Optical Sensor For The Detection Of Tryptophan And Melatonin

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Back to Journal »International Journal of Nanomedicine» Volume 16

Author Khan ZA, Hong PJS, Lee CH, Hong Y

Published on October 11, 2021, the 2021 volume: 16 pages 6861-6888

DOI https://doi.org/10.2147/IJN.S325099

Single anonymous peer review

Editor approved for publication: Professor Israel (Rudi) Rubinstein

Multimedia components 1. Use screen printing technology (GPT/WPE) to fabricate electrodes on waterproof paper.

Zeeshan Ahmad Khan,1–3 Paul Jung-Soo Hong,4,* Christina Hayoung Lee,5,* Yonggeun Hong1– 3,6,7 1 Department of Physical Therapy, School of Health Sciences and Engineering, Inje University, Gimhae, Gyeong-nam , 50834, South Korea; 2 Inje University Bio-Health Product Research Center (BPRC), Gimhae, Gyeongnam, 50834, South Korea; 3 Ubiquitous Healthcare & Anti-Aging Research Center (u-HARC), Inje University, Gimhae, Gyeong-nam, 50834, Korea; 4 Department of Chemistry, Newton South High School, Newton, Massachusetts, 02459, USA; 5 Department of Biology, Vanderbilt University College of Arts and Sciences, Nashville, Tennessee, 37212, USA; 6 Inje University Graduate School of Rehabilitation Sciences Department, Gimhae, Gyeongnam, 50834, South Korea; 7 Faculty of Medicine, Faculty of Hematology/Oncology, Harvard Medical School-Beth Israel Deaconess Medical Center, Boston, Massachusetts, 02215, USA *These authors did the same for this work Corresponding author: Yonggeun Hong Department of Rehabilitation Sciences, Graduate School of Inje University, 197 Inje-ro, Gimhae, Gyeong-nam, 50834, Korea Tel 82-55-320-3681 Fax 82-55-329-1678 Email [Electronics Mail is protected] Abstract: Tryptophan and melatonin are pleiotropic molecules, each of which can affect several cellular, biochemical and physiological reactions. Therefore, sensitive detection of tryptophan and melatonin in drugs and human samples is essential for human well-being. Mass spectrometry, high performance liquid chromatography, and capillary electrophoresis are common methods for the analysis of tryptophan and melatonin; however, these methods require a lot of time, money, and manpower. New electrochemical and optical detection tools have become the subject of in-depth research due to their ability to provide better signal-to-noise ratio, high specificity, ultra-high sensitivity and wide dynamic range. Recently, researchers have designed a sensitive and selective electrochemical and optical platform for the detection of tryptophan and melatonin by using new surface modification, micromachining technologies, and the decoration of a variety of nanomaterials with unique characteristics. However, few review articles deal with the latest developments in electrochemical and optical detection of tryptophan and melatonin. Here, we conducted a critical and objective review of the highly sensitive tryptophan and melatonin sensors developed in the past six years (after 2015). We reviewed the principles, performance and limitations of these sensors. We have also addressed key aspects such as sensitivity and selectivity, detection limits and scope, manufacturing process and time, durability and biocompatibility. Finally, we discussed the challenges associated with the detection of tryptophan and melatonin, and proposed future prospects. Keywords: tryptophan, melatonin, electrochemistry, sensor, voltammetry, optics

For decades, tryptophan and melatonin have been the subject of scientific research because of their pleiotropic properties, each of which can affect a variety of cellular, biochemical, and physiological responses. 1,2 Tryptophan is considered as an over-the-counter drug in many countries, crossing the effective blood-brain barrier, and as a nutritional supplement, sleep aid, appetite suppressant and antidepressant. 3 Tryptophan plays an important role in the treatment of many diseases, such as depression, obesity, cerebellar ataxia, persistent headache, fibromyalgia and insomnia. 4-6 Due to dietary dependence, biological influence and treatment Use, accurate detection of tryptophan levels in food, medicine and human samples is essential. Despite its basic properties, the measurement of tryptophan is often omitted in some studies because tryptophan analysis is usually tedious. A key challenge for tryptophan detection is that the acidic conditions used for the analysis of normal amino acids for hydrolyzed proteins can lead to massive or even complete oxidative degradation of tryptophan. 7 Alkaline hydrolysis is usually used to partially solve the problem of acid hydrolysis, but it will cause the formation of high concentrations of molecular oxygen which will interfere with the detection of tryptophan. Removal of oxygen from the sample can be very time-consuming and, depending on the nature of the sample, is very difficult. 8

Tryptophan is an important precursor for the synthesis of melatonin (5-methoxy-N-acetyltryptamine). The melatonin pathway is conservative, tryptophan→5-hydroxytryptophan→serotonin→N-acetylserotonin→melatonin is observed. 9-11 Melatonin is a timekeeping molecule in vertebrates; it is synthesized in a circadian rhythm to synchronize physiology, metabolism and behavior with the ambient light-dark cycle, light intensity and temperature. 12-14 In addition to acting as a circadian rhythm regulator, melatonin is also an effective antioxidant, immune enhancer, anti-inflammatory agent, and bone protectant, so it is considered a therapeutic adjuvant for the management and treatment of COVID-19. 15-17 The daily level of melatonin in human plasma ranges from a few pM during the day to 215-430 pM at night. 18,19 The measurement of melatonin levels has also become a means of diagnosing certain mental illnesses and is recommended Phase classification of patients with sleep and mood disorders. 20-22 Due to the low concentration of melatonin in biological fluids and the simultaneous presence of many other compounds in the blood, the routine determination of melatonin has always been an analytical challenge. 23,24 In addition, the dramatic changes in melatonin levels due to the natural and pro-catheter and the different complexity of the matrix where melatonin is found have forced researchers to develop new tools to extract melatonin before testing. Nevertheless, due to the amphiphilic and chemical reactivity of melatonin, the choice of extraction solvent is difficult. 23 Melatonin is also a commercial drug, which can be obtained in the form of a variety of pharmaceutical products, such as sustained-release tablets, sustained-release tablets, and sustained-release tablets. -Release tablets, ordinary tablets and capsules, and liquid medicines. The content of melatonin in medicines ranges from 0.1 to 10 mg per serving. 25,26 sensors that provide micromolar and nanomolar sensitivity are used to measure melatonin in medicines.

In the past three years, the need to measure tryptophan and melatonin levels in biological and pharmaceutical samples has increased significantly. Several new and improved detection methods have been developed to study the physiological and psychological effects of tryptophan and melatonin, including high performance liquid chromatography (HPLC), 27-29 mass spectrometry, 30,31 capillary electrophoresis, 32,33 And gas chromatography. 34,35 Although these methods have the advantages of sensitivity and accuracy, tryptophan and melatonin measurement tools are bulky, expensive, time-consuming, lack portability, and require laboratory maintenance and technicians. For cost and simplicity reasons, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are most commonly used for melatonin detection. 36 However, immunoassays are likely to cross-react with structurally similar compounds if the melatonin is not extracted correctly; the complete extraction of melatonin is a complicated and time-consuming process. The electrochemical and optical detection of tryptophan and melatonin is inexpensive, sensitive and portable, and easy to operate, which attracts researchers.

In recent years, the development of many sensors based on advanced functional nanomaterials for the detection of tryptophan and melatonin has steadily increased. One of the advanced functional materials development tools is molecular imprinting technology (MIT) or molecular template technology, whose purpose is to synthesize three-dimensional cross-linked polymers with specific molecular recognition capabilities. 37 MIT-based sensors have the following advantages: relatively stable storage, overcoming the key limitations of biometric elements such as enzymes or antibodies. 38,39 However, the manufacturing of MIT-based sensors is complicated and requires expert handling. In addition, MIT sensors can recognize but cannot transmit signals; therefore, they must be combined with other methods, such as electrochemical or optical settings, which also have their own limitations. Another important advanced nanomaterial sensor is the fluorescent "switch" sensor. Because of its simplicity, rapid response and high detection limit, fluorescent "switches" have shown promising ability to detect biomolecules. First, the oxidation-reduction potential of the analyte is used to change the oxidation state of the functional group on the fluorophore, thereby "turning on" the fluorescence of the dye. Subsequently, the "on" fluorophore is modified in a way that another redox agent binds to it, causing it to "off". 40 Although “on-off” sensors provide sensitivity and control, most traditional fluorophores with π- are bound in biological samples and are affected by aggregation-induced quenching, resulting in low fluorescence quantum yields, which limits their high Technology application. 41

In this review, we discussed the advantages and limitations of various electrochemical and optical tools for the detection of tryptophan and melatonin. The focus will be on human testing, but the application of plant and pharmaceutical sample testing will be discussed where appropriate. These publications are collected from PubMed, Google Scholar and Embase, and do not represent system searches. Discussed an overview of several emerging sensors (released in 2015 or later), including electrochemical and optical sensors, and their related precautions (Tables 1 and 2). Finally, the challenges related to the detection of tryptophan and melatonin and their future prospects are presented. Table 1 Recently developed (after 2015) tryptophan electrochemical and optical sensors Table 2 Recently developed melatonin electrochemical and optical sensors

Table 1 Recently developed (after 2015) tryptophan electrochemical and optical sensors

Table 2 Recently developed electrochemical and optical melatonin sensors

Electrochemical methods are promising because they are simple, easy to use, fast, require low-cost equipment, and provide relatively good sensitivity and limit of detection (LOD). In addition, electrochemical tools can be used to develop portable devices for in vivo and real-time sensing. Electrochemical detection consists of at least two electrodes (a working electrode and a counter electrode or auxiliary electrode), which are in contact with each other in two ways: a conductive medium (electrolyte, that is, a liquid that acts as an ionic conductor) and an external circuit (electronic conductor). The electrode is made of a special conductive material and has a catalytic effect, so certain chemical reactions can occur in the presence of the analyte. 42 The analyte is applied to the working electrode, and the analyte and the electrode interact under a specific current or voltage to cause an electrochemical reaction. The reaction is an oxidation reaction or a reduction reaction, depending on the type of analyte. Oxidation causes electrons to flow from the working electrode to the counter electrode through an external circuit. The reduction reaction transfers the flow of electrons from the counter to the working electrode. Either direction of electron flow will produce a current proportional to the analyte concentration. According to different modes of applied voltage and current response model, electrochemical detection can be carried out in a variety of ways through a variety of electrodes and analytes (Figure 1). Figure 1 Electrochemical techniques for the detection of tryptophan and melatonin. Schematic diagram of the electrochemical sensor and the conversion of these interactions into measurable signals. The asterisk () represents the analyte (melatonin/tryptophan). In the figure, the black line () represents voltage, and the blue line () represents current.

