Poly(thymine)-Templated Selective Formation of Copper

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Poly(thymine)-Templated Selective Formation of Copper Nanoparticles for Alkaline Phosphatase Analysis Aided by Alkyne-Azide Cycloaddition “Click” Reaction Dawei Yang, Zhenzhen Guo, Yuguo Tang, and Peng Miao ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00078 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017

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ACS Applied Nano Materials

Poly(thymine)-Templated Selective Formation of Copper Nanoparticles for Alkaline Phosphatase Analysis Aided by Alkyne-Azide Cycloaddition “Click” Reaction Dawei Yang,† Zhenzhen Guo,† Yuguo Tang,† and Peng Miao*,†,‡ †

CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and

Technology, Chinese Academy of Sciences, Suzhou 215163, People’s Republic of China ‡

University of Science and Technology of China, Hefei 230026, People’s Republic of China

ACS Paragon Plus Environment

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ABSTRACT: Alkaline phosphatase (ALP) is closely associated with many health disorders like liver diseases and bone diseases, which is a routine index of blood examination. In the current study, a fluorescent method for the detection of ALP is established based on poly(thymine)templated selective formation of copper nanoparticles (CuNPs) and alkyne-azide cycloaddition “click” reaction. Generally, in the presence of ALP, L-ascorbic acid-2-phosphate (AAP) is hydrolyzed and the generated ascorbic acid reduces Cu2+ to Cu+, which further catalyzes alkyneazide cycloaddition “click” reaction between two poly(thymine) segments. The two DNA fragments (18 thymine) are thus ligated, forming the DNA template (36 thymine), which is effective for the synthesis of CuNPs. Experimental results show that the fluorescence response of CuNPs increases with higher ALP concentration from 0.1 to 40 U/mL and the limit of detection is as low as 0.05 U/mL. Furthermore, the proposed method is highly selective and is capable of detecting ALP in biological fluid like serum, which suggests its wide biomedical applications.

KEYWORDS: alkaline phosphatase; copper nanoparticles; “click” reaction; DNA-templated synthesis; fluorescence assay.


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INTRODUCTION Alkaline phosphatase (ALP) is a widely distributed membrane-bound hydrolase which catalyzes the dephosphorylation process of DNA, carbohydrates, and proteins.1 It plays a crucial role in the regulation of intracellular processes like signal transduction pathways, cell growth and apoptosis.2-3 Since the level of ALP in serum is closely associated with several diseases, it has been regarded as an important indicator of human health. For example, ALP has been frequently used as a crucial diagnostic biomarker for diseases like hepatitis,4 prostatic cancer,5 and bone cancer.6 As a result, great efforts should be made to develop convenient, stable and cost-effective methods for the detection of ALP activity. A diversity of ALP assays have already been established, such as colorimetric,7-8 electrochemical,9 chemiluminescent,10 chromatographic,11 and surface-enhanced Raman scattering (SERS) assays.12 Nevertheless, these assays may need professional instrumentations and the sensitivity is always unsatisfactory. Therefore, simple and sensitive methods for ALP detection are still urgently needed. The technique of fluorescence (FL) measurement has attracted much attention due to the relatively high sensitivity, simplicity, and fast response.13-18 Recently, several fluorescent methods are developed for ALP analysis.19 He and coworkers established a sensitive analytical method based on λ exonuclease catalyzed cleavage reaction and the formation of G-quadruplex.20 Wang et al. developed a facile fluorescence turn-on approach for the detection of ALP activity utilizing resorufin.21 Cao et al. designed phosphorylated tetraphenylethylene probes with the property of aggregation-induced emission for the monitoring of ALP.22 Liu et al. reported a near-infrared (NIR) fluorescent probe for ALP trapping which was composed of a NIR-emitting fluorophore and a phosphate moieties.23 However, these strategies involve the use of expensive fluorescence dyes, which increases the cost and complexity of the


