Article pubs.acs.org/ac
Highly Sensitive Real-Time Assay of Inorganic Pyrophosphatase Activity Based on the Fluorescent Gold Nanoclusters Jian Sun,† Fan Yang,† Dan Zhao,†,‡ and Xiurong Yang*,† †
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡ University of Chinese Academy of Sciences, Beijing 100039, China S Supporting Information *
ABSTRACT: On the basis of the competition assay approach and fluorescent 11-mercaptoundecanoic acid-capped AuNCs (AuNCs@11-MUA) with unique optical properties, a convenient, reliable and highly sensitive real-time assay of pyrophosphatase (PPase) activity is established and developed for the first time. Pyrophosphate (PPi) could recover the Cu2+quenched AuNCs@11-MUA fluorescence selectively owing to the higher binding affinity between PPi and Cu2+ than that between 11-MUA and Cu2+. Whereas PPase could catalyze the hydrolysis of PPi, thus released Cu2+, leading to fluorescence requenching of the AuNCs@11-MUA. In the assay, a good linearity between the fluorescence response and PPase activity within a range from 1 to 20 mU is found, with a detection limit of less than 1 mU, which is better than other PPase assays using PPi as the substrate. Additionally, we demonstrate that our AuNCs@11-MUA-based fluorescent assay can be applied to assay the PPase activity in real biological samples such as the cell lysate. This strategy paves a new avenue for exploring the sensing applications of fluorescence AuNCs and improving the development of competition assay approach. quantum dots.19,20 Organic fluorophores with diversified chemical structures and fluorescent properties usually have limited water-solubility and photostability, which are prone to photobleaching. Meanwhile, the large physical size and potential toxicity of heavy metal may compromise the application of semiconductor quantum dots in biologically relevant detection and imaging. As a type of nontoxic element, Au-based nanoparticles are the most commonly used optical sensing nanomaterials because of their strong surface plasmon resonance (SPR) absorptions in the visible region, and the sensing events can be easily transformed into color changes in the AuNP-based colorimetric assays.21,22 However, the advent and development of fluorescent AuNCs with smaller size would be beneficial to improve detection limits and extend detection range, partly owing to the inherent advantage of fluorescent assays in the sensitivity in contrast to the colorimetric assays.18 AuNC-based fluorescent assays for the biologically important molecules, including the enzymatic activity, have drawn considerable attention.23,24 In most enzymatic activity assays, the stabilizers or templates for the AuNCs have usually been designed to be an active substrate, whereas the specific enzyme−substrate interaction could destabilize the AuNCs in
T
he ubiquitous inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential metal-dependent hydrolysis enzyme that specifically catalyzes the conversion of one molecule of inorganic pyrophosphate (PPi) into two orthophosphate (Pi) ions.1 The pyrophosphate hydrolysis is a highly exergonic reaction, and therefore this enzymatic hydrolysis process can be coupled to energetically less- or unfavorable biochemical reactions in order to drive these reactions to completion.2 PPases have been demonstrated to play key roles in many biological processes, including lipid metabolism,3 calcium absorption,4 bone formation5 and other biochemical transformations.6,7 To date, several methods including enzymatic,8,9 fluorometric10 and colorimetric11,12 techniques have been developed for the assays of PPase activity using PPi as the substrate. However, these methods are limited by the tedious, time-consuming preparation procedure, requirement of expensive precursors and/or specialized skills. Thus, there is an urgent need for simple, convenient and sensitive methods for real-time PPase activity assay. Recently, fluorescent noble metal nanoclusters, especially gold nanoclusters (AuNCs) with unique optical properties, good biocompatibility and photostability, have been developed as a new type of fluorophores,13,14 especially as optical reporters for sensitive biological detection.15,16 Current fluorescence applications mostly involve traditional fluorescent materials such as organic fluorescent dyes17,18 and semiconductor © 2014 American Chemical Society
Received: May 15, 2014 Accepted: July 17, 2014 Published: July 17, 2014 7883
