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A comparative study for the impact performance of shaped charge JET on UHPC targets
European Polymer Journal 112 (2019) 248–254
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A novel luminescent sensor for disaccharide detection in food: Synthesis and application of a water-soluble rod-coil ionic block copolymer Na Wu, Jie Li, Mi Zhou
T
⁎
College of Materials Science and Engineering, Zhejiang University of Technology, Zhejiang 310014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rod-coil ionic block copolymer Luminescent sensor Sucrose Maltose Lactose
A simple and low cost method for detecting disaccharide in food product is important for people who need control the consumption of saccharides in daily life. In this study, a water soluble rod-coil ionic block copolymer poly[2,7-(9,9-dihexyl-fuorene)]-b-cationic quaternized-poly[2-(dimethylamino)ethyl methacrylate] (PF-b-PDI) was employed as luminescent sensor for detection of lactose, sucrose and maltose in food. It was found that the PF-b-PDI could emit vision fluorescence with the existence of hydroquinone (HQ) in aqueous, but would be quenched by the oxidization of hydrogen peroxide (H2O2). The glucose oxidase (GOx) could be used for catalyzing the glucose to generate the H2O2. And the sucrase, maltase and lactase could respectively catalyze sucrose, maltose and lactose to glucose. With the selectivity and catalyst of enzyme, the PF-b-PDI aqueous blend with HQ showed fluorescence sensitive to sucrose, maltose and lactose with detection limit value of 1.36 μM, 1.09 μM and 3.08 μM, respectively. Moreover, the detection of lactose in milk samples by this method just revealed 0.44% of average deviation compared to standard lactose solution, which mean this method was suitable for detecting lactose in milk.
1. Introduction Among variety of saccharides, the disaccharides that is abundant in food, provide the most of energy for human life [1]. However, not all of people can eat the food which contains disaccharides. For instance, about 70 percent of the world's population are lactose intolerant, which may cause various symptoms such as cramps, nausea, excessive intestinal gas and diarrhea [3,4]. Whereas lactose is widely present in dairy products [2]. Another typical example is diabetes that is one of the most common chronic diseases in the world [5]. The patients could not eat excess glucose or disaccharide like sucrose and maltose for preventing a high level of blood glucose [6–8]. For these reasons, the detection of disaccharide in food products is of particular interest for monitoring the production process to producer and distinguishing the quality to consumer. At present, the quantitative determination of disaccharide in food product is usually performed in specialized laboratories, and requires long analysis times and expensive instruments for methods such as chromatography [9,10], spectrophotometry [11,12], mass spectrometry [13] and titrimetry [14]. However, a simple, fast and low cost method for detecting disaccharide in food product is required in daily life. Nowadays, fluorescent probe is considered as an effective and convenient tool to detect biomolecules due to its distinct advantages of ⁎
sensitivity, specificity, and rapidness [15–17]. In early time, researchers found the saccharides could be detected by boronic acid based molecular receptors with fluorescence sensing, but without selectivity for different kind of saccharide [18–20]. The luminescent sensor for lactose and sucrose was reported by Wang et al. [21] and Chiang et al. [22], respectively. However, a new probe for both detecting different disaccharides like sucrose, maltose and lactose with selectivity and sensitivity should be developed [23]. Conjugated polymers (CPs) have gain great interest in the application of fluorescent probe, because of its high fluorescence quantum yield, good stability, non-toxicity, as well as high sensitivity [24–26]. The water-soluble CPs which are decorated with pendant water-soluble ionic groups have been researched as chemical and biological sensors [27–29]. One the other side, the rod-coil block copolymers containing a rigid conjugated block and a random coil block are rarely reported to fluorescence sensors. Herein, we developed a water-soluble rod-coil ionic block copolymer as luminescent sensor for detection of lactose, sucrose and maltose in food products. The rod block is poly[2,7-(9,9dihexyl-fuorene)] (PF) and the coil block is poly[2-(dimethylamino) ethyl methacrylate] with CH3I decorated (PDI). It was found that the PF-b-PDI could emit vision fluorescence with the existence of hydroquinone (HQ) in aqueous, but would quenched by the oxidization of hydrogen peroxide (H2O2). By this way, we designed the PF-b-PDI
Corresponding author. E-mail address: [email protected] (M. Zhou).
https://doi.org/10.1016/j.eurpolymj.2019.01.005 Received 14 October 2018; Received in revised form 22 December 2018; Accepted 2 January 2019 Available online 07 January 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. (A) The synthetic route for PF-b-PDI; (B) The simple representation for PF-b-PDI fluorescence (FL) emission and quenching; (C)–(E) The fluorescence spectra and photograph (inserted) of (a) PF-b-PDI, (b) PF-b-PDI with HQ, (c) PF-b-PDI with HQ and H2O2, (d) PF-b-PDI with HQ, GOx and sucrase, (e) PF-b-PDI with HQ, GOx and maltase, (f) PF-b-PDI with HQ, GOx and lactase, (g) PF-b-PDI with HQ, GOx, sucrose, sucrase, (h) PF-b-PDI with HQ, GOx, maltose, maltase, (i) PF-b-PDI with HQ, GOx, lactose, lactase.
