Simple Colorimetric and Fluorometric Assay Based on 2,3-Naphthalenedialdehyde for Melatonin in Human Saliva
- 62 Downloads
In this paper, 2,3-naphthalenedialdehyde (2,3-Nda) was selected as color reagent with colorimetric and fluorometric assay for detecting melatonin in human saliva. 2,3-naphthalenedialdehyde reacted with melatonin under the catalysis of hydrochloric acid and Fe3+, which the color change was observed sensitively from colorless to yellow with the naked eye. On the other hand, the product of the reaction had a large conjugate structure with strong fluorescence for the fluorometric analysis. Under optimized experimental conditions, a concentration range of melatonin was 2.5–37.5 µM (R2 = 0.998) by colorimetry with the limit of detection for 1.288 µM (S/N = 3), and 0.01–0.1 µM (R2 = 0.997) by fluorometry with the limit of detection for 0.004 µM (S/N = 3). The two assays were successfully applied to the determination of melatonin in human saliva, which average recoveries were 97.80% and 95.96%, respectively, which has the potential in fast clinical examination of melatonin in human saliva.
KeywordsColorimetry Fluorometry 2,3-Naphthalenedialdehyde Melatonin
Melatonin (C13N2H16O2, MT) is a neuroregulatory hormone secreted by the pineal gland [1, 2]. It belongs to indoles heterocyclic compound, and its chemical name is N‐acetyl‐5‐methoxytryptamine, also known as pineal hormone or melatonin . MT is a kind of body hormone that induces natural sleep, and it can overcome sleep disorder and improve sleep quality by regulating people’s natural sleep [4, 5, 6]. And MT can directly act on the gonads, reduce the content of androgen, estrogen and progesterone [7, 8]. In addition, MT has strong neuroendocrine immune regulatory activity, which may become a new antiviral treatment method and approach [9, 10, 11, 12, 13]. And it has free radical scavenging antioxidant ability, which slow down the aging of human organs [14, 15, 16]. Most importantly, MT has a good effect of inhibiting cancer [17, 18, 19]. It was reported that people with abnormally low MT levels are more likely to have prostate cancer and breast cancer [20, 21]. What’s more, MT has an ability to reduce the toxicity during chemotherapy, which protects the health of human normal cells and organs, to a certain extent [22, 23]. The level of melatonin in the body is very low, and it exists in saliva at 2.4-12.4 pg mL−1 level . The secretion of melatonin at the night is 5–10 times more than that in the day, which reaches the peak from 24:00 to 5:00 am. The level of MT at night directly affects the quality of sleep [25, 26, 27]. So many assays of detecting melatonin have been developed continuously.
Many methods for the detection of melatonin in body fluids have been reported, such as high performance liquid chromatography (HPLC) coupled to fluorescence detector , gas chromatography mass spectrometry (GC–MS) [29, 30], HPLC–LC/MS [31, 32], capillary electrophoresis , radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) [35, 36], colorimetric sensor arrays [37, 38, 39]. Among them, ELISA kit for detection of melatonin is commercially available and the LOD was 0.5 pg mL−1 . However, there are few reports about fluorescence of determination melatonin in human body.
MT, as mentioned above, belongs to the Indoles heterocyclic compounds with a free C-2 position. It has been reported that the electron density of this position is strong. In the previous work , sensitive and accurate colorimetric and fluorometric methods were presented for the determination of melatonin in tablets and serum. The authors utilized the reactions of p-dimethylaminobenzaldehyde in hydrochloric acid (van Urk reagent)-ferric chloride in sulphuric acid for colorimetry and the reaction of melatonin with o-phthalaldehyde in acid medium which yields high fluorescent condensation product for fluorometry.
Aromatic compounds are usually view as fluorescent probes because of its conjugated structures. Aldehyde-decorated fluorescence probe is an important and excellent probe because of the activity of the aldehyde group, which can specially identify certain groups or ions [37, 41]. 2,3-Naphthalenedialdehyde (2,3-Nda) with the structure of naphthalene, has two aldehyde group as reactive positions, which are easy to be attacked by a nucleophile. Matsufuji’s group  developed a fluorometric method for angiotensins (ANG I, II, III) in human plasma based on 2,3- Nda with highly reproducible and precision, which excitation and emission wavelengths were set at 420 nm and 490 nm, respectively. There are also reports in the literature which 2,3-Nda was applied to detect cyanide based on the reaction of cyanide, 2,3-Nda and taurine. And the proposed method was applied successfully to blood analysis [43, 44]. It was also reported that several NDA derivatives was designed and synthesized, which had been successfully used to detect glutathione in HeLa cells .
