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Ultrasensitive and non-labeling fluorescence assay for biothiols using enhanced silver nanoclusters
Sensors and Actuators B 267 (2018) 174–180
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Ultrasensitive and non-labeling fluorescence assay for biothiols using enhanced silver nanoclusters Wenmiao Wang a,1 , Jian Li b,1 , Jialong Fan a , Weimin Ning a , Bin Liu a,∗ , Chunyi Tong a,∗ a b
College of Biology, Hunan University, Changsha, Hunan, 410082, China The Clinical laboratory of the third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, China
a r t i c l e
i n f o
Article history: Received 15 January 2018 Received in revised form 1 April 2018 Accepted 2 April 2018 Available online 4 April 2018 Keywords: Biothiol Silver nanoclusters Non-labeling Cysteine Fluorescence
a b s t r a c t A label-free, ultra-sensitive and turn-off fluorescence method for detecting cysteine (Cys) has been developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as the fluorescence probe. The method is based on the specific interaction between Cys and DNA-AgNCs via robust Ag-S bonds and the fluorescence quenching ability of Cys to DNA-AgNCs. Using this method for Cys assay, it was found that the change of fluorescent intensity has a good linear relationship with Cys concentration in the range from 0.1 nM to 100 nM (R2 = 0.991). The detection limit of Cys was 0.05 nM. Furthermore, the method was successfully used for the detection of Cys in human serums, the result of which was confirmed using clinical colorimetric assay. In summary, our study shows that this simple, sensitive and rapid detection method can be hopefully used for theranostics. © 2018 Elsevier B.V. All rights reserved.
1. Introduction As a biological thiol, Cys plays a critical role for its participation in the process of reversible redox reaction, detoxification and metabolism [1]. Many evidences have indicated that the imbalance of the cellular biothiols often resulted in a variety of disease [2]. For example, a lack of Cys can cause retarded growth in children, leukocyte loss, liver damage, hematopoiesis decrease, skin lesions and weakness, whereas excess Cys leads to neurotoxicity [3]. Thus, developing sensitive and specific methods for Cys assay in the human plasma or fluids is in high demand especially for the early diagnosis of a variety of diseases. Until now, a variety of developed methods including chromatography [4,5], capillary electrophoresis [6] and mass spectroscopy [7], have made great contribution for biothiols assay. However, these approaches involve cumbersome laboratory procedures, require expensive and sophisticated instrument in different degree. Furthermore, the relatively low sensitivity of some methods limited their practical application. Recently, new methods for biomolecules assay, which were based on nanomaterials, have attracted great attention due to their significant advantages. Among of them, DNA strand has become
∗ Corresponding authors. E-mail addresses: [email protected] (B. Liu), sw [email protected] (C. Tong). 1 These authors contributed to the work equally and should be regarded as co-first authors. https://doi.org/10.1016/j.snb.2018.04.010 0925-4005/© 2018 Elsevier B.V. All rights reserved.
an extremely favorable tool in nanotechnology and material science owing to its remarkable molecular recognition properties and flexible structure [8]. For example, a large amount of phosphate groups, amino groups and heterocyclic nitrogen atoms in DNA molecules offered multiple binding sites for several metal ions to form metallic cluster following the contour of the DNA template [9]. Moreover, the optical and physical properties of these metal clusters can be conveniently tuned by the change of base sequence, temperature and pH [10]. Recently, Yeh et al. synthesized a Ag cluster, the fluorescence intensity of which was enhanced only when placed in proximity to G-rich seqeunces [11]. Compared with the semiconductor quantum dots or dye molecules, this kind of nanomaterial showed low toxicity, ultrasmall size, prominent photostability, good biocompatibility and water solubility. Until now, silver nanoclusters have been widely used in many fields such as biosensing [12–14], molecule image[15–19] and anti-bacteria[20], multiplexed genes analysis [21]; DNA-targeted anticancer drugs in vitro [22] and latent fingerprint visualization [23]. However, these reported Ag clusters often exposed their weak anti-inference ability and low sensitivity when they were used for complicated biosample assay due to the weak fluorescence signal. In order to overcome these drawbacks and further widen application of Ag clusters, we synthesized a new Ag clusters nanomaterial with significantly improved fluorescence signal using G-richsequences and further developed a turn-off and label-free approach for biothiols detection.
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Scheme 1. Schematic illustration of the fluorescence assay for Cys by using DNA-AgNCs.
2. Experimental section
ples at 535 nm were recorded with the excitation wavelength of 450 nm.
2.1. Materials and regents The oligonucleotides sequences involved were listed in the Table S1 and all oligonucleotides were synthesized and purified by HPLC (Takara Biotechnology Inc., Dalian). Sodium borohydride (NaBH4 ), silver nitrate (AgNO3 ), dibasic sodium phosphate (Na2 HPO4 ) and sodium dihydrogen phosphate (NaH2 PO4 ), l-Cys and other amino acids were purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were of analytical reagent grade without further purification. All solutions were prepared using distilled water and stored at 4 ◦ C before use. 2.2. Apparatus Fluorescence measurements were carried out on the FL-2500 Fluorescence Spectrophotometer (Japan). CD spectra were determined using a MOS-500 Circular Dichroism Spectrometer (France). AFM images were carried out on the bioscope system Atomic Force Microscope (America). UV–vis absorption spectra were recorded by using UV-1800 Ultraviolet spectrophotometer (Japan). Fluorescence life assays were measured on FLS920 single-photon counting (TSCPC) spectrofluorometer (United Kingdom). 2.3. Preparation of fluorescence silver nanoclusters The ultrasmall DNA-AgNCs were prepared according to the literature reported method [24] with minor modification. The ssDNA-templated silver deposition was synthesized by reduction of AgNO3 with NaBH4 in the presence of DNA. Briefly, DNA was mixed with AgNO3 solution and vortexed for 30 s, then, incubated for 20 min in the dark. Finally, according to the molar ratio Ag:DNA:NaBH4 of 6:1:6, NaBH4 was added to the mixture to reduce Ag ions. The final mixture was kept in the dark for 12 h before use.
