Copper-Mediated DNA-Scaffolded Silver ... - ACS Publications

Subscriber access provided by Northern Illinois University

Article

Copper-Mediated DNA-Scaffolded Silver Nanocluster on-off Switch for Detection of Pyrophosphate and Alkaline Phosphatase Jin-Liang Ma, Bin-Cheng Yin, Xin Wu, and Bang-Ce Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02465 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Copper-Mediated DNA-Scaffolded Silver Nanocluster on-off Switch for Detection of Pyrophosphate and Alkaline Phosphatase Jin-Liang Ma †, Bin-Cheng Yin*,†, Xin Wu‡, and Bang-Ce Ye*,†ξ †

Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China ξ

School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China

‡

Department of Rheumatology and Immunology, Shanghai Changzheng Hospital, The Second Military Medical University, Shanghai, 200433, China Corresponding Author *Bin-Cheng Yin, [email protected]; *Bang-Ce Ye, [email protected], Fax no. 0086-21-64252094 ABSTRACT: We present a new copper-mediated on-off switch for detecting either pyrophosphate (PPi) or alkaline phosphatase (ALP) based on DNA-scaffolded silver nanoclusters (DNA/AgNCs) templated by a single-stranded sequence containing a 15-nt polythymine spacer between two different emitters. The switch is based on three favorable properties: the quenching ability of Cu2+ for DNA/AgNCs with excitation at 550 nm; the strong binding capacity of Cu2+ and PPi; and the ability of ALP to transform PPi into orthophosphate (Pi). The change in fluorescence of DNA/AgNCs depends on the concentrations of Cu2+, PPi, and ALP. Copper (II) acts as a mediator to interact specifically with the Probe, while PPi and ALP convert the signal of the Probe by removing and recovering Cu2+, operating as an on-off switch. In the presence of Cu2+ only, DNA/AgNCs exhibit low fluorescence because the combination of Cu2+ and DNA template disturbs the precise formation of DNA/AgNCs. When PPi is added to the system containing Cu2+, free DNA template is obtained due to the stronger interaction of PPi and Cu2+, leading to a significant fluorescence increase (ON state) which depends on the concentration of PPi. Further addition of ALP results in the release of free Cu2+ via ALP-catalysis of hydrolysis of PPi into Pi, thereby returning the system to the low fluorescence OFF state. The switch allows the analysis of either PPi or ALP by observation of the fluorescence status, with the detection limit of 112.69 nM and 0.005 U/mL for PPi and ALP, respectively. The AgNCs on-off switch provides the advantages of simple design, convenient operation, and low experimental cost without need of chemical modification, organic dyes, or separation procedures.

Phosphate species, especially, pyrophosphate ion (P2O74-, PPi) formed by a condensation reaction of two inorganic phosphate units, are important in biological metabolic process, such as hydrolysis of citrate and ATP, DNA replication,1 and the generation of cyclic adenosine monophosphate (c-AMP).2 The status of PPi in living cells plays a pivotal role in some disorders, such as chondrocalcinosis and calcium pyrophosphate dehydrate (CPPD) crystal deposition.2-4 Alkaline phosphatase (ALP), as a membrane-bound enzyme, is responsible for both catalyzing the hydrolysis of PPi, and transphosphorylation and dephosphorylation of other molecules containing phosphate esters.5,6 Also, this commonly studied enzyme is involved in signal transduction and intracellular regulation in the cell cycle of growth and apoptosis.7 The abnormal expression of ALP can mirror several diseases, including prostate cancer,8 bone disease,9 liver diseases,10,11 etc. As an important indicator, ALP is one of the most extensively assayed enzymes used as a biomarker for the clinic diagnosis and therapy of some diseases. Therefore, there is a need for convenient and facile approaches to sense PPi and ALP activity.

