鮮棗中糖精的快速、無標(biāo)簽檢測

吳昊 邵菲 郭小玉 吳一萍 楊海峰

Fund Project： The National Natural Science Foundation of China（21475088）

Biography： WU Hao（1990—），male，graduate student，research area：Raman application.E-mail：qingdaodaxueguzi@163.com;SHAO Fei（1995—），female，graduate student，research area：design of novel SERS substrates for detection application.E-mail：1446573055@qq.com

?Co-first authors：WU Hao carried out the preparation of materials，data acquisition and writing，SHAO Fei completed the characterization of part materials，detection of real sample and corresponding writing.These authors contributed equally to this work.

*Corresponding authors：WU Yiping（1987—），female，associate professor，research area：construction of Raman probe for biological and chemical applications.E-mail：yipingwu@shnu.edu.cn;YANG Haifeng（1968—），male，professor，research area：design of Raman detection strategies for biochemical application.E-mail：hfyang@shnu.edu.cn

引用格式： 吳昊，邵菲，郭小玉，等.鮮棗中糖精的快速、無標(biāo)簽檢測 [J].上海師范大學(xué)學(xué)報（自然科學(xué)版），2020，49（2）：121-133.

Citation format： WU H，SHAO F，GUO X Y，et al.Rapid and label-free detection of saccharin in fresh jujube fruit [J].Journal of Shanghai Normal University（Natural Sciences），2020，49（2）：121-133.

Rapid and label-free detection of saccharin in fresh jujube fruit

WU Hao^?， SHAO Fei^?， GUO Xiaoyu， WU Yiping^*， YANG Haifeng^*

（College of Chemistry and Materials Science，Shanghai Normal University，Shanghai 200234，China）

Abstract： Saccharin is one of the oldest artificial sweeteners used in food industries because it has no calories.However，the abuse of saccharin is illegal.According to the National Standard of China，the maximum permitted level is 8.189×10^-4mol·L^-1in food.In this work，inositol hexakisphosphate（IP₆） as protection agent was introduced to synthesize silver（Ag） nanoparticles（Ag NPs），designated as Ag NPs@IP₆，and a rapid method based on surface-enhanced Raman scattering （SERS） was explored for the determination of saccharin in food products.The minimal detectable concentration for saccharin in water by using the optimal SERS assay was 50 nmol·L^-1，which meets the requirement of National Food Safety Standard for tolerance level of food additives.This proposed Ag NPs@IP₆-based SERS method with the portable Raman system could be implemented for on-site detection of saccharin in food such as fresh jujube fruit，a kind of Chinese date product.

Key words： saccharin; silver（Ag） nanoparticles（Ag NPs）; surface-enhanced Raman scattering（SERS）; rapid detection

CLC number： CLC number：O 614.24? Document code： A? Article ID： 1000-5137（2020）02-0121-13

摘 要： 糖精是食品工業(yè)中最古老的人造甜味劑之一，因為沒有卡路里而被廣泛使用，但其濫用是非法的，食品中最大允許添加量為8.189×10^-4mol·L^-1.介紹了以六磷酸肌醇（IP₆）為保護(hù)劑合成的銀（Ag）納米粒子（Ag NPs），即Ag NPs@IP₆，并提出了一種基于表面增強(qiáng)拉曼散射（SERS）的快速方法.探討了食品中糖精的測定，用最佳SERS法測定水中糖精的最低可檢測濃度可達(dá)50 nmol·L^-1，符合食品添加劑耐受性水平的國家食品安全標(biāo)準(zhǔn).提出了基于便攜式拉曼的Ag NPs@IP₆的SERS方法，可用于現(xiàn)場檢測食品中的糖精，如新鮮棗果.