2.5. Detection of Cys Cys samples with different concentrations were freshly prepared before use. After mixing the 10 L of 5 M DNA-AgNCs with 89 L PBS (pH6.6), the mixture was equilibrated for 0.5 h. Then, different concentrations of Cys was added and incubated for 0.5 h. Fluorescence intensities of all samples at 535 nm were recorded with the excitation wavelength of 450 nm. In the following experiment, other amino acids including alanine (Ala), Cys, leucine (Leu), lysine (Lys), methionine (Met), proline (Pro), serine (Ser), tyrosine (Tyr) and valine (Val) were used to investigate the selectivity of DNA-AgNCs for Cys assay. Meanwhile, the stability of DNA-AgNCs was investigated by monitoring its fluorescence change in 7 days. 2.6. Analysis of human serum samples Human serum samples were obtained from the third Xiangya Hospital of Central South University. The disulfide bonds of samples were reduced according to the literature [1]. 40 L of hydrochloric acid (HCl, 0.2 M) and 20 L of triphenylphosphine (PPh3 ) (400 mM in H2 O- acetonitrile;methyl cyanide (CH3 CN) 20:80 v/v and 2 M HCl) were added to 500 L of plasma and incubated for 15 min at 25 ◦ C to hydrolyze the disulfide bonds. Then, the same volume (500 L) of CH3 CN was mixed with hydrolyzed plasma to precipitate plasma proteins [25,26] followed by centrifugation at 3000 g for 20 min. The supernatant containing the reduced biothiols was used for further analysis. In the recovery study experiment, Cys solutions with known concentration were added to the samples and the total biothiol concentration was determined combing with the constructed standard curve. Before measurement, the plasma samples were appropriately diluted with PB buffer so as to keep consistence with the dynamic range of our method. 3. Results and discussion
2.4. Optimization of temperature and pH 3.1. Strategy for Cys detection 10 L of 5 M DNA-AgNCs was added into 89 L phosphate buffer saline (PBS, pH6.6). The mixture solution was equilibrated for 0.5 h under different temperature or pH. Then, 1 L of 10 M Cys was added into the above solutions and incubated 0.5 h under different temperature or pH. Fluorescence intensities of all sam-
The working principle for Cys assay is represented in the Scheme 1. The detection system consists of DNA- templated AgNCs and detection target of Cys. The synthesis of AgNCs was achieved using a single-stranded DNA containing core sequences as the tem-
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Fig. 1. (A–C) AFM image and height profile of DNA, DNA-AgNCs and DNA-AgNCs in the presence of 500 nM Cys. All samples were deposited on mica substrates. (D) CD spectra of DNA-AgNCs alone (black line), DNA-Core (red line) and DNA-AgNCs in the presence of 500 nM Cys (blue line). (E) Absorption spectra of DNA-AgNCs, Inset: The excitation and maximum emission of DNA-AgNCs. [DNA-AgNCs] = 500 nM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
plate to control silver atom deposition at the presence of NaBH4. The synthesized nanoclusters with diameter of about 2 nm, the size of which approaches the Fermi wavelength of electrons, can emit strong fluorescence upon photo excitation in the UV–vis range. However, the fluorescence of DNA-AgNCs can be strongly quenched in the presence of Cys or other biothiols due to the generation of S-Ag bond. In our strategy, the DNA strand is the combination of a core sequence with a G-rich aptameric sequence. It has been reported [27] that the existence of G-rich sequence can significantly improve the fluorescence signal of AgNCs. As our expected, the fluorescence intensity of DNA-AgNCs contain aptameric sequence increased about 30-fold comparing with that of Core-AgNCs (Fig. S1). 3.2. Characterization of DNA-AgNCs AFM images of Fig. 1 revealed the topographic morphology and determined the thickness of DNA-AgNCs. The uniform size with a diameter of 2 nm and spherical morphology was observed (Fig. 1B). However, the diameter of DNA-AgNCs became 7 nm in the presence of Cys (Fig. 1C), which is consistent with previous study [28]. The increase of diameter (7 nm) is mainly attributed to the aggregation of AgNCs through the interaction of Cys with Ag+ [28]. In addition, we further evaluated the stability of AgNCs and found that the color of DNA-AgNCs dispersing in the PB solution is clear and transparent, while the color of solution prepared by the simple mixing
of DNA strand with AgNO3 is turbid and aggregates of nanoparticle tend to form (data not shown). This result clearly indicated the higher stability of AgNCs comparing with Ag nanoparticles. In addition, CD spectra assay was used to study the conformation of DNA strand in the DNA-AgNCs. As previous study has shown that silver atom deposition on DNA scaffolds can change CD signal of DNA [29]. Fig. 1D indicated that the CD spectra of DNA alone showed a positive band at 285 nm, whereas the peak at 280 nm became negative with red shift of 5 units after the formation of DNA-AgNCs. As a control, the CD spectra of the mixture containing Cys were very similar to that of bare DNA strand, which indicated the effective release of DNA strand. By monitoring the ultraviolet spectrum of DNAAgNCs, we found that no UV absorption peak between 300 nm and 500 nm appeared (Fig. 1E). These data strongly indicated that no silver nanoparticle was formed except for uniformly dispersed silver nanoclusters. It has been reported that the excitation wavelength and emission wavelength of silver nanoclusters often varied as the variation of sequences of DNA or the ratio of DNA:AgNO3 :NaBH4 [19]. In this study, the inset spectra of synthesized DNA-AgNCs displayed in Fig. 1E showed the maximum excitation and emission wavelength of 450 nm and 535 nm, respectively. 3.3. Feasibility assay of the study In order to confirm the feasibility of the proposed strategy, we monitored the change of fluorescence signal of DNA-AgNCs at the
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3.5. Fluorescence detection for Cys
Fig. 2. Fluorescence spectra of DNA-AgNCs in the presence of 500 nM Cys.
