Poly(thymine)-Templated Copper Nanoparticles as a Fluorescent

Jun 26, 2015 - However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed...
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Poly(thymine)-Templated Copper Nanoparticles as a Fluorescent Indicator for Hydrogen Peroxide and Oxidase-Based Biosensing Zhengui Mao,§ Zhihe Qing,§ Taiping Qing, Fengzhou Xu, Li Wen, Xiaoxiao He,* Dinggeng He, Hui Shi, and Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha, Hunan 410082, P. R. China S Supporting Information *

ABSTRACT: Biomineralized fluorescent metal nanoparticles have attracted considerable interest in many fields by virtue of their excellent properties in synthesis and application. Poly(thymine)-templated fluorescent copper nanoparticles (T-CuNPs) as a promising nanomaterial has been exploited by us recently and displays great potential for signal transducing in biochemical analysis. However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed for H2O2 and oxidase-based biosensing by exploiting T-CuNPs as an effective signal indicator. The mechanism is mainly based on the poly(thymine) length-dependent formation of T-CuNPs and the probe’s oxidative cleavage. In this assay, the probe T40 can effectively template the formation of T-CuNPs by a fast in situ manner in the absence of H2O2, with high fluorescent signal, while the probe is cleaved into short-oligonucleotide fragments by hydroxyl radical (·OH) which is formed from the Fenton reaction in the presence of H2O2, leading to the decline of fluorescence intensity. By taking advantage of H2O2 as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and has been detected, and its practical applicability has been investigated by assay of real clinical blood samples. Results demonstrate that the proposed strategy has not only good detection capability but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications. elemental fluorescent metal nanoparticles have been intensely investigated in the past decade, such as gold nanoclusters (AuNCs),32,33 silver nanoclusters (AgNCs),34,35 and copper nanoparticles (CuNPs).11,36 Lately, as a development of nucleic acid-templated fluorescent metal nanoparticles, we have systematically screened different DNAs for the formation of fluorescent CuNPs and found that single-stranded poly(thymine) DNA can effectively template fluorescent CuNPs which emit at 615 nm with an excitation of 340 nm.36,37 Attractively, compared with other nucleic acid-templated fluorescent metal nanoparticles, poly(thymine)-templated CuNPs (denoted as TCuNPs) exibit distinctive properties: (1) Its preparation is very simple, with only the requirement for mixing several reagents; (2) the synthesis conditions are mild in room temperature, without any rigorous operations including heating/cooling, vigorous agitation, and dark treatment, resulting in good

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ecause of mild reaction conditions and high efficiencies, fluorescent inorganic metal nanoparticles have been generously synthesized by a biomineralization processe, in which biomolecules are used as templates to mediate the formation of fluorescent nanoparticles.1−17 By virtue of their strong and excellent fluorescent properties, these biomineralized metal nanoparticles have contributed to biochemical analysis, including in vitro detection18−21 and in vivo imaging.22−25 Among these biomineralized fluorescent metal nanoparticles, nucleic acids represent particularly good candidates for template systems because of their unique nanosized structure, excellent programmable properties, and rich functional groups including heterocyclic nitrogen atoms, amino groups, and phosphate groups, which can bind with specific metal ions and provide nucleation sites for metallic nanoparticles. For example, nucleic acid-templated upconversion nanoparticles (UCNPs) have been recently developed by Qu and co-workers;26,27 nucleic acid-templated quantum dots (QDs) have been intensely investigated and applied in the past few years by Ma and co-workers.12,28−31 In addition, nucleic acid-templated © XXXX American Chemical Society