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assays. The requirement of point-of-care testing (POCT) cannot be met. Facile label-free assays using cost-effective fluorescent probes to monitor ALP activity should be investigated. Fluorescent nanomaterials have a number of unique optical merits, such as sharp and tunable emission spectra, high quantum yield, and robust photostability.24-26 Taking advantages of fluorescent nanomaterials, different ALP assays have been developed. For example, Tang et al. synthesized β-cyclodextrin-modified carbon quantum dots for ALP assay via host-guest recognition.27 He et al. recorded the fluorescence signals from single-stranded DNA (ssDNA)templated silver nanoclusters which can be inhibited by ALP triggered enzyme reactions.28 Willner’s group analyzed the activity of ALP and casein kinase (CK2) with the mechanism of CdSe/ZnS quantum dots based FRET.29 Copper nanoparticles (CuNPs) are another kind of fluorescent nanomaterials with inherent advantages of the convenient synthesis, inexpensive Cu resources and high fluorescence intensity.30-31 In addition, Cu is an essential micronutrient for all living animal, thus CuNPs are supposed to be much safer than commonly used noble metal NPs and semiconductor quantum dots in the application of ALP analysis. DNA is able to be used as the template for the synthesis or assembly of nanomaterials with the advantages of simple conformation, programmable sequence design, easy of chemical synthesis and modification, inherent molecular recognition ability and high biocompatibility.32-37 DNA-templated CuNPs have become excellent in situ nano-dyes for signal transducing and outputing. For example, Zhang et al. fabricated a label-free fluorescent method for ALP measurement utilizing the inhibition effect of pyrophosphate on the double-stranded DNA (dsDNA)-templated CuNPs.38 Liu et al. described another fluorescent sensor with dsDNA-templated CuNPs and λ exonuclease.39 Considering that the assembly of dsDNA requires extra hybridization events, the efficiency of which may not be always good


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enough, single-stranded DNA (ssDNA) is more promising materials for the fabrication of nanoscale materials. In 2013, Qing et al. pioneered a strategy for controlled preparation of CuNPs aided by specified sections of ssDNA.40 They found that the number of poly(thymine) played an important role in the formation of CuNPs. In the presence of longer poly(thymine) segment, larger CuNPs were synthesized, which had higher fluorescence quantum yield.41 Since then, ssDNA has been used as a simple scaffold for CuNPs growth, which retain excellent fluorescence property for ALP analysis.42-43 Cu+-catalyzed azide-alkyne cycloaddition (CuAAC) is considered as a typical example of “click” chemistry, which is a robust means of functionalizing biomolecules. CuAAC owns some important merits, which make it particularly attractive for biosensing. CuAAC proceeds well in aqueous medium and could be efficiently performed under physiological conditions. In addition, CuAAC is a highly selective reaction, which could also be used for modifying nucleic acids, polypeptides, and polysaccharides.44 In this contribution, we have designed a sensitive method for ALP assay based on CuAAC and ssDNA-templated CuNPs. An alkyne modified 18-nt poly(thymine) and an azide modified 18-nt poly(thymine) are designed. In the presence of ALP, L-ascorbic acid-2-phosphate (AAP) is hydrolyzed to ascorbic acid, which reduces Cu2+ to active Cu+ species. Cu+ is able to catalyze CuAAC “click” reaction.45 The two poly(thymine) fragments are thus ligated and the generated ssDNA is long enough to act as effective template for the formation of CuNPs. Compared with other existing approaches, this method exhibit several outstanding merits. First, it doesn’t require sophisticated design, fluorescence labeling or expensive precursor, which significantly reduces the complexity and cost of the detection system; second, the synthesis conditions of CuNPs are mild and the process is efficient which completes within 20 min; third, the long-wavelength


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emission is favorable for the application in complex biological samples; fourth, the reduced Cu+ can not only trigger CuAAC “click” reaction but also acts as the Cu resource for the formation of CuNPs, which simplifies the experimental procedure. The proposed method may not only have potential clinical utility in the future, but also encourages more in-depth studies and applications of ssDNA-templated CuNPs and CuAAC “click” reaction. EXPERIMENTAL SECTION Materials and Instruments. ALP, RecJf exonuclease, Nb.BbvCI nicking endonuclease, EcoRI endonuclease and Klenow fragment were received from New England Biolabs (Beijing, China). Sodium chloride (NaCl), sodium orthovanadate (Na3VO4), AAP and copper sulfate (II) were purchased from Sigma-Aldrich (USA). 3-(N-Morpholino)propanesulfonic acid (MOPS) was obtained from J&K Chemical (USA). 20 bp DNA Ladder was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Human serum samples were collected from local hospitals. Other reagents were of analytical grade and used as received. Oligonucleotides used in this work were HPLC purified, which were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). The sequences were shown in Table S1. All solutions were prepared with ultrapure water obtained from a Milli Q water purification system (Bedford, USA) with the resistivity of 18.2 MΩ. DNA storage buffer contained 10 mM Tris-HCl and 1 mM EDTA (pH 8.0). DNA reaction buffer contained 10 mM MOPS and 150 mM NaCl (pH 7.6). Fluorescence measurements were carried out on an F-4600 fluorescence spectrophotometer (Hitachi, Japan). Fluorescence emission spectra of CuNPs were recorded from 500 to 660 nm at room temperature with a 340 nm excitation wavelength. The slits for excitation and emission were set at 10 nm and 10 nm, respectively.


