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Abstract
In this paper, we explored a rapid and ultra-sensitive detection method of fucoxanthin in algae by surface-enhanced Raman scattering. Gold nanoparticles (Au NPs) were prepared by the reduction of chloroauric acid with citric acid. An effective Raman indicator molecule, Rhodamine 6G(R6G) dye molecule, was used to characterize Au NPs that were used in SERS analysis to detect fucoxanthin. It was found that the detection limit of fucoxanthin could reach 10−6 mol·L−1. The characteristic peak of fucoxanthin at a wave number of 1520 cm−1was selected as the working curve, and it presents good linearity of fucoxanthin concentration with R2 = 0.932 in the range from 5×10−4∼1×10−7 M. We first proposed the rapid detection of fucoxanthin by surface-enhanced Raman scattering, which took only one minute to detect. In addition, the Raman strength of the pure algae or kelp and the addition of fucoxanthin was compared under the gold nanoparticle substrate, so as to verify the application value of SERS technology in the rapid determination of fucoxanthin.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
As the most abundant marine carotenoid in nature, fucoxanthin is widely found in unicellular algae such as diatom, chrysophyta, and edible seaweeds such as U. pinnatifida [1], H. fusiformis [2]. Fucoxanthin has a unique structure including an allenic bond, conjugated carbonyl group in polyene chain [3] and a 5,6-monoepoxide in the molecule structure [4](Fig. 1). Among nearly 700 natural carotenoids, only about 40 kinds of carotene contain this propylene dienol bond [5], which contributes to the unique physiological functions of fucoxanthin. A large number of studies have proved that fucoxanthin can significantly promote human health. It has been found to have remarkable physiological activities and biological properties such as anti-inflammatory [6], antioxidant [7],antiobesity [8],radical scavenging [9],inhibition of angiogenesis [10], anti diabetes [11] and anticarcinogenic [12]. The latest animal experimental studies have shown that fucoxanthin also has a good effect in the treatment of kidney stones [13], testicular injury [14], asthma [15], alcoholic liver poisoning [16] and other diseases. From the physiological function of fucoxanthin which has been known at present, researchers generally believe that fucoxanthin has a broad application prospect in food health care, biomedicine and other fields in the future.
In food industry, fucoxanthin was initially added to food only as a colorant [17]. When the above vital physiological functions of fucoxanthin were discovered, researchers have followed up with functional studies of fucoxanthin in an effort to make it more widely available in the functional or nutritionally fortified food industry. For antioxidant, Molina [18] have devoted themselves to studying and comparing the antioxidant effects of fucoxanthin and vitamin C. The results show that the combination of the antioxidants had more antioxidant and anti-inflammatory effects than when they were applied alone. Fucoxanthin has been also proven to be one of the most effective anti-obesity natural products by stimulating lipolysis in both animal and human studies [19,20]. Guo [8] evaluate its role in modulation of gut microbiota composition and the results suggest that there is a differential effect of fucoxanthin on cecal and fecal microbiota in mice fed with diets with different fat contents. Fucoxanthin could be a promising microbiota-targeted functional-food ingredient. Kh [21] summarize findings from experimental studies on the effects of fucoxanthin on lipid metabolism, adiposity, and related conditions and discuss the possible underlying mechanisms. The review finally shows that the consumption of fucoxanthin and its derivatives as nutritional supplements is a promising option for the prevention and treatment of obesity and a variety of related pathological diseases, including metabolic syndrome, type 2 diabetes and heart disease. As a lipophilic molecule, the applications of fucoxanthin in functional or nutritionally fortified food are limited. To improve the dispersibility and intestinal absorption of fucoxanthin in water, fucoxanthin–oleic acid–protein complexes were constructed [22].