Received: May 5, 2015 Accepted: June 26, 2015

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buffer (10 mM MOPS, 150 mM NaCl, pH 7.8) was prepared in deionized water and used for the synthesis of fluorescent TCuNPs. Sodium acetate buffer (50 mM sodium acetate, pH 5.1) was used to dissolve glucose-oxidase. Deionized water used in this work was prepared by a Nanopure Infinity ultrapure water system (Barnstead/Thermolyne Corp.). The buffers’ pH was determined by a pH Benchtop meter (Orion 3-Star, Thermo Scientific). All fluorescence spectra and real-time scan were measured by a fluorescence spectrophotometer (F4500, Hitachi) at 20 °C; the temperature was controlled by an aqueous thermostat (Amersham) accurate to 0.1 °C. The Ex and Em slits of the spectrophotometer were both set at 10.0 nm; the PMT voltage was set at 700 V, and the response time was set at 2 s. The detected solution of 200 μL was placed in a 0.2 × 1 cm2 quartz cuvette, and fluorescence emission spectra from 520 to 660 nm were collected with an excitation wavelength of 340 nm. Fluorescence emission images were taken by a common digital camera from a WD-9403 imaging system (Shanghai, China) with a transmitted ultraviolet irradiation. Results of electrophoresis characterization were recorded by the Tanon-2500R gel imaging system (Shanghai, China). Electrophoresis Characterization of the Cleavage of the Probe. The cleavage reaction was carried out in a 20 μL system containing 5 μM T40, 200 μM ferrous chloride, and 2.2 mM hydrogen peroxide with an incubation of 30 min at 37 °C. Then, 10 μL of resulted solution was mixed with 2 μL of nucleic acid dye (100× SYBR Gold) and 2 μL of 6× loading buffer. After staining for 10 min, 10 μL of the solution was transferred into the gel for electrophoresis. A 2% agarose gel was prepared using 1× TAE buffer (40 mM Tris-glacial acetic acid, 20 mM ethylene diamine tetraacetic acid). The electrophoresis was carried out under 100 V for 5 min. The results were recorded by the gel imaging system with ultraviolet light irradiation. Poly(thymine)-Templated the Formation of Fluorescent Copper Nanoparticles. Poly(thymine)-templated fluorescent copper nanoparticles were synthesized according to our reported method.36 In a typical procedure, the stock solutions of poly(thymine) were first diluted to desired concentrations in MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.8). Then, 2 mM sodium ascorbate and 200 μM copper sulfate were added into the poly(thymine)-contained solutions, reaching a total volume of 200 μL for each sample. Their fluorescent signals were measured. H2O2 Sensing. Generally, a detection system of 20 μL containing 5 μM T40 and 200 μM Fe2+ was used to carry out the detection of H2O2. After introducing H2O2 with different concentrations into the detection system, an incubation was continued for 30 min. Then, 172 μL of MOPS buffer, 2 mM sodium ascorbate, and 200 μM copper sulfate were orderly added into the resulted solution. After another incubation of 10 min at room temperature (20 °C), the fluorescence spectra of the mixtures were measured on the spectrophotometer. Glucose Sensing. The feasibility investigation of glucose sensing was first carried out by detecting glucose which was dissolved in water. Typically, different mixtures of 5 μM T40, 200 μM Fe2+, 0.15 mg mL−1 GOD, and 2 mM glucose were incubated for 1 h at 37 °C. The formation of fluorescent TCuNPs was subsequently triggered by addition of 172 μL of MOPS buffer, 2 mM sodium ascorbate, and 200 μM copper sulfate. The fluorescence signal of the mixtures was subsequently recorded.

