Understanding the complex functions of a brain remains one of the grand challenges of biomedical research. One possible way is to develop in vivo biosensors for monitoring the activities and the signal pathways of neurotransmitters in brain. Neurotransmitters are essential chemicals that can transfer information between the neuron cells. Otto Loewi was one of the first people to discover one type of neurotransmitter called acetylcholine in 1921 [1
]. His work significantly impacted the human perception of how information is transmitted in animals. Subsequently, other types of neurotransmitters were later discovered, with over 100 different types known to date. Almost all neurotransmitters are essential to human mental and physical health and any abnormalities or changes in their activity may cause severe disease and mental disorders. Hence, monitoring of such neurotransmitters is critical for medical treatment and clinical analysis.
A biochemical sensor that can continuously monitor in real time the dynamic behaviors of the various neurotransmitters can be a powerful tool for monitoring and treating patients with neurological brain disorders such as Alzheimer’s disease, epilepsy, Parkinson’s disease, and addiction. For instance, in Parkinson’s disease (PD), the primary symptoms appear as a gradual deterioration of substantia nigra that controls motor functioning such as balance and movements [2
]. This region of brain is located deep in the brain stem [3
] where the dopamine produced in this area is responsible for neural communication between the striatum and the substantia nigra [5
]. Parkinsonian symptoms appear when dopaminergic neuronal death exceeds a critical threshold of 70–80% [6
]. The decreased level of dopamine is directly associated with the uncontrolled motor function, which leads to the inability to neutralize the imbalance in neurotransmitters [7
]. In particular, the motor function in the striatum is dependent on the balanced equilibrium between dopamine and acetylcholine. The disruption in the balance of these two neurotransmitters can bring about the progression of PD [8
]. It is also known that norepinephrine dysfunction is a contributing factor in PD, and serotonin and gamma(γ)-aminobutyric acid (GABA) may also affect the condition as secondary symptoms of PD [11
]. Furthermore, histamine, the initial neurotransmitter and immune mediator, has been reported to be significantly elevated in the brain with PD [13
]. The above findings illustrate that there is a complex interplay between various neurotransmitters that are closely related to the progression of neural diseases. Therefore, simultaneous detection of multiple neurotransmitters in vivo is urgently needed for the better understanding of mental disorders and for the development of treatment and therapy for such diseases.
The mesocorticolimbic dopamine system plays a crucial role in reward, motivation and learning [15
]. It is also severely affected by drug or substance addiction. The ventral tegmental area (VTA) is the midbrain region that has been implicated in the rewarding effect of various addictive drugs such as cocaine [18
], nicotine [19
] and opiates [20
]. Important neurotransmitter signaling pathways include limbic afferents to the nucleus accumbens (NAc), efferents from the NAc to VTA, dopaminergic projections from VTA to NAc, to prefrontal cortex (PFC) and to tegmental pedunculopontine nucleus (TPP) [16
] as shown in Figure 1
and Figure 2
. In the event of drug administration, a wide range of neurotransmitters will change their dynamics as a result of either overproduction or inhibition. The above scenario showcases the potential application where a multi-analyte neurotransmitter-sensing probe could be used to advance our understanding of the brain from a neurological signaling perspective. Hence, the development of an implantable multi-analyte sensor that can simultaneously monitor the dynamics of multiple neurotransmitters as they occur in real time can be an extremely power tool.
In vivo monitoring is challenging because the response time of the neurotransmitters is fast, rapidly releasing and clearing from the extracellular space [21
], and the concentrations involved are typically low [22
]. Two of the most widely used techniques for in vivo monitoring of neurotransmitters are microdialysis and fast-scan cyclic voltammetry (FSCV). In microdialysis, a semi-permeable probe is injected into the brain and the analyte that is present in the brain is perfused through the probe and collected for chemical analysis using techniques such as high-performance liquid chromatography (HPLC) with mass spectrometry (MS) or fluorescence as a detector [23
]. The main disadvantage of microdialysis is the low temporal resolution in the order of minutes since sample fluid must be drawn from the brain and collected for off-line analysis. FSCV has recently emerged as one of the leading techniques for in vivo neurotransmitter detection due to its fast sampling rate leading to high temporal resolution [21
]. The in vivo electrochemical monitoring of neurotransmitters in the brain have been effective for electroactive species (e.g., dopamine, norepinephrine, serotonin, adenosine, etc.). However, several challenges remain to be solved: (1) many chemical species in the brain have similar oxidation/reduction potentials and the presence of many interfering species makes it difficult to conduct multi-analyte detection in vivo. (2) non-electroactive species (such as glutamate, histamine, acetylcholine, etc.) require enzymes (e.g., glutamate oxidase, acetylcholinesterase, etc.) to be detected electrochemically. The limitations associated with enzyme-based sensors are their instability, degradation in enzymatic activities and complex immobilization protocols. Hence, a novel sensing technique that is reliable and allows simultaneous real-time multi-analyte detection with high specificity, temporal and spatial resolution is greatly needed.