Figure 1 Electrochemical techniques for the detection of tryptophan and melatonin. Schematic diagram of the electrochemical sensor and the conversion of these interactions into measurable signals. The asterisk () represents the analyte (melatonin/tryptophan). In the figure, the black line () represents voltage, and the blue line () represents current.

Voltammetry is an electrical analysis method in which the current is measured as a function of applied potential to qualitatively and quantitatively analyze chemicals and biomolecules. Voltammetry shows the reduction potential and electrochemical reactivity of the analyte, thereby facilitating the detection of the analyte. Examples of common voltammetry include Cyclic Voltammetry (CV), Linear Sweep Voltammetry (LSV), Step Voltammetry, Square Wave Voltammetry (SWV), Stereo Square Wave Stripping Voltammetry (OSWSV) and Differential Pulse Voltammetry (DPV). The voltammetry system usually includes three electrodes: a reference electrode, a working electrode and a counter electrode. In this system, the electrodes can be modified with active materials or nanoparticles, these materials can convert specific chemical ions into measurable electrons at a specific voltage. The specific voltage is obtained by applying a potential to the electrode and scanning back and forth within a given voltage range for a specified number of cycles. The peak potential when the peak current is obtained allows the user to analyze the electrochemical reversibility of the electrode surface reaction by changing the scan rate. 43,44 In the case of CV, the peak potential changes linearly with time. On the other hand, the peak potentials of SWV and DPV represent the continuous increase of square oscillation or rectangular pulse respectively. 45 In addition, the three-electrode system can also be used to characterize redox performance, stability and effective surface area. 43,44 In order to conduct a comprehensive and objective evaluation of the analyte, it is necessary to select the most appropriate electrochemical detection method according to the characteristics of the analyte's charge, electrocatalytic potential, redox potential, matrix and size. For example, through comparative experiments, Gholivand et al.46 compared the effects of CV, LSV and DPV in the detection of bovine serum albumin (BSA). In the process, they found that DPV is the most suitable method for BSA detection and applied the experimental results to clinical treatment. It should be noted that the choice of voltammetry technique also depends on the redox characteristics, shape and size of the electrode. Therefore, for the same analyte, depending on the type of electrode used, different voltammetry tools may be suitable. In addition, the voltammetric tool can be selected based on the rate of reaction between the electrode and the analyte. For example, DPV is commonly used in irreversible systems and systems that exhibit slow reaction kinetics. On the other hand, SWV is usually applied to reversible systems and fast reaction kinetic systems.

CV is a general technique that can be used for direct and indirect analysis in many research fields involving electron transfer processes. 47 Recently, Zhang et al.48 developed an electrochemical sensor containing nitrogen-doped ordered mesoporous carbon/Nafion/glassy carbon electrode (GCE) for the highly sensitive and selective detection of tryptophan. The cyclic voltammogram showed that the oxidation current increased linearly with the concentration of tryptophan in the range of 0.5-70.0 μM, and the LOD was 35.0 nM. In order to check the selectivity, tryptophan was measured in a mixture of 18 amino acids, and the recovery rate was between 99.30-103.60%. Although the sensor is highly sensitive and selective to tryptophan, it is very time-consuming to manufacture and may take up to 6 days. In addition, the paper lacks physical, chemical or microstructure characterization of modified materials, so it is difficult to understand the reasons behind the high sensitivity and selectivity.

It is also possible to use graphene-coated screen-printed carbon electrodes (G-SPE) for cyclic voltammetry to evaluate melatonin levels in pharmaceuticals. 49 The graphene coating provides a large surface area (theoretical single layer is 2630 square meters/g), excellent thermal conductivity (k = 5000 W/mK) and electrical conductivity (r = 64 mS/cm); 50,51 This results in a large number of available electroactive sites, thereby increasing the sensitivity of the carbon electrode. For this graphene-based electrochemical sensor, the linear detection range is 1-300 μM, and the LOD is 870 nM. The main limitation of this study is that it did not address the potential interference of indoles that are structurally and chemically similar to melatonin (such as tryptophan and serotonin). Therefore, it is not recommended to use G-SPE to evaluate melatonin in complex biological samples, which usually contain higher levels of tryptophan and serotonin than melatonin.

In vivo detection of melatonin remains a major challenge. In order to solve this problem, Hensley et al.52 developed an electrochemical technique to detect melatonin in living lymph nodes; they achieved sub-segmentation by fast scanning cyclic voltammetry (FSCV) using carbon fiber microelectrodes (CFME). Second-level time resolution. Initially, the detected oxidation peaks of melatonin were 1.0, 1.1, and 0.6 V, respectively. However, due to electrode contamination, a third peak was also observed. Biological contamination is a major challenge for analysis using electrochemical sensors, especially for in vivo analysis. This is because the oxidation products of biological analytes can be electropolymerized in the solution and absorbed on the electrode surface, resulting in the detection of [error] peaks during the electrochemical evaluation. Living tissue can further exacerbate this problem by physically connecting to the electrodes. Overcome the problem of biological fouling interference by developing customized waveforms. The optimized waveform of melatonin includes scanning from 0.2 to 1.3 V and returning at 600 V/s; this reduces the signal generated by electrode contamination while maintaining the sensitivity level. Using CFME and FSCV, the LOD obtained using this method is 24 ± 10 nM of melatonin. In addition, the CFME-FSCV method can successfully detect melatonin in the presence of biological interference, and can detect melatonin together with its biosynthetic precursor serotonin. The method was then validated by successfully measuring the level of melatonin in intact lymph node tissue, which made this the first report that FSCV used to analyze this tissue type. Despite this achievement, the durability of CFME is still an area of concern. Due to the fragility of CFME, these microelectrodes are easily damaged during in vivo analysis. Therefore, instead of using microelectrodes made of carbon fibers, applying a carbon coating to a metal electrode can produce the required durability while maintaining the biocompatibility and electrochemical properties of carbon. In addition, CFME/carbon-coated metal microelectrode arrays can be developed for simultaneous measurement of multiple analytes, such as melatonin biosynthetic precursors (such as tryptophan, serotonin, and N-acetyl serotonin); this may be The regulation of melatonin synthesis in the body provides useful insights.

The electrochemical detection of melatonin mainly uses solid, rigid electrodes, such as carbon paste electrodes (CPE), boron-doped diamond electrodes and GCE. 53-55 Recently, a type of boron oxide (B2O3) and graphene was developed using vacuum filtration, and then used to simultaneously detect melatonin and ascorbic acid (AA) in spiked serum samples. 56 To generate this paper, B2O3 nanostructures were fabricated using a hydrothermal method, and then boron-graphene oxide (B-GO) composite materials were synthesized by mixing B2O3 in GO dispersion. B2O3 reduced graphene oxide (B-rGO) paper was obtained by vacuum filtration of B-GO dispersion, and then the filtrate was chemically reduced with 57% (w/w) hydroiodic acid (HI) (Figure 2). Although the B-rGO paper electrode is flexible, durable and can be molded into the required shape, the cumbersome production of B-rGO composite paper and the use of toxic, highly corrosive and environmentally unfriendly HI have great limitations to this Work. In addition, the paper-based melatonin sensor has an LOD of 700 nM, which limits its applicability. Figure 2 Schematic diagram of boron oxide reduction graphene oxide (B-rGO) composite paper manufacturing. Note: Disperse graphene oxide (GO) and mix with boron oxide (B2O3) solution, and then perform ultrasonic treatment. The B2O3-GO composite material (B-GO) was then vacuum filtered and then dried to form B-GO paper. Next, the B-GO paper is immersed in a hydrochloric acid (HI) solution, then washed and air-dried to produce B2O3-reduced GO (B-rGO) composite paper. Adapted from Diamond and Related Materials, Volume 105, issued in 2020, Topçu E, Dağcı Kıranşan K, Electrochemical simultaneous sensing of melatonin and ascorbic acid on a new flexible B-RGO composite paper electrode, page 107811, copyright ( 2020), with permission from Elsevier.56

Figure 2 Schematic diagram of boron oxide reduction graphene oxide (B-rGO) composite paper manufacturing.

Note: Disperse graphene oxide (GO) and mix with boron oxide (B2O3) solution, and then perform ultrasonic treatment. The B2O3-GO composite material (B-GO) was then vacuum filtered and then dried to form B-GO paper. Next, the B-GO paper is immersed in a hydrochloric acid (HI) solution, then washed and air-dried to produce B2O3-reduced GO (B-rGO) composite paper. Adapted from Diamond and Related Materials, Volume 105, issued in 2020, Topçu E, Dağcı Kıranşan K, Electrochemical simultaneous sensing of melatonin and ascorbic acid on a new flexible B-RGO composite paper electrode, page 107811, copyright ( 2020), with permission from Elsevier.56

The main advantage of DPV is that it enhances the ability to distinguish Faraday (charging/capacitor) currents, thereby minimizing the background charging current. Therefore, compared with the traditional sampling DC voltammetry, the sensor using DPV is more sensitive to the charging current (electron passing in and out of the electrode). DPV produces small peaks of Faraday current superimposed on a staircase waveform instead of an S-shaped waveform; this results in improved resolution, reduced background, and better analyte quantification in multiple analyte systems. 57 Therefore, DPV is a promising tool for single and simultaneous molecular detection.