關(guān)鍵詞： 糖精; 銀（Ag）納米粒子（Ag NPs）; 表面增強(qiáng)拉曼散射光譜（SERS）; 快速檢測

1 Introduction

The abuse of the additives is a current problem in the field of food safety，which is a major issue of concern to the peoples healthcare^[1].Saccharin is one of the oldest artificial sweeteners used in food industries because it has no calories.In the 1970s，PRICE et al^[2] found that saccharin had close correlation with bladder cancer in rodents.Consequently，foods containing saccharin must be labeled with a warning to match the requirement of the “Saccharin Study and Labeling Act” of 1977.However，in the year 2000，due to some reports exploring the different rodents cellular microenvironment，involving high pH，high calcium phosphate，and high protein levels^[3-4]，from human situation，the United States removed the warning labels from the external packing of food containing saccharin.Next，some researches showed saccharin might give rise to the release of insulin in humans and rats，which has not been confirmed by the later control studies^[5-7].In 2012，Qin^[8-9]found the close relationship between inflammatory bowel disease and the intake amount of saccharin，meaning that the saccharin is health risk for human as food additives.In China，the acceptable daily intake （ADI） value of the saccharin is in the range from 8.189×10^-4to 2.729×10^-2 mol·L^-1for different foods.Thereupon，even if it remains controversy over the safety for saccharin as the food additive，some methods to detect saccharin have been developed.WANG et al^[10]proposed a competitive enzyme-linked immunosorbent assay to determine the sodium saccharin in food samples.This immunosorbent method showed an excellent specificity for sodium saccharin with the limit of detection （LOD） of 1.146×10^-8mol·L^-1by the diazo-reaction，but it needed more than 1.5 h for a whole test process and even 12 h in a preparatory process.Bergamo et al^[11]demonstrated an accurate analytical technique for simultaneous determination of different artificial sweeteners by using capillary electrophoresis with capacitively coupled contactless conductivity with the 30 kV separation voltages and 450 kHz operating condition，but it was not easy to actualize an on-site strategy.GREMBECKA et al^[12] reported a HPLC-CAD-UV/DAD protocol to analyze the mixture of artificial sweeteners with the LOD less than 3×10^-6 mol·L^-1and relative standard deviation （RSD） less than 2% but it has to perform the tedious pre-treatments，control exorbitant operating conditions，and require large-sized instrument.Therefore，it is necessary to explore some fast approaches for pre-screening in food on field or market.

The surface-enhanced Raman scattering（SERS） technique has become one of the most potential spectroscopic tools for label-free determination of the metal ions，bio-analytes or food additives^[13-16]，due to its extraordinary capability for signal enhancement and inherent narrow width of Raman peak.The amplified Raman intensities could be attributed to the contributions of electromagnetic （EM） field enhancement^[17-18]and chemical enhancement （CE）^[19].With the huge electromagnetic field from “hot spots” between neighbor noble metal nanoparticles by laser inducing^[20]，Raman signal for some especial molecules could be dramatically elevated even down to single molecule level^[21-22].The additional merits of SERS technique such as rapidness，no interference by water，and simple pre-treatment of sample have aroused great interests of many analysts in various disciplines.As above-mentioned，SERS-based methods have lots of applications in life science^[23-25]，biotherapy^[26]，and chemical analysis^[27].Also，SERS spectroscopy provides fingerprint vibrational information of molecule moieties adsorbed on a metallic surface，bringing an intrinsic selectivity.It is perspective that with the development of reasonably active and stable SERS substrates，Raman spectroscopy will play the key role in quality control application for goods and foods.

According to our previous work^[28-30]，inositol hexakisphosphate （IP₆） as a naturally non-toxic substance，which has the strong interaction with metallic ions，could be used to synthesize and stabilize the SERS substrates.In this work，tuning the ratio of IP₆and AgNO₃amounts for obtaining silver（Ag）nanoparticles （Ag NPs） （designated as Ag NPs@IP₆）with optimal sensitivity was explored.Herein，we proposed the Ag NPs@IP₆-based SERS method to determine saccharin in the food product of fresh jujube fruit.The lowest detectable concentration for saccharin was 50 nmol·L^-1，which meets the requirement of National Food Safety Standard for tolerance level of food additives.This SERS protocol with good reproducibility can be employed for on-market monitoring the food quality by using the portable Raman system.