MT can be detected in the blood, urine and saliva [46, 47]. There is no doubt that saliva sampling is the better choice because it is non-invasive, non-painful and easy to obtain . Salivary fluid is composed of water (more than 95%), and of various electrolytes, hormones, enzymes, immunoglobulins, cytokines, etc. . There are also glucose and nitrogenous products, such as urea and ammonia . Most of the mucins may be denatured by freezing and thawing the sample; the denatured mucins also tend to adsorb much of the debris from the sample and the freezing eliminates most of the froth associated with a fresh saliva sample . When saliva samples treated with anhydrous ethanol, these proteins denature. After centrifugation, the denatured protein is removed as a precipitate. The components become single and there are basically no interfering factors, supernatant becomes relatively simple.
In this paper, simple colorimetric and fluorometric assay was developed for melatonin in human saliva using 2,3-Nda as a chromogenic reagent. The reagent reacted with MT under acidic conditions with FeCl3 as the catalyst, which the color of solution changed from colorless into yellow. On the other hand, the product has a large conjugated system with strong fluorescence for the fluorometric analysis. Finally, the assay was applied to the determination of melatonin in saliva with the average recovery of colorimetry and fluorometry for 97.80% and 95.96%, respectively. The methods are simple and low cost for detection of MT.
2 Materials and Methods
2.1 Reagents and Instruments
Melatonin (98%) was purchased from Solarbio® (Beijing, China). Stock standard solution of melatonin (0.2000 mmol L−1) was prepared in ethanol and stored refrigerated at 4 °C in brown glass flasks. 2,3-Naphthalenedialdehyde was purchased from Tokyo Chemical Industry Co. Ltd (Japan). H2SO4, HCl and absolute ethyl alcohol were obtained from XiLong Scientific (Guangdong, China). All chemicals are analytical grade and do not require further treatment. Ultrapure water (18.25 MΩ, Millipore, USA) are used throughout the whole experiment. 2,3-Nda (2.0000 mmol L−1) was prepared in mixture of concentrated HCl (s.g.1.19) and ethanol (HCl: ethanol = 1:1), and FeCl3 (0.5 mmol L−1) was prepared in a mixture of sulfuric acid and H2O (H2SO4:H2O = 3:5). Above stock standard solution was advised to store refrigerated at 4 °C in brown glass flasks, and diluted into different concentrations that we need.
CR-I was prepared for colorimetry, which was made up of 2,3-Nda (2.00 mmol L−1) and FeCl3 (0.05 mmol L−1) with the ratio of 4:1. CR-II was prepared for fluorometry similarly, which was made up of 2,3-Nda (1.00 mmol L−1) and FeCl3 (8.0 × 10−5 mmol L−1) with the ratio of 1:4.
UV–vis absorption spectra were recorded using UV-2450 Shi-madzu Vis-spectrometer (Japan). The fluorescence spectra were performed on a F-4600 fluorescence spectrometer (Hitachi, Japan).
Colorimetric method 0.5 ml MT standard solution (140 µmol L−1)and 0.5 ml CR-I solution, together with 1 mL HCl were transferred to centrifuge tubes and incubated at room temperature for 4 min. Then the mixture solutions were monitored at 400 nm by UV–vis spectrometer.
Fluorometric method 10 μL standard solution (10 µmol L−1) MT was mixed with 50 μL CR-II, 80 μL HCl and 920 μL ultrapure water, then were transferred to centrifuge tubes and incubated at 45 °C for 30 s. Finally, the resulting solutions were monitored at 540 nm emission with excitation at 410 nm by the fluorescence spectra.
3 Results and Discussion
3.1 Principle of Colorimetric and Fluorometric Analysis
3.2 Optimization of sensing system
3.3 Linearity and limit of detection
3.4 Test of selectivity
3.5 Real samples detection
Determination of MT spiked in human saliva by colorimetric and fluorometric assay
(n = 3)
In summary, a simple and low-cost colorimetric and fluorometric assay for MT was proposed by use of 2,3- naphthalenedialdehyde as color reagent. The method show sensitivity and high-selectivity toward MT with the limit of detection of 0.004 µM, and has been applied to assay MT in the human saliva samples with the satisfactory results. The proposed assays are fast, sample and low cost, and the required instruments are easy to get. Above advantages prove the proposed colorimetry and fluorometry can be applied to fast clinical examination of melatonin in human saliva.
This work was financially supported by the National Natural Science Foundation of China (1765015, 21808099) and the Science and Technology Innovation Platform Project of Jiangxi Province (20192BCD40001).
- 9.Lissoni P, Pittalis S, Rovelli F, Vigorè L, Roselli MG, Brivio F (1995) J Biol Reg Homeos Ag 9:63–66Google Scholar
- 12.Skwarlo-Sonta K (2002) Neuro Endocrinol Lett 1:61–66Google Scholar
- 42.Matsufuji H, Matsui T, Oki T, Osajima Y, Kawasaki T (1995) Food Sci Biotechnol 44:783–788Google Scholar
- 46.Urbanowicz KEK, Wei F, Rao SL, Kim J, Shin H, Cheng J, Tu M, Wong DTW, Kim Y (2019) Bba-Rev Cancer 1872:49–59Google Scholar