In the past decades, a variety of strategies using colorimetry,12-14 fluorescence (organic dyes15 and CdSe/ZnS quantum dots16), surface-enhanced Raman scattering,17 and electrochemistry18 as measurable signals have been established for detecting PPi and ALP. However, they pose several challenges, such as the need for modification, discrimination against interferences, sophisticated instrumentation, and facilities for organic reactions. Therefore, the development of a cost-effective, label-free, and simple method for quantitative assessment of PPi and ALP is of great significance. Metal nanoclusters (NCs) with sizes comparable to the Fermi wavelength of electrons display molecule-scale discrete energy levels, leading to size-dependent fluorescence emission with unique characteristics different from nanoparticles.19,20 Among these molecule-like nanoclusters, silver nanoclusters (AgNCs) as attractive nanomaterials have attracted attention because of their excellent brightness and good photostability.21 In particular, DNA-scaffolded AgNCs (DNA/AgNCs), first reported by Braun and co-workers in 1998,22 are highly fluorescent, photostable, and cost-effective to synthesize, and they represent a fascinating option to produce label-free fluo-

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

rescence signals for biosensor and cellular imaging design.23-27 It is well-known that many metal ions (such as Ce4+,28 Ni2+,29 Fe3+,30 and Cu2+31,32) act as quenchers of nanocluster and quantum dot fluorescence via photoinduced electron transfer or excited state intra-molecular proton transfer. In particular, Cu2+ is utilized frequently as a mediator and quencher due to its quencher capacity and strong binding to some analytes. Based on combination of Cu2+-induced quenching and analyte-triggered recovery of molecular fluorescence, many designs have been applied for detection of various targets, such as phosphatecontaining metabolites30,31, histidine,32 and inorganic pyrophosphatase (PPase).33 In the presence of Cu2+ only, fluorescence of DNA/AgNCs is quenched. If PPi is also present, fluorescence is restored because the substrate displaces Cu2+ from interacting with DNA/AgNCs. Addition of relevant enzyme leads to the recurrence of quenching by hydrolysis of substrate binding to Cu2+, resulting in the detection of enzyme activity. For example, PPi and ALP detection based on coupling of Cu2+ and carbon quantum dots has been realized via strong Cu2+/PPi binding capacity and ALP-catalyzed removal of Cu2+/PPi complex, respectively.31 In the previous findings by our group, a single-stranded DNA containing darkish emitter domains at each terminus connected by a 15-nt polythymine spacer can act as a template to stabilize highly fluorescent DNA/AgNCs.34,35 We now have designed a novel type of copper-mediated on-off switch for PPi and ALP detection using the DNA template. The conceptual framework of the proposed method for label-free PPi and ALP detection is illustrated in Scheme 1. In the absence of PPi, the fluorescence intensity is low (OFF state), because the interaction of Cu2+ with the Probe (DNA template) disrupts the synthesis of DNA/AgNCs. Upon the addition of PPi, due to the stronger binding ability between PPi and Cu2+, free DNA template allows rigid formation of DNA/AgNCs, leading to highly fluorescent signal readout (ON state). Incorporation of ALP into the solution of Cu2+ and PPi again leads to quenching of DNA/AgNCs (OFF state) due to the recurrence of free Cu2+ via ALP-catalyzed hydrolysis of PPi into Pi. To summarize, fluorescence enhancement occurs in the presence of PPi and Cu2+, while fluorescence quenching reoccurs when ALP is also added. Since the degree of enhancement and quenching of DNA/AgNCs is directly related to the addition and consumption of PPi, this principle can be used for quantitative monitoring of either PPi or ALP activity. The proposed on-off strategy for PPi and ALP detection is label-free, low-cost, and easy to perform.

EXPERIMENTAL SECTION Reagents and Materials. The oligonucleotide used (Probe: CCCTTAATCCCCTTTTTTTTTTTTTTTCCCTAACTCCCC) was custom-synthesized by Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Silver nitrate (AgNO3) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sodium

