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Poly(Thymine)-Templated Fluorescent Copper Nanoparticles for Ultrasensitive Label-Free Nuclease Assay and Its Inhibitors Screening Zhihe Qing,† Xiaoxiao He,† Taiping Qing, Kemin Wang,* Hui Shi, Dinggeng He, Zhen Zou, Lvan Yan, Fengzhou Xu, Xiaosheng Ye, and Zhengui Mao State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, P. R. China ABSTRACT: Noble-metal fluorescent nanoparticles have attracted considerable interest on account of their excellent properties and potential applicable importance in many fields. Particularly, we recently found that poly(thymine) (poly T) could template the formation of fluorescent copper nanoparticles (CuNPs), offering admirable potential as novel functional biochemical probes. However, exploration of poly T-templated CuNPs for application is still at a very early stage. We report herein for the first example to develop a novel ultrasensitive label-free method for the nuclease (S1 nuclease as a model system) assay, and its inhibitors screening using the poly T-templated fluorescent CuNPs. In this assay, the signal reporter of poly T of 30 mer (T30) kept the original long state in the absence of nuclease, which could effectively template the formation of fluorescent CuNPs. In the presence of nuclease, poly T was digested to mono- or oligonucleotide fragments with decrease of fluorescence. The proposed method was low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. The assay process was very rapid with only 5 min for the formation of fluorescent CuNPs. The capabilities for target detection from complex fluids and screening of nuclease inhibitors were verified. A high sensitivity exhibited with a detectable minimum concentration of 5 × 10−7 units μL−1 S1 nuclease, which was about 1−4 orders of magnitude more sensitive than the developed approaches. ue to their strong and robust fluorescence emission for use for in vitro biochemical sensing and in vivo imaging, few-atom noble-metal nanoparticles have been developed in the past decade as ideal alternatives to organic dyes and quantum dots.1−8 For example, fluorescent gold nanoparticles and platinum nanoclusters were applied for fluorescence sensing of proteins and bioimaging of cancer cells.9−12 By virtue of its unique nanosized structure, excellent programmable properties, and great affinity for some noble-metal ions, deoxyribonucleotide (DNA) has attracted special research interest and has been screened as a template for fluorescent metal nanoparticles through the binding of metal ions on the DNA and subsequent chemical reduction of the DNA-complexed metal ions.5,7,8,13,14 These DNA-templated fluorescent nanoparticles display high photostability and tunable fluorescence emission that are suitable for diagnostic techniques of biochemical concern.15−22 For example, cytosine-rich DNA has been screened to be able to serve as an ideal template for the formation of highly fluorescent silver nanoclusters, 19,23,24 which have been successfully applied for single nucleotide mutation identification, ion detection, small molecule detection, protein detection, cancer cell detection, in vivo imaging, and ion-tuned logic device.18−22,25,26 Lately, as a development of DNA-templated fluorescent nanoparticles, we have found that single-stranded poly(thymine) (poly T) DNA can template fluorescent CuNPs.14

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The CuNPs formation templated by poly T is due to high affinity between thymine and Cu2+, and the thymine-complexed Cu2+ is chemically reduced to Cu0 by the reducing agent along the shape of the poly-T scaffold. Because of the quantum size effect,27−29 poly T-templated CuNPs can be excited with a maximum absorption wavelength (340 nm). Interestingly, the poly T-templated fluorescence produced is highly efficient and completes within several minutes after the reaction beginning under ambient conditions, which is much faster and more convenient than the synthesis produced by other fluorescent metal nanoparticles, such as silver nanoclusters (24 h in the dark at 4 °C),8 gold nanoclusters (2 days),2 and platinum nanoclusters (stirred for two weeks).12 Thus, the poly Ttemplated CuNPs is more propitious to in situ synthesis as a fluorescence probe for biochemical analysis. In addition, the MegaStokes shifting (275 nm) of fluorescent CuNPs (λex = 340 nm, λem = 615 nm) maybe provided an opportunity for detection of the target from complex biological media, as the MegaStokes shifting of fluorophore can enable the removal of the strong background signal of complex biological systems.30,31 Moreover, the poly T-templated formation of fluorescent Received: October 16, 2013 Accepted: November 15, 2013