2 Materials and methods

2.1 Chemicals and materials

Silver nitrate （AgNO₃），sodium salt of IP₆and saccharin 98% （mass fraction） were obtained from Sigma-Aldrich （USA）.Crystal violet，perchloric acid，acetic anhydride，sodium hydroxide（NaOH），Rhodamine 6G （R6G），hydroxyl-ammonium chloride （NH₂OH·HCl） and acetic acid were purchased from Sinopharm Chemical Reagent （Shanghai，China）.Ethanol was obtained from Shanghai Titan Scientific Co.，Ltd.Raw fresh jujube fruit （Raw-J） was purchased from a local supermarket，and retail jujube fruit （Retail-J） was bought from a local agricultural trade market.All reagents were of analytical grade and used without further purification.Deionized water （18 MΩ·cm） was produced using a Millipore water purification system.

The SERS experiment was carried after the mixture totally dried at the condition of 20 ℃ and 60% relative humidity.

2.4 Titration determination of saccharin

According to suggestion by the National Standard of China，the titration method was a routine technique to analyze the saccharin in foods.In detail，the sample containing saccharin was added into the 20 mL of acetic acid and 5 mL of acetic anhydride.Then，two drops of crystal violet （1.23×10^-2mol·L^-1） were injected as an indicator.Finally，the above solution was titrated by 0.1 mol·L^-1of perchloric acid until the color of the solution turned cyan and the experiment was repeated for three times.

3 Results and discussion

3.1 Preparation and characterizations of Ag NPs@IP₆

Silver substrates have their inherent surface plasmon resonance （SPR） phenomenon to enhance the Raman scattering of adsorbed species，and the enhancement factor is related to their geometry^[32-33]，chemical composition^[34]，and size distribution^[35].In this work，silver nanoparticles were picked out as SERS substrates to carry out the detection of saccharin residue on the surface of foods because of Raman signal-enhancing peculiarity of Ag NPs^[36].Unfortunately，silver nanoparticles are unstable under ambient condition.Normally，the citrate salt was used as reducing agent to obtain Ag NPs and citrate salt residue on the Ag NPs resulted in the Raman spectroscopic interference to the trace detection.We used NH₂OH·HCl with no Raman activity as the reduction reagent to prepare Ag NPs.IP₆molecules were introduced into the synthesis procedure to stabilize Ag NPs for real application requirement.Interestingly，tuning the ratio of AgNO₃and IP₆，the as-obtained Ag NPs@IP₆products showed different SERS effects （R6G as Raman probe）.As shown in figure 1（a），Raman intensity of R6G rises with the increase of the dosage of AgNO₃.However，the stability of Ag NPs becomes worse and results in a serious aggregation in the case of amount of AgNO₃increased from 5.0 to 7.0 mL.As a result，the optimal volume of 1×10^-2mol·L^-1AgNO₃solution is fixed at 5.0 mL.Additionally，the usage of IP₆is also carefully examined for the long-term stability of Ag NPs.As shown in figure 1（b），3.0 mL of 1.0×10^-3mol·L^-1IP₆may be the best one for constructing SERS substrate.The excessive amount of IP₆will increase the thickness of IP₆at the surface of Ag NPs，which suppresses the Raman signals of target sample.

Figure 1 SERS spectra of 5×10^-7mol·L^-1R6G based on the optimized conditions.（a） the volume of the AgNO₃，from line 1 to line 5：3，4，5，6，7 mL，respectively;（b） the volume of the IP₆，from line 1 to line 5：1，2，3，4，5 mL，respectively（λ_ex=785 nm，laser poweris 300 mW，t=5 s）

Figure 2 depicts the TEM images of the Ag NPs and Ag NPs@IP₆.It is found that their average diameters are around 50 nm.The thickness of IP₆shell for Ag NPs@IP₆ is about 6±2 nm.In figure 2（e），the SPR bands for Ag NPs and Ag NPs@IP₆are observed at 403 nm and 408 nm，respectively.Closely investigating the TEM image in figure 3 shows that the gap between Ag NPs @IP₆is less than 10 nm and the distribution of Ag NPs@IP₆exhibits more uniform than that of Ag NPs，which agrees with the narrow band in SPR spectrum of Ag NPs@IP₆.