Page 2 of 9

borohydride (NaBH4) was purchased from Tianlian Fine Chemical Co., Ltd. (Shanghai, China). Sodium pyrophosphate, bovine serum albumin (BSA), glucose oxidase (GOD), and lysozyme were purchased from Sigma Aldrich, Inc. (Saint Louis, MO, USA). ALP was purchased from Worthington Biochemical Corp. (Lakewood, USA). Trypsin was purchased from Thermo Fisher Scientific Co., Ltd. (Waltham, MA, USA). All chemicals used were of analytical reagent, obtained from commercial sources, and directly used without additional purification. The solutions were prepared using distilled water purified by a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA) with an electrical resistance of 18.2 MΩ⋅cm. Instrumentation. UV-vis absorption and fluorescence spectra were measured by a fluorescence microplate reader (Bio-Tek Instrument, Winooski, USA) using a transparent 96-well microplate and a black 96-well microplate (Corning Inc., NY, USA), respectively. Transmission electron microscopy (TEM) was performed on Jeol JEM-2100 instrument (JEOL Ltd., Japan). PPi Detection Procedure. In a typical experiment for PPi detection, a total volume of 200 μL, 500 nM Cu2+ and PPi with different concentration were added to MOPS buffer (20 mM MOPS, pH 7.0) containing 1 μM Probe solution. After incubation for 30 min at room temperature to enable sufficient combination of PPi and Cu2+, the AgNCs were prepared by adding an aliquot of 6 μM AgNO3 solution to the Cu2+/PPi/Probe reaction solution with Ag+/DNA molar ratio of 6:1. Then, the mixture was incubated in the dark at room temperature for 20 min. Subsequently, an aliquot of fresh 6 μM NaBH4 solution was added, followed by vigorous shaking for 5 s. The resultant mixture was kept in the dark at room temperature for 15 min before use. Unless noted otherwise, all experiments in this work were repeated three times. ALP Detection Procedure. A volume of 200 μL, 50 μM PPi and varying amounts of ALP were added to MOPS buffer (20 mM MOPS, pH 7.0), followed by reaction for 90 min at 37 °C according to the reported procedure with minor modification.36 After deactivation of the enzyme for 10 min at 90 °C, 500 nM Cu2+ and 1 μM Probe solution were added to the reaction system, followed by incubation for 30 min at room temperature to enable sufficient combination of PPi and Cu2+. Finally, AgNCs were prepared by the above procedure for PPi detection. PPi and ALP Detection in Bovine Serum. PPi or ALP with different concentration were spiked in 1% diluted bovine serum. The procedure for PPi detection in bovine serum was the same as that in buffer except the replacement of 30 μM Cu2+. For ALP detection in bovine serum, the detection procedure was also the same as that shown in the above experiment for ALP detection in buffer except 30 μM Cu2+ and 100 µM PPi. PPi Detection in Synovial Fluids. All synovial fluid samples and human serum samples were collected from Shanghai Changzheng Hospital. In synovial fluid samples, two were from arthritis patients while the others were from healthy controls. The human serum samples were


Page 3 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from hepatocellular carcinoma patients and healthy controls. Written informed consent was obtained from all study participants prior to enrollment, and the study was approved by the ethics committees from each institution involved. The synovial fluid samples were pretreated by loading into centrifugal filtration devices (Molecular weight cutoff or MWCO 50 kDa, Millipore Amico Ultra) to centrifuge at 6000 rpm for 20 min. For the fluorescent sensing of PPi in the synovial fluids, 10 μL 2-fold diluted synovial fluid pretreated was added to the solution containing 1 μM Probe and 5 μM Cu2+ in 20 mM MOPS (pH

7.0), incubated for 30 min. The obtained mixture was used for generation of AgNCs. To concentrate ALP, the serum samples were pretreated by loading into centrifugal filtration devices with MWCO of 10 kDa to centrifuge at 10, 000 rpm for 20 min. The subsequent operation is the same as the procedure for ALP detection in bovine serum.

Scheme 1. Schematic representation of the proposed label-free method for PPi and ALP detection using a copper-mediated on-off switch based on fluorescent DNA-scaffolded silver nanoclusters.

RESULTS AND DISCUSSION

2+

Figure 1. (A) UV−vis spectra of DNA/AgNCs before and after adding 50 μM Cu . (B) Excitation spectra of as-prepared 2+ DNA/AgNCs upon addition of Cu with four concentrations (0, 0.5, 5, and 50 μM) using emission at 604 nm (normalized to 1.0 2+ at 550 nm for 0 nM Cu ). (C) Emission spectra of DNA/AgNCs at excitation of 475 nm and 550 nm before and after adding 50


Analytical Chemistry 2+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 9

2+

μM Cu . (D) Fluorescence emission spectra of DNA/AgNCs after adding Cu at different final concentrations (from top to bottom: 0, 25, 100, 200, 400, 600, 800, and 1000 nM). The inset shows photographs of DNA/AgNCs under UV irradiation before (up2+ per) and after (bottom) adding Cu .

