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Rhodamine-assisted fluorescent detection for lysozyme based on the inner filter effect of gold nanoparticles
Microchemical Journal 150 (2019) 104118
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Rhodamine-assisted fluorescent detection for lysozyme based on the inner filter effect of gold nanoparticles
T
Cong Zhenga, Tao Wangb, Qi Kangc, Jianhong Xiaob, Li Yua,
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a
Key Laboratory of Colloid and Interface Chemistry, Shandong University, Ministry of Education, Jinan 250100, PR China Petroleum Engineering Technology Research Institute of Shengli Oilfield, Sinopec, Dongying 257000, PR China c College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Gold nanoparticles Lysozyme Rhodamine 6G Inner filter effect
This work proposed a rapid, sensitive and low-cost fluorescent assay to achieve lysozyme detection in the presence of gold nanoparticles (AuNPs) and rhodamine 6G (R6G). When R6G and AuNPs aqueous solutions were mixed, fluorescence of R6G was quenched via the inner filter effect (IFE). Whereas, upon addition of lysozyme into AuNPs solution, lysozyme with positive charge bound to the negatively-charged AuNPs, leading to the aggregation of AuNPs. After that, R6G as a fluorescence indicator was introduced into the above-mentioned mixture. In this case, weakened IFE occurred between AuNPs and R6G. It is found that the fluorescent intensity of R6G enhanced as the concentration of lysozyme increased. The optimum concentrations of R6G and AuNPs were studied by fluorescence spectrophotometer. Based on the above, lysozyme could be detected and its detection limit is about 1 ng/mL. Quantitative evaluation of lysozyme could be performed within a concentration range of 0.1–10 μg/mL. This strategy was also applied to monitor lysozyme in urine samples with a pleased result. It provides a great promise for the diagnosis and prognosis by fluorescence for lysozyme-related diseases.
1. Introduction Lysozyme is an important protein in the body and can promote the clearance of bacteria in the oral cavity, which is usually found in animal, plant tissue fluids and certain microorganisms [1]. It has a molecular weight of 14,400 and the isoelectric point of 11.1 [2]. For healthy adults, the normal level of lysozyme in urine and serum is about 0.18 and 1.7 μg/mL, respectively [3]. When its concentration is elevated to 15 or even 100 μg/mL, it is highly likely to incur leukemia, kidney disease and sarcoidosis [3,4]. Lysozyme is also a well-known biochemical indicator, including human kidney problems, immune system and leukemia [4–7]. It plays an essential role in the clinical diagnosis and prevention of related diseases. Therefore, sensitive and selective monitoring of lysozyme is highly demand for practical application. Currently, there are various well-established methods to detect lysozyme in biological samples, such as high-performance liquid chromatography [8,9], enzyme-linked immunosorbent assay [9,10], capillary electrophoresis [11], molecular imprinting technique [1,12], and electrochemical approach [13,14]. However, most of these methods involve long incubation time, costly reagents, sophisticated instrumentations, tedious sample preparation procedure and skilled operators. Thus, to make great efforts in seeking a novel method for
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lysozyme monitoring is indispensable. Fluorometry is a desirable and powerful analytical technique due to high sensitivity, simple operation, quick response and low cost [15,16]. Some fluorescence probes have been reported for lysozyme detection in recent years, such as Cu-In-S ternary quantum dots [15], CdTe-CdSe quantum dots [17], magnetic nanoparticles [18], silica nanoparticles [19], and graphene oxide [20]. These nanoprobes described above, however, generally require complex synthesis procedures or time-consuming operation. Gold nanoparticles (AuNPs) have attracted extensive attention and become useful for biological and chemical applications owing to excellent optoelectronic properties, unique biocompatibility, and facile synthesis [21–24]. Many of the sensing methods using AuNPs are based on the inner filter effect (IFE) [21,25]. More recently, several assays have appeared for the detection of pesticide [25,26], protein [24], ions [27,28], and micromolecule [29,30] with AuNPs as a absorber of IFE. In this study, we exploited R6G as a fluorescent indicator to sensitively monitor the level of lysozyme in the presence of AuNPs. As shown in Scheme 1, AuNPs combine with R6G because of electrostatic interaction, leading to the fluorescent quenching of R6G via IFE. While the positively-charged lysozyme molecules in PBS (pH=7.4) would bind to the AuNPs with negative charge and causes AuNPs to accumulate [21]. When R6G is introduced, the IFE between AuNPs and R6G was
Corresponding author. E-mail address: [email protected] (L. Yu).
https://doi.org/10.1016/j.microc.2019.104118 Received 27 June 2019; Received in revised form 19 July 2019; Accepted 19 July 2019 Available online 20 July 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Schematic illustration of the detection mechanism for lysozyme.
weakened. It is observed that the fluorescence intensity of R6G increases with the increasing content of lysozyme. Based on this, the proposed strategy achieves the simple, sensitive and rapid quantification detection of lysozyme. Moreover, the method was further applied for detection lysozyme in urine samples. 2. Experiments
Fluorescence spectra were determined with a FluoroMax-4 fluorescence spectrophotometer. The fluorescence signals (excitation at 520 nm with an Xe lamp) were recorded in the range of 535–700 nm. Slits for both excitation and emission measurements were 2 nm. BI-200SM/BI-9000 instrument (Brookhaven Co., USA) was employed to carry out the dynamic light scattering (DLS) studies and the data were acquired by using the CONTIN method.