3.2 The SERS performance

For the evaluation of the SERS effects of the Ag NPs@IP₆ and Ag NPs，1.0×10^-7mol·L^-1R6G solution was used as the Raman probe.It was found from figure 4 that the SERS signal of R6G from Ag NPs@IP₆is stronger than that from Ag NPs.The enhancement should arise from “hot-spot” formation via Ag NPs@IP₆bridging each other.In figure 5（a），in case of R6G，the lowest detectable concentration could be down to 1.0×10^-9mol·L^-1.As a consequence，the Ag NPs@IP₆ colloid is regarded as a promising substrate to elevate the SERS sensitivity for analyzing saccharin in real samples.

The stability of the as-made Ag NPs@IP₆was also monitored by the time-dependent SERS experiments.The statistical results as column diagrams in comparison with Ag NPs are given in figure 5（b）.Obviously，with a storage time within 3 weeks，as compared with the original signal intensity from newly prepared substrate，the SERS signal of Ag NPs@IP₆ could keep about 90% even under ambient condition （20 ℃ and 60% relative humidity） and it exhibits a long-term stability，which is beneficial to the real application.

Figure 2 TEM images of （a），（b）Ag NPs and （c），（d） Ag NPs@IP₆with different scales，and corresponding （e） UV-vis absorption spectra

Figure 3 The TEM image of Ag NPs@IP₆with large magnification

Figure 4 SERS spectra of 1×10^-7mol·L^-1R6G mixed with different substrates：Ag NPs@IP₆（blue curve） and Ag NPs （red curve）

Figure 5 SERS spectra of various concentrations of R6G and the stability of Ag NPs@IP₆and Ag NPs.（a） SERS spectra （1-5） of R6G at different concentrations of 1×10^-5，1×10^-6，1×10^-7，1×10^-8，and 1×10^-9mol·L^-1，acquired from Ag NPs@IP₆and spectrum 6 is acquired from blank Ag NPs @IP₆（λ_ex=785 nm，laser poweris 300 mW，t=5 s）;（b） Column diagrams of normalized intensity ratio （I/I₀） of R6G signals recorded on novel prepared substrates （Ag NPs （green color） or Ag NPs@IP₆（red color）） and after different storage time （error bar：each datum was acquired by repeating 6 times）

3.3 Detection of saccharin with portable Raman system

For the interpretation of the SERS bands，normal Raman spectrum of powder saccharin and SERS spectrum of saccharin （5.0×10^-4mol·L^-1） together with the structure formula were given in figure 6.Clearly，the diversities between normal Raman spectrum of solid saccharinand SERS spectrum could be due to the saccharin molecules adsorbed onto the surface of Ag NPs@IP₆.According to the calculation based on B3LYP/LANL2DZ level，the assignments to main vibration bands of saccharin were tabulated in table 1.SERS peak at 779 cm^-1is attributed toν_as（C-S-N） andν_s（C-C） and a band at 1 175 cm^-1could be due toδ（C-C-H） andρ（N-H）.SERS band at 1 287 cm^-1belongs to co-contributions ofρ（C-H） andν（C-N-O）.The strongest characteristic band at 1 385 cm^-1in SERS spectrum is from the asymmetric stretching modes of O=S=O group while it is a very weak band in normal Raman spectrum.This indicates that the saccharin molecules are anchored on the Ag NPs@IP₆surface via lone pairs of oxygen atom.

Figure 6 SERS spectrum of 5×10^-4mol·L^-1saccharin adsorbed on Ag NPs@IP₆（blue color） and normal Raman spectrum of solid saccharin （red color） （λ_ex=785 nm，laser power is 300 mW，t=5 s）

As shown in figure 7，concentration dependent SERS spectra of saccharin in the aqueous solution are recorded with the Ag NPs@IP₆ substrates.The linear relationship was plotted by the intensities of the Raman band at 1 385 cm^-1versus the varying concentrations of saccharin in the range from 1.0×10^-4to 1.0×10^-3mol·L^-1.The SERS substrate of blank Ag NPs@IP₆was also recorded with no interference on the spectrum of target molecule （figure 8）.The error bars given by six independent measurements are for the indication of the standard deviation.The lowest detectable concentration reaches 5.0×10^-8mol·L^-1，indicating that this optimized Ag NPs@IP₆-based SERS method could be performed for a practical analysis.

