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Luminol functionalized gold nanoparticles as colorimetric and chemiluminescent probes for visual, label free, highly sensitive and selective detection of minocycline
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 455502 (http://iopscience.iop.org/0957-4484/25/45/455502) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 202.28.191.34 This content was downloaded on 28/02/2015 at 08:07
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 25 (2014) 455502 (7pp)
doi:10.1088/0957-4484/25/45/455502
Luminol functionalized gold nanoparticles as colorimetric and chemiluminescent probes for visual, label free, highly sensitive and selective detection of minocycline Yi He1 and Rufang Peng2 1
School of National Defence Science & Technology, Southwest University of Science and Technology, Mianyang, 621010, People’s Republic of China 2 School of Materials Science and Engineering & State Key Laboratory Cultivation, Base for Nonmetal Composite and Functional Materials, Southwest University of Science and Technology, Mianyang, 621010, People’s Republic of China E-mail: [email protected] Received 2 June 2014, revised 8 September 2014 Accepted for publication 9 September 2014 Published 20 October 2014 Abstract
In this work, luminol functionalized gold nanoparticles (LuAuNPs) were used as colorimetric and chemiluminescent probes for visual, label free, sensitive and selective detection of minocycline (MC). The LuAuNPs were prepared by simple one-pot reduction of HAuCl4 with luminol, which exhibited a good chemiluminescence (CL) activity owing to the presence of luminol molecules on their surface and surface plasmon resonance absorption. In the absence of MC, the color of LuAuNPs was wine red and their size was relatively small (∼25 nm), which could react with silver nitrate, producing a strong CL emission. Upon the addition of MC at acidic buffer solutions, the electrostatic interaction between positively charged MC and negatively charged LuAuNPs caused the aggregation of LuAuNPs, generating a purple or blue color. Simultaneously, the aggregated LuAuNPs did not effectively react with silver nitrate, producing a weak CL emission. The signal change was linearly dependent on the logarithm of MC concentration in the range from 30 ng to 1.0 μg for colorimetric detection and from 10 ng to 1.0 μg for CL detection. With colorimetry, a detection limit of 22 ng was achieved, while the detection limit for CL detection modality was 9.7 ng. S Online supplementary data available from stacks.iop.org/NANO/25/455502/mmedia Keywords: minocycline, gold nanoparticles, colorimetry, chemiluminescence (Some figures may appear in colour only in the online journal) 1. Introduction
(MC) was synthesized semi-synthetically from natural TC antibiotics by Lederle laboratories in 1972, and has a broader spectrum than the other members of the group [4]. It is always used to treat many different bacterial infections such as urinary tract infections, respiratory infections, skin infections, chlamydia, and others. However, the dose-limiting side effect of MC has the possibility for renal and liver toxicity, which may cause autoimmune disorders such as drug related lupus and auto-immune hepatitis [5]. In order to acquire the best
Antibiotic pollution has attracted more and more attention because it is considered to be a serious threat to human health. Misuse or overuse of antibiotics does not just lead to drugresistant superbugs, it may also permanently eliminate the body’s good bacteria, which could contribute to the rise of chronic diseases such as obesity, asthma, and cancer [1–3]. As a broad-spectrum tetracycline (TC) antibiotic, minocycline 0957-4484/14/455502+07$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Nanotechnology 25 (2014) 455502
Y He and R Peng
2. Experimental section
therapeutic effect and weaken the toxicity of MC, some reliable and sensitive analytical techniques have been developed to detect MC, including high performance liquid chromatography (HPLC), capillary electrophoresis (CE), enzymelinked immunosorbent assay (ELISA), and fluorescence (FL) [6–9]. Nevertheless, these approaches are usually challenged with some drawbacks, such as expensive instruments for HPLC and CE, time-consuming and high cost for ELISA, and low sensitivity for FL. Thus, simple and convenient assays for the detection of MC are urgently needed. Gold nanoparticles (AuNPs) are emerging as potential probes in biomedical applications due to their size and distance-dependent surface plasmon resonance (SPR) absorption, high extinction coefficient, and good biocompatibility [10]. The sensors based on the self-assembly or aggregation of AuNPs have been proven to achieve high sensitivity in visual and colorimetric detection of DNA, protein, ions and small molecules [11]. For example, Qu’s group developed a colorimetric