presence of Cys. As given in Fig. 2, the fluorescence signal of samples decreased at the presence of 500 nM Cys. This result suggested that the addition of Cys can efficiently interact with DNA-AgNCs and cause DNA release after forming Ag-S bonds. The change of fluorescence signal of DNA-AgNCs in the presence of Cys can be produced through excited state reaction, collisional interaction (dynamic), static quenching or both or even through monomolecular mechanisms where fluorophore itself or other absorbing species attenuates the fluorescence intensity. In order to further explore the mechanism of silver cluster quenching, we measured the lifetime of DNA-AgNCs before and after Cys addition by using single photon counting (TCSPC) technique. According to the fluorescence decay curves (Fig. S2A), we found that both of the life time of DNAAgNCs in the absence and presence of Cys were 2.70 ± 0.26 ns. This data, which is consistent with previous report [1], confirmed the characteristic of static quenching of DNA-AgNCs. Meanwhile, no obvious red or blue shift of the maximum emission wavelength upon addition of Cys was observed as previously reported[1,28], which was caused by the change of the microenvironment of the DNA template. This result indicated that the interaction of DNA-AgNCs with Cys did not produce significant effect on the microenvironment of DNA template, which reversely improved the practicability of this kind of material. However, the reason should be further explored.
3.4. Optimization of detection conditions Temperature often decreases the fluorescence signal of DNAAgNCs by affecting the number and enrichment way of silver atoms on DNA [30]. By monitoring the effect of temperature on the signal intensity of DNA-AgNCs, we found that the fluorescence intensity of DNA-AgNCs decreased as the temperature increased from 25 ◦ C to 37 ◦ C (Fig. 3A) and Cys caused the maximal signal change of the DNA-AgNCs at 30 ◦ C (Fig. S3). Thus, this temperature was chosen as detection temperature in the following studies. In addition, the effect of pH on the fluorescence signal of DNA-AgNCs and the interaction of DNA-AgNCs with Cys were investigated, which were shown in Fig. 3B. From this figure, we found that pH was an important determine factor of the fluorescence signal of DNA-AgNCs and the alkaline solution (pH >7.5) led to the destruction of DNA-AgNCs, followed by the rapid fluorescence signal decrease. However, the fluorescence intensity of DNA-AgNCs kept stable at the condition of pH 6–7. Moreover, Cys caused the maximal signal change of DNAAgNCs at pH 6 (Fig. S4). Thus, pH 6 and 30 ◦ C were chosen as optimal conditions for the following Cys assay.
Under the optimal conditions, we monitored the fluorescence signal of DNA- AgNCs at the presence of different concentrations of Cys. From the wavelength scan curves of Fig. 4A, it was found that the fluorescence signal intensity of DNA-AgNCs at 535 nm gradually decreased with the increase of Cys concentration. We further investigated the relation of Cys concentration with fluorescence value of DNA-AgNCs and the result was shown as Fig. 4B. In this figure, F0 represents the fluorescence value of DNA-AgNCs, F1 represents the fluorescence value of DNA-AgNCs after adding Cys. This figure clearly indicated that the ratio increased as the concentration of Cys increase, which reflected the more fluorescence reduction. From this figure, it was found that the concentration of Cys in 0.1–100 nM (R2 = 0.991) has a linear relationship with the ratio of (F0 -F1 )/F0 and the detection limit reached 0.05 nM. By comparing the Cys detection limit of previously reported literatures, the new method showed the ultra-high sensitivity (at least 40-fold) due to the significant improved fluorescence signal of the new material (Table 1). 3.6. Selectivity and stability of the strategy The response of DNA-AgNCs to other amino acids in complex biosamples should be carefully considered before the method was used for biosample assay. Using Cys as a positive control, no significant change of the fluorescent signal of DNA-AgNCs was found at the presence of other amino acid (Fig. 5A), which indicated the high selectivity of DNA-AgNCs for Cys assay. In addition, we tested the stability of DNA-AgNCs in order to confirm the reliability of the new method for clinic use in a long period. From Fig. 5B, it was found that the fluorescence intensity of DNA-AgNCs kept stable at least for 7 days. Meanwhile, the assay fluorescence signal of DNA-AgNCs can keep stable at least 5 h at the presence of Cys (Fig. S2B). These data fully demonstrated the guarantee of this kind of nanomaterial for clinical diagnosis. 3.7. Cys detection in human serum Considering the superior sensitivity and specificity of the new method in the homogenous solution, we then explored its capability for Cys assay in the complicated samples. First, we evaluated the resistance capability of the DNA template to deoxyribonuclease I (DNase I, 25 ng/mL), the main unspecific nuclease of biosamples by monitoring the fluorescent change of DNA-AgNCs. As our expected that no fluorescence change of this nanomaterial was observed when the DNA-AgNCs was exposed to DNase I. This data further confirmed that the local high concentrations of metal ions around DNA-AgNCs can efficiently inhibit DNase I activity through steric hindrance effect. In the following experiment, various amounts of Cys in the 1% fatal bovine serun (FBS) solution were mixed with DNA-AgNCs and the fluorescence signals were measured to construct a standard curve for Cys assay under complicated conditions. Fig. S5 indicated that the standard curve for Cys obtained in serum were similar to that obtained in the buffer. This result demonstrated that the method can efficiently avoid disturb of other components. We also investigated the accuracy of the new method for Cys assay in the serum using standard addition method. The result in Table 2 showed that the recovery of Cys was about 101.3% to 104.2% and all relative standard deviations (RSD) were less than 10%. These results clearly confirmed the high accuracy and good anti-interference ability of the new method for Cys assay under complicated conditions. Finally, the method was used DNA-AgNCs to detect thiol compounds in serum samples. Fig. 6 showed that the concentration of thiol compounds of tested samples varied in some extent due to the personal difference. The average concen-
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Fig. 3. (A) Fluorescence decrease of DNA-AgNCs in the presence of Cys (gray bar) at different temperature. (B) Fluorescence decrease of DNA-AgNCs in the presence of Cys (gray bar) at different pH. The concentration of Cys and DNA-AgNCs were 100 nM and 500 nM respectively.