repeatability; (3) the poly(thymine)-templated formation of fluorescent CuNPs is highly efficient, the reaction can happen at very low concentrations of poly(thymine); (4) its formation of fluorescent CuNPs is fast, which is completed within several minutes after the reduction beginning; (5) since copper is a significant and essential micronutrient for all living animals and plants,38−40 CuNPs in applications should be safer than other heavy-metal nanoparticles and quantum dots.41,42 Thus, due to its simplicity, high efficiency, rapidity, and hypotoxicity, the fluorescent T-CuNPs hold great potential for signal transducing as an in situ synthetic nanoprobe. However, the application of the T-CuNPs for biochemical analysis is still at an early stage since our first report on its synthesis; therefore, it is very important and encouraging to exploit it in further application. In the present study, T-CuNPs are exploited as a fluorescent indicator for hydrogen peroxide (H2O2) detection. The detection is mainly based on our finding that the formation of the fluorescent T-CuNPs is highly dependent on the poly(thymine) length; only the relatively long poly(thymine) can template fluorescent CuNPs, while the fluorescence signal is negligible practically in the presence of poly(thymine) of less than 15 bases.36 H2O2 is an important biochemical target, which plays intrinsic roles in the regulation of multiple biological processes, such as aging and carcinogenesis.43−47 In the presence of a kind of transition metal ion which is at its low oxidation state (e.g., Fe2+), H2O2 can convert to hydroxyl radical (·OH) based on Fenton reaction,48 and the products can trigger oxidative damage of DNA,49−52 resulting in the poly(thymine) probe being cleaved into mono- or shortoligonucleotide fragments which lose the ability for templating the fluorescent T-CuNPs. Thus, H2O2 can be detected by the fluorescence change of T-CuNPs. With further application, this sensing platform is exploited to construct oxidase-based biosensors because oxidase can catalyze its specific substrate to hydrolysates and H2O2.53−57 As the proof-of-concept of the proposed approach for oxidase-based biosensors, glucose is chosen as a model system to be investigated in the presence of glucose oxidase.

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EXPERIMENTAL SECTION Chemicals and Apparatus. Poly(thymine) with varying length (T15 5′-TTT TTT TTT TTT TTT-3′, T20 5′-TTT TTT TTT TTT TTT TTT TT-3′, T30 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3′, T40 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T-3′, T50 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TT-3′) was purchased from SangonBiotech Company, Ltd. (Shanghai, China) and purified by HPLC. Twenty μM stock solutions of all poly(thymine) were obtained by dissolving them in sterile deionized water. 3-(N-Morpholino) propanesulfonic acid (MOPS), copper sulfate, sodium chloride, sodium ascorbate, and other salts were commercially obtained from Dingguo Biotechnology Company, Ltd. (Beijing, China). Hydrogen peroxide, ferrous chloride, glucose, and amino acids were purchased from Aladdin Industrial Inc. (Shanghai, China). They were of analytical grade at least and used with no treatment after purchase. Glucose-oxidase was purchased from Sigama Company (Shanghai, China). Human serum (AB type) was obtained from Laite (Beijing, China). The real blood sample was obtained from a health volunteer in Xiangya Hospital (Changsha, China), and the blood sample was stored in a vacutainer-separating gel procoagulant tube. The MOPS B

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the effect of the poly(thymine) length on the signal intensity is verified by using poly(thymine) of different lengths to induce the formation of fluorescent signal. As shown in Figure 1A,

In order to make the detection more realistic, the detection of glucose from human serum was carried out. For obtaining accurate concentration of glucose, the human serum was first treated with 10 mg mL−1 glucose oxidase for 12 h at 37 °C to exhaust the intrinsic glucose. Then, glucose with varying concentrations was spiked into the treated human serum, resulting in serum samples containing glucose of affirmatory concentrations. After the preparation of serum samples, the detection conditions were optimized, and the glucose detection was carried out according to the procedure as mentioned above. Finally, the practical applicability was evaluated by testing the real clinical blood sample which was collected from a health volunteer in Xiangya Hospital (Changsha, China). After agglutination, the supernatant was centrifuged (5000 rpm, 5 min, 20 °C) to dislodge the small-size aggregates and was detected by the proposed strategy. The detection procedure was the same as mentioned above.

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RESULTS AND DISCUSSION Sensing Principle. The principle of our proposed fluorescent strategy for hydrogen peroxide and oxidase-based biosensing is illustrated in Scheme 1. Its mechanism is mainly Scheme 1. Schematic Illustration of Our Proposed Strategy for Hydrogen Peroxide and Oxidase-Based Biosensing by Using Poly(thymine)-Templated CuNPs as a Fluorescent Indicator