In 2015, Tadayon et al. developed a DPV-based sensor for simultaneous detection of melatonin and tryptophan. 58 In order to detect multiple analytes simultaneously, each analyte in the suspension must be oxidized at a specific oxidation potential. Therefore, graphene is used as a substrate due to its high conductivity and surface area. In order to further improve the electrocatalytic activity simultaneously detected in the above research, the electrode surface was modified with a new composite material of graphene and copper. They constructed a CPE decorated with nitrogen-doped graphene nanosheets/copper-cobaltite (CuCo2O4) nanoparticles. They achieved an LOD of 4.9 nM for melatonin and 4.1 nM for tryptophan. The sensitivity of the CuCo2O4 sensor to tryptophan is sufficient for biological samples. However, in order to simultaneously detect melatonin and its biosynthetic precursors in biological samples, it is necessary to increase the sensitivity of melatonin detection to be applied to biological samples. In addition, graphene toxicity is a major issue for human and environmental health. 59-61

Zeinali et al62 later developed a more sensitive tool. They used a new type of DPV-based electrochemical sensor to jointly detect tryptophan and melatonin in interference environments (such as urine and human serum). Their sensor is an ionic liquid (IL) CPE whose surface is modified with rGO and tin oxide-cobalt oxide (SnO2-Co3O4) nanoparticles (SnO2-Co3O4@rGO/IL/CPE) (Figure 3). 62 Using the new SnO2 -Co3O4@rGO/IL/CPE sensor, Zeinali et al. 62 demonstrated that during the DPV oxidation of the analyte, melatonin undergoes a two-electron/single-proton reaction, while tryptophan undergoes a two-electron/single-proton reaction. Two-proton reaction; this enables the selective sensing of two biomolecules (Figure 3C-E). The large electroactive surface area and good conductivity of SnO2-Co3O4@rGO/IL/CPE produced a strong electrochemical response during melatonin measurement and melatonin/tryptophan combined measurement. The detection range is 0.02–6.00 μM, and the LODs of melatonin and tryptophan are 4.1 and 3.2 nM, respectively; the measurements are made using human and pharmaceutical samples. Given that melatonin performs a two-electron/single-proton reaction, and tryptophan performs a two-electron/two-proton reaction, this method is unlikely to be adapted to the detection of serotonin, which is similar in structure to the analyte and its intermediate products. The melatonin biosynthesis pathway, 63-65, thus limits the applicability of the SnO2-Co3O4@rGO/IL/CPE sensor. In addition, the synthesis of SnO2-Co3O4@rGO/IL/CPE is a time-consuming and complex multi-step process, which limits its widespread use (Figure 3). 62 Figure 3 Synthesis of SnO2-Co3O4@rGO for the detection of melatonin and tryptophan. Note: (A) SnO2-Co3O4@rGO is gradually prepared from graphene oxide (GO). (B) Electrochemical detection of melatonin (MEL) and tryptophan (Trp) in real samples. (C) Two-electron and single-proton oxidation of MEL. (D) and (E) Two-electron and two-proton oxidation of tryptophan. Adapted from Material Science and Engineering: C, Volume 71, Issued in 2017, Zeinali H, Bagheri H, Monsef-Khoshhesab Z, Khoshsafar H, Hajian A, Simultaneous determination of tryptophan by a new SnO2 modified ionic liquid carbon paste electrode Nanomolar-Co3O4@rGO nanocomposite with melatonin, pages 386-394, copyright (2017), with permission from Elsevier.62

Figure 3 Synthesis of SnO2-Co3O4@rGO for the detection of melatonin and tryptophan.

Note: (A) SnO2-Co3O4@rGO is gradually prepared starting from graphene oxide (GO). (B) Electrochemical detection of melatonin (MEL) and tryptophan (Trp) in real samples. (C) Two-electron and single-proton oxidation of MEL. (D) and (E) Two-electron and two-proton oxidation of tryptophan. Adapted from Materials Science and Engineering: C, Volume 71, issued in 2017, Zeinali H, Bagheri H, Monsef-Khoshhesab Z, Khoshsafar H, Hajian A, simultaneous determination of tryptophan by a new SnO2 modified ionic liquid carbon paste electrode Nanomolar-Co3O4@rGO nanocomposite with melatonin, pages 386-394, copyright (2017), with permission from Elsevier.62

It is well known that the electrochemical sensing of tryptophan shows sub-optimal performance due to low electron transfer efficiency and high overpotential. 66 Therefore, efforts have been made to develop new materials for manufacturing modified electrodes to promote electron transfer and reduce overpotentials. Carbon nanotubes (CNT) exhibit attractive properties, such as high electron transfer between the electroactive material and the electrode surface. In 2018, a modified electrode based on the in-situ addition of tetrabutylammonium bromide on β-cyclodextrin and multi-walled carbon nanotube modified GCE (TBABr/β-CD/MWCNTs/GCE) for quantitative analysis Tryptophan levels in the presence of uric acid (UA) and the concentration of AA. 67 DPV measurement shows a linear relationship with tryptophan concentration of 1.5-30.5 μM, LOD is 0.07 μM, and has excellent selectivity, good stability and reproducibility. The sensitivity and selectivity of TBABr/b-CD/MWCNTs/GCE for the detection of tryptophan are due to the good conductivity of MWCNTs, molecular recognition ability β-CD, hydrophobic interaction and electrostatic attraction between TBABr and tryptophan. The ion of TBABr is large in Bu4N, low in conductivity, and high in activation enthalpy of charge transfer. 68 Therefore, using another cationic surfactant, such as sodium tetrafluoroborate, 69 has a higher ionic conductivity, 68 can be used to improve the sensitivity of the electrode.

In order to make high-performance electrodes for tryptophan, the researchers used graphene (GR); because it is better than CNT. 70 Li et al. 71 and Haldorai et al. 72 respectively fabricated electrodes by mixing palladium-copper-copper oxide (Pd-Cu-Cu2O) and tin oxide nanoparticles (SnO2) on rGO. The DPV voltammogram shows that the LODs of Pd-Cu-Cu2O and SnO2 are 1.9 nM and 0.04 μM, respectively. Both groups used the hydrothermal route to develop tryptophan-sensitive electrodes. The main limitations of hydrothermal synthesis include the need for an expensive autoclave and the inability to observe crystal growth due to the steel tube of the autoclave. 73 In addition, Pd-Cu-Cu2O is only used to detect tryptophan samples in milk and urine, so it needs to be tested in the presence of other amino acids.

Generally speaking, there are two enantiomers of tryptophan (L-tryptophan and D-tryptophan)74 in nature, but their biological activities are different. 75,76 L-tryptophan has a significant impact on the normal function of life, and its deficiency may cause a variety of diseases. On the other hand, D-tryptophan was identified as a biologically active compound used in the development of the ovary of the Ryukyu Dugosia embryo. 77 Recently, a nanocomposite material was synthesized from rGO, carboxymethyl cellulose (CMC) and polyaniline (PANI) for the selective detection of L- and D-tryptophan by DPV measurement. 78 The developed nanocomposite material is decorated on GCE to obtain the electrochemical sensor rGO/PANI/CMC/GCE. The linear response range of the sensor is 0.01 to 5 mM, and the detection limits of L-tryptophan and D-tryptophan are 0.07 μM and 0.005 μM, respectively. In the absence of strong coordinating ions, since two different diastereomeric complexes are formed between the enantiomers and selectors with different binding energies, chiral recognition will occur. This difference in binding energy is reflected in the electrochemical signal to distinguish the two enantiomers. The enantioselectivity of rGO/PANI/CMC/GCE is 2.26. In addition, chemical substances such as epinephrine, dopamine, histamine, epinephrine, serotonin, tryptamine, tyramine and phenethylamine are also used for interference analysis. The results show that the presence of these chemicals has little effect on electrochemical chiral recognition. The main disadvantage of this sensor is the in-situ preparation of nanocomposite materials. In-situ synthesis uses a large amount of starting material chemicals and a short time to perform the polymerization process. 79 In addition, unreacted educts from the in-situ reaction may affect the properties of the final material, which can change the entire electrochemical process and hinder reproducibility. 80

Gomez et al81 detected melatonin and serotonin in drug samples. In this study, graphene was used as the base material due to its high conductivity and surface area. They used single-wall/multi-wall carbon nanotubes to modify the carbon screen-printed electrodes for their DPV method, which has an LOD of 0.4 μM serotonin and 1.1 μM melatonin. Try to detect both melatonin and serotonin in biological samples. However, this electrode is not sensitive enough to be applied to biological samples, and can only be used for drug sample analysis.