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nuclease onto the plastic wall in the dilution and reaction process. 3-(N-Morpholino) propanesulfonic acid (MOPS), sodium chloride, copper sulfate, and sodium ascorbate were commercially obtained from Dingguo Biotechnology Company, Ltd. (Beijing, China), they were at least analytical grade and used without further treatment. The MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.6) was used for the formation of fluorescent CuNPs. Deionized water was prepared through the Nanopure Infinity ultrapure water system (Barnstead/Thermolyne Corp.). All fluorescence measurements were carried out on a F4500 fluorescence spectrophotometer (Hitachi, Japan) equipped with an aqueous thermostat (Amersham) accurate to 0.1 °C. Excitation wavelength was set at 340 nm, the Ex and Em slits were set at 5.0 and 10 nm, respectively, with a 700 V PMT voltage. Fluorescence emission spectra were obtained by collecting the emission from 520 to 660 nm with a 0.2 × 1 cm2 quartz cuvette containing 100 μL of solution. All fluorescence emission images were recorded by a digital camera from a WD-9403 imaging system (Shanghai, China) with a transmitted ultraviolet light. Quantum Yield Determination. The quantum yields of poly T-templated CuNPs were determined by a comparative method.55 Because the dye pyrene has the same absorption wavelength (340 nm) as poly T-templated CuNPs, pyrene in ethanol was used as a reference (Φr = 0.65).56 To avoid inner filter effects, concentrations of the reference and CuNPs were regulated to yield an approximate absorption intensity (less than 0.05). An excitation wavelength of 340 nm was used, and fluorescence emission intensity was integrated from 360 to 660 nm. The quantum yields were calculated from the following formula:

CuNPs is highly length-dependent. Only the relatively long poly T can template fluorescent CuNPs formation, while the fluorescence induced by poly-T of less than 15 bases is negligible practically, which holds an immense potential of providing a novel approach for biochemical sensing based on length-change of nucleic acid. Nucleases can catalyze the cleavage reactions by hydrolyzing nucleic acid into mono- or oligonucleotide fragments.32−38 They are involved in a series of cellular functions, such as DNA replication, transformation, recombination, repair, and selfprotection.39−43 Besides their significant cellular functions, nucleases have also been widely employed as useful tools in biotechnology, including site-directed mutagenesis, genotyping, restriction mapping, molecular cloning, DNA sequencing, and even as therapeutic agents.34,44,45 Due to their intrinsic biological importance and use in a wide range of applications, development of sensitive methods for nuclease activity assay is essential in the fields of molecular biology, biomedicine, nanoscience, and biosensing. Many traditional methods for nuclease assays have been developed, such as polyacrylamide gel electrophoresis (PAGE), high-performance liquid chromatography (HPLC), radioactive labeling, and enzyme-linked immunosorbent assay (ELISA),46−49 which suffer from timeintensive, cost-expensive, laborious, and isotope labeling. Thus, it is still highly important and desirable to develop rapid, lowcost, efficient, and amenable strategies for assessing nuclease activity. The fluorescent method has attracted much attention due to its high sensitivity and fast analysis speed. However, most developed dye-based fluorescent methods for assessing nuclease activity required complex labeling or sophisticated synthesis processes.37,50−54 To address these challenges, based on our finding of poly T-templated fluorescent CuNPs, a labelfree and ultrasensitive fluorescent method for nuclease activity assay was proposed in this work. As the proof-of-concept of our approach, S1 nuclease was chosen as a model system to be investigated. A relatively long poly T DNA of 30 mer (T30) was used as a signal reporter. Because the poly T-templated formation of fluorescent CuNPs was highly length-dependent, the relatively long poly T could template the formation of CuNPs with bright fluorescence in the absence of nuclease. However, poly T would undergo enzymatic digestion to monoor short-oligonucleotide fragments in the presence of nuclease and then failed to template fluorescent CuNPs. Thus, the nuclease activity could be identified by CuNPs’ fluorescence changes.