We first explored the operational process of quenching between Cu2+ and the proposed fluorescent DNA/AgNCs. The absorption spectra of DNA/AgNCs in the absence and presence of Cu2+ were investigated to verify the effect of Cu2+ on the fluorescence properties of AgNCs. As shown in Figure 1A, with addition of Cu2+, the absorbance at 540 nm decreases, while the absorbance at 420 nm increases, demonstrating that Cu2+ disturbs the formation of the AgNCs species absorbing at 540 nm, but contributes to the production of the species corresponding to the 420 nm peak. The observation was further verified and characterized by transmission electron microscopy (TEM), which showed that DNA/AgNCs formed by the system without Cu2+ are homogeneous with size of ~3 nm in diameter, while after adding Cu2+ there are few particles because Cu2+ disrupts the generation of DNA/AgNCs (Figure S1). In addition, we investigated the excitation spectra of DNA/AgNCs after adding different concentrations of Cu2+ using emission at 604 nm. As shown in Figure 1B, the species excited at 550 nm decreases, while another species excited at 475 nm grows, which is because the presence of Cu2+ may lead to formation of new copper/silver nanoclusters (Cu/AgNCs), 37-39 resulting in competitive coordination of Cu/AgNCs and AgNCs with the DNA probe. When the reaction system is excited at 550 nm, the emission spectrum of DNA/AgNCs shows strong fluorescence intensity at 604 nm in the absence of Cu2+, but with 475 nm excitation, emission occurs at 560 nm, and increases in the intensity in the presence of Cu2+ (Figure 1C). Consistent with the excitation spectra, Cu2+ induces the system to produce lower fluorescence intensity at 604 nm and higher (yellow) emission at 560 nm. With addition of PPi to the solution containing Cu2+, as shown in Figure S2, fluorescence was partially restored because PPi preferentially binds to Cu2+ with the higher stability constant (K) of Cu2+/ PPi complex (log KCu-PPi= 12.45),40 and effectively competes with the interaction of Cu2+ and DNA template, leading to an obvious enhanced fluorescence readout. To rule out the possibility that the fluorescence enhancement of AgNCs arises directly from PPi, we investigated fluorescence change of DNA/AgNCs before and after adding PPi with different concentrations. As shown in Figure S3, negligible alteration in the fluorescence of AgNCs with addition of PPi was observed, indi-

cating that the fluorescence enhancement of AgNCs is not due to PPi, but rather the strong affinity of PPi and Cu2+. When ALP were introduced to the solution with PPi and Cu2+ to catalyze the hydrolysis of PPi into Pi, and subsequently incubated for 90 min at 37 °C and deactivated for 10 min at 90 °C, the AgNCs fluorescence was again quenched due to the binding of liberated Cu2+ to the DNA template (Figure S2). The fluorescence of AgNCs was also measured before and after adding ALP (no Cu2+ or PPi) at different concentrations from 0.06 to 6 U/mL, there was no change in the fluorescence of AgNCs, eliminating the possibility that quenching behavior results from ALP by itself (Figure S4). To achieve the greatest fluorescence ratio (F/F0), where F and F0 are the fluorescence intensities at 604 nm in the presence and absence of PPi, respectively, three DNA/AgNCs synthesis conditions (choice of reaction buffer, and the concentrations of Cu2+, Na+) were investigated. First, by carrying out PPi detection in three widely used buffers (phosphate, Tris, and MOPS), as shown in Figure S5, MOPS buffer offers excellent conditions for obtaining the best fluorescence enhancement ratio. Using MOPS as reaction buffer, we investigated the effect of Cu2+ concentration on the assay system because the amount of Cu2+ directly affects the background signal and the concentrations of PPi need for recovery of fluorescence (i.e., the sensitivity for PPi). As indicated in Figure 1D, when Cu2+ with different concentration from 0 to 1000 nM were mixed with DNA template for preparation of DNA/AgNCs, the fluorescence intensity of DNA/AgNCs gradually decreased to a minimum value. Using the ratio of initial fluorescence intensity before adding Cu2+ to minimum after adding Cu2+, a maximum quenching efficiency was achieved up to ∼99.42%. Then the detection of 5 µM PPi was studied using four concentrations of Cu2+: 100, 250, 500, and 5000 nM. Figure 2A shows the fluorescence emission spectra with different Cu2+ concentrations in the absence and presence of PPi. As shown in Figure 2B, 500 nM Cu2+ achieved the best signal-to-background value (F/F0). The effect of Na+ was also studied, and as shown in Figure S6, the best performance was achieved in the absence of Na+. Therefore, the optimum conditions for PPi detection are as follows: operation of the reaction system in MOPS buffer using 500 nM Cu2+ in the absence of Na+.