2. Experimental section
2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM) were mixed with 100 μL GOx (90 U/mL). Afterwards, the mixture was incubated at 37 °C water bath for 60 min. Finally fluorescence was recorded at the excitation wavelength of 386 nm.
2.1. Materials
2.5. Detection of maltose
Glucose oxidase (GOx), glucose, hydroquinone (HQ), sucrose, maltose, lactose and hydrogen peroxide (H2O2, 30%) were purchased from Sigma-Aldrich. Sucrase, maltase and lactase were provided by Solarbio. All the chemicals were analytical grade and were used without further purification. The milk was bought from local supermarket.
2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM) were mixed with 100 μL GOx (90 U/mL). Then maltose solutions with different concentration were separately mixed with 50 μL (60 U/mL) maltase solution in turn. After the mixed solution was incubated at 37 °C for 60 min, the fluorescence was recorded at the excitation wavelength of 386 nm.
blended with HQ as fluorescent probe for detecting sucrose, maltose and lactose with the enzyme generating H2O2.
2.2. Detection of H2O2 2.6. Detection of lactose Initially, 2.7 mL of PF-b-PDI (0.1 mg/mL) and 200 μL of HQ (300 μM) were mixed with 100 μL of the stock solution of H2O2 at different concentrations. Following that, the mixed solution was reacted at 25 °C. After reaction for 20 min, the fluorescence intensity was recorded by a Hitachi F-4600 fluorescent spectrophotometer. The fluorescence intensity of solution without H2O2 was defined as F0, and F was the fluorescence intensity with the presence of H2O2. Fluorescence spectra were recorded with the excitation wavelength at 386 nm.
Different volumes of lactose aqueous solutions (concentration ∼ 300 μM) were mixed with 2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM). Then 50 μL GOx (180 U/mL) and 50 μL lactase (36 U/mL) were added to the complex aqueous solution and incubated at 37 °C for 60 min. Finally, the fluorescence was recorded at the excitation wavelength of 386 nm. 2.7. Detection of milk samples
2.3. Detection of glucose Firstly, the lactose in milk was determined by HPLC. For our developed detection method, different concentration of lactose in milk were mixed with 2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM). Then 50 μL GOx (180 U/mL) and 50 μL lactase (36 U/mL) were added to the complex aqueous solution and incubated at 37 °C for 60 min and taken into the analysis.
Glucose detection procedure: 2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM) were mixed with 100 μL GOx (90 U/mL). Then, 100 μL of the stock solution of glucose at different concentrations was added in turn to the above reaction solution, and the obtained solutions were incubated at 37 °C for 60 min. Finally, the fluorescence intensity of the mixed solution was measured. In the absence of glucose, the fluorescence intensity was defined as F0, and F was the fluorescence intensity in the presence of glucose. Fluorescence spectra were recorded with the excitation wavelength at 386 nm.
3. Results and discussion The rod-coil ionic block copolymer PF-b-PDI was prepared by the ionization modification reaction between PF-b-PDMAEMA and CH3I (Fig. 1A). The PF-b-PDMAEMA was synthesized by the method of our previous work (see Scheme S1) [30]. Briefly, with Pd2(dba)3/t-Bu3P and bromobenzyl alcohol complex as the initiator, phenylboronic acid as the quencher and 7-bromo-9,9-dihexylfluoren-2-ylboronic acid
2.4. Detection of sucrose Sucrose solutions with different concentrations were separately mixed with 50 μL (3600 U/mL) sucrase solution and then added into 249
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Fig. 2. (A) Relative fluorescence responses after adding different concentrations of sucrase; (B) The effect of concentration of sucrase on fluorescence intensity. Experimental conditions: 0.1 mg/mL PF-b-PDI; 20 μM HQ; 3 U/mL GOx. (C) Relative fluorescence responses after adding different concentrations of maltase; (D) The effect of concentration of maltase. Experimental conditions: 0.1 mg/mL PF-b-PDI; 20 μM HQ; 3 U/mL GOx. F0 is the intensity in the absence of H2O2, and F is the intensity in the presence of H2O2; (E) Relative fluorescence responses after adding different concentrations of lactase; (F) The effect of concentration of lactase on fluorescence intensity. Experimental conditions: 0.1 mg/mL PF-b-PDI; 20 μM HQ; 3 U/mL GOx.