Fig. 4. (A)The wavelength scan curves of DNA-AgNCs in the presence of Cys (0–1000 nM). (B) Plots of the ratio ((F0 –F1 )/F0 ) as a function of Cys concentration. The inset showed the linear region of Cys. F0 and F1 represent the florescence intensity of DNA-AgNCs in the absence and presence of Cys at 535 nm, respectively. Table 1 Comparison of the linear range and detect limit for Cys using other methods. Methods
Linear range
Detection limit
Reference
Oligonucleotide-stabilized fluorescent silver nanoclusters Combination of graphene oxide and DNA metallization Ag/Au bimetallic nanoclusters Cu2+ ensemble Colorimetric assay based on DNA-Ag/Pt nanoclusters DNA-functionalized silver nanoclusters DNA-AgNCs
8–100 nM 0–1000 nM 0.25–7 M 5–200 nM 0.5–4.5 M 0.1–100 nM
4 nM 2 nM 111 nM 2 nM 0.134 M 0.05 nM
[1] [3] [31] [32] [28] this study
Table 2 Determination of thiol compounds in human serum. Sample
Determined thiol compounds (M)
Added Cys (M)
Measured (M)
Recovery (%)
RSD (n = 3%)
1
15.5 15.5 10.7 10.7 59.3 59.3
20.0 40.0 20.0 40.0 20.0 40.0
36.1 56.6 32.0 52.5 80.3 101.3
101.7 102.0 104.2 103.6 101.3 102.0
5.77 8.10 7.86 4.79 4.37 3.47
2 3
tration of thiol compounds was 18.49 M in 14 samples. We also adopted the clinical colorimetric method to confirm the reliability of the new method and the result indicated that the average concentration of these samples of 13.41 M, the average concentration
of which was slightly lower than that of DNA-AgNCs assay (Fig. S6). However, it should be noted that the relative level of these samples was wholly similar between the two methods. As to the reason of concentration difference existed between the two methods, it can
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Fig. 5. (A) The specificity analysis of DNA-AgNCs. Fluorescence response profiles of DNA-AgNCs toward amino acid (1 M). (B)The fluorescence value of DNA-AgNCs change against the time.
that can be used for fast and low cost pre-clinical diagnosis of various related diseases. Acknowledgments This work was partially supported by the Natural Science Foundation of China (81374062, 81673579 and 31672457), the Natural Science Foundation of Hunan Province (h14JJ2049), Hunan Province Universities 2011 Collaborative Innovation Center of Protection and Utilization Of Hu-xiang Chinese Medicine Resources and the National Standardization Project of Traditional Chinese Medicine (Grant ZYBZH-Y-HUN-23) Appendix A. Supplementary data
Fig. 6. The detection results of thiol compounds in human serum using DNA-AgNCs. The blue bars stand for the concentration of thiol compounds. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
be attributed to the difference of treatment condition of samples, reagent and detect equipment. The further experiments are in processing. In summary, these results suggested that the simple and low cost method showed the applicability for the thiol compounds assay of serum especially in those developing countries. 4. Conclusions In this paper, an enhanced silver cluster nanomaterial with strong fluorescence signal was synthesized through optimization of DNA template. By using the fluorescence quenching capability of Cys to silver cluster through the interaction between silver ions and Cys thiol groups, we developed a simple method for Cys detection. The method showed a linear detection range of 0.1–100 nM for Cys with a detection limit of 0.05 nM. In addition, we successfully demonstrated the discriminability of silver cluster probe for Cys under complicated conditions. Furthermore, the method showed solid reliability for detecting Cys/thiol compounds of serum. In our point, the simple method for Cys with high sensitivity and low cost will provide great opportunities for Cys-related disease diagnosis, prognosis and response to treatment. Assuming that the method can be combined with point-of-care testing high throughput diagnostic equipment, the detection of Cys allows for feasible system
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Biographies Wenmiao Wang received her B.E degree from School of Life Science and Technology, Central South University of Forestry and Technology. She is commencing her master study in College of Biology, Hunan University from 2016 under the guidance of Prof. Bin Liu. Her research focus on the molecular diagnosis and the construction of drug delivery system. Jian Li obtained his M.M. degree from Xiangya School of Medicine, Central South University during which time he spent three years on laboratory medicine. He is commencing his Ph.D study in the Third Xiangya Hospital, Central South University from 2016 under the guidance of Prof. Xinmin Nie. His research interests lie in new method for clinical diagnosis and the construction of nano-drug carrier. Jialong Fan earned his B.S degree from School of Life Science and Technology, Central South University of Forestry and Technology. He is currently a MSBE candidate under the supervision of Profs. Chunyi Tong and Bin Liu in College of Biology, Hunan University. His current research interests include biosensing assay. Weining Ning received her B.E degree from School of Biological Engineering, HuaiHua University. She is commencing her master study in College of Biology, Hunan University from 2016 under the guidance of Profs. Chunyi Tong and Bin Liu. Her research focus on the synthesis and application of new type of sliver nanomaterials. Bin Liu is currently an associate professor in Hunan University in PR China. He received his PhD degree in Analytical Chemistry from Hunan University at 2007. From 2007 to 2009, he was a post-doctor and research associate in Internal Medicine School, Health Sciences Center, Texas Tech University. His major research interests focus on the biosensors and nanotheranostics. Chunyi Tong is currently an associate professor in Hunan University in PR China. He received his B.S. and PhD degree in Analytical Chemistry from Hunan University in 2003 and 2008, respectively. From 2016–2017, he was a visiting scholar at the University of Pennsylvania. His major research interests focus on biosensors and nanotheranostics.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Ultrasensitive and non-labeling fluorescence assay for biothiols using enhanced silver nanoclusters Wenmiao Wang a,1 , Jian Li b,1 , Jialong Fan a , Weimin Ning a , Bin Liu a,∗ , Chunyi Tong a,∗ a b
College of Biology, Hunan University, Changsha, Hunan, 410082, China The Clinical laboratory of the third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, China
a r t i c l e
i n f o
Article history: Received 15 January 2018 Received in revised form 1 April 2018 Accepted 2 April 2018 Available online 4 April 2018 Keywords: Biothiol Silver nanoclusters Non-labeling Cysteine Fluorescence
a b s t r a c t A label-free, ultra-sensitive and turn-off fluorescence method for detecting cysteine (Cys) has been developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as the fluorescence probe. The method is based on the specific interaction between Cys and DNA-AgNCs via robust Ag-S bonds and the fluorescence quenching ability of Cys to DNA-AgNCs. Using this method for Cys assay, it was found that the change of fluorescent intensity has a good linear relationship with Cys concentration in the range from 0.1 nM to 100 nM (R2 = 0.991). The detection limit of Cys was 0.05 nM. Furthermore, the method was successfully used for the detection of Cys in human serums, the result of which was confirmed using clinical colorimetric assay. In summary, our study shows that this simple, sensitive and rapid detection method can be hopefully used for theranostics. © 2018 Elsevier B.V. All rights reserved.