2.1. Materials
2.3. Preparation procedures of AuNPs
Chloroauric acid hydrated (HAuCl4·3H2O) was purchased from Sahn chemical technology (Shanghai) Co., Ltd. of China. β-CD (96%) was provided by Aladdin Chemistry Co., Ltd. of China. α-Amylase (AMS) (≥150 units/mg protein (Biuret), aspergillus oryzae), phosphate buffered saline (PBS) (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl; pH=7.4), and lipase (100–150 units/mg protein, porcine pancreas) were obtained from Sigma-Aldrich. Pepsin (≥98%, porcine stomach mucose), trypsin (10 units/mg, bovine pancreas), lysozyme (20,000 units/mg, chicken egg), and γ-glutamyl transpeptidase (γ-GT, 0.2 units/mg, porcine pancreas) were bought from Shanghai Shifeng Biological Technology Co., Ltd. China. Sodium hydroxide (NaOH, ≥96.0%) was provided by Sinopharm Chemical Reagent Co., Ltd. Rhodamine 6G was purchased from J&K Scientific Co., Ltd. China. All chemicals were used without further purification. Ultrapure water (resistivity, 18.25 MΩ cm) was obtained from a Ulupure system and used in all experimental procedures. All the solution of various enzymes (AMS, lipase, pepsin, trypsin, lysozyme and γ-GT) were freshly prepared in PBS. The other chemicals were dissolved in water.
AuNPs were prepared by the β-CD reduction of HAuCl4, following the procedure reported earlier [31,32]. Herein, 39 mL of deionized water, 10 mL of 0.01 M β-CD, and 1 mL of 0.01 M HAuCl4 were added into the three-neck flask and mixed evenly. Since gold ions can be reduced in basic solution, 0.1 mL of 1 M NaOH was then utilized to adjust pH to 10.5. Furthermore, the mixed solution was continuously heated for 3 h at 60 °C under stirring. In the course of reaction, the color of solution changed from pale yellow to red, purple and finally wine red, indicating the formation of AuNPs. Afterwards, the solution was centrifuged for 20 min at a speed of 9000 r/min. The as-prepared AuNPs was redispersed with ultrapure water and stored at 4 °C for later use. 2.4. Fluorescence quenching experiments of R6G by AuNPs R6G solution (500 μL, 0.2 μM) was mixed with AuNPs solution (500 mL, 0.7 nM) under stirring at room temperature. That is, the final concentration of R6G and AuNPs was 0.1 μM and 0.35 nM, respectively. After that, fluorescence spectra of both the mixture and R6G solution were measured with excitation at 520 nm. In the presence of AuNPs, the fluorescence intensity of solution decreased significantly, indicating that R6G was bound to the AuNPs.
2.2. Instruments UV–vis absorption spectra were obtained using a U-4100 UV–vis spectrometer (Hitachi) in a quartz cell (light path of 5 mm). The background signal was subtracted from the sample signal with water as a reference. Transmission electron microscopy (TEM) images were obtained by a HT-7700 instrument with an accelerating voltage of 120 kV.
2.5. Detection assays of lysozyme The detecting process of lysozyme is described as follows: AuNPs solution (350 μL, 1.05 nM) was added to 350 μL of lysozyme solution 2
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with various contentions (1×10−3, 0.01, 0.1, 0.5, 1, 5, 10, 100 μg/mL). The homogeneous mixtures were incubated in water bath (37 °C) for 5 min. Subsequently, R6G (350 μL, 0.3 μM) was introduced to obtain the final solution which was employed to determine the fluorescence intensity. In order to evaluate the selectivity of lysozyme detection, interference study was conducted by preparing 1 μg/mL of pepsase, γ-GT, AMS, lipase and trypsin, respectively. Each of protein solution was added into AuNPs solution (350 μL, 1.05 nM) under stirring. A certain amount of R6G (50 μL, 0.3 μM) was then mixed into the above-mentioned solution. After culturing for 5 min, all the resulting mixtures were measured using a fluorescence spectrophotometer. 2.6. Detection of lysozyme in urine samples The urine samples, collected from healthy volunteers, were diluted 10 times with PBS without other treatment before the further experiments. In order to minimize the interference of background signal, the standard addition method was used to detect the content of lysozyme in urine [33]. Briefly, 175 μL of human urine was added to equal volume lysozyme with different concentration in PBS solution. Then, 350 μL of 1.05 nM AuNPs was spiked and incubated for 5 min at 37 °C. The resulting solutions were further mixed with R6G (350 μL, 0.3 μM) solution, followed by determination of fluorescence property and confirmation of the lysozyme concentration in urine.
Fig. 2. UV–vis absorption spectrum of the AuNPs.