show fluorescence under the UV lamp perhaps due to the quenching of I- (Fig. 1C). The PF block recovered blue fluorescence with the addition of HQ, but quenched again after added H2O2 (Fig. 1B and C). Since HQ is a kind of reductant, it may be oxidized to benzoquinone by H2O2, resulting the quenching of PF-b-PDI (Fig. 1B). Thus, the PF-b-PDI aqueous solution was sensitive to H2O2 under the presence of HQ·H2O2 is one of the most import intermediate species involved in many biological processes especial enzymatic reaction [31]. The glucose could be oxidized to produce H2O2 by GOx and the disaccharide could be oxidized to glucose by the specific disaccharidase [32,33]. So, it is feasible to detect disaccharide by PF-b-PDI aqueous solution with the presence
pinacol ester as monomer, the polymerization via the controlled chaingrowth mechanism was performed successfully to obtain the conjugate polymer PF-OH. Subsequently, the PF-OH was modified by α-bromoisobutyryl bromide and then the PF-Br enabled the atom transfer radical polymerization (ATRP) to prepare PF-b-PDMAEMA in the presence of CuBr/PMDETA as catalyst. The 1H NMR spectrum (Fig. S1) has determined the structure of PF-b-PDMAEMA and PF-b-PDI. The gel permeation chromatography (GPC) spectroscopy has been used to calculate molar mass dispersity and Mn of PF-b-PDMAEMA (Fig. S2). The PF-b-PDI could be dissolved in water easily because of the long ionization chains. However, the PF-b-PDI aqueous solution did not
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Fig. 3. (A) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of sucrose in the presence of GOx and sucrase; (B) The relationship between F0-F and the concentration of sucrose. The inset is the linear plot in the range of 10–900 μM; (C) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of maltose in the presence of GOx and maltase; (D) Linear correlation between decreased fluorescence intensity and maltose concentration (10, 30, 70, 90, 100, 300, 500, 700, 900 μM); (E) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of lactose in the presence of GOx and lactase; (F) Linear correlation between decreased fluorescence intensity and lactose concentration (10, 30, 70, 90, 100, 300, 500, 700, 900 μM). λex = 422 nm.
sucrase and GOx were all essential to quench the PF-b-PDI with the presence of HQ, since the exclusion of either component would not produce H2O2 which is essential to induce the decrease of fluorescence (Fig. S3). Meanwhile, maltose and lactose are associated with this state (Fig. 1E). The appearance of fluorescence quenching demonstrated that the corresponding enzyme catalyzed the disaccharide and then GOx oxidized glucose to produce H2O2. The kinetics of glucose detection have been given in Supporting
of disaccharidase and GOx. In order to determine the response of PF-b-PDI, HQ and GOx complex towards disaccharide. The fluorescence response of PF-b-PDI to GOx and sucrose with sucrase was firstly investigated. As shown in Fig. 1D, both the GOx and disaccharidase (sucrase, maltase and lactase) could not quench the fluorescence of PF-b-PDI. However, under the addition of both sucrose and sucrase, the fluorescence intensity decreased. The controlled experimental results showed that the sucrose,
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Fig. 4. (A) Fluorescence responses of 0.1 mg/mL PF-b-PDI and 20 μM HQ containing 3.0 U/mL GOx, 0.6 U/mL lactase and 1 mM; (a–g) Ca2+, Na+, Mg2+, vitamin, phosphorus, protein, lactate; (B) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of milk in the presence of GOx and lactase; (C) Nonlinear fitting of fluorescence intensity as function of concentration of lactose in standard lactose solution (black squares) and milk (green squares). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Information (Fig. S4). As can been seen, the fluorescence intensity decreased with time going on due to enzymatic glucose oxidation, and was almost constant when incubation time was above 60 min. As a sequence, 60 min of reaction was employed in further study. The optimum conditions for the determination of disaccharide were decided by varying the concentration of the corresponding enzyme. We adopted F0-F as a signal for the determination of disaccharide. As shown in Fig. 2A, with the sucrase concentration increase from 10 to 100 U/mL, the fluorescence intensity quenched and then F0-F reaches the maximum value at the concentration of sucrase was 60 U/mL. Therefore, 60 U/mL of sucrase was chosen as the optimum sucrase concentration. It can be seen from Fig. 2B that the optimum the concentration of maltase was 1.0 U/mL. In order to optimize the concentration of lactase, the concentration of lactase adopted in this study were 0.1–1.0 U/ mL (Fig. 2C). The results indicated that the F0-F increased to a maximum value with an increase of concentration to 0.6 U/mL. And then, the value decreased when the concentration of lactase was further increased. Hence, the optimum concentration of lactase was decided as being 0.6 U/mL. Under the optimal conditions, the quantitative behavior of the proposed method for the detection of disaccharide was assessed. As shown in Fig. 3A, the measured fluorescence intensity decreased markedly upon increasing the concentration of sucrose. Fig. 3B showed the relative fluorescence intensity versus the concentration of sucrose. Good linear relationship was obtained in the range of 10–900 μM of sucrose. The equation of the calibration curve was F0F = 0.68C + 155.74 (where C is the concentration of sucrose, F0 is the intensity in the absence of sucrose, and F is the intensity in the presence