Later, a platform using a simpler and faster nanocomposite production process was developed, in which self-assembled gold nanoparticles (AuNPs) connected to molybdenum disulfide (MoS2) nanosheets (Au-MoS2) were prepared by probe ultrasound. The rapid synthesis of Au-MoS2 composites was carried out by sonicating a mixture of MoS2 nanoflake and AuNP at an amplitude of 30% for 5 minutes. The Au-MoS2 composite material was drip-cast onto the GCE to create the Au-MoS2-GCE sensing platform. Due to the high electrochemically active surface area, Au-MoS2-GCE exhibits a high specific surface area (high catalytic activity). The Au-MoS2-GCE platform is used to simultaneously detect UA and melatonin 82, the LOD of UA is 18.2 nM, and the LOD of melatonin is 15.7 nM. Although the Au-MoS2-GCE sensor produced reproducible results and showed good operation and storage stability, this study did not prove the clinical utility of this dual UA/melatonin detection system, because the samples analyzed are Urine sample mixed with UA and melatonin. Quantifying the level of melatonin in urine is not common in clinical studies, because only a small amount (~5%) of endogenous melatonin is present in urine samples; the level of a6MTs in urine samples is usually 83,84, Because the concentration of a6MTs in urine is 2-3 times higher than melatonin, the concentration of a6MTs in urine reflects the concentration of endogenous melatonin. 85-87

In 2017, Rajkumar et al. 88 used microwave reduction to synthesize highly stable palladium nanoparticles on porous carbon aerogels (Pd/CA), and they used them for electrochemical sensing of melatonin and dopamine. Due to its high surface area (851.8 m2/g) and pore volume (3.021 cm3/g), Pd/CA nanocomposites show excellent electrocatalytic activity and selectivity for both melatonin and dopamine. Use Pd/CA electrodes to apply various voltammetry, such as DPV, LSV and OSWSV, to analyze melatonin and dopamine samples in the presence of interfering biomolecules (including AA and UA). The electrochemical detection of melatonin (0.02–500 μM) and dopamine (0.01–100 μM) on the Pd/CA modified electrode with DPV showed an impressive linear response range, with LODs of 2.6 and 7.1 nM, respectively. Pd/CA nanocomposites are produced using a relatively simple microwave reduction method; however, the synthesis of carbon aerogel and the curing of Pd/CA require carbonization procedures under N2 gas at 900°C and 400°C, respectively. 88 Efforts should be made to simplify the Pd/CA manufacturing process to promote electrode amplification.

In 2018, the combined measurement of acetaminophen, epinephrine and melatonin was performed for the first time, using the DPV method and a CPE sensor modified with zinc ferrite nanoparticles (ZnFe2O4NPs/CPE). 89 These zinc ferrite nanoparticles have a high surface-to-volume ratio and show good electrocatalytic activity for melatonin, paracetamol and epinephrine. For melatonin, acetaminophen and epinephrine, the best electrocatalytic activity is obtained at working voltages of 0.55, 0.35 and 0.09 V, respectively. The linear detection range and LOD of melatonin are 6.5-145 μM and 3 μM, respectively, acetaminophen is 6.5-135 μM and 0.4 μM, and epinephrine is 5-100 μM and 0.7 μM; the analyzed samples are spiked Human serum samples and medicines. Although the ZnFe2O4NPs/CPE sensor has low interference to other biomolecules, the LOD of the ZnFe2O4NPs/CPE sensor is relatively high, so it has nothing to do with human samples.

In 2019, the same research team that developed the ZnFe2O4NPs/CPE sensor89 developed a more sensitive electrochemical platform that can simultaneously analyze melatonin, dopamine, and acetaminophen. 90 By using a CPE sensor coated with alumina-loaded palladium nanoparticles to apply DPV to the sample (PdNP/Al2O3), the detection ranges for dopamine, acetaminophen and melatonin are 50 nM–1.45 mM, 40 nM–1.4 mM and 6.0 nM–1.4 mM. The LOD of dopamine is 36.5 nM, acetaminophen is 36.5 nM, and melatonin is 21.6 nM.

The synthesis of ZnFe2O4NPs/CPE and PdNP/Al2O3 electrodes, especially the preparation method of paste, is laborious; this poses a future challenge for scale-up and large-scale production. In addition, the use of Pd increases the production cost of the electrode, thereby hindering its public use. Therefore, the research team studied the use of expensive metals to replace cheaper alternatives to produce melatonin sensors. Similarly, by decorating cheap carbon nanofibers (CNF) with FeCo bimetallic alloys, and then depositing them on GCE ([email protection]/GCE), a high-performance melatonin sensor is produced. 91 FeCo shows high electrocatalytic and electron transfer activity, which makes fast and continuous melatonin analysis possible. Using [email protection]/GCE to analyze melatonin-containing drug samples, the detection limit is 2.7 nM; the detection limit is sufficient for the analysis of drug samples. Similar to the other electrochemical sensors discussed above, the main limitation of the [email protected]/GCE sensor is its long and complicated manufacturing process; it takes 24 hours to synthesize [email protected] alone. In addition, it takes 1 hour for nanofibers to stabilize at 280°C, and then carbonize at 800°C for 2 hours. A short and simple process should be developed to produce bimetallic nanowires.

Recently, Liu et al. developed a sensitive DPV melatonin sensor based on integrated 2D materials. 92 The electrode of the melatonin sensor is made by decorating the surface of SPE with a ternary compound of tin disulfide (SnS2) nanosheets, GO and β-CD (SnS2/GO/β-CD/SPE). The melatonin detection linear range of 92 DPV method and SnS2/GO/β-CD/SPE sensor combination is 1 nM to 100 μM, and the LOD is 0.17 nM. The SnS2/GO/β-CD/SPE electrochemical sensor has been successfully applied to detect melatonin in drug samples and human saliva, and both have a good correlation with the corresponding ELISA results. The SnS2/GO/β-CD/SPE sensor has a fast response time, is made of cheap tin and graphene materials, and is relatively easy to operate, thus overcoming the limitations of traditional melatonin sensors. The sensitivity of the SnS2/GO/β-CD/SPE sensor is high enough to analyze nighttime biological samples; however, its sensitivity must be increased to expand its applicability to daytime samples. In addition, the manufacturing time of the SnS2/GO/β-CD/SPE sensor is close to 24 hours, including 12 hours of hydrothermal synthesis at 200°C, which highlights another potential area for improvement.

In plants, melatonin is an effective antioxidant and growth promoter that can prevent oxidative stress and promote adaptation to harsh environmental conditions. 92 Plants operate under a wide range of pH conditions, so it is difficult to develop electrochemical sensors to monitor biologically active molecules in plants. 93 In order to solve this problem, a DPV system using copper oxide (CuO)-poly(L-lysine)/graphene electrodes as sensors was developed to evaluate the melatonin and vitamin B6 (pyridoxine) in plants in situ. Pyridoxine, PN) levels. 94 Melatonin PN detection relies on CuO and poly(L-lysine), and the conductivity of the 3D graphene layer amplifies the catalytic current. Between pH 6.8-7.4, the current-time curve shows that the detection range of melatonin is 0.016-1110 μM, and the LOD is 12 nM. The linear detection range of PN is 3-2076 μM, and the LOD is 2.3 μM. Since the pH of fruits varies by species and maturity, further research is needed to improve the pH range of the working electrode.

SWV is a fast voltammetric technique, often used for quantitative analysis. Although SWV was first reported as early as 1995 in 1957, its practical application is limited by the current state of the art. The latest advances in analog and digital electronics have made it possible to integrate SWV into modern polarographic analyzers. SWV is much faster than ordinary pulse or DPV; DPV usually exhibits a scan rate of 1-10 mV/s, while the scan rate of SWV is usually >1 V/s. 96,97

Due to the low background current (compared to solid graphite or other precious metal electrodes), easy synthesis, the feasibility of adding various substances in the paste preparation process, large potential window, cheap, direct surface renewal process and easy to small CPE is widely used in electrochemical research and electrical analysis. 98-102 In 2015, Beitollahi et al. 103 used 2-chlorobenzoylferrocene/Ag-ZnO nanoplates (2CBFAGZCPE) to improve CPE for the detection of tryptophan and captopril. The SWV peak current increases linearly with the concentration of tryptophan in the range of 0.05 to 20.0 μM, and the detection limit is 0.02 μM. The electrochemical analysis on the surface of the modified electrode 2CBFAGZCPE showed that it catalyzed electrooxidation at pH 7.0. This result indicates that the manufactured sensor 2CBFAGZCPE may be sensitive to pH, which will limit its applicability for the detection of tryptophan in various patient samples such as acidosis, acute pancreatitis, and poisoning. 104-106 Although the application of transition metal complexes (eg, Ag-ZnO) for the development of tryptophan sensors has shown electrocatalytic performance, 2CBFAGZCPE has a low linear range for detecting tryptophan because the electron transfer process is slow and the color The overpotential of direct oxidation of acid on CPE is higher. 107,108

In 2016, nickel oxide (NiO) and copper oxide (CuO) nanoparticles and modified GR were decorated on GCE to create a sensor that simultaneously measures tryptophan, dopamine, and acetaminophen using square wave voltammetry. 109 GCE is superior to CPE in terms of promoting electron transfer and improving sensitivity. 110 GR is modified with citric acid to generate more functional groups, which is conducive to the deposition of dispersed metal particles. The NiO-CuO/GR modified electrode is prepared by electrodeposition. Then, modified electrodes were prepared by electrodepositing NiO-CuO and citrate modified GR (NiO-CuO/GR/GCE) on GCE. The NiO-CuO/GR/GCE electrode shows that the linear response ranges for the detection of tryptophan, dopamine and paracetamol are 0.3-40 μM, 0.5-20 μM and 4-400 μM, respectively, and the detection limit is 0.1 μM, 0.17 μM And 1.33 μM. Similarly, He et al. 111 used GCE and modified it with a nanocomposite of cuprous oxide and rGO (Cu2O-rGO/GCE). Cuprous oxide Copper oxide is a p-type semiconductor, has a better ability to promote the electron transfer process, is low in price, relatively low in toxicity, and has effective antibacterial activity. The oxidation peak current of 112-115 tryptophan and its SWV analysis range is 0.02-20 μM, and the LOD is 0.01 μM. The tryptophan sensor Cu2O-rGO/GCE is used to quantify tryptophan in human serum samples and commercial amino acid injections. The authors show that the sensor has a high recovery rate of 97.0-102.3% and can be used to determine tryptophan in real samples such as human serum. However, the Cu2O-rGO/GCE sensor uses sulfuric acid as a supporting electrolyte to detect tryptophan, which is a very strong and corrosive acid. Sulfuric acid can decompose lipids and proteins by hydrolysis of esters and amides when it comes into contact with living tissues such as skin or meat. Sulfuric acid exhibits strong dehydration properties for carbohydrates, releases extra heat, and causes secondary heat damage. 116,117 The use of sulfuric acid as the supporting electrolyte for tryptophan detection limits the practical applicability of Cu2O-rGO/GCE. In addition, by modifying the metal oxide on the GCE, the LOD can be further improved.