3.2. Fluorescence quenching of R6G by AuNPs Firstly, feasibility of the fluorescent method was explored for lysozyme sensing based on AuNPs. We separately measured the fluorescence spectra of free R6G, and (R6G+AuNPs) in the absence and presence of lysozyme. As shown in Fig. 3a, R6G molecules as a fluorescent indicator exhibit a strong fluorescent emission peak at 552 nm. Furthermore, it is found that the fluorescent signal of R6G was nearly quenched by AuNPs. The possible reasons are ascribed to two aspects as follows: R6G with positively-charged amino groups can adsorb onto the surface of the negatively-charged AuNPs due to electrostatic interaction, which can lead to accumulation of AuNPs (Fig. S1a). Besides, the emission spectrum of R6G (Fig. 3a) can overlap with the absorption spectrum of AuNPs (Fig. 2) to some extent, manifesting that AuNPs can act as an IFE-absorber to cause IFE with R6G. Whereas, as the positively-charged lysozyme solution was mixed with the AuNPs solution, they combine together to form aggregated AuNPs (Fig. S1b). Moreover, in the absorption spectra, the aggregated AuNPs have large shift from 520 nm to 600 nm after the mixing with the lysozyme in Fig. S2. The overlap between the plasmonic absorption of AuNPs and the emission of R6G reduced. Then, when R6G was introduced into the above mixture, the IFE weakened, Therefore lysozyme may be detected based on the emission of R6G. We also carried out experiments to determine UV–vis absorption spectra of R6G, R6G+AuNPs and R6G+AuNPs+Lysozyme at the same
3. Results and discussion 3.1. Characterizations of AuNPs To prepare AuNPs, we introduced β-CD, as a reductor and stabilizer, to reduce HAuCl4 in basic solution [32]. TEM image indicates that the AuNPs are well mono-dispersed and spherical with an average size of about 17 nm (Fig. 1a). DLS measurement presents their average hydrodynamic diameter is ~20 nm (Fig. 1b), consistent with the TEM result. UV–vis absorption spectrum of AuNPs (Fig. 2) shows that there is an obvious peak at approximately 520 nm, indicative of a good dispersion degree and typical feature of AuNPs [32]. As reported previously [34], the extinction coefficient of AuNPs at 520 nm for 17 nm AuNPs in diameter is approximately 3.24×108 M−1 cm−1. Therefore, the concentration of AuNPs solution was calculated to be about 8 nM by Lambert-Beer law. AuNPs solution with a concentration of 0.7 nM and 1.05 nM utilized in this work was obtained by diluting the solution above.
Fig. 1. TEM image (a) and size distribution (b) of the synthesized AuNPs. 3
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Fig. 3. Fluorescence spectra (a) and UV–vis absorption spectra (b) of different systems. (The concentration of R6G, AuNPs and lysozyme is 0.1 μM, 0.35 nM and 1 μg/ mL, respectively.)
concentrations and conditions as fluorescence spectra (Fig. 3a) and presented the results in Fig. 3b. It is found in Fig. 2 that AuNPs aqueous solution exhibits a distinct peak at ~520 nm. While for R6G, only a weak absorption at ~525 nm was observed (Fig. 3b). As depicted in Fig. 3b, for (R6G+AuNPs) and (R6G+AuNPs+Lysozyme) systems, there is an absorption peak at 528 nm and 534 nm, respectively. Concomitantly, the absorption peak widens significantly. As comparison with AuNPs, addition of either R6G or (R6G+lysozyme) results in an obvious change both in the peak position and peak width. According to the past report [35], agglomeration of AuNPs caused absorption peak broadening and red shift. Hence, we speculate that presence of either R6G or (R6G+lysozyme) may lead to accumulation of AuNPs.
concentration-dependence fluorescence intensity at 552 nm was depicted in Fig. 4b. The inset in Fig. 4b reveals within the concentration range of lysozyme (0.1–10 μg/mL), there is a good linear relationship between the fluorescence intensity and logarithm concentration of lysozyme (LgC) (with correlation coefficient of R2=0.997). To further explore the sensitivity of assay, we also determined the fluorescence spectra of R6G (0.1 μM) in the presence of AuNPs (0.35 nM) and 0.5 ng/ mL lysozyme. For the sake of comparing, the fluorescent intensities in the case of 1 ng/mL (1×10−3 μg/mL) lysozyme and in the absence of lysozyme (Fig. 4a) were also presented in one histogram and displayed in Fig. S5. As can be seen from Fig. S5, the fluorescent intensity for the sample containing 0.5 ng/mL lysozyme is similar to that without lysozyme. While when introducing 1 ng/mL lysozyme into the test system, the fluorescent intensity of R6G enhances remarkably, showing that the LOD of lysozyme is ~1 ng/mL. Table S1 summarizes the detection limit and responsive time of lysozyme detection in this work and other various assays in previous reports. Almost all of the earlier detection methods of lysozyme require either long incubation time or tedious sample preparation procedure. Whereas fluorescent spectroscopy is a desirable and powerful analytical technique due to high sensitivity, simple operation, and quick response. Hence as shown in Table S1, the strategy proposed here is superior to the other approaches for lysozyme detection in sensitivity and detection time. To evaluate the specificity of this assay for lysozyme monitoring, several typical proteins in human body including pepsin, γ-GT, AMS, lipase, and trypsin were utilized to conduct control experiments. As illustrated in Fig. S6, under the same experimental conditions, only lysozyme can cause remarkable relative fluorescent intensity ((I-I0)/I0, I and I0 refer to the fluorescence intensity of the system in the presence and absence of lysozyme, respectively). The results as above substantially indicate that the developed method can open a selective and sensitive way toward lysozyme detection.