Currently, the need is great for establishing a new route to monitor the complex intercommunication of neurotransmitters in the brain. In particular, a measurement technology that is capable of parallel, rapid, and specific quantification of numerous analytes for the requisite extended time period in a living brain, without negatively impacting the implanted region is in demand. Existing in vivo measurement techniques are incapable of (1) multi-analyte detection with high temporal resolution in real time and (2) long-term monitoring without device failure.
Many works related to neurotransmitter detection have been published in recent years, with emphasis on both in vivo and ex vivo sensing. Various sensing mechanisms have been explored with a variety of nanomaterials used. For example, micromachined electrode array (MEA) is widely used for neurotransmitter detection in vivo because it provides facilitated communication between the sensor and the neuron [24
]. The assembly of the microelectrode could greatly enhance the surface area for capturing the released neurotransmitters from cells, thereby minimizing the diffusional delay [25
]. Several parameters are crucial in evaluating the performance of the sensor: the sensitivity and the limit of detection (LOD) of the sensor must be sufficient for the level of concentration for the target neurotransmitter in serum. Also, the selectivity of the sensor must be high enough because much interference may be present in the real sample. Reproducibility is also crucial for robust analysis. The electrodes may foul due to the adhering or adsorbing of the proteins in real samples [26
]. Fouling can greatly impact the sensor response, hence it is necessary to develop electrode surfaces that are resistant to bio-fouling [27
]. Detecting multiple neurotransmitters simultaneously is a very challenging task because many neurotransmitters possess similar molecular structures and physicochemical properties which make them difficult to differentiate one from the other. In this review, we summarize the primary materials used for sensor electrode development as well as the strategies for the in vivo and ex vivo detection of neurotransmitters. We also discuss the current state of the art in simultaneous detection of multiple species of neurotransmitters.
4. Simultaneous Detection of Multiple Species
Most work published so far has focused on individual detection of a single neurotransmitter species. However, in practical applications and for in vivo measurements, simultaneous determination of multiple species of analytes is the subject of great interest but also a grand challenge. Traditionally, multi-analyte sensing has been performed by developing an array of sensors commonly known as an electronic nose (e-nose) for gas phase samples and electronic tongue (e-tongue) for liquid phase detection [121
]. Microdialysis is another commonly used sampling technique for analyzing multiple species [122
]. In microdialysis, a small volume (typically a few microliters) of extracellular fluid is sampled through a semipermeable membrane located at the tip of the implanted probe at a fixed time interval. The sampled fluids are each collected in a separate container, stored in sequential order, for analysis offline using advanced techniques such as high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC/MS). The advantages of microdialysis include accurate analysis of multiple species due to high-precision analytical tools (i.e., HPLC, LC/MS, etc.), long-term detection capability without the issue of electrode fouling, and minimally invasive sampling tools. However, the disadvantages are low temporal resolution (on the order of minutes), inability to measure in vivo, and expensive analysis methods. Hence, a novel multi-analyte sensor with high sampling rate, low cost, and in vivo measurement capability is needed. When developing simultaneous and multi-analyte sensors, the following concerns must be addressed: first, a single sensing technique must be applied to all the sensors in the array; second, each modified sensor must be sufficiently selective and sensitive toward the analyte; third, for in vivo detection, the developed sensor must be resistant to fouling in order to achieve long-term measurement. In previous work, dopamine (DA), uric acid (UA), and ascorbic acid (AA) were commonly chosen as the analytes, since they often coexist in the real biological samples. Zhang et al. developed a Poly(l-lysine)/graphene oxide-modified sensor for the simultaneous detection of DA and UA [123
]. The polymerization of l-lysine is performed electrochemically through potential cycling on the graphene oxide-modified glassy carbon electrode. Their sensor shows two distinct oxidation peaks when DPV was applied to detect DA and UA simultaneously from the same sample solution. Also, the addition of one analyte had minimally interfered with the detection of the other. The detection limits of DA and UA are 21 nM and 74 nM, respectively.