In 2021, GCE will be modified with a nanocomposite (PT-CuO/GCE) of CuO nanosheets anchored by polythiophene. 118 Although copper oxide has the above-mentioned exciting properties in sensing, it has few limitations, such as low conductivity and aggregation. In order to improve these shortcomings, CuO-PT nanocomposites were prepared. Polythiophene is a special organic conductive polymer that can effectively transfer the charge generated by the biochemical reaction to the electronic circuit. Compared with the previous report, the use of the PT-CuO/GCE electrode did not show a 15 nM LOD enhancement and a linear detection range of 0.1 μM to 1 mM for tryptophan. Further research is needed to explore new materials for environmental protection and sensitive detection of tryptophan.

Several SWV-based sensing platforms have been developed for the detection of melatonin and other related biomolecules. Molaakbari et al.119 fabricated zinc oxide (ZnO) nanorods to coat CPE (ZnO/CPE) to produce sensors for simultaneous assessment of dopamine, melatonin, methionine, and caffeine levels. 119 The LOD of SWV-ZnO/CPE combined technology is 5.0 μM, 750.0 μM, 700.0 μM and 450.0 μM for dopamine, melatonin, methionine and caffeine; the linear detection range is 0.3–0.1 mM. The sensitivity of using ZnO/CPE to detect dopamine may be sufficient; however, the LOD of melatonin is relatively poor. Therefore, ZnO/CPEs cannot be used to detect melatonin in most biological fluids.

The electrocatalytic activity of chemically modified electrodes depends on the electrode material. In order to improve sensitivity, graphene decorated with magnetite (Fe3O4) magnetic nanoparticles (G/Fe3O4/CPE) was used to modify the CPE, and then SWV was applied to simultaneously detect melatonin and dopamine. 120 The synergy of graphene and Fe3O4 magnetic nanoparticles creates a large surface area, as well as good electrical conductivity and electrocatalysis. Compared with ZnO/CPE, G/Fe3O4/CPE shows a wider linear range and higher LOD: the detection range of melatonin and dopamine (0.02–5.80 μM) is the same, but the LOD of each is different (8.40 and 8.40 μM). 6.50 nM means melatonin and dopamine respectively). Although the sensitivity of G/Fe3O4/CPEs is higher than that of ZnO/CPEs, G/Fe3O4/CPEs show poor molecular specificity. The 12-fold excess tryptophan severely interferes with the melatonin measurement using G/Fe3O4/CPE. Under standard physiological conditions, the level of tryptophan in the human body is more than 1,000 times higher than melatonin, and may be higher in certain disease situations, such as schizophrenia. 121–123 Therefore, tryptophan interference is an important limitation of G/Fe3O4/CPE sensors.

In another study, the simultaneous detection of melatonin and serotonin was achieved by combining SWV with an electrochemical sensor by depositing acetylene black chitosan nanoparticles (AB-C) onto gold Made on electrodes (AB-C/GEs). 124 The author's study claims that biopolymer-based sensors are economical; nevertheless, the use of gold electrodes invalidates this claim. The LOD using AB-C/GE is 0.16 μM serotonin and 1.9 μM melatonin. The AB-C/GE sensor can detect melatonin in the presence of equimolar concentrations of dopamine and AA. More sensors should be developed using biologically derived economic compounds (such as chitosan, agar, and cellulose), but cheaper electrodes (such as CPE) should be used.

In 2019, a sensitive and selective electrochemical sensor was developed for the simultaneous sensing of melatonin, norepinephrine and nicotine. The sensor contains nicotinamide adenine dinucleotide (NAD) immobilized on gamma (γ)-irradiated tungsten trioxide nanoparticles (WO3), which are used to coat GCE (Figure 4). 125 Nicotinamide coenzyme is needed to catalyze the reversible NAD into NADH redox reaction, which is the main source of energy generated during cell respiration. 126 The strong redox properties of deposited NAD differentiate the electrooxidation of melatonin, norepinephrine, and nicotine, enabling the identification and quantification of the three analytes. SWV is used because of its applicability, selectivity and sensitivity; this technology facilitates the electrocatalytic analysis of melatonin, norepinephrine and nicotine in the presence of various biomolecules, which may cause interference to samples in vivo , Such as dopamine, serotonin, epinephrine, cotinine, adenosine diphosphate, adenosine triphosphate, glucose, folic acid, tyrosine, tryptophan, UA, cysteine and AA. 125 The LODs of melatonin, norepinephrine, and nicotine are 2.6 nM, 1.4 nM, and 1.7 nM, respectively. In the same research, a sensitive GCE modified with γ-ray ethylenediaminetetraacetic acid-WO3 nanoparticles containing immobilized NAD was developed for melatonin detection. However, due to its toxicity, the use of gamma rays in electrode manufacturing is a major problem (Figure 4A); short-term exposure to gamma rays can easily cause DNA mutations, leading to tumor formation or cancer. 127 Therefore, methods that are not harmful to biology and the environment should be developed to manufacture electrochemical sensors. Figure 4 NAD/GI synthesis and detection of nicotine, melatonin and norepinephrine EDTA-WO3/GCE. Abbreviations: EDTA, ethylenediaminetetraacetic acid; GI, gamma radiation; WO3, tungsten trioxide nanoparticles.

Note: (A) Gamma radiation-mediated immobilization of nicotinamide adenine dinucleotide (NAD) on a glassy carbon electrode (GCE). (B) The mechanism of electrochemical determination of nicotine (NIC) and melatonin (MEL) by one proton and one electron oxidation and the electrochemical determination of norepinephrine (NEP) by two proton and two electron oxidation. Adapted from Biosensors and Bioelectronics, Volume 143, 2019, Anithaa AC, Asokan K, Lavanya N, Sekar C, tungsten trioxide nanoparticles immobilized with nicotinamide adenine dinucleotide for simultaneous detection of norepinephrine , Melatonin and nicotine, page 111598, copyright (2019), with permission from Elsevier.125

Figure 4 NAD/GI EDTA-WO3/GCE synthesis and detection of nicotine, melatonin and norepinephrine.

Abbreviations: EDTA, ethylenediaminetetraacetic acid; GI, gamma radiation; WO3, tungsten trioxide nanoparticles.

In response to health and environmental issues related to electrode manufacturing, electrodes made of biodegradable and biocompatible waterproof paper have been developed to produce a disposable electrochemical sensing platform for the quantification of melatonin and paracetamol And qualitative assessment. 128 In order to achieve this goal, conductive ink is synthesized using graphite powder dispersed in nail polish (GPT), and then coated on waterproof paper using screen printing technology to generate GPT/waterproof paper electrodes (GPT/WPE) (Multimedia component 1). The reference electrode is decorated with silver ink. The combination of SWV and GPT/WPE was used to analyze melatonin samples, and the linear detection range was 0.80-100 μM, and the LOD was 32.5 nM. Then use the SWV-GPT/WPE method to detect paracetamol and melatonin in synthetic saliva, sweat and urine samples. Although this waterproof paper-based electrochemical device is simple, disposable, and cost-effective, it has not been tested with real biological samples and tryptophan interference. 120 In addition, the detection range and detection limit of GPT/WPE should be improved before it can be used on a large scale. Other conductive polymers such as poly(3,4-ethylenedioxythiophene) and poly(styrene sulfonate) are more conductive than graphite, so they can be used to further improve sensitivity and LOD.

As mentioned above, the specific detection of melatonin in complex biological samples is a major challenge. Some recently developed methods showed good selectivity but compromised in terms of LOD and detection range, while other methods did not test for molecular interference in real biological samples. Manufacturing molecular recognition materials for voltammetric analysis can increase the specificity of electrodes. Molecular imprinting technology is a tool for synthesizing powerful molecular recognition materials for the detection of hormones, proteins, nucleotides, drugs, pollutants, food and other molecules. The principle of MIT relies on the formation of a complex between an analyte as a template and a functional monomer. In the presence of the crosslinking agent, a three-dimensional polymer network is formed between the analyte and the monomer. After polymerization, the analyte (template) is stripped from the polymer, leaving a specific recognition site complementary to the analyte in size, shape, and chemical function (Figure 5). 129 Molecular recognition usually occurs through intermolecular interactions (for example, hydrogen bonds), ionic bonds, and dipole-dipole interactions between analyte molecules and functional groups present in the polymer matrix. Therefore, the resulting molecular pattern embedded in the polymer can selectively recognize and bind the desired analyte. MIT has been successfully applied to modified electrodes for electrochemical sensing. The advantage of this method is that it is relatively stable during storage and exhibits a low rate of denaturation; instability and denaturation are the main limitations of biometric elements (such as enzymes and antibodies). Figure 5 shows the scheme of using graphene and copolymer composite materials to fabricate a molecularly imprinted melatonin sensor. Abbreviations: GCE, glassy carbon electrode; ANHSA, 4-amino-3-hydroxy-1-naphthalenesulfonic acid; MM, melamine.

Note: The graphene is dropped on the GCE surface and dried overnight at room temperature. Films containing imprinted copolymers are prepared by electropolymerization. The template is then removed to obtain a molecularly imprinted surface for the detection of melatonin. Adapted with permission from the Royal Society of Chemistry, a molecularly imprinted sensor based on graphene and copolymer composite materials for the ultra-trace determination of melatonin in human biological fluids, Gupta P, Goyal RN, RSC Advances, Volume 5, 50th edition, copyright (2015); permission communicated through Copyright Clearance Center, Inc. 130

Figure 5 shows the scheme of using graphene and copolymer composite materials to fabricate a molecularly imprinted melatonin sensor.