So as a potential drug source obtained from marine algae, Fucoxanthin could be used in the food industry. The application of fucoxanthin in functional foods is paid much attention at home and abroad. And it is sold in the market as a nutritional and health product and added to over-the-counter drugs. Related nutraceutical [23] on the market emerge in endlessly as well. There are food-related patents in which fucoxanthin is made into tablet candy and microcapsules, and some of them are made into health products that protect eyesight, resist oxidation and anti-aging, lose weight, and resist the cardiotoxicity of adriamycin. In addition to pure fucoxanthin, there are many health products that compound other natural products, such as undaria dietary fiber biscuits, lipid-lowering and liver-protecting food. The special food used by tumor patients is in line with the special diet.
To sum up, it can be seen that the demand for fucoidin in the food market is gradually increasing. An accurate and rapid method to assess the fucoxanthin content is a prerequisite for the healthy development of the fucoxanthin health food industry. However, there is a lack of a accurate and rapid method for the detection of fucoxanthin in the world, which is not conducive to the realization of effective market supervision and safeguard the legitimate rights and interests of consumers.
As mentioned above, fucoxanthin is insoluble in water and other polar solvents but soluble in non-polar organic solvents under neutral conditions, which is a natural fat-soluble pigment. Methods for fucoxanthin determination involving ultraviolet spectrum(UV-vis) [24], high performance chromatography (HPLC) [25,26], high performance thin layer chromatography (HPTLC) [27], high performance liquid chromatography system equipped with a photodiode array (HPLC-PDA) [28] liquid chromatography tandem mass spectrometry (LC-MS/MS) [29], inductively coupled plasma(ICP)-MS [30] and atmospheric pressure chemical ionization mass spectrometry (APCI-LC/MS) [2] have been developed for the separation and identification carotenoids in seaweed. Although the separation effect of chromatographic method is operation stable and result accurate, the sample processing is too cumbersome, which is not conducive to rapid and effective monitoring. Spectral method is easily interfered by another chromophore in the sample, which is not conducive to accurately detecting the real signal. It is very important to establish a rapid and accurate method for the analysis of fucoxanthin in order to ensure the healthy development of the industry, realize effective market supervision and safeguard the legitimate rights and interests of consumers.
Surface-enhanced Raman scattering, or SERS, is a commonly used sensing technique in which inelastic light scattering by molecules is greatly enhanced (by factors up to 108 or even larger, enabling single-molecule (SM) SERS in some cases) when the molecules are adsorbed onto silver or gold nanoparticles (NPs) [31–33]. For analytical applications, SERS could be distinguished from many other technologies through the rich vibration spectral information provided by it, which led to its applications in several different directions, including electrochemistry, catalysis, biology, medicine, art protection, material science, etc [34]. In the last decade, Raman spectroscopy and SERS has been applied in the determination of various microbial pigments. For example, Portable Raman instrumentation with 532 nm excitation allows the detection of carotenoids in different colored layers [35]. SERS also can be used to identify lycopene and red carotene in different mature stages [36]. But up to now, there is no relevant report on the detection of fucoxanthine by SERS.
In this study, we are the first to report the detection of fucoxanthin by surface-enhanced Raman spectroscopy (SERS). SERS can provides non-destructive trace detection, and gold nanoparticles have good chemical stability and are not easily oxidized. In this paper, we synthesized Au NPs in situ reduction with the use of trisodium citrate as reducing agent. The obtained Au NPs was used to detect Rhodamine 6G(R6G) [37] and fucoxanthin by SERS, and it exhibited much higher detection sensitivity and distinguishability. Kelp and five kinds of potential algae are used. The detection of fucoxanthin from raw materials is equivalent to controlling the source, and the quality inspection will be more standardized. The earliest raw materials for extracting fucoflavin are large seaweed, such as kelp, sargassum fusiforme and undaria [38]. However, there are many problems, such as high cost of large seaweed culture, seasonal growth, only cortical cells on the surface of algae and low content. Large-scale seaweed also contains a lot of fucoidin, single-celled microalgae grow fast, easy to culture, artificially controllable, a variety of advantages can solve the problem of raw materials for industrial production [39]. Fucoxanthin is expected to become the focus of the food market, so the detection of fucoidin in algae has become a primary and critical task.