Figure 7 SERS spectra of various concentraction of saccharin and calibration plot based on Raman intensity at 1 385 cm^-1.（a） SERS spectra of saccharin obtained from Ag NPs@IP₆（the concentration of saccharin from top to bottom：5×10^-2，5×10^-3，5×10^-4，5×10^-5，5×10^-6，5×10^-7，5×10^-8，and 5×10^-9mol·L^-1，respectively）;（b） SERS intensities at 1 385 cm^-1versus the different concentrations of saccharin（inset is the linear relationship between SERS intensity and the concentration ranging from 1.0×10^-4to 1.0×10^-3mol·L^-1in aqueous solution，λ_ex=785 nm，laser power is 300 mW，t=5 s）

Figure 8 SERS spectrum of Ag NPs@IP₆ without saccharin

The investigation of SERS reproducibility of saccharin on the Ag NPs@IP₆was given in figure 9 and the RSD is about 10.4%.The RSD value of Ag NPs@IP₆is less than 20.0%，remarking the good reproducibility^[37]of such Ag NPs@IP₆substrates.

3.4 Detection of saccharin in real sample

The SERS-based protocol to determine saccharin might have interference of food matrix，such as acesulfame potassium，sucrose and so on.Especially，in food production process，acesulfame potassium is routinely mixed with saccharin to offer sweeter tastes by some enterprises^[38].Herein，SERS spectra of saccharin，acesulfame potassium，sorbitol，sucrose，and glucose together with the mixture of above 5 species were recorded，which were shown in figure 10.It is confirmed that the characteristic Raman band of saccharin at 1 385 cm^-1without interfering could be distinguished from the others.

Figure 9 30 sequential 3D SERS spectra of 1×10^-4mol·L^-1saccharin mixed with Ag NPs@IP₆for examining thereproducibility of SERS substrates

Figure 10 SERS spectra of （a） mixture of 5 species，（b） saccharin，（c） acesulfame potassium，（d） sorbitol，（e） sucrose and （f） glucose

Pitifully in news report，some fresh jujube fruits were added with excessive saccharin by bad vendors to obtain illegal economic benefits.For conducting SERS detection，the pre-treatment approach for sample has an important process^[39-42].We used 20%（volume fraction）ethanol aqueous solution to extract the raw jujubes（Raw-J），the retail jujubes（Retail-J），and the spiked jujubes （Spiked-J）.All of the SERS signals were recorded from the ethanol extract solution after they were mixed with Ag NPs@IP₆substrates.In figure 11（line 1），no obvious Raman signal from the extract solution of Raw-J is visible，while in figure 11（line 2），the Raman spectrum of the extract solution from Spiked-J shows the characteristic bands of saccharin.The SERS spectrum of the extract from Retail-J presents the characteristic bands at 779 and 1 385 cm^-1of saccharin as shown in figure 11（line 3），meaning that Retail-J might be added by saccharin.We also tested the same sample of Retail-J according to the titration method recommended by GB 4578-2008.As demonstrated in table 2，the results obtained by the? titration method are not exactly same as those from the SERS method，but the SERS results are still in acceptable values，especially for quick on-market analysis with the help of the portable Raman system.

Figure 11 Ag NPs@IP₆-based SERS spectra of the Raw-J（line 1，green），the Spiked-J（line 2，red） and the Retail-J（line 3，blue）extract solutions （λ_ex=785 nm，laser power is 300 mW，t=5 s）

Through preparing an optimal Ag NPs@IP₆with an IP₆shell of around 6 nm in thickness，the Ag NPs@IP₆-based SERS approach for the rapid determination of saccharin in fresh jujube fruit was developed.The Ag NPs@IP₆showed a much stronger Raman scattering enhancement factor and long-term stability.The lowest detectable concentration of saccharin down to 50 nmol·L^-1was achieved.For real application，by using the ethanol solution，after a facile pre-treatment method was done to extract the saccharin from the food products，the SERS test could be conducted.Although the SERS result had about 25% RSD in comparison with the result from the titration method recommended by the National Standard of China，as a perspective，this Ag NPs@IP₆-based SERS approach with the aid of portable Raman spectrometer could realize rapid，sensitive and on-site detection of saccharin for food quality control.

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