∫ Is A r ⎛ ns ⎞2 Φs = ⎜ ⎟ Φr ∫ Ir A s ⎝ nr ⎠ Where the subscripts s and r denote sample and reference; Φ, ∫ I, A, and n are fluorescence quantum yield, integrated fluorescence emission intensity, absorbance, and refractive index of the solvent, respectively. In this case, ns = 1.333 and nr = 1.200. Nuclease Assay by Fluorescent CuNPs. First, S1 nuclease was diluted to corresponding low concentrations using the S1 nuclease buffer. Twenty microliters reaction system containing 2.5 μM T30 and S1 nuclease of various concentrations was used to carry out the enzymatic cleavage reaction. After incubation for a corresponding time at 37 °C in the PCR tube, the cleavage reaction was stoped by heating at 95 °C for 5 min. Finally, 80 μL MOPS buffer was mixed with the prepared solutions, 5 μL sodium ascorbate of 100 mM, and 2 μL of copper sulfate of 10 mM were introduced and triggered the formation reaction of fluorescent CuNPs; after another incubation of 5 min, the fluorescence spectra and emission images of the mixtures were recorded at room temperature (20 °C). Dynamic Monitoring of Cleavage Reaction. Twelve PCR tubes containing 2.5 μM T30 and 0.05 units μL−1 S1 nuclease in 20 μL reaction system stayed at 37 °C for 0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, and 30 min, respectively. After incubation for a predetermined period, the cleavage reaction was ended by heating at 95 °C for 5 min. Then, the formation reaction of fluorescent CuNPs was carried out by subsequent addition of MOPS buffer, sodium ascorbate, and copper sulfate. After

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EXPERIMENTAL SECTION Chemicals and Apparatus. Oligonucleotides with specific length (T10 5′-TTT TTT TTT T-3′; T15 5′-TTT TTT TTT TTT TTT-3′; T20 5′-TTT TTT TTT TTT TTT TTT TT-3′; T25 5′-TTT TTT TTT TTT TTT TTT TTT TTT T-3′; T30 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3′) were synthesized by SangonBiotech (Shanghai) Company, Ltd. and purified by HPLC. Ten micromolar stock solutions of all poly T were obtained by dissolving them in sterile deionized water. RPMI 1640 cell medium was also obtained from SangonBiotech (Shanghai) Company, Ltd. S1 nuclease (100 units μL−1) and BSA were purchased from Thermo Fisher Scientific Inc. The S1 nuclease buffer (2 mM NaAc, 15 mM NaCl, and 0.1 mM ZnSO4, pH 4.5) was used for S1 dilution and enzymatic digestion reaction. 0.5 mg mL−1 BSA was mixed into the S1 nuclease buffer to reduce the absorption of S1 B

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further incubation for 5 min, the fluorescence spectra and emission images of the resulting solutions were recorded at room temperature (20 °C). The inhibition experiments were the same as the above procedure, except for 5 min preferential incubation of inhibitor with S1 nuclease before the addition of T30. Preparation of Complex Samples. Analysis of the target from complex samples was usually used to test the practical application capability of the proposed method. In the work, RPMI 1640 cell medium was used as the complex fluid and spiked with S1 at different concentrations. Because S1 is a Zn2+dependent enzyme, 0.2 mM zinc acetate was added into the cell medium to protect the enzyme activity. Subsequently, S1 detection was carried out by the proposed method and the recovery was calculated.

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RESULTS AND DISCUSSION Principle Design. By taking advantage of the fact that poly T-templated formation of fluorescent CuNPs is highly lengthdependent with an immense potential application for biochemical sensing, a novel fluorescent method for nuclease assay based on poly T-templated CuNPs was proposed in this work. The sensing mechanism of the method was shown in Scheme 1. When in the absence of nuclease, the signal reporter Scheme 1. Schematic Illustration of the Label-Free Strategy for Nuclease Assay Based on Poly T-Templated Fluorescent CuNPs

Figure 1. (A) Fluorescence spectra of CuNPs templated by 500 nM poly T with different length. (B) Fluorescence spectra of CuNPs templated by different resulting digestion solutions of T30. [S1] = 0.05 units μL−1.

failed to induce fluorescence practically, which maybe provide an ultrasensitive method for nuclease assay without requirement for thorough digestion of nucleic acid probe to monooligonucleotide or very short fragments. Then, 20 μL of nuclease buffer containing 2.5 μM T30 and 0.05 units μL−1 S1 nuclease was used to investigate the enzymatic cleavage reaction. After cleavage for 30 min and subsequent heating at 95 °C for 5 min to terminate the reaction, MOPS buffer, copper sulfate, and sodium ascorbate were added to induce fluorescent CuNPs formation. The resulting reaction solutions were studied by fluorescence measurements. As shown in Figure 1B, the solution in the absence of S1 nuclease produced obvious fluorescence in the spectra region of about 615 nm with UV excitation of 340 nm (black curve), while the fluorescence of the solution containing S1 nuclease was negligible (red curve). We aimed to further test whether the S1 protein itself brought influence on the fluorescence of CuNPs. The denatured S1 nuclease (heating at 95 °C for 5 min) was used for the T30 cleavage reaction instead of the active S1 nuclease, the resulting solution exhibited high fluorescence (green curve), which was almost equivalent to that without S1 nuclease (black curve). Thus, we concluded that fluorescence change of CuNPs was due to the cleavage reaction of T30 catalyzed by S1 nuclease. As shown in Figure 2, the fluorescence intensity as a function of cleavage time was also investigated as the dynamic monitoring of the T30 digestion reaction. The emission maximum of CuNPs at 615 nm was gradually decreased with the cleavage time from 0 to 30 min. It should be noted that the fluorescence decreased quickly in the first 6 min (over 80% decline), which potentially indicated that the proposed method was fast and ultrasensitive to the nuclease. Thus, the excellent feasibility demonstrated