assay for label-free detection of glucose based on glucose-fueled conformational switch of i-motif DNA and non-crosslinking AuNPs aggregation [12]. This assay was simple in operation and the sensitivity was better than colorimetric glucose sensors. On the other hand, AuNPs have been employed as catalysts for various chemiluminescence (CL) systems. The catalytic activity of AuNPs is always related to their size. For instance, the catalytic activity of aggregated AuNPs for the luminol and hydrogen peroxide CL reaction is better than that of dispersed AuNPs. On this basis, two assays for the detection of DNA and proteins were designed by Li’s group [13, 14]. However, the assays needed a high hybridization temperature and unstable reagent (hydrogen peroxide), which may reduce the stability of AuNPs and generate false signals, leading to bad accuracy. Therefore, the construction of CL assays on the basis of AuNPs and stable CL systems under mild experimental conditions still remains a great challenge. To further enlarge the application fields of AuNPs, it is necessary to functionalize AuNPs with suitable functional molecules such as luminol, an important chemiluminescent reagent. In previous work, luminol functionalized AuNPs (LuAuNPs) were synthesized, and exhibited a unique CL and electrochemiluminescence (ECL) activity. They have been labeled with biomolecules as bioprobes for ECL detection of various targets, including protein and DNA [15–17]. Herein, we use LuAuNPs as colorimetric and chemiluminescent probes for visual, label free, sensitive and selective detection of MC for the first time. As MC bears two tertiary amine groups, it can be protonated in acidic solutions. The protonated MC was demonstrated to be able to directly cause the aggregation of LuAuNPs through electrostatic interaction between positively charged MC and negatively charged LuAuNPs. The status of aggregated LuAuNPs could be reflected by either colorimetric change or CL change. The signal changes were employed to detect MC. This strategy combines the advantages of the high sensitivity of CL assays and the convenience of colorimetric assays.
2.1. Chemicals and materials
Luminol was obtained from Sigma-Aldrich. MC, TC, clenbuterol (CB), cefazolin (CZ), penicillin (PN), chloromycetin (CP), glucose, and arginine were supplied by Sangon Biotech (Shanghai, China). HAuCl4, NaCl, NaOH, MgCl2, KCl, AgNO3, (NH4)2SO4, H3PO4, HAc, H3BO3, and cetrimonium bromide were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Other chemicals were analytical grade and used without purification. Mill-Q water (18.2 MΩ) was used throughout. 2.2. Apparatus and characterizations
The morphology of LuAuNPs was observed by a JEM-2100 field emission transmission electron microscope (TEM) operating at 200 KV (Japan Electronic Company, Japan). The UV–vis extinction spectra were recorded on a UV–vis spectrophotometer (Shimadzu UV-1800, Japan). CL measurements were carried out on a microplate luminometer (Centro LB 960, Berthold, Germany). 2.3. Preparation of LuAuNPs
LuAuNPs were synthesized according to the literature with little modification [15]. Briefly, a 100 mL portion of HAuCl4 solution (0.01%, weight/weight) was heated to boiling point. While stirring vigorously, 2 mL of luminol solution (0.01 mol L−1) was added rapidly to the solution above. The mixture solution was maintained at boiling point for 27 h, during which time a color change from yellow to black to purple was observed before a wine red or purple color was reached. After cooling down to room temperature, the asprepared LuAuNPs were centrifuged at 12 500 rpm for 5 min and re-dispersed in ultrapure water (18.2 MΩ) and stored at 4 °C for further use. The concentration of LuAuNPs (ca. 4.78 nM) was calculated by Beer’s law, using an extinction coefficient of ca. 6 × 108 M−1 cm−1 at 523 nm [18]. 2.4. UV–vis spectroscopic analysis
The LuAuNPs-based colormetric detection was performed at room temperature. In a typical experiment, 300 μL of LuAuNPs was added into 300 μL of Britton-Robinson (BR) buffer solution (pH 3.0) containing different concentrations of MC and kept for 15 mins before the UV–vis spectra were recorded. The presence of MC caused the absorption peak of LuAuNPs redshift from 523 to 659 nm, thus the quantitative determination was based on the absorption ratio (A659/A523). 2.5. CL analysis
CL determination of MC was performed in the AuNPsluminol-AgNO3 reaction system at room temperature. Typically, 50 μL of LuAuNPs solution, and 50 μL of BR buffer solution (pH 3.0) containing different concentrations of MC were sequentially added to a well in a polystyrene 96-well 2
Nanotechnology 25 (2014) 455502
Y He and R Peng
Figure 1. (a) The molecular structures of MC, luminol, and 3-aminophthalate. (b) Schematic of LuAuNPs utilized as colorimetric and
chemiluminescent probes for visual and label free detection of MC.