3.3. Optimal doses of R6G and AuNPs for lysozyme assay Next, effect of reaction concentrations on fluorescence intensity in this system was optimized. The fluorescence spectra of R6G within 0.01–100 μM were determined and shown in Fig. S3a. The results indicate that the fluorescent intensity enhances with the increasing concentration of R6G among 0.01–1 μM and then subsequently declines in the range of 10–100 μM. As reported [36,37], when the concentration of fluorescent substance is too high, self-quenching phenomenon will occur. In order to avert this, concentration of R6G below 10 μM (namely 1, 0.1 and 0.01 μM) was employed to conduct further study. As illustrated in Fig. S3b–d, at the same concentration of R6G, the fluorescence intensity weakens with increment of the investigated AuNPs concentration. The fluorescence of 1, 0.1 and 0.01 μM R6G was almost completely quenched by 1.61, 0.35 and 0.05 nM AuNPs, respectively. Then, the fluorescence spectra of (R6G+AuNPs+lysozyme) system were measured in which the corresponding concentrations of R6G and AuNPs were as noted above in the absence or presence 1 μg/mL lysozyme (Fig. S4). Apparently, when the concentration of R6G and AuNPs was 0.1 μM and 0.35 nM, respectively, there is a greater difference in the fluorescence intensity between the two systems with and without lysozyme, which can provide a relatively high sensitivity for lysozyme quantification.
3.5. Application of the proposed method for lysozyme monitoring in human urine samples
3.4. Fluorescent assay for lysozyme detection
We employed this method to monitor lysozyme in human urine specimens from healthy subjects by standard addition method [12]. Urine samples were spiked with serial concentrations of lysozyme (1, 5 and 10 μg/mL) for the detection. The found concentration of lysozyme can be obtained by estimation of the recoveries based on fluorescence spectra. The corresponding results were illustrated in Table 1. The recoveries of lysozyme range from 84.20% to 114.85% with the relative standard deviation (RSD) < 0.75%. All the above results indicate that the method could provide a reliable access to monitor lysozyme in urine, which is promising for the real applications.
Based on the above, fluorescence spectra of 0.1 μM R6G in the presence of 0.35 nM AuNPs and different concentration of lysozyme were determined (Fig. 4a). It is found that the fluorescent intensity of R6G enhances distinctly when introducing 1 ng/mL or higher concentration of lysozyme into the test system. This may be due to the weakening of the IFE of R6G by AuNPs, resulting in enhanced fluorescence intensity of R6G. Based on the Fig. 4a, the limit of detection (LOD) of lysozyme for the strategy proposed here is 1 ng/mL. The 4
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Fig. 4. (a) The fluorescence spectra of R6G (0.1 μM) in the presence of AuNPs (0.35 nM) and different concentrations of lysozyme (0, 1×10−3, 0.01, 0.1, 0.5, 1, 5, 10, 100 μg/mL). (b) The fluorescence intensity at 552 nm of the assay toward different concentrations of lysozyme. Inset: The linear fitting curve between fluorescence intensity and lysozyme concentration. Table 1 Performance of lysozyme detection in urine samples. Sample
Spiked concentration (μg/mL)
Found concentration (μg/mL)
Recovery (%)
RSD (%) (n = 3)
1 2 3
1 5 10
0.84 5.08 11.49
84.20 101.50 114.85
0.75 0.49 0.41
[4] [5] [6] [7]
4. Conclusions
[8]
A sensitive, rapid, and inexpensive strategy based on AuNPs was designed for lysozyme detection with R6G as a fluorescence indicator. Without lysozyme, AuNPs could lead to the fluorescent signal quenching of R6G via IFE. When solutions of lysozyme and AuNPs with opposite charges were mixed, they bound to each other by electrostatic interaction and resulted in the aggregation of AuNPs. After adding R6G into the above mixture, the presence of lysozyme prevented the combination of R6G and AuNPs, as well as advent of IFE, Under such circumstances, stronger fluorescence intensity was observed for R6G in the presence of higher lysozyme concentration. The detection of limit for lysozyme was as low as 1 ng/mL. More importantly, this assay was demonstrated to detect lysozyme in the urine samples. This approach offers a prospect on the clinical diagnose of related diseases in the future.
[9] [10] [11] [12]
[13] [14] [15]
Acknowledgments
[16]
This work was supported by the National Natural Science Foundation of China (No. 21373128), Scientific and Technological Projects of Shandong Province of China (No. 2018GSF121024).