Sun et al. have developed an electrochemical sensor for simultaneous detection of DA, UA, and AA [124
]. Gold nanoparticles and MoS2
nanosheets were chosen as the sensing material to amplify the DPV response of the three analytes. They have reported that bare GCE, AuNPs/GCE or MoS2
/GCE alone were not able to distinguish among the three analytes. However, the combination of AuNPs and MoS2
nanosheets was able to individually identify and also quantify the three species from the mixture of analytes. Figure 10
shows the DPV response and calibration curves when detecting DA, UA, and AA, simultaneously. The detection limits for DA, UA, and AA were 0.05 μM, 10 μM, and 100 μM, respectively.
A colorimetric sensor was developed by Jafarinejad et al. for the detection of dopamine (DA), norepinephrine (NE), and epinephrine (EP) [125
]. They have designed a Au/Ag core-shell nanostructure, and it was able to individually quantify the analytes by obtaining the various absorbance spectra. A solution of gold nanorods and silver nitrate were mixed to develop a sensing solution. The addition of neurotransmitters works to reduce mediators, causing silver growth on the surface of the gold nanorods and leads to color changes in the solution. Unique color patterns were collected using UV-vis spectra after the sensing solutions reacted with different analytes. They also successfully detected DA, NE, and EP simultaneously using the colorimetric sensor array.
Wojnicz et al. reported a multi-analyte sensor using liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis approach [126
]. They were able to detect eight different neurotransmitters simultaneously including DA, NE, EP, Glutamate, 5-HT, γ-aminobutyric acid (GABA), 5-hydroxyindole acetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) However, the limitations of this approach are the complexity and the high cost of the LC–MS/MS system.
Zhang et al. have successfully improved the sensitivity of the liquid chromatography/mass spectrometry (LC/MS) analysis using N-α-Boc-l-tryptophan hydroxysuccinimide ester (Boc-TRP) derivatives when detecting multiple neurotransmitters simultaneously. Their approach is capable of detecting GABA, glutamate, glycine, and citrulline with a detection limit of several nanomolar concentrations. Multiple amino acid neurotransmitters are also detected in rat brain microdialysates [127
Barman et al. designed a AgNPs–penicillamine modified gold electrode electrochemical sensor for the simultaneous determination of DA and EP [128
]. They first prepared the penicillamine (PCA)-modified gold electrode by immersing the gold electrode in 1.0 mM PCA solution. After the self-assembly process, the electrode was dipped into AgNPs solution forming a AgNPs–penicillamine modified electrode. The sensor also showed high selectivity towards DA and EP in the presence of AA and UA. Another work reported by Tezerjani et al. introduced a graphene oxide nanosheets-based electrochemical sensor for the detection of DA, EP, and acetaminophen [129
]. The detection limit of EP was calculated as 0.65 μM.
5. Optical Sensing of Neurotransmitters
Most papers reviewed in this article are based on electrochemical sensing techniques. However, many of them exhibit the level of detection limits that are still too high for in vivo measurements, and the electroactive species are prone to the interference problem in the signal acquisition. In terms of in vivo sensing of neurotransmitters, another popular and promising sensing technique besides electrochemical methods is the optical detection approaches. In most cases, optical sensors exhibit highly reproducible and sensitive readings with the limit of detection often reaching a nanomolar range or less [130
]. Also, the interference from other chemical species could be minimized by utilizing a broad range of optical spectrum. Most importantly, the main benefit of optical sensors is that they provide high spatial resolution. Furthermore, it does not require electrical wiring or electrodes at the implanted probe for signal acquisition since the optical signals are generally transmitted through fiber optic cables. Spatial and temporal resolutions are equally important for in vivo detection of neurotransmitters since the release and uptake of such chemicals occur in a short time period, typically on a millisecond range [21
], and in a highly localized fashion [131
]. Therefore, a sensor should possess a sampling rate that is high enough to capture the concentration changes that occur in a millisecond timescale and also be small enough to identify which neurons are involved in such release and uptake of the chemical signals. Carbon nanotubes can be used as active materials in optical sensing due to their unique electrical and optical properties [132
]. Kruss et al. have utilized a polymer functionalized single-walled carbon nanotubes (SWCNTs) to measure the changes in the near-IR fluorescence signal modulated by various neurotransmitters, which they have termed corona phase molecular recognition. They showed that the unique polymer composition (DNA, RNA, phospholipids, amphiphilic polymers, etc.) in combination with its close proximity to the SWCNT to which the polymers are wrapped around, have resulted in a selective detection of neurotransmitters with high spatial resolution [133
]. Using this optical technique, a fluorescent nanosensor array for dopamine sensing with high spatial and temporal resolution was also demonstrated [134
]. The sensor array was based on fluorescent carbon nanotubes, and able to monitor dopamine concentration from PC12 neuroprogenitor cells. The spatial and temporal resolutions have reached 20,000 sensors per cell and 100 ms, respectively.