2. Material and methods
2.1 Reagents and aparatus
Fucoxanthin (Sigma,USA). Chloroauric acid (HAuCl4) was purchased from Shenbo Chemical Co., Ltd. (Shanghai, China). Trisodium citrate (C6H5Na3O7) and rhodamine 6G (R6G) were supplied by Macklin Biochemical Co., Ltd. (Shanghai, China). Aluminum metal sheet was obtained from Sinopharm (Shanghai, China). Analyses were conducted using UV-Vis spectrometry (UV 1902, Lengguang Tech., Shanghai, China), scanning electron microscopy (SEM) (Regulus 8100, HITACHI, Tokyo, Japan) and Renishaw confocal Raman instrument (London, UK).
All solutions except fucoxanthin solution were prepared using distilled water and fucoxanthin solution was prepared with absolute methanol. All chemicals were used as received without further purification.
Chaetoceros muelleri, Thalassiosira weissflogii, Isochrysis galbana, Phaeodactylum tricornutum, Dinophycae Symbiodinium and Laminaria japonica preserved by the Southern Institute of Oceanography Fujian Normal University.
2.2 Preparation of gold nanoparticles
For synthesis of Au NPs, 10 mL HAuCl4 (2.4×10−3M) and 90 mL distilled water was added to 250 mL erlenmeyer flask followed by rapid stirring. The solution was boiling and as soon as the solution started boiling, 1ml of 1 wt% Na3Ct solution was added. Subsequently, boiling was continued for 15min to allow the Au NPs to mature. The solution is naturally cooled to room temperature.
2.3 Sample preparation and measurement
Sample preparation:First, the sample is freeze-dried, and sample pretreatment: the freeze-dried sample was extracted with methanol, and then centrifuged to remove the supernatant, and then extracted and detected fucoidin with methanol. The purpose of pretreatment is to eliminate the interference of pigments such as chlorophyll and lutein. Untreated: algae powder was extracted with methanol and fucoidin was detected directly.
The obtained Au NPs were precipitated by centrifugation (10000 rpm10 min) and the supernatant liquid was removed. 1.5 µL fucoxanthin solutions with different concentrations and 1.5 µL Au NPs was mixed on the clean aluminum sheet and allowed to dry at room temperature. Then, SERS spectra were acquired from a Renishaw InVia Raman microscope (Renishaw, Sheffield, England) on the aluminum sheet using 785 nm irradiation (0.34 mW). Each spectrum was obtained with 10s exposure time and the 520.7 cm−1 peak of a silicon wafer was selected for calibration. The detection power of the laser irradiated on the sample was adjusted according to the Raman enhancement signal. The Raman spectrum test was carried out under the microscope with a 50 yield lens, and the Raman detection range was from 400 cm−1 ∼ 1800cm−1. A Vancouver Raman algorithm based on a fifth-order polynomial fitting method was used to remove fluorescence background and noise signals for all the raw SERS spectra.
2.4 Statistical analyses
Data statistical processing was via SPSS 19.0 software used for, and single-factor analysis of variance was used for significant difference analysis. The measurement results were expressed as mean ${\pm} $ standard deviation $({\bar{x} \pm \textrm{s}} )$. When P<0.05, it was statistically significant, and a significant difference was considered.
3. Results and discussion
The as-prepared Au NPs was characterized by scanning electronic microscope (SEM) and involving ultraviolet spectrum (UV-vis) absorption spectra. It can be seen clearly (Fig. 2), the obtained Au NPs dispersed uniformly, and their mean size was about 25 nm. The UV–Vis spectrum of Au NPs showed that the maximal absorption peak at 525 nm and the peak width was narrow [40].