[17]
Appendix A. Supplementary data
[18]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.microc.2019.104118.
[19]
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Rhodamine-assisted fluorescent detection for lysozyme based on the inner filter effect of gold nanoparticles
T
Cong Zhenga, Tao Wangb, Qi Kangc, Jianhong Xiaob, Li Yua,
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a
Key Laboratory of Colloid and Interface Chemistry, Shandong University, Ministry of Education, Jinan 250100, PR China Petroleum Engineering Technology Research Institute of Shengli Oilfield, Sinopec, Dongying 257000, PR China c College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Gold nanoparticles Lysozyme Rhodamine 6G Inner filter effect
This work proposed a rapid, sensitive and low-cost fluorescent assay to achieve lysozyme detection in the presence of gold nanoparticles (AuNPs) and rhodamine 6G (R6G). When R6G and AuNPs aqueous solutions were mixed, fluorescence of R6G was quenched via the inner filter effect (IFE). Whereas, upon addition of lysozyme into AuNPs solution, lysozyme with positive charge bound to the negatively-charged AuNPs, leading to the aggregation of AuNPs. After that, R6G as a fluorescence indicator was introduced into the above-mentioned mixture. In this case, weakened IFE occurred between AuNPs and R6G. It is found that the fluorescent intensity of R6G enhanced as the concentration of lysozyme increased. The optimum concentrations of R6G and AuNPs were studied by fluorescence spectrophotometer. Based on the above, lysozyme could be detected and its detection limit is about 1 ng/mL. Quantitative evaluation of lysozyme could be performed within a concentration range of 0.1–10 μg/mL. This strategy was also applied to monitor lysozyme in urine samples with a pleased result. It provides a great promise for the diagnosis and prognosis by fluorescence for lysozyme-related diseases.
1. Introduction Lysozyme is an important protein in the body and can promote the clearance of bacteria in the oral cavity, which is usually found in animal, plant tissue fluids and certain microorganisms [1]. It has a molecular weight of 14,400 and the isoelectric point of 11.1 [2]. For healthy adults, the normal level of lysozyme in urine and serum is about 0.18 and 1.7 μg/mL, respectively [3]. When its concentration is elevated to 15 or even 100 μg/mL, it is highly likely to incur leukemia, kidney disease and sarcoidosis [3,4]. Lysozyme is also a well-known biochemical indicator, including human kidney problems, immune system and leukemia [4–7]. It plays an essential role in the clinical diagnosis and prevention of related diseases. Therefore, sensitive and selective monitoring of lysozyme is highly demand for practical application. Currently, there are various well-established methods to detect lysozyme in biological samples, such as high-performance liquid chromatography [8,9], enzyme-linked immunosorbent assay [9,10], capillary electrophoresis [11], molecular imprinting technique [1,12], and electrochemical approach [13,14]. However, most of these methods involve long incubation time, costly reagents, sophisticated instrumentations, tedious sample preparation procedure and skilled operators. Thus, to make great efforts in seeking a novel method for
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lysozyme monitoring is indispensable. Fluorometry is a desirable and powerful analytical technique due to high sensitivity, simple operation, quick response and low cost [15,16]. Some fluorescence probes have been reported for lysozyme detection in recent years, such as Cu-In-S ternary quantum dots [15], CdTe-CdSe quantum dots [17], magnetic nanoparticles [18], silica nanoparticles [19], and graphene oxide [20]. These nanoprobes described above, however, generally require complex synthesis procedures or time-consuming operation. Gold nanoparticles (AuNPs) have attracted extensive attention and become useful for biological and chemical applications owing to excellent optoelectronic properties, unique biocompatibility, and facile synthesis [21–24]. Many of the sensing methods using AuNPs are based on the inner filter effect (IFE) [21,25]. More recently, several assays have appeared for the detection of pesticide [25,26], protein [24], ions [27,28], and micromolecule [29,30] with AuNPs as a absorber of IFE. In this study, we exploited R6G as a fluorescent indicator to sensitively monitor the level of lysozyme in the presence of AuNPs. As shown in Scheme 1, AuNPs combine with R6G because of electrostatic interaction, leading to the fluorescent quenching of R6G via IFE. While the positively-charged lysozyme molecules in PBS (pH=7.4) would bind to the AuNPs with negative charge and causes AuNPs to accumulate [21]. When R6G is introduced, the IFE between AuNPs and R6G was
Corresponding author. E-mail address: [email protected] (L. Yu).
https://doi.org/10.1016/j.microc.2019.104118 Received 27 June 2019; Received in revised form 19 July 2019; Accepted 19 July 2019 Available online 20 July 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Schematic illustration of the detection mechanism for lysozyme.
weakened. It is observed that the fluorescence intensity of R6G increases with the increasing content of lysozyme. Based on this, the proposed strategy achieves the simple, sensitive and rapid quantification detection of lysozyme. Moreover, the method was further applied for detection lysozyme in urine samples. 2. Experiments
Fluorescence spectra were determined with a FluoroMax-4 fluorescence spectrophotometer. The fluorescence signals (excitation at 520 nm with an Xe lamp) were recorded in the range of 535–700 nm. Slits for both excitation and emission measurements were 2 nm. BI-200SM/BI-9000 instrument (Brookhaven Co., USA) was employed to carry out the dynamic light scattering (DLS) studies and the data were acquired by using the CONTIN method.