poly T kept the original long state, which could effectively template the formation of fluorescent CuNPs by addition of sodium ascorbate and copper sulfate. However, in the presence of nuclease, the poly T cleavage reaction was triggered, yielding mono- or small oligonucleotide fragments, which failed to template the formation of fluorescent CuNPs. Thus, through the fluorescence change of CuNPs, nuclease activity might be successfully identified. Feasibility Verification of the Proposed Method. Following the design, the feasibility of the proposed method for nuclease assay was investigated. First, a series of poly T with different lengths were tested for templating fluorescent CuNPs whose quantum yields and fluorescence intensity signals were recorded. As a result, the fluorescence quantum yields decreased with a decreasing length of poly T, reaching a value of 0.068 for T30-templated CuNPs and a value of 0.031 for T20-templated CuNPs representatively. The fluorescence intensity signals were shown in Figure 1A and very sensitive to the length of poly T; the poly-T of less than 15 bases almost C

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obtained from three repeated experiments. A good linear relationship (R2 = 0.992) in the concentration range from 5 × 10−6 to 2 × 10−3 units μL−1 was obtained and indicated as the inset in Figure 3B. Besides, it should be also noted that 5 × 10−7 units μL−1 S1 nuclease could be sensitively detected, with an obvious fluorescence signal. Simultaneously, compared with other types of enzymes and proteins (polymerase, ligase et al.), the proposed method was specific for nuclease (Figure 4).

Figure 2. Fluorescence intensity changes of CuNPs as a function of cleavage time. Inset: corresponding fluorescence spectra.

that the ultrasensitive label-free fluorescent assay of nuclease activity based on poly T-templated CuNPs could be carried out. Ultrasensitive Nuclease Assay Based on Fluorescent CuNPs. To evaluate the dynamic response range and detectable minimum concentration of nuclease in buffer solution by CuNPs’ fluorescence response, the effects of different concentrations of S1 on the fluorescence intensity of the CuNPs formed were recorded. As the concentration of S1 nuclease increased from 5 × 10−7 to 1 × 10−2 units μL−1, the fluorescence intensity of CuNPs was continually decreased (Figure 3A), which indicated a gradual cleavage of T30. The plot of fluorescence intensity of the CuNPs versus S1 concentration is shown in Figure 3B with a saturation point near 5 × 10−3 units μL−1 S1, and the standard deviation was

Figure 4. Selectivity of the assay system for nuclease. S1 was at a concentration of 5 × 10−3 units μL−1. Bst polymerase, Thrombin, Exo III, and E. coli ligase were at a concentration of 5 × 10−2 units μL−1. BSA was at a concentration of 5 mg mL−1.

Detection of Nuclease from Complex Biological Fluids. Most of the fluorescent dyes have a Stokes shift of only around 20 nm, which are vulnerable to the interference from the background fluorogenic molecules in complex medium. Exhilaratingly, poly T-templated fluorescent CuNPs have a MegaStokes shifting (275 nm) with excitation wavelength of 340 nm and emission wavelength of 615 nm, which maybe provide an ideal opportunity for the use of a target detection from complex biological media. In this work, RPMI 1640 cell medium was selected as the complex fluid, and the S1-spiked cell medium was used to investigate the feasibility for nuclease detection from complex biological fluids. From Table 1, we could find that the recovery and the relative Table 1. Detection of S1 Nuclease from RPMI 1640 Cell Medium added (units μL−1) −4

5 × 10 1 × 10−3 2 × 10−3

detected (units μL−1) −4

4.8 × 10 1.04 × 10−3 1.87 × 10−3

recovery (%)