R6G molecule is a special dye used for characterizing SERS performance with its obvious characteristic peak, which is a probe molecule for identifying the enhancement effect of SERS substrate. It shows a Raman spectrum of R6G powders and a SERS spectrum of 10−5M R6G solutions measured by Au NPs substrate (Fig. 3). The enhancement factor calculation is performed on the obtained SERS data, and the calculation details are as follows [41].
Fig. 3. Normal Raman spectra of R6G and the SERS spectra of R6G(10−5M) measured by Au NPs substrate.
V is the volume of the droplet of 1.5µL, C is the molar concentration for analyte solution, Scan is the area of Raman scanning, Ssub=3.14 mm2 (d=1mm, regard a circular) Sscan=6.25 µm2 and Ssub is the area of the substrate; ARaman is the laser spot diameter, which was calculated for 785 nm Using the formula, ARaman=2.4 µm and h is the confocal depth with 20X objective, with the laser spot diameter=(1.22λ/NA), NA=0.4. h=(2λ/NA2) = 9.8µm. IRS=1010, ISERS=2120 at 1361cm−1, C=1 × 10−5M, so that, EF=3.55 × 106 at 1361cm−1.
The powder form and the solution of R6G exhibit similar Raman peaks at about 770 cm−1, 1182 cm−1, 1310 cm−1, 1362 cm−1,1508 cm−1, 1575 cm−1, and 1647 cm−1 [42]. However, SERS spectra of the 10−5M R6G solution displayed more visible peaks than the normal Raman spectra of R6G powders. It indicated that the Au NPs substrate prepared by reduction method could well enhance the Raman signal of R6G, showing a better surface Raman enhancement effect. Therefore, this substrate was employed for the detection of trace amount of fucoxanthin.
SERS spectra (n=3) of different concentrations fucoxanthin in absolute ethanol from 5×10−4 M to 1×10−6 M depicted in (Fig. 4(A))shows the three main carotenoid bands, which are located approximately at 1520, 1160 and 1016 cm−1, and the limit of detection was calculated to be 1×10−6 M. As shown in Fig. 4(B), the band at 1520 cm−1 was chosen as the normalization benchmark, and a good linear correlation (y= - 1732log c+11057, R2 = 0.9927) was established by plotting the log values of fucoxanthin concentration and the SERS intensity. The band at 1520 cm−1 arises from stretching vibrations of C = C double bonds. Its frequency depends on the length of the π-electron conjugated chain and on the molecular configuration of the carotenoid [43]. The band at 1160cm−1 assignment C-C stretchering. The band at 1019cm−1 is attributed to O-CH3 and C-H tensile vibration. The band at 529cm−1 arises from -OH out-of-plane bending. The results show that SERS coupled with Au NPs could be a sensitive determination method to detect fucoxanthin content in organism. To evaluate the practicability of this method, we used it to detect the fucoxanthin content in six kinds of algae, including Chaetoceros muelleri, Thalassiosira weissflogii, Isochrysis galbana, Phaeodactylum tricornutum, Dinophycae Symbiodinium and Laminaria japonica.
Fig. 4. (A) SERS spectra of different concentrations of fucoxanthin. (B) Peak intensity at 1520cm−1 as a function of fucoxanthin concentration from 5×10−4to1×10−6M.
From Table 1, we can clearly see that the SERS method can be used to detect fucoxanthin with more sensitive and rapid than other methods. The HPLC-PDA detection time can take up to 3 hours, while for Raman it takes only one minute to achieve fast and efficient detection. Fucoxanthin has the property of being easily decomposed by light preheating, and requires rapid low temperature when it is detected. SER detection can be used for rapid and effective detection. The detection limit of SERS can reach 1 × 10−6M.