dx.doi.org/10.1021/ac501814u | Anal. Chem. 2014, 86, 7883−7889
Analytical Chemistry
Article
(TEM) and high resolution TEM (HRTEM) measurements were carried out by using an FEI TECNAI F20 EM with an accelerating voltage of 200 kV. Photostability tests were performed using both 6 W, 365 nm and 6 W, 254 nm ultraviolet lamps (ZF-5, Jiapeng Instrument Co., Ltd., Shanghai, China), which were placed 10 cm away from the AuNCs@11MUA in aqueous solution at room temperature. Preparation of AuNCs. All glassware was thoroughly cleaned with aqua regia (HNO3/HCl, 1:3, v/v) and rinsed extensively with water prior to use (precautions must be taken while handling aqua regia, which is highly toxic and corrosive). The fluorescent AuNCs were synthesized according to a modified protocol described previously.36 In brief, 11-MUA (13.1 mg) and NaOH (6.0 mg) were dissolved in water (18.5 mL). The mixture was added with an aqueous solution of HAuCl4 (1.5 mL, 10 mM), then left to stand for 20 h at room temperature without stirring, during which the solution remained colorless. As-prepared AuNCs were dialyzed with an 8−14 kDa cutoff dialysis membrane, against water for more than 24 h with a water change every 6 h, to remove all small molecule impurities. The larger precipitation produced in the dialysis procedure was removed by passing through 0.45 μm filters (Pall Corporation, MI, USA). The as-purified AuNCs@ 11-MUA solution was stored at room temperature before use. Fluorescent PPase Activity Assay. The as-prepared AuNCs solution was diluted 100 times, named AuNCs solution (1/100), in the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM, pH 7.4) and the fluorescence intensities were recorded from 540 to 750 nm at an excitation wavelength of 260 nm at 25 °C. For the real-time enzyme assays, a 10 μL volume of PPase with different activities ranging from 1 to 40 mU was respectively added to 2 mL of the aforementioned AuNCs solution (1/100) containing Cu2+ (1 μM), PPi (20 μM) and Mg2+ (10 μM). After a brief mixing (30 s), the fluorescence spectra of the mixture were recorded in 1 min intervals.
aqueous solution, resulting in the aggregation and subsequent fluorescence quenching.25,26 The fluorescent AuNCs typically have a core−shell structure where the gold core is
Highly Sensitive Real-Time Assay of Inorganic Pyrophosphatase Activity Based on the Fluorescent Gold Nanoclusters Jian Sun,† Fan Yang,† Dan Zhao,†,‡ and Xiurong Yang*,† †
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡ University of Chinese Academy of Sciences, Beijing 100039, China S Supporting Information *
ABSTRACT: On the basis of the competition assay approach and fluorescent 11-mercaptoundecanoic acid-capped AuNCs (AuNCs@11-MUA) with unique optical properties, a convenient, reliable and highly sensitive real-time assay of pyrophosphatase (PPase) activity is established and developed for the first time. Pyrophosphate (PPi) could recover the Cu2+quenched AuNCs@11-MUA fluorescence selectively owing to the higher binding affinity between PPi and Cu2+ than that between 11-MUA and Cu2+. Whereas PPase could catalyze the hydrolysis of PPi, thus released Cu2+, leading to fluorescence requenching of the AuNCs@11-MUA. In the assay, a good linearity between the fluorescence response and PPase activity within a range from 1 to 20 mU is found, with a detection limit of less than 1 mU, which is better than other PPase assays using PPi as the substrate. Additionally, we demonstrate that our AuNCs@11-MUA-based fluorescent assay can be applied to assay the PPase activity in real biological samples such as the cell lysate. This strategy paves a new avenue for exploring the sensing applications of fluorescence AuNCs and improving the development of competition assay approach. quantum dots.19,20 Organic fluorophores with diversified chemical structures and fluorescent properties usually have limited water-solubility and photostability, which are prone to photobleaching. Meanwhile, the large physical size and potential toxicity of heavy metal may compromise the application of semiconductor quantum dots in biologically relevant detection and imaging. As a type of nontoxic element, Au-based nanoparticles are the most commonly used optical sensing nanomaterials because of their strong surface plasmon resonance (SPR) absorptions in the visible region, and the sensing events can be easily transformed into color changes in the AuNP-based colorimetric assays.21,22 However, the advent and development of fluorescent AuNCs with smaller size would be beneficial to improve detection limits and extend detection range, partly owing to the inherent advantage of fluorescent assays in the sensitivity in contrast to the colorimetric assays.18 AuNC-based fluorescent assays for the biologically important molecules, including the enzymatic activity, have drawn considerable attention.23,24 In most enzymatic activity assays, the stabilizers or templates for the AuNCs have usually been designed to be an active substrate, whereas the specific enzyme−substrate interaction could destabilize the AuNCs in