of sucrose). The relative standard deviation for ten repeated measurements of 100 μM was 0.308. The detection limit value of sucrose was 1.36 μM obtained from the experiment (S/N = 3). Fig. 3C depicts that the fluorescence can be continuously quenched by the addition of maltose in a large concentration range of 10–900 μM. As shown in Fig. 3D, the probe exhibited an excellent linear response to maltose concentration ranging from 10 to 100 μM with a detection limit of 1.09 μM (S/N = 3). A linear relationship between the extent of the quenching and lactose concentration was obtained over the range of 10–100 μM with the detection limit of 3.08 μM (Fig. 3F) (S/N = 3). The equation of the calibration curve was F0-F = 0.30C + 160.98. Recent reports from Wang et al. [21] and Noratto et al. [32] showed a limit of detection of 30 μM and 43.3 μM to lactose, respectively. Here, our probe system is more sensitive in detecting lactose. Besides, the amount of materials such as PF-b-PDI, hydroquinone (HQ) and disaccharidase is very small for detecting a single sample. The concentration of PF-b-PDI and HQ is 0.1 mg/mL and 20 μM, respectively. The disaccharidase is 60 U/mL, 1.0 U/mL and 0.6 U/mL respective for sucrase, maltase and lactase. Moreover, the solution is only 3 mL for a sample. And our system is suitable for sucrose, maltose and lactose analysis in real samples following simple dilution. The highly selective and sensitive sensing of lactose of PF-b-PDI stimulated us to further investigate our designed probe under more realistic conditions. Commercially available dairy products and milk were tested to detect the lactose content. Under the optimum assay conditions, we investigated the fluorescence signal response in the presence of other components in milk. As displayed in Fig. 4A, only lactose can lead to obvious fluorescence quenching, while other 252
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substances have nearly no response at the same concentration. It can be attributed to the high selectivity of lactase to lactose. The results show that the method has high selectivity for lactose. In the case of the milk, the concentration of lactose quantified using HPLC was 245.6 mM. For our method, the simple measurement can only be performed by directly adding the milk sample to the solution of PF-b-PDI, HQ, GOx and lactase. This is similar to the response result of the addition of same amounts of lactose standard solution. And as shown in Fig. 4B, the nonlinear least-squares fittings of fluorescence intensity as a function of concentration of lactose in standard lactose solution and milk matched well, just despite there is about 0.44% of the average deviation between the two concentrations measured.
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4. Conclusion The detection of disaccharide often suffer interference with various substances in food and usually require HPLC separation. Here, a novel fluorescent probe was proposed for the detection of disaccharide based on the fluorescence quenching of PF-b-PDI in the presence of hydroquinone. The detection and selectivity mechanism was based on the enzymatically generated H2O2 which resulted in the decrease of their fluorescence. Under optimal conditions, we observed that the fluorescence quenching signal showed good linearity with the sucrose concentration in the range of 10–100 μM, and the detection limit of this assay was 1.36 μM. Meanwhile, the linear concentration range for maltose was 10–100 μM, and the lower limit of detection was 1.09 μM. And it not only exhibited a highly selective and sensitive (LOD = 3.08 μM) sensing of lactose, but also could easily and exactly detect lactose in milk samples with the average deviation value 0.44% compared to the standard lactose solution. As a result, this work presents a new fluorescent probe and may provide a new promising platform for food detection. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Notes The authors declare no competing financial interest. Acknowledgements The author Mi Zhou thanks Prof. Jinying Yuan and Prof. Yingwu Yin from Tsinghua University to the advisement and help in his research. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.01.005. References [1] T.K. Lindhorst, J.F. Kennedy, L. Zheng, Essentials of carbohydrate chemistry and biochemistry, 116–116, Carbohydr. Polym. 55 (2004), https://doi.org/10.1016/ S0144-8617(01)00274-0. [2] A.C. Adam, M. Rubiotexeira, J. Polaina, Lactose: the milk sugar from a biotechnological perspective, Crit. Rev. Food Sci. Nutr. 44 (2004) 553–557, https:// doi.org/10.1080/10408690490931411. [3] H.A. Büller, R.J. Grand, Lactose intolerance, Annu. Rev. Med. 41 (1989) 141–148, https://doi.org/10.1146/annurev.me.41.020190.001041.