Page 5 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


+

2+

Figure 2. (A) Fluorescence emission spectra of the reaction system containing 50 mM Na using different concentrations of Cu in the absence and presence of 5 μM PPi . (B) Relative fluorescence intensity (F/F0) responses to the different concentrations of 2+ Cu . F and F0 are the fluorescence intensities at 604 nm in the presence and absence of PPi, respectively. The error bars were calculated from three independent experiments.

Figure 3. Sensitivity investigation of the proposed AgNCs on-off switch for detection of PPi. (A) Fluorescence emission response to PPi at increased concentrations (0, 0.25, 0.5, 2.5, 5, 10, 25, 50, 100 μM). (B) Plot of fluorescence change ratio (F/F0–1) vs concentration of PPi, F and F0 are the fluorescence intensities at 604 nm in the presence and absence of PPi, respectively. Inset: plot of linear region from 0.25 to 10 μM.

Figure 4. Selectivity investigation of the proposed AgNCs on-off switch for detection of PPi. The fluorescence change ratio (F/F0−1) values in response to 5 μM samples of different targets, where F and F0 are the fluorescence intensities at 604 nm in the presence and absence of tested target, respectively.

Using the above optimized experimental conditions, PPi over a concentration range from o.25 to 100 μM was added to the reaction solution containing 500 nM Cu2+. As shown in Figure 3A, the fluorescence intensity gradually increased with an increase in the concentration of PPi from 0 to 100 μM, indicating that recovery of fluorescence proceeded in a dose-dependent response to PPi concentration. Figure 3B shows the fluorescence change ratio (F/F0–1) as a function of PPi concentration. In the linear region (0.25, 0.5, 2.5, 5, and 10 μM), the regression equation is F/F0–1=2.010[PPi] + 0.626 with a correlation coefficient R2 of 0.997. The detection limit of PPi was estimated to be 112.69 nM (3σb/slope, σb is the standard deviation of the blank samples). To assess the performance of PPi detection, we compared the assay with other nanomaterial-based methods. As shown in Table S1, the detection limit of PPi based on carbon quantum dots,29 gold nanoparticles,12 and gold nanoclusters33 are 2.56 μM, 130 nM, and 1 μM, respectively. Compared to these methods, our method was more sensitive; and it was simple and labelfree.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

To validate the specificity of the interaction of Cu2+ with PPi, we prepared several different targets including PPi, ATP, ADP, AMP, HPO42-, H2PO4-, PO43-, SO42-, CO32-, and HCO3-. Figure 4 shows the fluorescence change ratio (F/F0−1) values towards the different targets at 5 μM. Compared to other tested targets, the highest fluorescence change ratio value was observed for PPi. Also, these results demonstrate that the possible products 0f PPi hydrolyzed by ALP, such as PO43-, HPO42 and H2PO4-, do not affect the competitive assay. Thus, the degree of the fluorescence requenching upon the addition of ALP is directly related to the consumption of PPi, confirming the feasibility of an ALP activity assay based on on-off fluorescence of AgNCs. When mixed with PPi and Cu2+, ALP catalyzes the hydrolysis of PPi into Pi. According to the experimental protocol described in the Experimental Section, the sensitivity of the proposed on-off switch for ALP detection was carefully investigated. The fluorescence spectra of the sensing system were recorded upon the addition of ALP at different concentrations ranging from 0.03 to 3 U/mL (Figure 5A). As a result of the liberation of Cu2+, the fluorescence intensity gradually decreased with increasing concentration of ALP from 0 to 3 U/mL, indicating that re-quenching of fluorescence is ALP dose-dependent. To obtain the linear relationship between the fluorescence of AgNCs from Probe and the activity of ALP, the fluorescence intensity ratios (F/F0) at 604 nm were plotted as a function of ALP activity. As shown in Figure 5B, the fluorescence change ratio (F/F0) as a function of ALP concentration (0.03, 0.06, 0.18, 0.12, and 0.24 U/mL) was obtained. The linear equation is F/F0=0.985–3.791[ALP] with a correlation coefficient R2 of 0.984. The detection limit of 0.005 U/mL was achieved according to the definition of detection limit = 3σb/slope. This detection limit was comparable to that of 1.428 and 1.1 U/L31 using carbon quantum dot-based methods (Table S1). More importantly, our proposed method was first example to achieve a sensitive detection of ALP by virtue of DNA/AgNCs. Also, compared with the other methods based on copper nanoparticles (CuNPs)41 and Cu/AgNCs,38 our proposed AgNCsbased method has unique advantages of a rapid rate for AgNCs synthesis within 15 min, higher fluorescence signal, and superior sensitivity for turn-on PPi detection. To confirm the suitability and reliability of the proposed on-off switch applied in biological samples, we first investigated the analytical performance of this assay for