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Poly(thymine)-Templated CuNPs Formation. 200 µL of poly(thymine) (T36) with the concentration of 3 µΜ, 200 µL of CuSO4 (0.6 mM) and 200 µL of ascorbic acid (6 mM) were mixed and allowed to react for about 5 min in the dark at room temperature. Gel Electrophoresis Analysis. DNA samples were prepared and analyzed by polyacrylamide gel electrophoresis. The measurement was carried out in the solution (90 mM Tris-boric acid and 1 mM EDTA, pH 8.0) at 100 V for about 40 min. Subsequently, the gel was stained with ethidium bromide, which was then photographed under UV light by Gel DocTM XR+ Imaging System (Bio-Rad, USA). ALP Catalyzed AAP Hydrolyzation and CuAAC. Standard ALP solutions with different concentrations were firstly prepared, which were then incubated with 200 µL of AAP (6 mM) at 37 °C for 20 min. Then, 100 µL of T1 (6 µM) and 100 µL of T2 (6 µM) were added to the mixture. After that, 200 µL of CuSO4 was added into the mixture with the concentration of 0.6 mM. Finally, the fluorescence spectra were recorded at a fixed reaction time of 20 min in the dark. Selectivity and Practical Utility Investigation. To testify the selectivity of this biosensor, different enzymes (RecJf, Nb.BbvCI, EcoRI, and Klenow fragment) were employed in the detection system for fluorescence measurement. The concentration of ALP was 3.2 U/mL, while the concentrations of the other control enzymes were 32 U/mL. To confirm the practical utility of this biosensor in real samples, different concentrations of ALP were spiked into diluted human serum (1%). The proposed method was then challenged with these real samples.


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RESULTS AND DISCUSSION Working Mechanism. The synthesis and sensing principles are illustrated in Scheme 1. ALP hydrolyzes AAP to active ascorbic acid, which reduces Cu2+ to Cu+. Since azide and alkyne groups are modified at the 3’ end and 5’ end of two 18-nt poly(thymine) segments respectively, the resulted Cu+ catalyzes CuAAC “click” reaction and concatenated the two DNA fragments into a longer poly(thymine) sequence, which acts as an effective DNA template. In addition, the Cu+ is also used as the Cu resources for the synthesis of CuNPs in the presence of ascorbic acid and DNA template. By analyzing the fluorescence peak intensity of the formed CuNPs, initial concentration of ALP can be evaluated. The fluorescence emission is excited at the wavelength of 340 nm and the peak is around 630 nm. The mega-Stokes shift and long-wavelength emission are favorable for the elimination of interference from background of complex biological systems. However, our fluorescence spectrophotometer is not able to eliminate frequency doubling peak, which occurs frequently when using fluorescent materials with larger Stokes shift. To avoid possible interfering frequency doubling peak, fluorescence emission in the range of 500 to 660 nm is recorded.


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Scheme 1. Schematic illustration of the principle for ALP activity analysis. Confirmation of CuNPs Synthesis. To verify the feasibility of the synthesis of CuNPs, we have firstly measured the fluorescence emission spectra of CuNPs with the templates of 18-nt and 36-nt poly(thymine), respectively. When the 18-nt poly(thymine) serves as the template, the fluorescence of solution is weak (Figure 1A). However, the fluorescence of the product is enhanced significantly with the employment of 36-nt poly(thymine) (Figure 1B) and obvious red emissive fluorescence is observed (Inset in Figure 1). The phenomenon demonstrates that shorter DNA fragments are not good candidates of templates unless they are ligated forming a long enough DNA fragment (Figure 1C). In addition, the successful formation of CuNPs is further confirmed by the transmission electron microscopic examination, which shows well dispersed and homogeneous nanoparticles (Figure S1).