Figure 1. (A) Effect of poly(thymine) length on the fluorescence intensity of T-CuNPs. The concentrations of T15, T20, T30, T40, and T50 are 1333, 1000, 667, 500, and 400 nM, respectively, making the concentration of thymine the same (20 μM) for each strand. (B) Electrophoresis demonstration of the cleavage of the probe T40, which is induced by as-generated ·OH from the Fenton reaction. a: T40; b: T40 + Fe2+; c: T40 + H2O2; d: T40 +Fe2+ + H2O2. [T40] = 5 μM; [Fe2+] = 200 μM; [H2O2] = 2.2 mM.

although the amount of thymine is the same in five cases, the signal intensity decreases obviously with the decrease of the poly(thymine) length, indicating that the signal formation is highly length dependent on poly(thymine). Then, the H2O2induced cleavage of the probe poly(thymine) in the presence of Fe2+ is verified by electrophoresis. As shown in Figure 1B, after treatment with only H2O2 (lane 2) or only Fe2+ (lane 3), the bands of poly(thymine) are the same as that without any treatment (lane 1), while the band disappears after treatment with both H2O2 and Fe2+ (lane 4), which indicates that poly(thymine) can be effectively cleaved by the product of the Fenton reaction. From the fluorescence spectra (Figure 2A), in the presence of H2O2 or Fe2+, there is almost no signal change, indicating that H2O2 and Fe2+ themselves have no influence on the formation of signal, while a distinct decline of fluorescent intensity can be observed when in the presence of both H2O2 and Fe2+, indicating that the signal change is due to ·OHinduced cleavage of poly(thymine). The corresponding fluorescence emission images with UV-light excitation are also consistent with the above results (inset in Figure 2A). From the real-time monitoring of the fluorescence (Figure 2B), after adding copper ion into the detection system, the poly(thymine)-templated formation of CuNPs’ fluorescent signal is very fast, reaching saturation in several minutes. The results indicate that T-CuNPs can be used as an effective indicator in sensing, and 10 min was selected for the formation of T-CuNPs in further detection experiments. Thus, good feasibility has been demonstrated for H2O2 sensing.

based on the length-dependent formation of T-CuNPs on poly(thymine) and the hydroxyl radical (·OH)-triggered oxidative damage of DNA. In the absence of corresponding targets, the probe poly(thymine) can effectively template the formation of T-CuNPs by a fast in situ manner, and obvious fluorescent signal can be recorded. However, when in the presence of the target H2O2, ·OH can be formed from the Fe2+catalyzed Fenton reaction. The probe will then be cleaved into mono- and/or short-oligonucleotide fragments which lose the ability for templating the fluorescent T-CuNPs. Because the declining degree of fluorescence intensity is proportional to the concentration of H2O2, H2O2 can be successfully detected through the fluorescence change of T-CuNPs. In addition, by taking advantage of H2O2 as a mediator, this sensing platform is exploited for constructing oxidase-based biosensors. When in the presence of a biological substrate, an oxidation reaction can be catalyzed by its specific oxidase, accompanied by the generation of H2O2, which can lead to the subsequent change of fluorescence intensity. H2O2 Sensing. According to the principle, the feasibility of the proposed strategy for H2O2 sensing is investigated. First, C

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Figure 3. (A) Fluorescence spectra of the detection system at different concentrations of H2O2. The arrow indicates the signal changes with the increase in H2O2 concentration. (B) Relationship between the fluorescence intensity and the H2O2 concentration. Inset shows the linear response of the detection system to H2O2.

Figure 2. Feasibility verification of our proposed strategy for H2O2 sensing. (A) Fluorescence spectra of the resulted solution under different conditions. The inset is the corresponding fluorescence emission image with UV-light excitation. (B) Real-time monitoring of the formation of fluorescent signal. The arrow marks the addition of Cu2+. a: T40; b: T40 + Fe2+; c: T40 + H2O2; d: T40 +Fe2+ + H2O2. [T40] = 5 μM; [Fe2+] = 200 μM; [H2O2] = 2.2 mM.

from control substances of higher concentration and good selectivity displays for H2O2 sensing. Oxidase-Based Biosensing. Many biological substrates, which play important roles in physiological reaction and human health, can be oxidized by their specific oxidases, and H2O2 will be formed in the oxidation reaction following the chemical equation:

In order to achieve a better sensing performance, experimental conditions (probe length and Fe2+ concentration) were optimized by the ratio of signal change ((F0 − F)/F0), where F0 is the fluorescence emission intensity of the T-CuNPs at 615 nm in the absence of H2O2 and F is that in the presence of H2O2. As shown in Figures S1 and S2, Supporting Information, the poly(thymine) of 40 mer (denoted as T40) is chosen as the detection probe, and 200 μM Fe2+ is chosen to catalyze the Fenton reaction in the H2O2 sensing. After the feasibility investigation and optimization of conditions, the detection capability of the proposed strategy for H2O2 sensing is evaluated. The dynamic response range and the detectable minimum concentration of H2O2 are indentified by T-CuNPs’ fluorescence. As shown in Figure 3A, with the increase of H2O2 concentration, the fluorescence intensity decreases gradually, indicating that the cleavage of T40 is concentration-dependent on H2O2. The relationship between the concentration of H2O2 and the fluorescence intensity of TCuNPs is demonstrated in Figure 3B. There is a good linear response (R2 = 0.9934) in the concentration range from 0.55 to 110 μM (inset in Figure 3B), with a detectable minimum concentration of 0.55 μM. The standard deviation is obtained from three independent repeated experiments. In addition, the selectivity is also tested (Figure S3, Supporting Information) by detecting some control substances. There are little interference

Thus, oxidase-based biosensing will be feasible based on the produced H2O2 and the length-dependent formation of TCuNPs. Here, as the proof-of-concept of the oxidase-based biosensing, glucose is chosen as the model system to be studied. Glucose oxidase can specifically catalyze glucose to form gluconic acid and H2O2. The produced H2O2 can then trigger the subsequent cleavage reaction of the probe, leading to fluorescent signal response. First, the feasibility is tested by detecting glucose which is dissolved in water. As shown in Figure 4A, Fe2+, glucose, and glucose oxidase themselves have little influence on the formation of T-CuNPs’ fluorescent signal, while there is an obvious fluorescence decline in the presence of Fe2+, glucose, and glucose oxidase (blue curve), indicating that the signal change is due to the glucose-triggered cascade oxidation. The cleavage of the probe T40 is further verified by electrophoresis (Figure S4, Supporting Information). The band of the probe T40 disappears only when Fe2+, glucose, and glucose oxidase coexist in the detection system, demonstrating that the signal change is traceable to the target-induced cleavage of the probe. Simultaneously, the selectivity of the oxidase-based glucose sensing is investigated by detecting different carbohydrates and other control substances (Figure 4B). Good selectivity is exhibited for the target glucose. Therefore, these results are D

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Figure 5. (A) Fluorescence spectra of the detection system at different concentrations of glucose. The arrow indicates the signal changes with the increase in glucose concentration. (B) Relationship between the fluorescence intensity and the glucose concentration. Inset shows the linear response of the detection system to glucose.

mM. Because the glucose concentration of diabetes patients is at a level ≥7 mM,58,59 the detection capability of this strategy can meet the requirement for clinical application. The practical applicability of the present strategy is tested by detecting glucose from real clinical blood samples. The real whole blood was collected from a healthy volunteer. After agglutination and centrifugation, the upper serum and three glucose-spiked serums are assayed following the proposed strategy and glucometer. The results are shown in Table 1. The

Figure 4. Feasibility verification of our proposed strategy for glucose sensing. (A) Fluorescence spectra of the resulted solution under different conditions. The inset is the corresponding fluorescence emission image. a: T40 + Fe2+; b: T40 + Fe2+ + glucose; c: T40 + Fe2+ + glucose oxidase; d: T40 + Fe2+ + glucose + glucose oxidase. [T40] = 500 nM; [Fe2+] = 200 μM; [GOD] = 0.15 mg mL−1; [glucose] = 2 mM. (B) The selectivity of the strategy for glucose sensing. Glucose is at a concentration of 2 mM; other control substances are at a concentration of 5 mM.