2.1. Materials
2.3. Preparation procedures of AuNPs
Chloroauric acid hydrated (HAuCl4·3H2O) was purchased from Sahn chemical technology (Shanghai) Co., Ltd. of China. β-CD (96%) was provided by Aladdin Chemistry Co., Ltd. of China. α-Amylase (AMS) (≥150 units/mg protein (Biuret), aspergillus oryzae), phosphate buffered saline (PBS) (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl; pH=7.4), and lipase (100–150 units/mg protein, porcine pancreas) were obtained from Sigma-Aldrich. Pepsin (≥98%, porcine stomach mucose), trypsin (10 units/mg, bovine pancreas), lysozyme (20,000 units/mg, chicken egg), and γ-glutamyl transpeptidase (γ-GT, 0.2 units/mg, porcine pancreas) were bought from Shanghai Shifeng Biological Technology Co., Ltd. China. Sodium hydroxide (NaOH, ≥96.0%) was provided by Sinopharm Chemical Reagent Co., Ltd. Rhodamine 6G was purchased from J&K Scientific Co., Ltd. China. All chemicals were used without further purification. Ultrapure water (resistivity, 18.25 MΩ cm) was obtained from a Ulupure system and used in all experimental procedures. All the solution of various enzymes (AMS, lipase, pepsin, trypsin, lysozyme and γ-GT) were freshly prepared in PBS. The other chemicals were dissolved in water.
AuNPs were prepared by the β-CD reduction of HAuCl4, following the procedure reported earlier [31,32]. Herein, 39 mL of deionized water, 10 mL of 0.01 M β-CD, and 1 mL of 0.01 M HAuCl4 were added into the three-neck flask and mixed evenly. Since gold ions can be reduced in basic solution, 0.1 mL of 1 M NaOH was then utilized to adjust pH to 10.5. Furthermore, the mixed solution was continuously heated for 3 h at 60 °C under stirring. In the course of reaction, the color of solution changed from pale yellow to red, purple and finally wine red, indicating the formation of AuNPs. Afterwards, the solution was centrifuged for 20 min at a speed of 9000 r/min. The as-prepared AuNPs was redispersed with ultrapure water and stored at 4 °C for later use. 2.4. Fluorescence quenching experiments of R6G by AuNPs R6G solution (500 μL, 0.2 μM) was mixed with AuNPs solution (500 mL, 0.7 nM) under stirring at room temperature. That is, the final concentration of R6G and AuNPs was 0.1 μM and 0.35 nM, respectively. After that, fluorescence spectra of both the mixture and R6G solution were measured with excitation at 520 nm. In the presence of AuNPs, the fluorescence intensity of solution decreased significantly, indicating that R6G was bound to the AuNPs.
2.2. Instruments UV–vis absorption spectra were obtained using a U-4100 UV–vis spectrometer (Hitachi) in a quartz cell (light path of 5 mm). The background signal was subtracted from the sample signal with water as a reference. Transmission electron microscopy (TEM) images were obtained by a HT-7700 instrument with an accelerating voltage of 120 kV.
2.5. Detection assays of lysozyme The detecting process of lysozyme is described as follows: AuNPs solution (350 μL, 1.05 nM) was added to 350 μL of lysozyme solution 2
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with various contentions (1×10−3, 0.01, 0.1, 0.5, 1, 5, 10, 100 μg/mL). The homogeneous mixtures were incubated in water bath (37 °C) for 5 min. Subsequently, R6G (350 μL, 0.3 μM) was introduced to obtain the final solution which was employed to determine the fluorescence intensity. In order to evaluate the selectivity of lysozyme detection, interference study was conducted by preparing 1 μg/mL of pepsase, γ-GT, AMS, lipase and trypsin, respectively. Each of protein solution was added into AuNPs solution (350 μL, 1.05 nM) under stirring. A certain amount of R6G (50 μL, 0.3 μM) was then mixed into the above-mentioned solution. After culturing for 5 min, all the resulting mixtures were measured using a fluorescence spectrophotometer. 2.6. Detection of lysozyme in urine samples The urine samples, collected from healthy volunteers, were diluted 10 times with PBS without other treatment before the further experiments. In order to minimize the interference of background signal, the standard addition method was used to detect the content of lysozyme in urine [33]. Briefly, 175 μL of human urine was added to equal volume lysozyme with different concentration in PBS solution. Then, 350 μL of 1.05 nM AuNPs was spiked and incubated for 5 min at 37 °C. The resulting solutions were further mixed with R6G (350 μL, 0.3 μM) solution, followed by determination of fluorescence property and confirmation of the lysozyme concentration in urine.
Fig. 2. UV–vis absorption spectrum of the AuNPs.