Page 6 of 9

PPi and ALP spiked in 1% diluted bovine serum, respectively. The complex components in bovine serum may influence the working concentration of Cu2+. Thus it is necessary to re-optimize the concentration of Cu2+. As shown in Figure S7, the best performance can be achieved at 30 μM Cu2+. Using the optimized Cu2+ concentration, PPi with different concentration in bovine serum can be detected and the regression equation is F/F0– 1=0.454[PPi]–7.905 with a correlation coefficient R2 of 0.998 (Figure S8). For the detection of ALP in 1% diluted bovine serum, using 100 μM PPi as working concentration (Figure S9), the working linear equation for ALP detection is F/F0=0.845–1.849[ALP] (Figure 6). In the spike-in experiment, quantitative detection of ALP was achieved with an acceptable recovery of 95.65∼116.40% (Table S2). These results indicate that our proposed assay has a potential to operate in biological samples. To further demonstrate the potential of application of our method in real sample, we detected PPi and ALP in synovial fluids and human serums from patients and healthy controls. The detection of PPi in synovial fluid from arthritis patients and healthy controls was performed using optimized Cu2+ concentration with 5 μM (Figure S10). To rule out the possibility that synovial fluid disturbs the fluorescence of AgNCs, fluorescence spectra of AgNCs with and without synovial fluid were compared. As shown in Figure S11, no obvious fluorescence change was observed, demonstrating that synovial fluids don’t affect the synthesis of AgNCs. Figure S12 shows the fluorescence intensity of the reaction system after adding synovial fluid samples from arthritis patients are ∼2.7 and ∼2.6 fold higher than that of healthy controls. According to the above calibration curve (F/F0–1=2.010[PPi] + 0.626), the initial value of PPi level in synovial fluids of arthritis patients was determined to be 34.74±12.02 and 30.92±13.08, respectively, which are consistent with physiologic PPi concentration in arthritis patients as measured in the previous methods (Table S3).12,42,43 When applying our method to detect ALP in human serums from hepatocellular carcinoma patients and healthy controls, quantitative results for ALP detection were successfully achieved based on the above working linear equation (F/F0=0.845– 1.849[ALP]) (Table S4). It should be note that no difference was observed in ALP levels between patients and healthy controls (Figure S13). We speculated that ALP level perhaps is not an indicator in our tested clinical samples.


Page 7 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Sensitivity investigation of the proposed AgNCs on-off switch for ALP detection. (A) Fluorescence emission spectra response in the presence of ALP at increasing concentrations (0, 0.03, 0.06, 0.12, 0.18, 0.24, 0.3, 0.6, 1.8, 3 U/mL). (B) The fluorescence ratio (F/F0) versus the concentration of ALP in the range from 0.03 to 3 U/mL, where F and F0 are the fluorescence intensities at 604 nm in the presence and absence of ALP, respectively. Inset: the linear curve between the fluorescence ratio (F/F0) value and ALP concentration ranging from 0.03 to 0.24 U/mL.

Figure 6. The sensitivity investigation of the reaction system for detecting ALP spiked in 1% diluted bovine serum. (A) Fluorescence emission spectra response in the presence of ALP at increasing concentration (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1 U/mL). (B) The fluorescence ratio (F/F0) versus the concentration of ALP in the range from 0.1 to 1 U/mL, where F and F0 are the fluorescence intensities at 604 nm in the presence and absence of ALP, respectively. Inset: the linear curve between the fluorescence ratio (F/F0) values and ALP concentration ranging from 0.1 to 0.4 U/mL. Figure 7. Selectivity investigation of the proposed AgNCs onoff switch for ALP detection. The fluorescence ratio (F/F0) values in respond to different tested targets, where F and F0 are the fluorescence intensities at 604 nm in the presence and absence of ALP, respectively. The concentration is 0.0006 mg/mL for ALP and 0.01 mg/mL for other targets.