1. Introduction As a biological thiol, Cys plays a critical role for its participation in the process of reversible redox reaction, detoxification and metabolism [1]. Many evidences have indicated that the imbalance of the cellular biothiols often resulted in a variety of disease [2]. For example, a lack of Cys can cause retarded growth in children, leukocyte loss, liver damage, hematopoiesis decrease, skin lesions and weakness, whereas excess Cys leads to neurotoxicity [3]. Thus, developing sensitive and specific methods for Cys assay in the human plasma or fluids is in high demand especially for the early diagnosis of a variety of diseases. Until now, a variety of developed methods including chromatography [4,5], capillary electrophoresis [6] and mass spectroscopy [7], have made great contribution for biothiols assay. However, these approaches involve cumbersome laboratory procedures, require expensive and sophisticated instrument in different degree. Furthermore, the relatively low sensitivity of some methods limited their practical application. Recently, new methods for biomolecules assay, which were based on nanomaterials, have attracted great attention due to their significant advantages. Among of them, DNA strand has become
∗ Corresponding authors. E-mail addresses: [email protected] (B. Liu), sw [email protected] (C. Tong). 1 These authors contributed to the work equally and should be regarded as co-first authors. https://doi.org/10.1016/j.snb.2018.04.010 0925-4005/© 2018 Elsevier B.V. All rights reserved.
an extremely favorable tool in nanotechnology and material science owing to its remarkable molecular recognition properties and flexible structure [8]. For example, a large amount of phosphate groups, amino groups and heterocyclic nitrogen atoms in DNA molecules offered multiple binding sites for several metal ions to form metallic cluster following the contour of the DNA template [9]. Moreover, the optical and physical properties of these metal clusters can be conveniently tuned by the change of base sequence, temperature and pH [10]. Recently, Yeh et al. synthesized a Ag cluster, the fluorescence intensity of which was enhanced only when placed in proximity to G-rich seqeunces [11]. Compared with the semiconductor quantum dots or dye molecules, this kind of nanomaterial showed low toxicity, ultrasmall size, prominent photostability, good biocompatibility and water solubility. Until now, silver nanoclusters have been widely used in many fields such as biosensing [12–14], molecule image[15–19] and anti-bacteria[20], multiplexed genes analysis [21]; DNA-targeted anticancer drugs in vitro [22] and latent fingerprint visualization [23]. However, these reported Ag clusters often exposed their weak anti-inference ability and low sensitivity when they were used for complicated biosample assay due to the weak fluorescence signal. In order to overcome these drawbacks and further widen application of Ag clusters, we synthesized a new Ag clusters nanomaterial with significantly improved fluorescence signal using G-richsequences and further developed a turn-off and label-free approach for biothiols detection.
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Scheme 1. Schematic illustration of the fluorescence assay for Cys by using DNA-AgNCs.
2. Experimental section
ples at 535 nm were recorded with the excitation wavelength of 450 nm.
2.1. Materials and regents The oligonucleotides sequences involved were listed in the Table S1 and all oligonucleotides were synthesized and purified by HPLC (Takara Biotechnology Inc., Dalian). Sodium borohydride (NaBH4 ), silver nitrate (AgNO3 ), dibasic sodium phosphate (Na2 HPO4 ) and sodium dihydrogen phosphate (NaH2 PO4 ), l-Cys and other amino acids were purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were of analytical reagent grade without further purification. All solutions were prepared using distilled water and stored at 4 ◦ C before use. 2.2. Apparatus Fluorescence measurements were carried out on the FL-2500 Fluorescence Spectrophotometer (Japan). CD spectra were determined using a MOS-500 Circular Dichroism Spectrometer (France). AFM images were carried out on the bioscope system Atomic Force Microscope (America). UV–vis absorption spectra were recorded by using UV-1800 Ultraviolet spectrophotometer (Japan). Fluorescence life assays were measured on FLS920 single-photon counting (TSCPC) spectrofluorometer (United Kingdom). 2.3. Preparation of fluorescence silver nanoclusters The ultrasmall DNA-AgNCs were prepared according to the literature reported method [24] with minor modification. The ssDNA-templated silver deposition was synthesized by reduction of AgNO3 with NaBH4 in the presence of DNA. Briefly, DNA was mixed with AgNO3 solution and vortexed for 30 s, then, incubated for 20 min in the dark. Finally, according to the molar ratio Ag:DNA:NaBH4 of 6:1:6, NaBH4 was added to the mixture to reduce Ag ions. The final mixture was kept in the dark for 12 h before use.