Table 1. The comparison of detection methods for fucoxanthin
In our work, we tested the fucoxanthin content in algae and kelp untreated and pretreated. According to the standard curve in Fig. 4(B), the concentration of fucoxanthin contained in each substance is obtained according to the Raman intensity, and the characteristic peak at 1520 cm−1 is used as a standard to make Fig. 5. It can be seen from Fig. 5 that our pretreated of Chaetoceros muelleri, Isochrysis galbana, and Laminaria japonica has a better effect and, the difference was statistically significant (P <0.05). After pretreated, the fucoxanthin contents are all 1 to 2 orders of magnitude smaller, while the changes in Thalassiosira weissflogii, Phaeodactylum tricornutum, and Dinophycae Symbiodinium are not significant(P>0.05).
Fig. 5. Comparison of changes of fucoxanthin content in unicellular algae and kelp after treatment (CM-Chaetoceros muelleri, TW-Thalassiosira weissflogii, IG-Isochrysis galbana, PT-Phaeodactylum tricornutum, DS- Dinophycae Symbiodinium, LJ- Laminaria japonica (Different letters in the figure indicate significant differences at the 0.05 level)
The above results show that for the three algae, Chaetoceros muelleri, Isochrysis galbana, and Laminaria japonica, the results obtained untreated are better, further verifying the convenience and speed of Raman spectroscopy, and the fucoxanthin content can be directly measured
The fucoxanthin was added to different algae and kelp for SERS detection, and the changes before and after the addition of fucoxanthin were compared from Fig. 6(A). It can be seen that the recovery rates in Fig. 6(B) are higher for Isochrysis galbana, Phaeodactylum tricornutum, and Laminaria japonica, among them, the recovery rate of Isochrysis galbana and Laminaria japonica is the highest, both reaching more than 100%. Followed by Phaeodactylum tricornutum, achive to 85% while the recovery rates for Chaetoceros muelleri, Thalassiosira weissflogii, and Dinophycae Symbiodinium are lower. Based on the results of Fig. 5 and 6, further analysis shows that SERS detection method is more suitable for Isochrysis galbana and Laminaria japonica algae.
Fig. 6. (A)Changes in fucoxanthin content after algae and kelp plus fucoxanthin; (B) Recovery rate. (CM-Chaetoceros muelleri, TW-Thalassiosira weissflogii, IG-Isochrysis galbana, PT-Phaeodactylum tricornutum, DS- Dinophycae Symbiodinium, LJ- Laminaria japonica;Different letters in Fig.(A) and (B) indicate significant differences at the 0.05 level)
4. Conclusion
In this work, Au NPs was found that the detection limit of fucoxanthin could reach 10−6M. The characteristic peak of 1520cm−1 was selected as the working curve, it presents good linearity of fucoxanthin concentration with R2 = 0.9927 in the range from 5×10−4 ∼1×10−7 M. Furthermore, fucoxanthin in different algae including Chaetoceros muelleri, Thalassiosira weissflogii, Isochrysis galbana, Phaeodactylum tricornutum, Dinophycae Symbiodinium, Laminaria japonica could be detected by Au NPs substrate. This method shows simple, rapid and sensitive properties. It can be extended to rapid detection of fucoxanthin in various seaweed-made food, cosmetics, drugs and health products.
Funding
Scientific Research Innovation Program Xiyuanjiang River Scholarship of College of Life Sciences (FZSKG2019014); 13th Five-Year Plan for the Marine Innovation and Economic Development Demonstration Projects (FZHJ14).
Acknowledgments
This study was supported by the 13th Five-Year Plan for the Marine Innovation and Economic Development Demonstration Projects (FZHJ14) and the scientific research innovation program Xiyuanjiang River Scholarship of College of Life Sciences(FZSKG2019014). Author Contributions: Conceived and designed the experiments: Yu-dong Lu, Lu-qiang Huang, Performed the experiments: Yu-qin Liao. Analyzed the data and discussed the results: Jia-ze Zhong, Yu-qin Liao. Wrote the paper: Yu-dong Lu, Lu-qiang Huang.
Disclosures
The authors declare that there are no conflicts of interest.
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