Abbreviations: GCE, glassy carbon electrode; ANHSA, 4-amino-3-hydroxy-1-naphthalenesulfonic acid; MM, melamine.

Recently, an electrochemical microfluidic chip for melatonin detection was developed using MIT. A composite material containing a copolymer of graphene, 4-amino-3-hydroxy-1-naphthalenesulfonic acid (AHNSA) and melamine is used to make a new molecularly imprinted polymer (MIP) sensor to detect human serum and urine samples Of melatonin (Figure 5). 130 MIP film is produced by depositing graphene on the surface of GCE, and then electropolymerizing ANHSA and melamine in the presence of melatonin. In order to release the imprinted melatonin molecules from the composite, the modified electrode was cycled in 0.5 M sulfuric acid for 25 cycles at a scan rate of 100 mV/s between -1.0 and 1.0 V. SWV analysis using the MIP sensor shows that the melatonin detection range is 0.05–100 μM, and the LOD is 60 pM.

MIT is a promising tool that can quickly replace biomolecules (for example, antibodies, aptamers, and enzymes) for molecular sensing. Choosing the right materials and preparation schemes is essential for the production of effective MIP. 131 However, there is currently a lack of cost-effective materials for the development of MIT-based sensors. 132 In addition, MIT technology utilizes free radical polymerization and sol-gel processes. Free radical polymerization involves multiple mechanical grinding and sieving steps to obtain tiny particles, which may result in lower than expected binding affinity. 131 In addition, free radical polymerization can only be carried out in bulk, so a large amount of template material is required, which makes the process expensive.

Amperometry is an electrochemical technique in which current is measured as a function of independent variables such as electrode potential or time. Han et al.133 pyrolyzed dandelion crests and cast them on GCE to develop an economical nanoporous carbon sensor to detect tryptophan. Due to the porous structure and large specific surface area of nanoporous carbon, it exhibits excellent electrocatalytic activity for the oxidation of tryptophan, while reducing the overpotential and improving the current response. The current response of sensors based on nanoporous carbon electrodes is linear in the range of 1 μM to 10 mM, and the LOD is 0.5 μM. This nanoporous carbon electrode-based sensor evaluated the tryptophan content in composite amino acid injection and fetal bovine serum, and showed a good correlation, ranging from 98.17% to 103.93%. The nanoporous carbon electrode sensor is biodegradable and economical, but the drip casting method used to manufacture the sensor has considerable limitations, such as the coffee ring effect (CRE). Deegan et al.134 observed and explained CRE, and they found that the periphery of the ring was concentrated by non-volatile solute particles compared to the center of the stain. Significant voltammetric results require the formation of a uniformly modified surface, but drip casting can cause CRE, which significantly limits the reproducibility of drip casting surfaces. A practical way to reduce CRE is to explore the combination of a variety of solvents and nanoporous carbon electrodes, and then microscopically image the cast surface. 135 The combination that provides the lowest CRE should be used to avoid reproducibility problems.

A major focus of today's electrical analysis research is to combine current detection with liquid flow analysis. This combination will ensure enhanced sensitivity and selectivity, as well as an increase in analytical throughput (Figure 6). 136 In addition to miniaturization and automation, flow injection also reduces reagents, samples, and wasted volume. In addition, flow injection enhances the diffusion of the sample into the reagent flow, thereby achieving high sensitivity. 137 Shaidarova et al.138 developed a flow injection-based amperometric detection platform for the determination of tryptophan by electrodepositing AuNP on a screen-printed electrode. The dependence of the analytical signal on the logarithmic coordinates of tryptophan and pyridoxine concentrations is linear in the range of 0.5 μM to 5 mM. The current response using the Au-SPE electrode under flow injection analysis shows a theoretical throughput of up to 180 samples/hour. Surprisingly, the author did not report LOD. In addition, flow injection is not fully utilized, for example, samples are manually prepared by dissolving accurately weighed parts in a supporting electrolyte solution. A fully automated flow system can be generated by coupling microfluidic channels (such as tree micromixers or tandem laminated mixers) to automatically mix and dispense tryptophan in multiple test chambers. 139,140 Figure 6 Schematic diagram of a simple flow injection analysis system for electrochemical detection.

Figure 6 Schematic diagram of a simple flow injection analysis system for electrochemical detection.

In 2015, Gomez et al.141 developed a single-walled carbon nanotube pressure transfer electrode (SW/PTE) to analyze melatonin, tryptophan and serotonin using a microchip electrophoresis platform. The microchip electrophoresis platform enables analysis to be performed quickly with low sample and reagent volumes and minimal waste. 142,143 In addition, the combination of electrochemical detection and microchip electrophoresis can achieve rapid analysis, high sensitivity, and miniaturization without affecting performance. By combining SW/PTE with microchip electrophoresis and current detection, melatonin, tryptophan, and serotonin were quickly detected in <150 seconds, with LODs of 4, 1, and 5 μM, respectively. The linear detection range of melatonin and tryptophan is 50-500 μM, and the linear detection range of serotonin is 10-200 μM. Although this research is promising, analyte separation must be performed before sample analysis, which may interfere with the results. In addition, Gomez et al.141 claimed that their device is portable (due to miniaturization) and that their electrodes are disposable. However, the durability and accuracy of their equipment was not specified. For new detection methods based on microchip electrophoresis, the number of analyses that can be performed before the chip needs to be replaced should be reported. 144

Random sensors have recently attracted considerable interest because they can provide fast, sensitive, and reconfigurable multi-analyte detection. Random sensors are known for their ability to conduct qualitative and quantitative research on materials of interest in complex samples. 145,146 The function of the random sensor is to measure the ion transmission of the analyte through the nano-scale pore group in the insulating film. 147 Random sensors are based on several parallel time-resolved measurements, designed to detect, identify, and count discrete macromolecular events, rather than reading the average response. Mihai et al.148 used modified graphene materials to fabricate four random microsensors, such as β-CD and nitrogen-doped graphene (β-CD/nGR), 2,2-diphenyl-1-picrylhydrazine and exfoliated Graphene (DPPH/exfGR), protoporphyrin IX and nGR (PIX/nGR) and PIX heat-treated nGR (PIX-nGR-TT) are used for the enantiomeric analysis of tryptophan in whole blood samples. The current development principle of random sensors is based on channel conductivity. The current development is carried out in two steps: the first step, when the molecule enters the channel/pore, it is completely or mostly blocked (the current drops to zero), and the second step is the chiral-dependent binding and redox reaction. , Leading to the enantioselective determination of tryptophan. The novelty of this screening method is that a random sensor can be used to simultaneously detect the two enantiomers of tryptophan without pretreatment of the whole blood sample. For L-tryptophan, the linear detection range and LOD of β-CD/nGR, DPPH/exfGR, PIX/nGR and PIX-nGR-TT sensors are 10 fM–1 mM and 10 fM, 1 fM–0.10 mM and 1 fM, 100 fM–0.10 mM and 100 fM, 0.10 nM–0.10 mM and 0.10 nM. Similarly, for D-tryptophan, the linear detection range and LOD of β-CD/nGR, DPPH/exfGR, PIX/nGR and PIX-nGR-TT sensors are 1 pM– nM and 1 pM, 1 pM–1 mM and 1 is pM, 0.10 pM–1 mM and 0.10 pM, 1 pM–1 nM and 1 pM, respectively. When the developed random sensor was used for the enantiomer analysis of tryptophan in blood samples, highly sensitive and reliable results were obtained. One disadvantage of random sensors is their poor durability, which limits their use in the laboratory. 149 Its characteristic is that the diameter of the random micro sensor is only a few hundred microns, and the deposited nanoparticles/electrodes usually break or leak current after several hours of use. In the future, random microsensors should be combined with microfluidics to develop durable lab-on-a-chip technology.

Recently, Staden et al. 150 designed two random sensors: one was modified with rGO (rGO-TiO2) modified with titanium oxide, and the other was modified with graphene (rGO/TiO2/AuNP) modified with TiO2 and AuNPs; both Both are mixed with the complex of protoporphyrin IX and cobalt. 150 Both sensors are used to detect melatonin in pharmaceuticals and biological fluids (such as breast milk and whole blood); 150 both show the same linear range (9.98 aM–0.99 pM and LOD 9.98 are). Even in complex samples, the LOD of random sensors is excellent, thus proving their suitability for biological sample testing. The main limitation of the melatonin random sensor is the complicated manufacturing process and poor repeatability due to the difficulty in producing the same pore size.

Optical sensors are a suitable platform for monitoring various disease-related parameters in a personalized way. Optical sensors may revolutionize the diagnosis and treatment of diseases. Colorimetric/fluorescence readings are particularly exciting for sensing applications, because changes in sample color or changes in emitted light intensity can be quantitatively correlated with the concentration of the target analyte. In the past 6 years, compared with electrochemical methods, fewer new optical detection methods have been reported for tryptophan and melatonin.