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Figure 1. Fluorescence emission spectra of CuNPs synthesized using the template of (A) T18, (B) T36, and (C) ligated T1 and T2. Inset shows the picture of T18 templated (left) and T36 templated CuNPs excited by a 365 nm UV lamp. CuAAC and ALP Catalyzed Dephosphorylation. We have then investigated CuAAC occurred between an azide modified 18-nt poly(thymine) named T1 and an alkyne modified 18nt poly(thymine) named T2. Before the “click” reaction, AAP and L-ascorbic acid are used, trying to convert Cu2+ into Cu+, which is the crucial catalyst for the reaction. Polyacrylamide gel electrophoresis experiments are performed to indicate the size of different DNA samples (Figure S2). In the gel image, the bands of T1 and T2 are slightly higher than 20 bp due to their modifications. In the presence of AAP, Cu+ cannot be produced to catalyze CuAAC. Therefore, T1 and T2 is not ligated and the corresponding band is similar to that of T1 and T2 samples. However, L-ascorbic acid is able to reduce Cu2+ to Cu+ for the subsequent CuAAC reaction and the formation of longer DNA fragment, which is reflected by the higher band. The size is larger than 40 bp, which is also due to the modification groups on T1 and T2. The 36-nt poly(thymine) can be used as the template for CuNPs synthesis. However, AAP cannot initial the formation of


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fluorescent CuNPs while L-ascorbic acid can, which is confirmed by the fluorescence emission spectra (Figure S3). Since ALP is able to catalyze the dephosphorylation process, converting AAP to L-ascorbic acid, by comparing the fluorescence spectra, a novel detection method for ALP can be established. In the presence of ALP, active L-ascorbic acid is formed from AAP, which produces sufficient Cu+ for the CuAAC reaction and 36-nt poly(thymine) templated formation of CuNPs. Finally, significant fluorescent response appears apparently (Figure S4). Preliminary Experiments. To achieve the best signal-to-noise level, preliminary experiments are carried out. First, the concentration of Cu2+ is an important parameter. The reduced Cu+ not only plays the catalyst role for ligation reaction, but also is used as the Cu resource for the synthesis of CuNPs. Larger amount of Cu2+ is supposed to contribute to larger fluorescence signal. The optimized value is determined to be 200 µM by comparing the peak FL intensity (Figure 2A). Second, the incubation time of ALP and AAP has been investigated. Experimental results reveal that the peak FL intensity increases from 1 min to 20 min, but decreases when the incubation time is above 20 min (Figure 2B). The trend is in good accordance with the previous report.46 Therefore, 20 min is chosen for the ALP catalyzed dephosphorylation.


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Figure 2. (A) Optimization of Cu2+ concentration for the synthesis of CuNPs (50, 75, 100, 150, 200, 250, 350, and 500 µM). (B) Effect of incubation time of ALP and AAP before the following CuAAC and DNA-templated synthesis reaction (1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, and 55 min). ALP Quantification. To probe the sensitivity of the ALP detection method, we have examined the fluorescence spectra by employing different concentrations of ALP under optimized experimental conditions. As shown in Figure 3A, the elevated concentration of ALP from 0.1 U/mL to 12.8 U/mL causes a gradual increase in the peak FL intensity. Nevertheless, ALP with the concentration higher than 12.8 U/mL does not further improve the signal intensity.


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Figure 3B shows the calibration curve that represents the detailed relationship between peak FL intensity and ALP concentration. It is observed that fluorescent signal is linearly related to ALP concentration across the range from 0.1 U/mL to 1.6 U/mL, which is suitable for quantitative analysis of ALP in biological samples. The fitting equation of the linear curve is y = 0.2059 + 0.3318 x, where y is the normalized peak FL intensity and x is the concentration of ALP (U/mL). The limit of detection (LOD) is calculated to be 0.05 U /mL based on signal to noise ratio of 3 (S/N = 3). The analytical performances of the proposed method are comparable to currently developed ALP assays (Table 1).


