pt site	0.016±0.001 nA/µM	1.82±0.17 µM	second	[240]
	Electrochemical microsensor	0.0034±0.001 nA/ µM	1-3 µM	seconds	[105]
	Electrode CP coated	14.0±0.2 nA/ µM	0.1±0.03 µM	seconds	[241]
	Electrode Pt-Ir	0.1 nA/ µM	2 µM	second	[212]
	Electrode Pt-NAE	194.6±9.2 µAmM-1cm-2	1 µM	seconds	[242]
	Pt-PPD-GluOx-GA		3.0±0.6 µM		[243]
	Biosensor basedon covalent immobilization of GluOx on polypyrrole nanoparticles-polyaniline modified gold electrode		0.1 nM	seconds	[244]
	Biosensors and microelectrodes	793 pA/mM	0.493 mM	seconds	[46]
	Graphene quantum dots		5.2x10-8 M	seconds	[54]
	Self-referencing Electrochemical biosensor	473±57 µA/ µM	0.9±0.3 µM	seconds	[246]
	Microsensors and amperometric monitoring	126±5 nA/ µM	2.1±0.2 µM	second	[8]
	Sensor enzyme free Ni nanowire array electrode	65-96 µA/mM	68-88 µM		[242]