2.5. Detection of Cys Cys samples with different concentrations were freshly prepared before use. After mixing the 10 L of 5 M DNA-AgNCs with 89 L PBS (pH6.6), the mixture was equilibrated for 0.5 h. Then, different concentrations of Cys was added and incubated for 0.5 h. Fluorescence intensities of all samples at 535 nm were recorded with the excitation wavelength of 450 nm. In the following experiment, other amino acids including alanine (Ala), Cys, leucine (Leu), lysine (Lys), methionine (Met), proline (Pro), serine (Ser), tyrosine (Tyr) and valine (Val) were used to investigate the selectivity of DNA-AgNCs for Cys assay. Meanwhile, the stability of DNA-AgNCs was investigated by monitoring its fluorescence change in 7 days. 2.6. Analysis of human serum samples Human serum samples were obtained from the third Xiangya Hospital of Central South University. The disulfide bonds of samples were reduced according to the literature [1]. 40 L of hydrochloric acid (HCl, 0.2 M) and 20 L of triphenylphosphine (PPh3 ) (400 mM in H2 O- acetonitrile;methyl cyanide (CH3 CN) 20:80 v/v and 2 M HCl) were added to 500 L of plasma and incubated for 15 min at 25 ◦ C to hydrolyze the disulfide bonds. Then, the same volume (500 L) of CH3 CN was mixed with hydrolyzed plasma to precipitate plasma proteins [25,26] followed by centrifugation at 3000 g for 20 min. The supernatant containing the reduced biothiols was used for further analysis. In the recovery study experiment, Cys solutions with known concentration were added to the samples and the total biothiol concentration was determined combing with the constructed standard curve. Before measurement, the plasma samples were appropriately diluted with PB buffer so as to keep consistence with the dynamic range of our method. 3. Results and discussion
2.4. Optimization of temperature and pH 3.1. Strategy for Cys detection 10 L of 5 M DNA-AgNCs was added into 89 L phosphate buffer saline (PBS, pH6.6). The mixture solution was equilibrated for 0.5 h under different temperature or pH. Then, 1 L of 10 M Cys was added into the above solutions and incubated 0.5 h under different temperature or pH. Fluorescence intensities of all sam-
The working principle for Cys assay is represented in the Scheme 1. The detection system consists of DNA- templated AgNCs and detection target of Cys. The synthesis of AgNCs was achieved using a single-stranded DNA containing core sequences as the tem-
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Fig. 1. (A–C) AFM image and height profile of DNA, DNA-AgNCs and DNA-AgNCs in the presence of 500 nM Cys. All samples were deposited on mica substrates. (D) CD spectra of DNA-AgNCs alone (black line), DNA-Core (red line) and DNA-AgNCs in the presence of 500 nM Cys (blue line). (E) Absorption spectra of DNA-AgNCs, Inset: The excitation and maximum emission of DNA-AgNCs. [DNA-AgNCs] = 500 nM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
plate to control silver atom deposition at the presence of NaBH4. The synthesized nanoclusters with diameter of about 2 nm, the size of which approaches the Fermi wavelength of electrons, can emit strong fluorescence upon photo excitation in the UV–vis range. However, the fluorescence of DNA-AgNCs can be strongly quenched in the presence of Cys or other biothiols due to the generation of S-Ag bond. In our strategy, the DNA strand is the combination of a core sequence with a G-rich aptameric sequence. It has been reported [27] that the existence of G-rich sequence can significantly improve the fluorescence signal of AgNCs. As our expected, the fluorescence intensity of DNA-AgNCs contain aptameric sequence increased about 30-fold comparing with that of Core-AgNCs (Fig. S1). 3.2. Characterization of DNA-AgNCs AFM images of Fig. 1 revealed the topographic morphology and determined the thickness of DNA-AgNCs. The uniform size with a diameter of 2 nm and spherical morphology was observed (Fig. 1B). However, the diameter of DNA-AgNCs became 7 nm in the presence of Cys (Fig. 1C), which is consistent with previous study [28]. The increase of diameter (7 nm) is mainly attributed to the aggregation of AgNCs through the interaction of Cys with Ag+ [28]. In addition, we further evaluated the stability of AgNCs and found that the color of DNA-AgNCs dispersing in the PB solution is clear and transparent, while the color of solution prepared by the simple mixing
of DNA strand with AgNO3 is turbid and aggregates of nanoparticle tend to form (data not shown). This result clearly indicated the higher stability of AgNCs comparing with Ag nanoparticles. In addition, CD spectra assay was used to study the conformation of DNA strand in the DNA-AgNCs. As previous study has shown that silver atom deposition on DNA scaffolds can change CD signal of DNA [29]. Fig. 1D indicated that the CD spectra of DNA alone showed a positive band at 285 nm, whereas the peak at 280 nm became negative with red shift of 5 units after the formation of DNA-AgNCs. As a control, the CD spectra of the mixture containing Cys were very similar to that of bare DNA strand, which indicated the effective release of DNA strand. By monitoring the ultraviolet spectrum of DNAAgNCs, we found that no UV absorption peak between 300 nm and 500 nm appeared (Fig. 1E). These data strongly indicated that no silver nanoparticle was formed except for uniformly dispersed silver nanoclusters. It has been reported that the excitation wavelength and emission wavelength of silver nanoclusters often varied as the variation of sequences of DNA or the ratio of DNA:AgNO3 :NaBH4 [19]. In this study, the inset spectra of synthesized DNA-AgNCs displayed in Fig. 1E showed the maximum excitation and emission wavelength of 450 nm and 535 nm, respectively. 3.3. Feasibility assay of the study In order to confirm the feasibility of the proposed strategy, we monitored the change of fluorescence signal of DNA-AgNCs at the
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3.5. Fluorescence detection for Cys
Fig. 2. Fluorescence spectra of DNA-AgNCs in the presence of 500 nM Cys.