López-Valenzuela et al. have introduced fluorescence detection system to detect Glutamate in the hippocampus, and the fluorescence measurement was able to reach one second resolution [135
]. In their approach, glutamate oxidase was used to generate H2
, which was quantified with Amplex Red, a fluorogenic probe. This probe reacts with H2
to produce resorufin which fluoresces at 590 nm when excited at 560 nm.
Kim et al. developed a wireless optical sensor for dopamine recognition in vivo [136
]. The optical sensor contained a fluorescence sensing probe, micro-spectrometer, and a system electronics module. CdSe/ZnS quantum dots were attached on the optical probe for dopamine detection. The fluorescent sensing probe is able to detect dopamine with a detection limit of 100 nM. Their sensor also shows excellent selectivity over uric acid and ascorbic acid.
Baluta et al. reported a fluorescence-based sensor to detect dopamine [137
]. Graphene quantum dots and low-temperature co-fired ceramics (LTCC) technology was introduced for dopamine recognition and sensor fabrication. The detection limit of their sensor was calculated to be 22 nM. Their sensor was able to achieve in vivo measurement, and the fabrication process was simpler compared to other optical sensors.
An ultra-sensitive optical sensor for GABA determination was reported by Huang et al. [138
]. The sensor was based on raspberry-like meso-SiO2
nanosphere functionalized silica microfibers. Silver or gold nanoparticles were further introduced to enhance the local electric field in near infrared. The concentration of GABA was monitored based on the transmission wavelength shift, and the detection limit of the sensor was reported to be
Gupta et al. have used a Sulphur-doped carbon dots, with an average particle size of 6 nm, for the detection of DA from PC12 cells [139
]. A significant quenching of the carbon dot fluorescence occurred at 425 nm when excited at 310 nm upon exposure to DA molecules. Table 7
below summarizes the recent progress in optical neurotransmitter sensors.
The ability to identify and measure various neurotransmitters with high sensitivity and low-cost will provide a powerful tool for use in clinical diagnostics and neuroscience research. Many recently published works have reported on the nanostructured materials for highly sensitive detection. The use of carbon-based sensors in conjunction with electrochemical sensing strategies have dominated this field due to the promising electrical property, stability and low cost. Polymers are also widely used for enhancing the biocompatibility and the redox properties of the sensor. Although aptamer-based sensors exhibit ultra-selectivity and high sensitivity towards the analyte, the complicated modification procedure and chemistry have limited the commercialization of the sensor. However, aptamer-based biosensors show great potential to become a practical sensing device in the near future. For improving the performance of the sensor, catalysts such as metal and metal oxide nanoparticles can be utilized to enhance the sensitivity and selectivity. The miniaturization of the sensing device is also critical for enhancing the spatiotemporal resolution especially in in vivo environment. Furthermore, nanomolar detection limit with high sensitivity and reproducibility are essential for the sensor to be used in clinical setting.
Electrochemical sensors and optical sensor are the two most commonly applied methods for in vivo monitoring of neurotransmitters due to the many advantages they offer. However, the simultaneous detection of multiple neurotransmitters still remains a major challenge for both techniques. It is expected that (1) real-time continuous monitoring, (2) in vivo detection, (3) high spatiotemporal resolution, (4) simultaneous multi-analyte sensing, and (5) long-term stability of the implanted sensors will continue to be the main objectives of research in the field of neurotransmitter sensors in the foreseeable future.