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A novel luminescent sensor for disaccharide detection in food: Synthesis and application of a water-soluble rod-coil ionic block copolymer Na Wu, Jie Li, Mi Zhou
T
⁎
College of Materials Science and Engineering, Zhejiang University of Technology, Zhejiang 310014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rod-coil ionic block copolymer Luminescent sensor Sucrose Maltose Lactose
A simple and low cost method for detecting disaccharide in food product is important for people who need control the consumption of saccharides in daily life. In this study, a water soluble rod-coil ionic block copolymer poly[2,7-(9,9-dihexyl-fuorene)]-b-cationic quaternized-poly[2-(dimethylamino)ethyl methacrylate] (PF-b-PDI) was employed as luminescent sensor for detection of lactose, sucrose and maltose in food. It was found that the PF-b-PDI could emit vision fluorescence with the existence of hydroquinone (HQ) in aqueous, but would be quenched by the oxidization of hydrogen peroxide (H2O2). The glucose oxidase (GOx) could be used for catalyzing the glucose to generate the H2O2. And the sucrase, maltase and lactase could respectively catalyze sucrose, maltose and lactose to glucose. With the selectivity and catalyst of enzyme, the PF-b-PDI aqueous blend with HQ showed fluorescence sensitive to sucrose, maltose and lactose with detection limit value of 1.36 μM, 1.09 μM and 3.08 μM, respectively. Moreover, the detection of lactose in milk samples by this method just revealed 0.44% of average deviation compared to standard lactose solution, which mean this method was suitable for detecting lactose in milk.
1. Introduction Among variety of saccharides, the disaccharides that is abundant in food, provide the most of energy for human life [1]. However, not all of people can eat the food which contains disaccharides. For instance, about 70 percent of the world's population are lactose intolerant, which may cause various symptoms such as cramps, nausea, excessive intestinal gas and diarrhea [3,4]. Whereas lactose is widely present in dairy products [2]. Another typical example is diabetes that is one of the most common chronic diseases in the world [5]. The patients could not eat excess glucose or disaccharide like sucrose and maltose for preventing a high level of blood glucose [6–8]. For these reasons, the detection of disaccharide in food products is of particular interest for monitoring the production process to producer and distinguishing the quality to consumer. At present, the quantitative determination of disaccharide in food product is usually performed in specialized laboratories, and requires long analysis times and expensive instruments for methods such as chromatography [9,10], spectrophotometry [11,12], mass spectrometry [13] and titrimetry [14]. However, a simple, fast and low cost method for detecting disaccharide in food product is required in daily life. Nowadays, fluorescent probe is considered as an effective and convenient tool to detect biomolecules due to its distinct advantages of ⁎
sensitivity, specificity, and rapidness [15–17]. In early time, researchers found the saccharides could be detected by boronic acid based molecular receptors with fluorescence sensing, but without selectivity for different kind of saccharide [18–20]. The luminescent sensor for lactose and sucrose was reported by Wang et al. [21] and Chiang et al. [22], respectively. However, a new probe for both detecting different disaccharides like sucrose, maltose and lactose with selectivity and sensitivity should be developed [23]. Conjugated polymers (CPs) have gain great interest in the application of fluorescent probe, because of its high fluorescence quantum yield, good stability, non-toxicity, as well as high sensitivity [24–26]. The water-soluble CPs which are decorated with pendant water-soluble ionic groups have been researched as chemical and biological sensors [27–29]. One the other side, the rod-coil block copolymers containing a rigid conjugated block and a random coil block are rarely reported to fluorescence sensors. Herein, we developed a water-soluble rod-coil ionic block copolymer as luminescent sensor for detection of lactose, sucrose and maltose in food products. The rod block is poly[2,7-(9,9dihexyl-fuorene)] (PF) and the coil block is poly[2-(dimethylamino) ethyl methacrylate] with CH3I decorated (PDI). It was found that the PF-b-PDI could emit vision fluorescence with the existence of hydroquinone (HQ) in aqueous, but would quenched by the oxidization of hydrogen peroxide (H2O2). By this way, we designed the PF-b-PDI
Corresponding author. E-mail address: [email protected] (M. Zhou).
https://doi.org/10.1016/j.eurpolymj.2019.01.005 Received 14 October 2018; Received in revised form 22 December 2018; Accepted 2 January 2019 Available online 07 January 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. (A) The synthetic route for PF-b-PDI; (B) The simple representation for PF-b-PDI fluorescence (FL) emission and quenching; (C)–(E) The fluorescence spectra and photograph (inserted) of (a) PF-b-PDI, (b) PF-b-PDI with HQ, (c) PF-b-PDI with HQ and H2O2, (d) PF-b-PDI with HQ, GOx and sucrase, (e) PF-b-PDI with HQ, GOx and maltase, (f) PF-b-PDI with HQ, GOx and lactase, (g) PF-b-PDI with HQ, GOx, sucrose, sucrase, (h) PF-b-PDI with HQ, GOx, maltose, maltase, (i) PF-b-PDI with HQ, GOx, lactose, lactase.