presence of Cys. As given in Fig. 2, the fluorescence signal of samples decreased at the presence of 500 nM Cys. This result suggested that the addition of Cys can efficiently interact with DNA-AgNCs and cause DNA release after forming Ag-S bonds. The change of fluorescence signal of DNA-AgNCs in the presence of Cys can be produced through excited state reaction, collisional interaction (dynamic), static quenching or both or even through monomolecular mechanisms where fluorophore itself or other absorbing species attenuates the fluorescence intensity. In order to further explore the mechanism of silver cluster quenching, we measured the lifetime of DNA-AgNCs before and after Cys addition by using single photon counting (TCSPC) technique. According to the fluorescence decay curves (Fig. S2A), we found that both of the life time of DNAAgNCs in the absence and presence of Cys were 2.70 ± 0.26 ns. This data, which is consistent with previous report [1], confirmed the characteristic of static quenching of DNA-AgNCs. Meanwhile, no obvious red or blue shift of the maximum emission wavelength upon addition of Cys was observed as previously reported[1,28], which was caused by the change of the microenvironment of the DNA template. This result indicated that the interaction of DNA-AgNCs with Cys did not produce significant effect on the microenvironment of DNA template, which reversely improved the practicability of this kind of material. However, the reason should be further explored.
3.4. Optimization of detection conditions Temperature often decreases the fluorescence signal of DNAAgNCs by affecting the number and enrichment way of silver atoms on DNA [30]. By monitoring the effect of temperature on the signal intensity of DNA-AgNCs, we found that the fluorescence intensity of DNA-AgNCs decreased as the temperature increased from 25 ◦ C to 37 ◦ C (Fig. 3A) and Cys caused the maximal signal change of the DNA-AgNCs at 30 ◦ C (Fig. S3). Thus, this temperature was chosen as detection temperature in the following studies. In addition, the effect of pH on the fluorescence signal of DNA-AgNCs and the interaction of DNA-AgNCs with Cys were investigated, which were shown in Fig. 3B. From this figure, we found that pH was an important determine factor of the fluorescence signal of DNA-AgNCs and the alkaline solution (pH >7.5) led to the destruction of DNA-AgNCs, followed by the rapid fluorescence signal decrease. However, the fluorescence intensity of DNA-AgNCs kept stable at the condition of pH 6–7. Moreover, Cys caused the maximal signal change of DNAAgNCs at pH 6 (Fig. S4). Thus, pH 6 and 30 ◦ C were chosen as optimal conditions for the following Cys assay.
Under the optimal conditions, we monitored the fluorescence signal of DNA- AgNCs at the presence of different concentrations of Cys. From the wavelength scan curves of Fig. 4A, it was found that the fluorescence signal intensity of DNA-AgNCs at 535 nm gradually decreased with the increase of Cys concentration. We further investigated the relation of Cys concentration with fluorescence value of DNA-AgNCs and the result was shown as Fig. 4B. In this figure, F0 represents the fluorescence value of DNA-AgNCs, F1 represents the fluorescence value of DNA-AgNCs after adding Cys. This figure clearly indicated that the ratio increased as the concentration of Cys increase, which reflected the more fluorescence reduction. From this figure, it was found that the concentration of Cys in 0.1–100 nM (R2 = 0.991) has a linear relationship with the ratio of (F0 -F1 )/F0 and the detection limit reached 0.05 nM. By comparing the Cys detection limit of previously reported literatures, the new method showed the ultra-high sensitivity (at least 40-fold) due to the significant improved fluorescence signal of the new material (Table 1). 3.6. Selectivity and stability of the strategy The response of DNA-AgNCs to other amino acids in complex biosamples should be carefully considered before the method was used for biosample assay. Using Cys as a positive control, no significant change of the fluorescent signal of DNA-AgNCs was found at the presence of other amino acid (Fig. 5A), which indicated the high selectivity of DNA-AgNCs for Cys assay. In addition, we tested the stability of DNA-AgNCs in order to confirm the reliability of the new method for clinic use in a long period. From Fig. 5B, it was found that the fluorescence intensity of DNA-AgNCs kept stable at least for 7 days. Meanwhile, the assay fluorescence signal of DNA-AgNCs can keep stable at least 5 h at the presence of Cys (Fig. S2B). These data fully demonstrated the guarantee of this kind of nanomaterial for clinical diagnosis. 3.7. Cys detection in human serum Considering the superior sensitivity and specificity of the new method in the homogenous solution, we then explored its capability for Cys assay in the complicated samples. First, we evaluated the resistance capability of the DNA template to deoxyribonuclease I (DNase I, 25 ng/mL), the main unspecific nuclease of biosamples by monitoring the fluorescent change of DNA-AgNCs. As our expected that no fluorescence change of this nanomaterial was observed when the DNA-AgNCs was exposed to DNase I. This data further confirmed that the local high concentrations of metal ions around DNA-AgNCs can efficiently inhibit DNase I activity through steric hindrance effect. In the following experiment, various amounts of Cys in the 1% fatal bovine serun (FBS) solution were mixed with DNA-AgNCs and the fluorescence signals were measured to construct a standard curve for Cys assay under complicated conditions. Fig. S5 indicated that the standard curve for Cys obtained in serum were similar to that obtained in the buffer. This result demonstrated that the method can efficiently avoid disturb of other components. We also investigated the accuracy of the new method for Cys assay in the serum using standard addition method. The result in Table 2 showed that the recovery of Cys was about 101.3% to 104.2% and all relative standard deviations (RSD) were less than 10%. These results clearly confirmed the high accuracy and good anti-interference ability of the new method for Cys assay under complicated conditions. Finally, the method was used DNA-AgNCs to detect thiol compounds in serum samples. Fig. 6 showed that the concentration of thiol compounds of tested samples varied in some extent due to the personal difference. The average concen-
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Fig. 3. (A) Fluorescence decrease of DNA-AgNCs in the presence of Cys (gray bar) at different temperature. (B) Fluorescence decrease of DNA-AgNCs in the presence of Cys (gray bar) at different pH. The concentration of Cys and DNA-AgNCs were 100 nM and 500 nM respectively.