3.2. Fluorescence quenching of R6G by AuNPs Firstly, feasibility of the fluorescent method was explored for lysozyme sensing based on AuNPs. We separately measured the fluorescence spectra of free R6G, and (R6G+AuNPs) in the absence and presence of lysozyme. As shown in Fig. 3a, R6G molecules as a fluorescent indicator exhibit a strong fluorescent emission peak at 552 nm. Furthermore, it is found that the fluorescent signal of R6G was nearly quenched by AuNPs. The possible reasons are ascribed to two aspects as follows: R6G with positively-charged amino groups can adsorb onto the surface of the negatively-charged AuNPs due to electrostatic interaction, which can lead to accumulation of AuNPs (Fig. S1a). Besides, the emission spectrum of R6G (Fig. 3a) can overlap with the absorption spectrum of AuNPs (Fig. 2) to some extent, manifesting that AuNPs can act as an IFE-absorber to cause IFE with R6G. Whereas, as the positively-charged lysozyme solution was mixed with the AuNPs solution, they combine together to form aggregated AuNPs (Fig. S1b). Moreover, in the absorption spectra, the aggregated AuNPs have large shift from 520 nm to 600 nm after the mixing with the lysozyme in Fig. S2. The overlap between the plasmonic absorption of AuNPs and the emission of R6G reduced. Then, when R6G was introduced into the above mixture, the IFE weakened, Therefore lysozyme may be detected based on the emission of R6G. We also carried out experiments to determine UV–vis absorption spectra of R6G, R6G+AuNPs and R6G+AuNPs+Lysozyme at the same
3. Results and discussion 3.1. Characterizations of AuNPs To prepare AuNPs, we introduced β-CD, as a reductor and stabilizer, to reduce HAuCl4 in basic solution [32]. TEM image indicates that the AuNPs are well mono-dispersed and spherical with an average size of about 17 nm (Fig. 1a). DLS measurement presents their average hydrodynamic diameter is ~20 nm (Fig. 1b), consistent with the TEM result. UV–vis absorption spectrum of AuNPs (Fig. 2) shows that there is an obvious peak at approximately 520 nm, indicative of a good dispersion degree and typical feature of AuNPs [32]. As reported previously [34], the extinction coefficient of AuNPs at 520 nm for 17 nm AuNPs in diameter is approximately 3.24×108 M−1 cm−1. Therefore, the concentration of AuNPs solution was calculated to be about 8 nM by Lambert-Beer law. AuNPs solution with a concentration of 0.7 nM and 1.05 nM utilized in this work was obtained by diluting the solution above.
Fig. 1. TEM image (a) and size distribution (b) of the synthesized AuNPs. 3
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Fig. 3. Fluorescence spectra (a) and UV–vis absorption spectra (b) of different systems. (The concentration of R6G, AuNPs and lysozyme is 0.1 μM, 0.35 nM and 1 μg/ mL, respectively.)
concentrations and conditions as fluorescence spectra (Fig. 3a) and presented the results in Fig. 3b. It is found in Fig. 2 that AuNPs aqueous solution exhibits a distinct peak at ~520 nm. While for R6G, only a weak absorption at ~525 nm was observed (Fig. 3b). As depicted in Fig. 3b, for (R6G+AuNPs) and (R6G+AuNPs+Lysozyme) systems, there is an absorption peak at 528 nm and 534 nm, respectively. Concomitantly, the absorption peak widens significantly. As comparison with AuNPs, addition of either R6G or (R6G+lysozyme) results in an obvious change both in the peak position and peak width. According to the past report [35], agglomeration of AuNPs caused absorption peak broadening and red shift. Hence, we speculate that presence of either R6G or (R6G+lysozyme) may lead to accumulation of AuNPs.
concentration-dependence fluorescence intensity at 552 nm was depicted in Fig. 4b. The inset in Fig. 4b reveals within the concentration range of lysozyme (0.1–10 μg/mL), there is a good linear relationship between the fluorescence intensity and logarithm concentration of lysozyme (LgC) (with correlation coefficient of R2=0.997). To further explore the sensitivity of assay, we also determined the fluorescence spectra of R6G (0.1 μM) in the presence of AuNPs (0.35 nM) and 0.5 ng/ mL lysozyme. For the sake of comparing, the fluorescent intensities in the case of 1 ng/mL (1×10−3 μg/mL) lysozyme and in the absence of lysozyme (Fig. 4a) were also presented in one histogram and displayed in Fig. S5. As can be seen from Fig. S5, the fluorescent intensity for the sample containing 0.5 ng/mL lysozyme is similar to that without lysozyme. While when introducing 1 ng/mL lysozyme into the test system, the fluorescent intensity of R6G enhances remarkably, showing that the LOD of lysozyme is ~1 ng/mL. Table S1 summarizes the detection limit and responsive time of lysozyme detection in this work and other various assays in previous reports. Almost all of the earlier detection methods of lysozyme require either long incubation time or tedious sample preparation procedure. Whereas fluorescent spectroscopy is a desirable and powerful analytical technique due to high sensitivity, simple operation, and quick response. Hence as shown in Table S1, the strategy proposed here is superior to the other approaches for lysozyme detection in sensitivity and detection time. To evaluate the specificity of this assay for lysozyme monitoring, several typical proteins in human body including pepsin, γ-GT, AMS, lipase, and trypsin were utilized to conduct control experiments. As illustrated in Fig. S6, under the same experimental conditions, only lysozyme can cause remarkable relative fluorescent intensity ((I-I0)/I0, I and I0 refer to the fluorescence intensity of the system in the presence and absence of lysozyme, respectively). The results as above substantially indicate that the developed method can open a selective and sensitive way toward lysozyme detection.