2. Experimental section
2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM) were mixed with 100 μL GOx (90 U/mL). Afterwards, the mixture was incubated at 37 °C water bath for 60 min. Finally fluorescence was recorded at the excitation wavelength of 386 nm.
2.1. Materials
2.5. Detection of maltose
Glucose oxidase (GOx), glucose, hydroquinone (HQ), sucrose, maltose, lactose and hydrogen peroxide (H2O2, 30%) were purchased from Sigma-Aldrich. Sucrase, maltase and lactase were provided by Solarbio. All the chemicals were analytical grade and were used without further purification. The milk was bought from local supermarket.
2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM) were mixed with 100 μL GOx (90 U/mL). Then maltose solutions with different concentration were separately mixed with 50 μL (60 U/mL) maltase solution in turn. After the mixed solution was incubated at 37 °C for 60 min, the fluorescence was recorded at the excitation wavelength of 386 nm.
blended with HQ as fluorescent probe for detecting sucrose, maltose and lactose with the enzyme generating H2O2.
2.2. Detection of H2O2 2.6. Detection of lactose Initially, 2.7 mL of PF-b-PDI (0.1 mg/mL) and 200 μL of HQ (300 μM) were mixed with 100 μL of the stock solution of H2O2 at different concentrations. Following that, the mixed solution was reacted at 25 °C. After reaction for 20 min, the fluorescence intensity was recorded by a Hitachi F-4600 fluorescent spectrophotometer. The fluorescence intensity of solution without H2O2 was defined as F0, and F was the fluorescence intensity with the presence of H2O2. Fluorescence spectra were recorded with the excitation wavelength at 386 nm.
Different volumes of lactose aqueous solutions (concentration ∼ 300 μM) were mixed with 2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM). Then 50 μL GOx (180 U/mL) and 50 μL lactase (36 U/mL) were added to the complex aqueous solution and incubated at 37 °C for 60 min. Finally, the fluorescence was recorded at the excitation wavelength of 386 nm. 2.7. Detection of milk samples
2.3. Detection of glucose Firstly, the lactose in milk was determined by HPLC. For our developed detection method, different concentration of lactose in milk were mixed with 2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM). Then 50 μL GOx (180 U/mL) and 50 μL lactase (36 U/mL) were added to the complex aqueous solution and incubated at 37 °C for 60 min and taken into the analysis.
Glucose detection procedure: 2.7 mL of PF-b-PDI (0.1 mg/mL) and 100 μL of HQ (600 μM) were mixed with 100 μL GOx (90 U/mL). Then, 100 μL of the stock solution of glucose at different concentrations was added in turn to the above reaction solution, and the obtained solutions were incubated at 37 °C for 60 min. Finally, the fluorescence intensity of the mixed solution was measured. In the absence of glucose, the fluorescence intensity was defined as F0, and F was the fluorescence intensity in the presence of glucose. Fluorescence spectra were recorded with the excitation wavelength at 386 nm.
3. Results and discussion The rod-coil ionic block copolymer PF-b-PDI was prepared by the ionization modification reaction between PF-b-PDMAEMA and CH3I (Fig. 1A). The PF-b-PDMAEMA was synthesized by the method of our previous work (see Scheme S1) [30]. Briefly, with Pd2(dba)3/t-Bu3P and bromobenzyl alcohol complex as the initiator, phenylboronic acid as the quencher and 7-bromo-9,9-dihexylfluoren-2-ylboronic acid
2.4. Detection of sucrose Sucrose solutions with different concentrations were separately mixed with 50 μL (3600 U/mL) sucrase solution and then added into 249
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Fig. 2. (A) Relative fluorescence responses after adding different concentrations of sucrase; (B) The effect of concentration of sucrase on fluorescence intensity. Experimental conditions: 0.1 mg/mL PF-b-PDI; 20 μM HQ; 3 U/mL GOx. (C) Relative fluorescence responses after adding different concentrations of maltase; (D) The effect of concentration of maltase. Experimental conditions: 0.1 mg/mL PF-b-PDI; 20 μM HQ; 3 U/mL GOx. F0 is the intensity in the absence of H2O2, and F is the intensity in the presence of H2O2; (E) Relative fluorescence responses after adding different concentrations of lactase; (F) The effect of concentration of lactase on fluorescence intensity. Experimental conditions: 0.1 mg/mL PF-b-PDI; 20 μM HQ; 3 U/mL GOx.