Fig. 4. (A)The wavelength scan curves of DNA-AgNCs in the presence of Cys (0–1000 nM). (B) Plots of the ratio ((F0 –F1 )/F0 ) as a function of Cys concentration. The inset showed the linear region of Cys. F0 and F1 represent the florescence intensity of DNA-AgNCs in the absence and presence of Cys at 535 nm, respectively. Table 1 Comparison of the linear range and detect limit for Cys using other methods. Methods
Linear range
Detection limit
Reference
Oligonucleotide-stabilized fluorescent silver nanoclusters Combination of graphene oxide and DNA metallization Ag/Au bimetallic nanoclusters Cu2+ ensemble Colorimetric assay based on DNA-Ag/Pt nanoclusters DNA-functionalized silver nanoclusters DNA-AgNCs
8–100 nM 0–1000 nM 0.25–7 M 5–200 nM 0.5–4.5 M 0.1–100 nM
4 nM 2 nM 111 nM 2 nM 0.134 M 0.05 nM
[1] [3] [31] [32] [28] this study
Table 2 Determination of thiol compounds in human serum. Sample
Determined thiol compounds (M)
Added Cys (M)
Measured (M)
Recovery (%)
RSD (n = 3%)
1
15.5 15.5 10.7 10.7 59.3 59.3
20.0 40.0 20.0 40.0 20.0 40.0
36.1 56.6 32.0 52.5 80.3 101.3
101.7 102.0 104.2 103.6 101.3 102.0
5.77 8.10 7.86 4.79 4.37 3.47
2 3
tration of thiol compounds was 18.49 M in 14 samples. We also adopted the clinical colorimetric method to confirm the reliability of the new method and the result indicated that the average concentration of these samples of 13.41 M, the average concentration
of which was slightly lower than that of DNA-AgNCs assay (Fig. S6). However, it should be noted that the relative level of these samples was wholly similar between the two methods. As to the reason of concentration difference existed between the two methods, it can
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Fig. 5. (A) The specificity analysis of DNA-AgNCs. Fluorescence response profiles of DNA-AgNCs toward amino acid (1 M). (B)The fluorescence value of DNA-AgNCs change against the time.
that can be used for fast and low cost pre-clinical diagnosis of various related diseases. Acknowledgments This work was partially supported by the Natural Science Foundation of China (81374062, 81673579 and 31672457), the Natural Science Foundation of Hunan Province (h14JJ2049), Hunan Province Universities 2011 Collaborative Innovation Center of Protection and Utilization Of Hu-xiang Chinese Medicine Resources and the National Standardization Project of Traditional Chinese Medicine (Grant ZYBZH-Y-HUN-23) Appendix A. Supplementary data
Fig. 6. The detection results of thiol compounds in human serum using DNA-AgNCs. The blue bars stand for the concentration of thiol compounds. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
be attributed to the difference of treatment condition of samples, reagent and detect equipment. The further experiments are in processing. In summary, these results suggested that the simple and low cost method showed the applicability for the thiol compounds assay of serum especially in those developing countries. 4. Conclusions In this paper, an enhanced silver cluster nanomaterial with strong fluorescence signal was synthesized through optimization of DNA template. By using the fluorescence quenching capability of Cys to silver cluster through the interaction between silver ions and Cys thiol groups, we developed a simple method for Cys detection. The method showed a linear detection range of 0.1–100 nM for Cys with a detection limit of 0.05 nM. In addition, we successfully demonstrated the discriminability of silver cluster probe for Cys under complicated conditions. Furthermore, the method showed solid reliability for detecting Cys/thiol compounds of serum. In our point, the simple method for Cys with high sensitivity and low cost will provide great opportunities for Cys-related disease diagnosis, prognosis and response to treatment. Assuming that the method can be combined with point-of-care testing high throughput diagnostic equipment, the detection of Cys allows for feasible system
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Biographies Wenmiao Wang received her B.E degree from School of Life Science and Technology, Central South University of Forestry and Technology. She is commencing her master study in College of Biology, Hunan University from 2016 under the guidance of Prof. Bin Liu. Her research focus on the molecular diagnosis and the construction of drug delivery system. Jian Li obtained his M.M. degree from Xiangya School of Medicine, Central South University during which time he spent three years on laboratory medicine. He is commencing his Ph.D study in the Third Xiangya Hospital, Central South University from 2016 under the guidance of Prof. Xinmin Nie. His research interests lie in new method for clinical diagnosis and the construction of nano-drug carrier. Jialong Fan earned his B.S degree from School of Life Science and Technology, Central South University of Forestry and Technology. He is currently a MSBE candidate under the supervision of Profs. Chunyi Tong and Bin Liu in College of Biology, Hunan University. His current research interests include biosensing assay. Weining Ning received her B.E degree from School of Biological Engineering, HuaiHua University. She is commencing her master study in College of Biology, Hunan University from 2016 under the guidance of Profs. Chunyi Tong and Bin Liu. Her research focus on the synthesis and application of new type of sliver nanomaterials. Bin Liu is currently an associate professor in Hunan University in PR China. He received his PhD degree in Analytical Chemistry from Hunan University at 2007. From 2007 to 2009, he was a post-doctor and research associate in Internal Medicine School, Health Sciences Center, Texas Tech University. His major research interests focus on the biosensors and nanotheranostics. Chunyi Tong is currently an associate professor in Hunan University in PR China. He received his B.S. and PhD degree in Analytical Chemistry from Hunan University in 2003 and 2008, respectively. From 2016–2017, he was a visiting scholar at the University of Pennsylvania. His major research interests focus on biosensors and nanotheranostics.