3.3. Optimal doses of R6G and AuNPs for lysozyme assay Next, effect of reaction concentrations on fluorescence intensity in this system was optimized. The fluorescence spectra of R6G within 0.01–100 μM were determined and shown in Fig. S3a. The results indicate that the fluorescent intensity enhances with the increasing concentration of R6G among 0.01–1 μM and then subsequently declines in the range of 10–100 μM. As reported [36,37], when the concentration of fluorescent substance is too high, self-quenching phenomenon will occur. In order to avert this, concentration of R6G below 10 μM (namely 1, 0.1 and 0.01 μM) was employed to conduct further study. As illustrated in Fig. S3b–d, at the same concentration of R6G, the fluorescence intensity weakens with increment of the investigated AuNPs concentration. The fluorescence of 1, 0.1 and 0.01 μM R6G was almost completely quenched by 1.61, 0.35 and 0.05 nM AuNPs, respectively. Then, the fluorescence spectra of (R6G+AuNPs+lysozyme) system were measured in which the corresponding concentrations of R6G and AuNPs were as noted above in the absence or presence 1 μg/mL lysozyme (Fig. S4). Apparently, when the concentration of R6G and AuNPs was 0.1 μM and 0.35 nM, respectively, there is a greater difference in the fluorescence intensity between the two systems with and without lysozyme, which can provide a relatively high sensitivity for lysozyme quantification.
3.5. Application of the proposed method for lysozyme monitoring in human urine samples
3.4. Fluorescent assay for lysozyme detection
We employed this method to monitor lysozyme in human urine specimens from healthy subjects by standard addition method [12]. Urine samples were spiked with serial concentrations of lysozyme (1, 5 and 10 μg/mL) for the detection. The found concentration of lysozyme can be obtained by estimation of the recoveries based on fluorescence spectra. The corresponding results were illustrated in Table 1. The recoveries of lysozyme range from 84.20% to 114.85% with the relative standard deviation (RSD) < 0.75%. All the above results indicate that the method could provide a reliable access to monitor lysozyme in urine, which is promising for the real applications.
Based on the above, fluorescence spectra of 0.1 μM R6G in the presence of 0.35 nM AuNPs and different concentration of lysozyme were determined (Fig. 4a). It is found that the fluorescent intensity of R6G enhances distinctly when introducing 1 ng/mL or higher concentration of lysozyme into the test system. This may be due to the weakening of the IFE of R6G by AuNPs, resulting in enhanced fluorescence intensity of R6G. Based on the Fig. 4a, the limit of detection (LOD) of lysozyme for the strategy proposed here is 1 ng/mL. The 4
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Fig. 4. (a) The fluorescence spectra of R6G (0.1 μM) in the presence of AuNPs (0.35 nM) and different concentrations of lysozyme (0, 1×10−3, 0.01, 0.1, 0.5, 1, 5, 10, 100 μg/mL). (b) The fluorescence intensity at 552 nm of the assay toward different concentrations of lysozyme. Inset: The linear fitting curve between fluorescence intensity and lysozyme concentration. Table 1 Performance of lysozyme detection in urine samples. Sample
Spiked concentration (μg/mL)
Found concentration (μg/mL)
Recovery (%)
RSD (%) (n = 3)
1 2 3
1 5 10
0.84 5.08 11.49
84.20 101.50 114.85
0.75 0.49 0.41
[4] [5] [6] [7]
4. Conclusions
[8]
A sensitive, rapid, and inexpensive strategy based on AuNPs was designed for lysozyme detection with R6G as a fluorescence indicator. Without lysozyme, AuNPs could lead to the fluorescent signal quenching of R6G via IFE. When solutions of lysozyme and AuNPs with opposite charges were mixed, they bound to each other by electrostatic interaction and resulted in the aggregation of AuNPs. After adding R6G into the above mixture, the presence of lysozyme prevented the combination of R6G and AuNPs, as well as advent of IFE, Under such circumstances, stronger fluorescence intensity was observed for R6G in the presence of higher lysozyme concentration. The detection of limit for lysozyme was as low as 1 ng/mL. More importantly, this assay was demonstrated to detect lysozyme in the urine samples. This approach offers a prospect on the clinical diagnose of related diseases in the future.
[9] [10] [11] [12]
[13] [14] [15]
Acknowledgments
[16]
This work was supported by the National Natural Science Foundation of China (No. 21373128), Scientific and Technological Projects of Shandong Province of China (No. 2018GSF121024).
[17]
Appendix A. Supplementary data
[18]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.microc.2019.104118.
[19]
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