Table 1. Detection of Glucose from Real Blood Samples

accordant with our proposed sensing mechanism and demonstrate that there is a good potential for selective oxidase-based biosensing. Then, glucose in human serum was detected by the strategy. In order to achieve a better sensing performance, the concentrations for the probe T40, glucose oxidase, and Fe2+ are optimized by the ratio of signal change ((F0 − F)/F0), where F0 is the fluorescence emission intensity of the T-CuNPs at 615 nm in the absence of glucose and F is that in the presence of glucose. As shown in Figures S5, S6, and S7, Supporting Information, experimental results demonstrate that a concentration of 1000 nM for T40, 0.4 mg mL−1 for glucose oxidase, and 600 μM for Fe2+ can provide better performance for the sensing system, which are chosen as the optimal conditions for further detection. After optimizing the experimental conditions, we measure the fluorescence emission spectra of T-CuNPs in the presence of glucose at varying concentrations (Figure 5A). There is continual decreasement in fluorescence intensity with an increase of the glucose concentration, which indicates that the signal change is concentration-dependent on glucose. The plot of fluorescence intensity versus glucose concentration is shown in Figure 5B; the linear response range is from 0.05 to 1.6 mM (inset in Figure 5B), with a detectable minimum concentration of 0.05

sample

spiked (mM)

1 2 3 4

0 2 4 8

a

detected (mM) (meana ± SDb) 4.80 7.08 8.87 11.52

± ± ± ±

0.0800 0.0340 0.2120 0.4900

glucometer (mM) (meana ± SDb)

recovery (%)

± ± ± ±

114.0 101.8 84.0

4.93 7.23 9.36 12.70

0.1500 0.1150 0.0580 0.1000

Mean value of three determinations. bStandard deviation.

concentration of glucose in serum determined by the proposed strategy is 4.80 ± 0.0800 mM that is in accordance with the fact that the concentration of glucose is 3.9−6.1 mM in normal physiological plasma.58,59 The results determined by our proposed strategy are very close to that provided by the glucometer. Thus, these results indicate that it is feasible and reliable for the proposed strategy to be used in practical applicability, such as the assay of real clinical samples.

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CONCLUSIONS In summary, by taking advantage of its simplicity in synthesis and good optical properties, T-CuNPs has been first exploited as an effective signal indicator for constructing a new fluorescent strategy for H2O2 sensing. The sensing mechanism E

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is mainly based on the poly(thymine) length-dependent formation of T-CuNPs and the oxidative damage of DNA probe triggered by hydroxyl radical (·OH) which is produced from the Fenton reaction in the presence of H2O2. The results demonstrate that the sensing strategy is simple, cost-efficient, sensitive, and selective. More meaningfully, the sensing strategy may be exploited as a platform for selective detection of other biological substrates (e.g., glucose, choline, cholesterol, xanthine, and lactic acid) based on specific oxidase-catalyzed oxidation reaction, from which H2O2 can be formed.53−57 As the proof-of-concept of the oxidase-based biosensing, glucose in human serum is chosen as the model system to be investigated. The good detection capability is achieved. In addition, the practical applicability is demonstrated by the assay of a real clinical blood sample. Therefore, we believe that this proposed strategy may pave a new way to develop simple, low-cost, and effective sensors for biochemical and clinical applications.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

* Supporting Information S

More experimental results and figures as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01700. Corresponding Authors

*E-mail: [email protected]. Phone: +86-731-88821566. Fax: +86-731-88821566. *E-mail: [email protected]. Phone: +86-731-88821566. Fax: +86-731-88821566. Author Contributions §

Z.M. and Z.Q. as the cofirst authors contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the Project of Natural Science Foundation of China (Grant Nos. 21175039, 21322509, 21305035, 21305038, 21190044, and 21221003), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20110161110016), and the project supported by Hunan Provincial Natural Science Foundation and Hunan Provincial Science and Technology Plan of China (Grant No. 2012TT1003).

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