To validate the specificity of the proposed switch for ALP detection, we prepared several different targets including ALP, GOD, lysozyme, BSA, and trypsin. Figure 7 exhibits the fluorescence ratio (F/F0) values towards the different targets with the concentrations of 0.01 mg/mL. We found that the fluorescence ratio (F/F0) value generated by ALP showed the largest difference relative to the background value of 1.0 among the tested targets. These results demonstrate that the proposed method is selective for distinguishing other proteins.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CONCLUSION In summary, we have proposed a label-free and simple copper-mediated AgNCs on-off switch. Coupling Cu2+induced quenching of DNA/AgNCs fluorescence with specific Cu2+-PPi binding ability and the degradation of PPi by ALP, this switch allows quantitative analysis of either PPi or ALP using the turn-on-off signal as readout. The label-free and separation-free strategy shows promise for the development of a simple, cost-effective, and portable detection platform. Thus, we believe our design expands the diversity of DNA/AgNCs applications and broadens the capabilities of fluorescence biosensors using on-off switches.

ASSOCIATED CONTENT Supporting Information Details about Figures S1-S13, and Table S1-S4. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Bin-Cheng Yin, [email protected]; *Bang-Ce Ye, [email protected], Fax no. 0086-21-64252094

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was jointly supported by the National Natural Science Foundation of China (Grants 21335003, 21575089, 21675052), the Key Grant Project of Chinese Ministry of Education (Grant 313019), and the Fundamental Research Funds for the Central Universities.

REFERENCES (1) Hirose, M.; Abe-Hashimoto, J.; Ogura, K.; Tahara, H.; Ide, T.; Yoshimura, T. J. Cancer Res. Clin. Oncol. 1997, 123, 337-344. (2) Caswell, A.; Guilland-Cumming, D.; Hearn, P.; McGuire, M.; Russell, R. Ann. Rheum. Dis. 1983, 42, 27. (3) Terkeltaub, R. A. Am. J. Physiol-Cell Ph. 2001, 281, C1-C11. (4) Timms, A.; Zhang, Y.; Russell, R.; Brown, M. Rheumatology 2002, 41, 725-729. (5) Coleman, J. E. Annu. Rev. Biophys. Biomol. Struct. 1992, 21, 441-483. (6) Lallès, J. P. Nutr. Rev. 2014, 72, 82-94. (7) Julien, S. G.; Dubé, N.; Hardy, S.; Tremblay, M. L. Nat. Rev. Cancer 2011, 11, 35-49. (8) Lorente, J. A.; Valenzuela, H.; Morote, J.; Gelabert, A. Eur. J. Nucl. Med. 1999, 26, 625-632. (9) Couttenye, M.; D'haese, P.; Van Hoof, V.; Lemoniatou, E.; Goodman, W.; Verpooten, G.; De Broe, M. Nephrol. Dial. Transplant. 1996, 11, 1065-1072. (10) Colombatto, P.; Randone, A.; Civitico, G.; Gorin, J. M.; Dolci, L.; Medaina, N.; Oliveri, F.; Verme, G.; Marchiaro, G.; Pagni, R. J. Viral Hepat. 1996, 3, 301-306. (11) Ooi, K.; Shiraki, K.; Morishita, Y.; Nobori, T. J. Clin. Lab. Anal. 2007, 21, 133-139. (12) Deng, J.; Yu, P.; Yang, L.; Mao, L. Anal. Chem. 2013, 85, 2516-2522.