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Figure 3. (A) Fluorescence emission spectra of CuNPs synthesized in the presence of various concentrations of ALP: 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 20, 40 U/mL. (B) Calibration curve representing the relationship between peak FL intensity and ALP concentration. Inset shows the linear range. Error bars represent standard deviations of three independent measurements.

Table 1. Analytical performance comparison of this work with other reported ALP assays. Detection range Technique

LOD

Strategy

Ref. (U/mL)

(U/mL)

0.032 to 0.1

0.032

47

0 to 0.3

-

48

1 to 20

0.1

49

0 to 0.2

0.04

50

0 to 0.8

0.01

51

DNA and perylene probe colorimetric assay

regulated AuNPs aggregation

amperometric assay

electrochemical detection of phenol λ exonuclease catalyzed

chronocoulometry reaction supramolecular assembly fluorescent assay

between β-cyclodextrin polymer and pyrene

fluorescent assay

β-cyclodextrin modified


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quantum dots CuAAC and DNA fluorescent assay

this 0.1 to 1.6

templated CuNPs

0.05 work

Selectivity and Inhibition Investigations. To check the selectivity of this method, four enzymes including RecJf, Nb.BbvCI, EcoRI and Klenow fragment have been used as interference species for the assay. The concentration of ALP is 3.2 U/mL, while the other enzymes are 32 U/mL. Although their concentrations are ten times higher than that of ALP, the produced fluorescent responses are negligible, demonstrating the high selectivity of the ALP detection method (Figure 4). The inhibition of ALP catalysis is also studied by introducing Na3VO4, a common ALP inhibitor. The fluorescent intensities for ALP assay are compared in the presence of different concentrations of Na3VO4. As shown in Figure 5, the recorded fluorescent intensity decreases with the increase of Na3VO4, suggesting that ALP catalyzed hydrolyzation gradually decreases with more Na3VO4. Real Sample Analysis. The levels of serum ALP are always much higher in the cases of certain diseases. For example, a large-scale study conducted in peritoneal dialysis patients showed that the risk of mortality increased when ALP level was larger than 0.15 U/mL.52 To further explore the application of the method for ALP detection in complex samples, we have used this method to detect ALP in serum samples. As is shown in Figure 6, different amount of ALP are spiked in buffer and serum samples separately. Analogical fluorescent signals for the analysis of ALP concentrations are observed, verifying that the proposed method performs well in complex biological matrix.


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Figure 4. Comparison of the FL responses of the assay method towards ALP and other control proteins (RecJf, Nb.BbvCI, EcoRI, and Klenow fragment). The concentration of ALP is 3.2 U/mL and all other proteins are 32 U/mL.

Figure 5. Plot of the inhibition efficiency versus the concentration of Na3VO4.


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Figure 6. Comparison of the FL responses of the biosensor for the detection of different amount of ALP in buffer and serum, respectively.

CONCLUSIONS In summary, we have designed a novel fluorescent strategy based on poly(thymine)templated formation of CuNPs via CuAAC “click” reaction. This strategy owns several distinctive advantages for the detection of ALP. First, the synthesis conditions of poly(thymine)templated CuNPs are mild without any rigorous operations including changing reaction temperature, dark treatment, “aging” process, and vigorous stirring. Thus, good repeatability is promised; second, the formation of fluorescent CuNPs is quite fast, which meets the requirement of POCT; third, mega-Stokes shift is achieved and the nanomaterials are suitable for the application in complex biological samples; fourth, since Cu is a significant and essential


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micronutrient for all living animal, CuNPs are supposed to be much safer in biomedical applications compared with other noble metal nanoparticles and semiconductor quantum dots; fifth, in this assay, the reduced Cu+ not only acts as a catalyst to trigger CuAAC “click” reaction but also is used as the Cu resource for the formation of CuNPs, which avoids the spiking of additional reagents. In view of the above advantages, this new strategy might hold a great potential for further applications in biomedical research and clinical diagnosis.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Additional results of TEM images of CuNPs, fluorescence emission spectra with different conditions, optimization experiments and the table summarizing DNA sequences used in this work (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (P.M.); Tel: +86-512-69588279. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant no. 81771929), the National Key Instrument Developing Project of China (Grant no. ZDYZ2013-1), China Postdoctoral Science Foundation (Grant no. 2017M611911) and the Science and Technology Program of Suzhou (Grant no. SYG201605).


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Poly(thymine)-Templated Selective Formation of Copper

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