T
he ubiquitous inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential metal-dependent hydrolysis enzyme that specifically catalyzes the conversion of one molecule of inorganic pyrophosphate (PPi) into two orthophosphate (Pi) ions.1 The pyrophosphate hydrolysis is a highly exergonic reaction, and therefore this enzymatic hydrolysis process can be coupled to energetically less- or unfavorable biochemical reactions in order to drive these reactions to completion.2 PPases have been demonstrated to play key roles in many biological processes, including lipid metabolism,3 calcium absorption,4 bone formation5 and other biochemical transformations.6,7 To date, several methods including enzymatic,8,9 fluorometric10 and colorimetric11,12 techniques have been developed for the assays of PPase activity using PPi as the substrate. However, these methods are limited by the tedious, time-consuming preparation procedure, requirement of expensive precursors and/or specialized skills. Thus, there is an urgent need for simple, convenient and sensitive methods for real-time PPase activity assay. Recently, fluorescent noble metal nanoclusters, especially gold nanoclusters (AuNCs) with unique optical properties, good biocompatibility and photostability, have been developed as a new type of fluorophores,13,14 especially as optical reporters for sensitive biological detection.15,16 Current fluorescence applications mostly involve traditional fluorescent materials such as organic fluorescent dyes17,18 and semiconductor © 2014 American Chemical Society
Received: May 15, 2014 Accepted: July 17, 2014 Published: July 17, 2014 7883
dx.doi.org/10.1021/ac501814u | Anal. Chem. 2014, 86, 7883−7889
Analytical Chemistry
Article
(TEM) and high resolution TEM (HRTEM) measurements were carried out by using an FEI TECNAI F20 EM with an accelerating voltage of 200 kV. Photostability tests were performed using both 6 W, 365 nm and 6 W, 254 nm ultraviolet lamps (ZF-5, Jiapeng Instrument Co., Ltd., Shanghai, China), which were placed 10 cm away from the AuNCs@11MUA in aqueous solution at room temperature. Preparation of AuNCs. All glassware was thoroughly cleaned with aqua regia (HNO3/HCl, 1:3, v/v) and rinsed extensively with water prior to use (precautions must be taken while handling aqua regia, which is highly toxic and corrosive). The fluorescent AuNCs were synthesized according to a modified protocol described previously.36 In brief, 11-MUA (13.1 mg) and NaOH (6.0 mg) were dissolved in water (18.5 mL). The mixture was added with an aqueous solution of HAuCl4 (1.5 mL, 10 mM), then left to stand for 20 h at room temperature without stirring, during which the solution remained colorless. As-prepared AuNCs were dialyzed with an 8−14 kDa cutoff dialysis membrane, against water for more than 24 h with a water change every 6 h, to remove all small molecule impurities. The larger precipitation produced in the dialysis procedure was removed by passing through 0.45 μm filters (Pall Corporation, MI, USA). The as-purified AuNCs@ 11-MUA solution was stored at room temperature before use. Fluorescent PPase Activity Assay. The as-prepared AuNCs solution was diluted 100 times, named AuNCs solution (1/100), in the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM, pH 7.4) and the fluorescence intensities were recorded from 540 to 750 nm at an excitation wavelength of 260 nm at 25 °C. For the real-time enzyme assays, a 10 μL volume of PPase with different activities ranging from 1 to 40 mU was respectively added to 2 mL of the aforementioned AuNCs solution (1/100) containing Cu2+ (1 μM), PPi (20 μM) and Mg2+ (10 μM). After a brief mixing (30 s), the fluorescence spectra of the mixture were recorded in 1 min intervals.
aqueous solution, resulting in the aggregation and subsequent fluorescence quenching.25,26 The fluorescent AuNCs typically have a core−shell structure where the gold core is