Page 8 of 9

(13) Deng, J.; Jiang, Q.; Wang, Y.; Yang, L.; Yu, P.; Mao, L. Anal. Chem. 2013, 85, 9409-9415. (14) Wei, H.; Chen, C.; Han, B.; Wang, E. Anal. Chem. 2008, 80, 7051-7055. (15) Dong, L.; Miao, Q.; Hai, Z.; Yuan, Y.; Liang, G. Anal. Chem. 2015, 87, 6475-6478. (16) Freeman, R.; Finder, T.; Gill, R.; Willner, I. Nano Lett. 2010, 10, 2192-2196. (17) Ruan, C.; Wang, W.; Gu, B. Anal. Chem. 2006, 78, 33793384. (18) Ino, K.; Kanno, Y.; Arai, T.; Inoue, K. Y.; Takahashi, Y.; Shiku, H.; Matsue, T. Anal. Chem. 2012, 84, 7593-7598. (19) Zheng, J.; Nicovich, P. R.; Dickson, R. M. Annu. Rev. Phys. Chem. 2007, 58, 409-431. (20) Jin, R. Nanoscale 2010, 2, 343-362. (21) Lin, C. A. J.; Lee, C. H.; Hsieh, J. T.; Wang, H. H.; Li, J. K.; Shen, J. L.; Chan, W. H.; Yeh, H. I.; Chang, W. H. J. Med. Biol. Eng. 2009, 29, 276-283. (22) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775-778. (23) Guo, W.; Yuan, J.; Dong, Q.; Wang, E. J. Am. Chem. Soc. 2010, 132, 932-934. (24) Huang, Z.; Pu, F.; Hu, D.; Wang, C.; Ren, J.; Qu, X. Chemistry 2011, 17, 3774-3780. (25) Ma, K.; Cui, Q.; Liu, G.; Wu, F.; Xu, S.; Shao, Y. Nanotechnology 2011, 22, 305502. (26) Li, J.; Zhong, X.; Cheng, F.; Zhang, J. R.; Jiang, L. P.; Zhu, J. J. Anal. Chem. 2012, 84, 4140-4146. (27) Latorre, A.; Somoza, Á. ChemBioChem 2012, 13, 951-958. (28) Qian, Z.; Chai, L.; Tang, C.; Huang, Y.; Chen, J.; Feng, H. Anal. Chem. 2015, 87, 2966-2973. (29) Qian, Z.; Chai, L.; Zhou, Q.; Huang, Y.; Tang, C.; Chen, J.; Feng, H. Anal. Chem. 2015, 87, 7332-7339. (30) Li, P. H.; Lin, J. Y.; Chen, C. T.; Ciou, W. R.; Chan, P. H.; Luo, L.; Hsu, H. Y.; Diau, E. W. G.; Chen, Y. C. Anal. Chem. 2012, 84, 5484-5488. (31) Qian, Z. S.; Chai, L. J.; Huang, Y. Y.; Tang, C.; Shen, J. J.; Chen, J. R.; Feng, H. Biosens. Bioelectron. 2015, 68, 675-680. (32) Zheng, X.; Yao, T.; Zhu, Y.; Shi, S. Biosens. Bioelectron. 2015, 66, 103-108. (33) Sun, J.; Yang, F.; Zhao, D.; Yang, X. Anal. Chem. 2014, 86, 7883-7889. (34) Ma, J. L.; Yin, B. C.; Ye, B. C. RSC Adv. 2015, 5, 98467– 98471. (35) Ma, J. L.; Yin, B. C.; Le, H. N.; Ye, B. C. ACS Appl. Mater. Interfaces 2015, 7, 12856–12863. (36) Chen, Y.; Li, W.; Wang, Y.; Yang, X.; Chen, J.; Jiang, Y.; Yu, C.; Lin, Q. J. Mater. Chem. C 2014, 2, 4080-4085. (37) Su, Y. T.; Lan, G. Y.; Chen, W. Y.; Chang, H. T. Anal. Chem. 2010, 82, 8566-8572. (38) He, Z.; Ye, T.; Li, C.; Su, C.; Ji, X. RSC Adv. 2015, 5, 5533655339. (39) Lan, G. Y.; Huang, C. C.; Chang, H. T. Chem. Commun. 2010, 46, 1257-1259. (40) English, J. B.; Martell, A. E.; Motekaitis, R. J.; Murase, I. Inorg. Chim. Acta 1997, 258, 183-192. (41) Zhang, L.; Zhao, J.; Duan, M.; Zhang, H.; Jiang, J.; Yu, R. Anal. Chem. 2013, 85, 3797-3801. (42) Medicine, O. E. M. I. Arthritis Rheum. 1973, 16, 171–178. (43) Russell, R. G. G.; Bisaz, S.; Fleisch, H.; Currey, H. L. F.; Rubinstein, H. M.; Dietz, A. A.; Boussina, I.; Micheli, A.; Fallet, G. Lancet 1970, 2, 899-902.


Page 9 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only


9

Copper-Mediated DNA-Scaffolded Silver ... - ACS Publications

Recommend Documents