SD (%), n = 3

96.08 103.84 93.58

5.14 9.51 6.28

standard deviation (RSD) for different concentrations of S1 were satisfactory, suggesting that the method was reliable and practical for the assay of the target from real complex fluids. Application for Nuclease Inhibitors Screening. As an enzymatic reaction can be weakened or prohibited by its enzyme inhibitor, some enzyme inhibitors have been utilized as drugs for disease therapy or as tools for adjusting the reaction rate in molecular engineering experiments. To demonstrate that our proposed method had potential application for nuclease inhibitors screening, the effects of some model molecules (ATP, glucose, and KCl) on S1-catalyzed cleavage reaction of T30 were investigated. As shown in Figure 5A, glucose and KCl had no influence on the digestion reaction. However, in the presence of 1.5 mM ATP, equivalent fluorescence to that without S1 was recorded, which indicated that S1 activity could

Figure 3. (A) Fluorescence spectra of the assay system at various concentrations of S1 nuclease. (B) Relationship of the fluorescence intensity of CuNPs with the S1 concentration. Inset shows the linear response of the assay system to S1 nuclease. D

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Table 2. Comparison of Our Proposed Method with Reported S1 Nuclease Detection Methods method colorimetric fluorescence fluorescence fluorescence fluorescence fluorescence fluorescence fluorescence fluorescence

tool positively charged gold nanoparticles cationic conjugated polymer and fluorescein-labeled DNA probe fluorophore and quencher-labeled molecular beacon conjugated polymer and DNA/ intercalating dye complex aggregation-induced emission (AIE) of silole G-quadruplex and protoporphyrin IX double-stranded DNA-templated copper nanoparticles C-rich DNA-templated silver nanoclusters poly T-templated fluorescent CuNPs

detection limit (U μL−1) ≈ 4.3 × 10

−3

2.8 × 10

−6

ref 36 50

≈ 3.0 × 10−3

51

2.6 × 10−6

52

≈ 7.5 × 10−3

57

4.0 × 10−5

58

3.0 × 10−4

59

≈1.0 × 10−3

60

5.0 × 10−7

this work

poly T-templated fluorescent CuNPs for biochemical application, which might pave the way to apply the fluorescent CuNPs as novel signal transducers for a wide range of fields, such as bioimaging, biomedicine, and more sensing systems.

Figure 5. The capability of the assay system for nuclease inhibitors screening. (A) Fluorescence spectra of CuNPs templated by digestion products of T30 in the presence of different inhibitors. (B) Fluorescence emission images as a function of cleavage time in the absence of inhibitor (up) and in the presence of inhibitor ATP (down). [S1] = 0.05 units μL−1, [ATP] = 1.5 mM, [glucose] = 5 mM, [KCl] = 5 mM.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-731-88821566. Fax: +86-731-88821566.

be prohibited by ATP. The inhibitory effect of ATP on S1 was further confirmed by optical imaging. From fluorescence emission images, the fluorescence emission intensity of CuNPs decreased obviously as a function of cleavage time in the absence of inhibitor (Figure 5B, up), while there was almost no change of fluorescence intensity in the presence of 1.5 mM ATP (Figure 5B, down). These results were in accordance with the reported fact that ATP was an inhibitor of S1 nuclease.36,37 Thus, our proposed method could be used not only for the label-free ultrasensitive nuclease activity assay but also for the screening of nuclease inhibitors.

Author Contributions †

Z.Q. and X.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the Key Project of Natural Science Foundation of China (Grants 21175039, 21322509, and 21190044), Research Fund for the Doctoral Program of Higher Education of China (Grant 20110161110016), and the project supported by Hunan Provincial Natural Science Foundation and Hunan Provincial Science and Technology Plan of China (Grant 2012TT1003).

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CONCLUSIONS In summary, based on the previous finding that the formation of fluorescent CuNPs could be templated by poly T and was highly length-dependent, a novel ultrasensitive label-free method for nuclease assay and its inhibitors screening was proposed and verified. Our method had multifaceted advantages: (1) it was low-cost and simple in its operation without any requirement for complex labeling of DNA substrates or sophisticated synthesis of fluorescent compound, displaying the capability for making the nuclease assay popular. (2) As the excellent property of MegaStokes shifting of fluorescent CuNPs, this method was practical for the assay of the target from real complex fluids. (3) The detection process was very rapid, the formation of fluorescent CuNPs took 5 min only, presenting a promising platform for high-throughput nuclease detection and nuclease inhibitors screening. (4) The assay presented here was ultrasensitive and reliable. Table 2 summarized different S1 detection methods; our fluorescent CuNPs-based system exhibited higher sensitivity and was more cost-effective in comparison with the developed methods. Finally, this was a groundbreaking example of using the novel

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