A simple colorimetric method was developed for the quantitative determination of free tryptophan in 96-well plate-level throughput. Wu et al. 151 used purified tryptophanase to enzymatically convert tryptophan to indole (Figure 7). The indole further reacts with hydroxylamine to form a pink product with a maximum absorption wavelength of 530 nm (Figure 7). 151 The formation of pink is directly proportional to the content of tryptophan in the sample. Therefore, a simple spectrophotometric method can be used to obtain quantitative results in two hours. This indole-hydroxyamine tryptophan sensor is very stable in complex biological samples. The authors used tyrosine, phenylalanine and two dipeptides as competing chemicals to demonstrate the specificity of this method for free tryptophan. They determined that, compared with other non-separation colorimetric methods, proteins in biological samples would not interfere with the determination, but this tryptophan detection based on the indole-hydroxylamine reaction showed a low LOD of 100 μM and a linear range of up to 600 μM . Figure 7 Schematic diagram of high-throughput tryptophan determination. Note: Tryptophan is enzymatically converted to indole by purified tryptophanase, and then the reactivity of indole with hydroxylamine is used to form a pink product. Adapted from Talanta, Volume 176, 2018, Wu Y, Wang T, Zhang C, Xing XH, a fast and specific free tryptophan quantitative colorimetric method, pages 604-609, copyright (2018) , With permission from Elsevier. 151

Figure 7 Schematic diagram of high-throughput tryptophan determination.

Note: Tryptophan is enzymatically converted to indole by purified tryptophanase, and then the reactivity of indole with hydroxylamine is used to form a pink product. Adapted from Talanta, Volume 176, 2018, Wu Y, Wang T, Zhang C, Xing XH, a rapid and specific tryptophan quantitative colorimetric method, pages 604-609, copyright (2018) , With permission from Elsevier. 151

In order to improve the sensitivity of optical detection of tryptophan, a water-stable metal organic framework (MOF) ZJU-108 was synthesized as a luminescence sensor. 152 As the singlet level of tryptophan is lower than other amino acids, the singlet-single Förster energy transfer (S-SFET) mechanism has been applied to design luminescence sensors. The luminescent MOF ZJU-108 was prepared by selecting 6-(4-pyridyl)-terephthalic acid, a singlet ligand suitable for tryptophan, and Zn2, a biocompatible and inexpensive metal ion. As expected, ZJU-108 showed good selective luminescence enhancement of tryptophan with an LOD of 42.9 nM. In addition, ZJU-108 exhibits excellent thermal stability (decomposition temperature of 400°C), water stability (the integrity of its frame after immersing in water for 1 month) and pH stability (pH range of 1.8-11.7, Lasts 12 hours). Despite its excellent stability and good LOD, ZJU-108 is an enhancement-based sensor, which means that it responds to other amino acids, but has a lower signal compared to tryptophan. In addition, only a single amino acid and tryptophan are used to evaluate the luminescence of ZJU-108, not a mixture of various amino acids and tryptophan. Evaluating tryptophan in amino acid mixtures can cause high background noise and false results. Therefore, further research is needed to develop a tryptophan-specific sensor.

A selective and sensitive colorimetric method based on surface-enhanced Raman scattering (SERS) and diazo coupling reactions was developed to quantify trace amounts of tryptophan in complex biological samples. Surface enhanced Raman scattering technology provides sharp spectral peaks of small molecules at very low concentrations. 152,153 can increase the signal intensity by 6-14 times. Tryptophan has low SERS intensity even at high concentrations. Tryptophan and naphthalene ethylene diamine (NEDA) molecules form a larger complex through the -N2 bridge after diazotization, which increases the contact surface of tryptophan and indirectly amplifies the SERS signal intensity of tryptophan. 154 LOD is 20 nM, with linearity. The detection range of 50 nM to 1 mM is achieved by this method. The developed sensor is used to analyze the difference in serum tryptophan concentration between healthy people and colorectal cancer patients. Compared with the data obtained by HPLC, the results show a high degree of correlation. The main chemicals used in the diazo coupling reaction include sodium nitrite and ammonium sulfamate, which are highly toxic to the environment and humans. 155 In particular, sodium nitrite is an extremely powerful oxidant, which may cause hypotension, arrhythmia, changes in consciousness, and restrict the transport and delivery of oxygen in the body through the formation of methemoglobin. 156 The use of such highly toxic chemicals should be strongly opposed, and biocompatible chemicals must be used to ensure sustainable development.

Recently, fluorescent chiral sensors with direct signals (such as enantioselective fluorescence enhancement) have aroused interest in the determination of amino acid enantiomers. Wu et al.157 synthesized a polymeric chiral ionic liquid-4 (PCIL-4) by free radical polymerization for the enantioselective detection of tryptophan. First, by using 2-azoisobutyronitrile (AIBN) as the initiator and cetyltrimethylammonium bromide (CTAB), polyethylene (PSVP-3) is selected as the main chain. CTAB forms micelles to control the homogeneity in ethanol and control the open growth of polymers. Then, the phenyl and pyridine groups are physically separated, but at the same time, allowing the production of fluorescent polymers through steric delocalization effects. Second, the phenylalanine derivative, poly(ionic liquid)(S)-PCIL-4 (chiral molecule) and alkyl chloride are linked to the pyridyl nitrogen group to form a pyridinium cation. The constructed sensor can provide photoluminescence in the presence of spatial π-π and ion-π interactions. Then, (S)-PCIL-4 can be used as the fluorescence switch off/on of the sensor for chiral recognition of tryptophan in the presence of Cu2. The linear detection range of tryptophan is 0-200 mM and 0-175 mM, and the enantiomeric fluorescence difference ratio is 1.08. Nevertheless, this research opens up possible ways to synthesize fluorescent sensors with different chiral properties. Subsequently, Pundi et al.158 synthesized a chiral carbazole sensor (CCS) with a "urea carboxylic acid" binding site for the quantitative fluorescence detection of tryptophan. The CCS sensor can be used as a reversible fluorescence quenching sensor for Fe3. Then, after adding tryptophan, the quenched fluorescence of the CCS-Fe3 complex can be restored, so that the tryptophan in the aqueous solution can be quantitatively detected. CCS showed that the LOD of tryptophan was low at 0.31 μM. In addition, the content of tryptophan in three commercial sleep-improving capsules was analyzed with high precision through CCS. Further experiments are needed to approve the sensor for the evaluation of tryptophan in complex biological matrix-like serum, urine and food samples.

The colorimetric/fluorescence detection of biomolecules requires spectrophotometric measurement, which is relatively expensive. In order to reduce the cost of inspection, the scanning measurement method is used to evaluate the color change. The scanner uses a flatbed scanner to measure the reflection of light in the solution. Compared with spectrophotometry, another advantage of scanning measurement method is that the sharpness of the maximum wavelength of the analyte is not a serious problem, because the measured color intensity is analyzed according to different color values (red, green, blue, etc.) , And therefore provide good results even if the sample is turbid. Jafari et al.159 used scanning measurement to develop a fast and inexpensive colorimetric sensor for chiral identification of tryptophan enantiomers through chitosan-terminated silver nanoparticles (CS-AgNPs). In the presence of D-tryptophan, no color change was observed in the CS-AgNPs solution, while in the presence of L-tryptophan, the color changed from yellow to brown. A scanner was used to take images of the colored solution, and Photoshop software was used to obtain the corresponding color values, which were then used as the analysis signal for the optimization of experimental parameters. Two types of color value systems are studied: RGB (red, green, and blue values) and CMYK (cyan, magenta, yellow, and black values). The color value indicates that L-tryptophan has a better interaction with chitosan-terminated silver nanoparticles than D-tryptophan. The author speculates that due to the double helical conformation of the CS molecule, the selective formation of hydrogen bonds between CS and tryptophan isomers may play a role in the chiral recognition of the enantiomers. In the concentration range of 13-460 μM, there is a good linear relationship between the concentration of L-tryptophan and the effective intensity of the color-developing product, and the LOD is 2.1 μM. Manufacturing custom-designed 3D printed accessories compatible with smartphones and appropriate software applications can be used to transform scanner-based tryptophan colorimetric analysis into a more sensitive, portable, inexpensive, and easy-to-use smartphone-based system.

In this section, we will discuss colorimetric/fluorescence assays that do not rely on antibodies, with the exception of chip-based immunochromatographic assays. Array sensors using nanomaterials have become impressive tools for detecting analytes such as proteins, antioxidants, explosives, metal ions, and toxic gases. 160-164 Nanomaterial-based array sensors use cross-reactive sensing elements to generate unique responses. Color or fluorescent patterns are formed in the presence of different analytes; the statistical analysis of the patterns is helpful for qualitative and quantitative analysis of various molecules.

In two independent studies by Huang et al., 165 manganese oxide (MnO2)-3,3',5,5'-tetramethylbenzidine (TMB) nanosheets (MnO2-TMB) and sodium hypochlorite (NaClO)-TMB Nanosheet (NaClO-TMB) 166 was synthesized to develop a multicolor sensor array for the analysis of various antioxidants, including L-glutathione, melatonin, L-cysteine, UA and AA. The different reducing abilities of the antioxidants listed in 165,166 cause different colorimetric response patterns, which can be at wavelengths of 370, 450 and 650 nm. Principal component analysis confirmed that the two colorimetric assays can effectively distinguish the antioxidants in buffer and antioxidant-added fetal bovine serum (FBS). For the MnO2-TMB and NaClO-TMB sensors, the LOD of the antioxidant suspended in FBS is 20 μM and 1 nM, respectively. It is worth noting that these economical MnO2-TMB and NaClO-TMB sensors can distinguish various antioxidants present in FBS, and the results are visible to the naked eye; however, this technology does not work at clinically relevant concentrations. If these sensors are to be successfully applied to disease monitoring and detection, the sensitivity of these sensors needs to be significantly improved, which is the main purpose of most visual biomolecular sensors.