show fluorescence under the UV lamp perhaps due to the quenching of I- (Fig. 1C). The PF block recovered blue fluorescence with the addition of HQ, but quenched again after added H2O2 (Fig. 1B and C). Since HQ is a kind of reductant, it may be oxidized to benzoquinone by H2O2, resulting the quenching of PF-b-PDI (Fig. 1B). Thus, the PF-b-PDI aqueous solution was sensitive to H2O2 under the presence of HQ·H2O2 is one of the most import intermediate species involved in many biological processes especial enzymatic reaction [31]. The glucose could be oxidized to produce H2O2 by GOx and the disaccharide could be oxidized to glucose by the specific disaccharidase [32,33]. So, it is feasible to detect disaccharide by PF-b-PDI aqueous solution with the presence
pinacol ester as monomer, the polymerization via the controlled chaingrowth mechanism was performed successfully to obtain the conjugate polymer PF-OH. Subsequently, the PF-OH was modified by α-bromoisobutyryl bromide and then the PF-Br enabled the atom transfer radical polymerization (ATRP) to prepare PF-b-PDMAEMA in the presence of CuBr/PMDETA as catalyst. The 1H NMR spectrum (Fig. S1) has determined the structure of PF-b-PDMAEMA and PF-b-PDI. The gel permeation chromatography (GPC) spectroscopy has been used to calculate molar mass dispersity and Mn of PF-b-PDMAEMA (Fig. S2). The PF-b-PDI could be dissolved in water easily because of the long ionization chains. However, the PF-b-PDI aqueous solution did not
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Fig. 3. (A) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of sucrose in the presence of GOx and sucrase; (B) The relationship between F0-F and the concentration of sucrose. The inset is the linear plot in the range of 10–900 μM; (C) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of maltose in the presence of GOx and maltase; (D) Linear correlation between decreased fluorescence intensity and maltose concentration (10, 30, 70, 90, 100, 300, 500, 700, 900 μM); (E) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of lactose in the presence of GOx and lactase; (F) Linear correlation between decreased fluorescence intensity and lactose concentration (10, 30, 70, 90, 100, 300, 500, 700, 900 μM). λex = 422 nm.
sucrase and GOx were all essential to quench the PF-b-PDI with the presence of HQ, since the exclusion of either component would not produce H2O2 which is essential to induce the decrease of fluorescence (Fig. S3). Meanwhile, maltose and lactose are associated with this state (Fig. 1E). The appearance of fluorescence quenching demonstrated that the corresponding enzyme catalyzed the disaccharide and then GOx oxidized glucose to produce H2O2. The kinetics of glucose detection have been given in Supporting
of disaccharidase and GOx. In order to determine the response of PF-b-PDI, HQ and GOx complex towards disaccharide. The fluorescence response of PF-b-PDI to GOx and sucrose with sucrase was firstly investigated. As shown in Fig. 1D, both the GOx and disaccharidase (sucrase, maltase and lactase) could not quench the fluorescence of PF-b-PDI. However, under the addition of both sucrose and sucrase, the fluorescence intensity decreased. The controlled experimental results showed that the sucrose,
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Fig. 4. (A) Fluorescence responses of 0.1 mg/mL PF-b-PDI and 20 μM HQ containing 3.0 U/mL GOx, 0.6 U/mL lactase and 1 mM; (a–g) Ca2+, Na+, Mg2+, vitamin, phosphorus, protein, lactate; (B) Fluorescence spectra of PF-b-PDI with HQ after adding different concentrations of milk in the presence of GOx and lactase; (C) Nonlinear fitting of fluorescence intensity as function of concentration of lactose in standard lactose solution (black squares) and milk (green squares). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Information (Fig. S4). As can been seen, the fluorescence intensity decreased with time going on due to enzymatic glucose oxidation, and was almost constant when incubation time was above 60 min. As a sequence, 60 min of reaction was employed in further study. The optimum conditions for the determination of disaccharide were decided by varying the concentration of the corresponding enzyme. We adopted F0-F as a signal for the determination of disaccharide. As shown in Fig. 2A, with the sucrase concentration increase from 10 to 100 U/mL, the fluorescence intensity quenched and then F0-F reaches the maximum value at the concentration of sucrase was 60 U/mL. Therefore, 60 U/mL of sucrase was chosen as the optimum sucrase concentration. It can be seen from Fig. 2B that the optimum the concentration of maltase was 1.0 U/mL. In order to optimize the concentration of lactase, the concentration of lactase adopted in this study were 0.1–1.0 U/ mL (Fig. 2C). The results indicated that the F0-F increased to a maximum value with an increase of concentration to 0.6 U/mL. And then, the value decreased when the concentration of lactase was further increased. Hence, the optimum concentration of lactase was decided as being 0.6 U/mL. Under the optimal conditions, the quantitative behavior of the proposed method for the detection of disaccharide was assessed. As shown in Fig. 3A, the measured fluorescence intensity decreased markedly upon increasing the concentration of sucrose. Fig. 3B showed the relative fluorescence intensity versus the concentration of sucrose. Good linear relationship was obtained in the range of 10–900 μM of sucrose. The equation of the calibration curve was F0F = 0.68C + 155.74 (where C is the concentration of sucrose, F0 is the intensity in the absence of sucrose, and F is the intensity in the presence
of sucrose). The relative standard deviation for ten repeated measurements of 100 μM was 0.308. The detection limit value of sucrose was 1.36 μM obtained from the experiment (S/N = 3). Fig. 3C depicts that the fluorescence can be continuously quenched by the addition of maltose in a large concentration range of 10–900 μM. As shown in Fig. 3D, the probe exhibited an excellent linear response to maltose concentration ranging from 10 to 100 μM with a detection limit of 1.09 μM (S/N = 3). A linear relationship between the extent of the quenching and lactose concentration was obtained over the range of 10–100 μM with the detection limit of 3.08 μM (Fig. 3F) (S/N = 3). The equation of the calibration curve was F0-F = 0.30C + 160.98. Recent reports from Wang et al. [21] and Noratto et al. [32] showed a limit of detection of 30 μM and 43.3 μM to lactose, respectively. Here, our probe system is more sensitive in detecting lactose. Besides, the amount of materials such as PF-b-PDI, hydroquinone (HQ) and disaccharidase is very small for detecting a single sample. The concentration of PF-b-PDI and HQ is 0.1 mg/mL and 20 μM, respectively. The disaccharidase is 60 U/mL, 1.0 U/mL and 0.6 U/mL respective for sucrase, maltase and lactase. Moreover, the solution is only 3 mL for a sample. And our system is suitable for sucrose, maltose and lactose analysis in real samples following simple dilution. The highly selective and sensitive sensing of lactose of PF-b-PDI stimulated us to further investigate our designed probe under more realistic conditions. Commercially available dairy products and milk were tested to detect the lactose content. Under the optimum assay conditions, we investigated the fluorescence signal response in the presence of other components in milk. As displayed in Fig. 4A, only lactose can lead to obvious fluorescence quenching, while other 252
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substances have nearly no response at the same concentration. It can be attributed to the high selectivity of lactase to lactose. The results show that the method has high selectivity for lactose. In the case of the milk, the concentration of lactose quantified using HPLC was 245.6 mM. For our method, the simple measurement can only be performed by directly adding the milk sample to the solution of PF-b-PDI, HQ, GOx and lactase. This is similar to the response result of the addition of same amounts of lactose standard solution. And as shown in Fig. 4B, the nonlinear least-squares fittings of fluorescence intensity as a function of concentration of lactose in standard lactose solution and milk matched well, just despite there is about 0.44% of the average deviation between the two concentrations measured.
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4. Conclusion The detection of disaccharide often suffer interference with various substances in food and usually require HPLC separation. Here, a novel fluorescent probe was proposed for the detection of disaccharide based on the fluorescence quenching of PF-b-PDI in the presence of hydroquinone. The detection and selectivity mechanism was based on the enzymatically generated H2O2 which resulted in the decrease of their fluorescence. Under optimal conditions, we observed that the fluorescence quenching signal showed good linearity with the sucrose concentration in the range of 10–100 μM, and the detection limit of this assay was 1.36 μM. Meanwhile, the linear concentration range for maltose was 10–100 μM, and the lower limit of detection was 1.09 μM. And it not only exhibited a highly selective and sensitive (LOD = 3.08 μM) sensing of lactose, but also could easily and exactly detect lactose in milk samples with the average deviation value 0.44% compared to the standard lactose solution. As a result, this work presents a new fluorescent probe and may provide a new promising platform for food detection. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Notes The authors declare no competing financial interest. Acknowledgements The author Mi Zhou thanks Prof. Jinying Yuan and Prof. Yingwu Yin from Tsinghua University to the advisement and help in his research. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.01.005. References [1] T.K. Lindhorst, J.F. Kennedy, L. Zheng, Essentials of carbohydrate chemistry and biochemistry, 116–116, Carbohydr. Polym. 55 (2004), https://doi.org/10.1016/ S0144-8617(01)00274-0. [2] A.C. Adam, M. Rubiotexeira, J. Polaina, Lactose: the milk sugar from a biotechnological perspective, Crit. Rev. Food Sci. Nutr. 44 (2004) 553–557, https:// doi.org/10.1080/10408690490931411. [3] H.A. Büller, R.J. Grand, Lactose intolerance, Annu. Rev. Med. 41 (1989) 141–148, https://doi.org/10.1146/annurev.me.41.020190.001041.
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