Another colorimetric/fluorescence sensor is used to detect melatonin in saliva, using 2.3-naphthalenedialdehyde (2,3-Nda) as a color reagent. 167 Under acidic conditions, 2.3-Nda reacts with melatonin as a chloride catalyst in the presence of iron (III); the color of the solution changes from colorless to yellow in a concentration-dependent manner. In addition, due to the large conjugated system of the reaction product, the application of different concentrations of the reactant will produce fluorescence. For the colorimetric method, the detection range of melatonin is 2.5-37.5 μM, and the LOD is 1.288 μM; the melatonin detection range of the fluorescence method is 0.01–0.1 μM, and the LOD is 0.004 μM. Compared with other non-enzymatic melatonin detection assays, this analysis provides higher sensitivity. A potential limitation of this assay is the use of hydrochloric acid to create acidic conditions for the reaction; hydrochloric acid is very corrosive and harmful to the environment and users. 168 Another disadvantage is the presence of reactive aldehyde groups on 2.3-Nda, which are easily attacked by nucleophiles present in biological samples, such as sulfhydryl/cysteine residues and thiol side chains of thiolates. 169,170 To ensure the reliability of this assay, further studies are needed on the effects of different concentrations of bionucleophiles.

Recently, an immunochromatographic method (ICA) was developed that uses AuNP conjugated to mouse monoclonal antibodies for rapid quantitative melatonin detection. The LOD generated by 171 ICA was 0.185 µM, which was determined by the naked eye, and the LOD determined by the strip reader was 215.25 nM, which was used for melatonin samples measured under optimized experimental conditions. The linear detection range is 215.25 nM to 42 μM, which is comparable to standard liquid chromatography. The ICA may be an excellent tool for determining the level of melatonin in food; however, the development of a lateral flow ICA device for melatonin detection with higher sensitivity is essential. The main culprit for the low sensitivity of ICA is the transient response caused by capillary action on the nitrocellulose membrane (NCM). This transient reaction results in a low binding rate between the probe-labeled target and the antibody immobilized on the nitrocellulose membrane. Modifying immobilized capture antibodies may increase their binding efficiency, which is currently limited by the protein binding capacity of nitrocellulose membranes. Therefore, the development of new materials with higher binding affinity may overcome the limited protein binding capacity of nitrocellulose membranes and further improve the LOD and detection range of ICA.

Due to the wide availability of tryptophan in several proteins, there is still a need to develop highly specific sensors that are insensitive to matrix and pH. On the other hand, the biggest challenge facing melatonin researchers is to evaluate circulating concentrations of <8.6 pM when metabolites with similar molecular structures are present in highly diverse and complex matrices. Despite recent efforts, there is still a need for major improvements to the LOD currently detected to provide cheap and simple point-of-care (POC) detection. The emerging sensors discussed in this review have many advantages over traditional tryptophan and melatonin testing methods, such as simplicity, co-detection, and portability; however, reproducibility, stability, detection range, and LOD are still worthwhile Concerns. In order to improve the reliability of the test, researchers should report the inter-test and intra-test variability of test sensitivity.

The latest developments in electrochemical sensors reflect the importance of this field and the power, flexibility and convenience that electrochemistry can provide for biomolecular detection. However, every electrochemical sensor faces the common problem of biological contamination of the electrode surface, especially during the analysis of complex biological samples. In order to evaluate biomolecules electrochemically, the electrode surface must physically interact with the working solution, which includes clinical samples such as plasma, serum, sweat, saliva, or urine. The interaction between the electrode and the solution will immediately or eventually cause the adsorption and adhesion of biomolecules to the electrode surface; this unavoidable biological fouling will interfere with the sensitivity and selectivity of the sensor. 172,173 Therefore, a key requirement for the development of robust and sensitive electrochemical sensors is the synthesis or discovery of powerful antifouling materials. The new antifouling material can be combined with the sensing/electroactive material to make a stable sensor. In order to develop anti-fouling bioelectrodes, the electrode surface is co-deposited with polydopamine and hyaluronic acid. The deposition of polydopamine promotes the absorption of hyaluronic acid with antifouling properties on the electrode surface. 174 Alternatively, new materials with electroactive and antifouling activity can also be synthesized. For example, newly discovered conjugated polymers such as poly(sulfobetaine-3,4-ethylenedioxythiophene) exhibit excellent electrical conductivity and antifouling/antibacterial properties. 175 Another outstanding problem facing the production of electrochemical sensors is the lack of research on their shelf life. Generally, electrodes for electrochemical detection are developed using new nanocomposite materials, but the shelf life of nanocomposite materials is rarely reported. Comprehensive analysis should be conducted to check the shelf life of nanocomposite materials and electrodes decorated with nanocomposites in various solutions. In addition, the development of nanocomposites requires skilled handling and expensive materials (such as Pd); 88 therefore, simpler protocols and cheaper materials should be developed to reduce the overall price and complexity of analyte detection. Before using electrochemical sensors based on nanocomposites, their biocompatibility and cytotoxicity must also be studied to ensure their applicability in the body.

Although there are reliable techniques for the detection of tryptophan and melatonin, most of the techniques are laboratory-based, expensive, and require technical training. The clinical significance of tryptophan and melatonin has only recently been underestimated. However, recent studies have increasingly demonstrated their key functions in human health and disease. Mental health and sleep disorders, jet lag, and shift work have brought new problems related to modern life; their related diseases are usually only discovered after they enter the chronic phase.

In addition, artificial light is an effective circadian disruptor, which reduces melatonin levels and has serious consequences. 177-180 POC measurement of tryptophan and melatonin levels can enable early diagnosis of physical and psychological disorders related to circadian rhythm disorders. In addition, real-time sensing of melatonin and tryptophan may provide key insights for understanding their role in immune regulation, inflammatory diseases, and inflammatory responses to epidemic diseases such as COVID-19.180-183. Therefore, nanoelectrodes for real-time detection are easy to synthesize, and there is an urgent need for durability, cheapness, eco-friendliness, biocompatibility, selectivity and sensitivity. Recently, micro-electromechanical nerve tools for non-invasive ultrasound brain stimulation have been developed to treat brain diseases. 184 Similarly, with the help of big data analysis and artificial intelligence, wearable micro/nano electromechanical sensors can be developed to study sleep-wake cycles and accurately quantify circadian rhythm disturbances. 185 Strategies that use wearable sensors to accurately diagnose circadian rhythm disorders may lead to new therapeutic interventions. 185,186 Another potentially fruitful approach to research on the detection of tryptophan and melatonin is the development of lateral flow ICA. In recent years, lateral flow ICA has achieved considerable commercial success and has had a positive impact on disease diagnosis. However, due to the need for very high sensitivity, there are few lateral flow ICAs available for melatonin detection. Lateral flow ICA is usually performed by visually inspecting color changes. Optical (colorimetric, fluorescence, chemiluminescence and surface-enhanced Raman scattering), thermal (thermal imaging, photoacoustic and speckle imaging), magnetism (quantification of magnetic nanoparticles, giant magnetoresistance and tunnel magnetoresistance) and electrochemistry (each A variety of voltammetry and electro-chemiluminescence) techniques can be used to develop high-sensitivity lateral flow ICA to quantify melatonin in biological samples for POC testing. 187

In this review, we objectively summarized electrochemical and optical sensors for the detection of tryptophan and melatonin, providing a scientific perspective for the future of this field. Given the large number of publications in this field, we focus on the highly sensitive methods developed in the past 6 years. Even with this focus, we will inevitably exclude some emerging technologies inadvertently. In the past few decades, people have developed a strong interest in the development of tryptophan and melatonin detection technologies using immunoassay, spectroscopy, HPLC, MS, colorimetry, fluorescence and electroanalysis sensors. Among them, electroanalysis detection tools have many advantages, including low LOD and high range, low interference, cost-effectiveness, fast response time, and suitable for quantitative and analytical applications. The future work of electrochemical sensors may involve optimization of LOD and sensitivity, development of integrated detection systems, and monitoring of biomolecules in the body. In addition, in order to make up for the lack of MIT output signal, fluorescent or highly electroactive materials should be combined with the high selectivity of MIT to develop ultra-sensitive and selective sensors for the detection of tryptophan and melatonin. In summary, MIT needs further work to achieve mass production. Research on the mechanism and characterization of "on-off" fluorophore sensors is still very limited, and further development is needed. Although the development of electrochemical sensors has made significant progress recently, we should still actively try to design and explore new technologies for environmentally friendly, cheaper, durable and faster sensing tools. In order for colorimetric/fluorescence sensors and lateral flow devices to be commercially successful, a significant increase in sensitivity is required. We also want to emphasize that, in addition to the modification of immunoassays, there is a serious lack of research on the optical detection of tryptophan and melatonin. There is an urgent need to develop new colorimetric and fluorescent materials for optical sensing of tryptophan and melatonin. Finally, commercialization efforts and application-driven cooperation in different disciplines are essential for future development.

AA, ascorbic acid; cELISA, competitive enzyme-linked immunosorbent assay; CV, cyclic voltammetry; Cys, cysteine; DPV, differential pulse voltammetry; DSI-MS, droplet spray ionization mass spectrometry; EIS, electrochemistry Impedance spectroscopy; FSCV, fast scanning cyclic voltammetry; FBS, fetal bovine serum; GC-MS, gas chromatography-mass spectrometry; GSH, glutathione; HPLC, high performance liquid chromatography; ICA, immunochromatography; LOD , Detection limit; LC-MS/MS, liquid chromatography tandem mass spectrometry; PS, pharmaceutical samples; RIA, radioimmunoassay; SWV, square wave voltammetry; TMB, 3.3',5,5'-tetramethylbenzidine ; UHPLC/HRMS, ultra-high performance liquid chromatography combined with high-resolution mass spectrometry; UA, uric acid.

The author would like to thank the members of the "Biological Clock and Aging Control" laboratory of Inje University in South Korea for their valuable support and criticism.

All authors have made significant contributions to the work of the report, whether in terms of concept, research design, execution, data acquisition, analysis and interpretation, or in all these areas; participating in drafting, revising or critically reviewing articles; final approval requirements Published version; agreed on the journal to which the article was submitted; and agreed to be responsible for all aspects of the work.

There are no funds to report.

The author has no potential conflicts of interest to disclose.

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