microtiter plate and incubated at room temperature for 15 mins. Subsequently, 50 μL of 0.01 M NaOH and 50 μL of 1 mM AgNO3 were injected into each well successively to initiate the CL reaction. The CL emission was collected by a microplate luminometer. The voltage of the photomultiplier tube was set at 1000 V. The quantitative determination was based on the peak height of the total CL emission.
effectively catalyze the reaction between luminol and silver nitrate, generating a strong CL emission [20]. The CL mechanism of this system involves the following process. Luminol is oxidized by AgNO3 by virtue of catalysis of AuNPs to produce Ag and luminol radical, which react with the dissolved oxygen resulting in CL emission. The small AuNPs show better catalytic activity than their counterparts because they have a large surface and high particle concentration leading to higher CL intensity [20, 21]. In the presence of protonated MC with positive charge, the surface charge of LuAuNPs would be neutralized, which contributed to increased van der Waals attractive force among MC-coated LuAuNPs, producing a rapid aggregation and a purple color. Meanwhile, the size of the aggregated LuAuNPs increased significantly and no longer effectively catalyzed the corresponding CL reaction, leading to a weak CL emission, thus enabling LuAuNPs to serve as colorimetric and chemiluminescent probes for visual and label free detection of MC.
3. Results and discussion 3.1. Principle of LuAuNPs based sensing MC
LuAuNPs were synthesized by one pot reduction of HAuCl4 with luminol in aqueous solution at reflux temperature. It was demonstrated that luminol and its oxidation product 3-aminophthalate (AP2−) (figure 1(a)) were capped on the surface of AuNPs as stabilizers via Au-N covalent interaction [15]. The as-prepared LuAuNPs were well-dispersed because the electrostatic repulsion between the surface-bound AP2− ions prevented the strong van der Waals attraction between AuNPs from causing them to aggregate. Additionally, as previously reported, the tertiary amine groups of the MC molecule can be protonated in an acidic solution [19], leading to the formation of a species with one positive charge (MC+, 2.8 < pH < 5) or two positive charges (MC2+, pH < 2.8). As shown in figure 1(b), the well-dispersed LuAuNPs are wine red in color, and the size of LuANPs is relatively small, which can
3.2. Colorimetric and visual detection of MC
Initially, the LuAuNPs were centrifuged at 12 500 rpm for 5 mins and re-dispersed in ultrapure water (18.2 MΩ) to remove the free luminol molecules, and the color of the well dispersed colloid was wine red due to their SPR absorption at 523 nm. Upon exposure of the LuAuNPs to 1 μg MC dispersed in BR buffer solution (0.04 M H3PO4−0.04 M HAc −0.04 M H3BO3), the color of the gold colloid changed from 3
Nanotechnology 25 (2014) 455502
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absence of MC revealed uniform monodisperse particles with an average size of ∼25 nm in diameter (figure 3(a)), while obvious aggregation of LuAuNPs occurred in the presence of 3 μg and 7 μg MC, with a few hundred nanometers to micrometers in diameter of the aggregates (figures 3(c) and (d)). The TEM results gave direct evidence for MC-induced aggregation of LuAuNPs. To optimize the conditions for the detection of MC, some important experimental factors, including pH of BR buffer solution and the concentration of LuAuNPs, were evaluated. The effect of pH of BR buffer solution was examined in the range of 2.0–8.0. At pH 3.0, the highest absorption ratio (A659/A523) was obtained (figure S1). MC was protonated at this pH so that the MC was positively charged, which led to the efficient electrostatic interaction with the negatively charged LuAuNPs. Thus, the pH of the BR buffer solution was chosen as 3.0. The effect of the concentration of LuAuNPs was also studied. The results showed that the highest sensitivity was obtained when using 2.4 nM LuAuNPs (figure S2). Therefore, 2.4 nM was selected as the final concentration of LuAuNPs. Under the optimized conditions above, the UV–vis spectra of LuAuNPs in the presence of MC with different concentrations were recorded. As shown in figure 4(a), the absorption of LuAuNPs at 523 nm decreased, and the absorption at 659 nm gradually increased by increasing the MC concentration. The absorption ratio (A659/A523) exhibited a linear correlation to the logarithm of MC concentration in the range from 30 ng to 1.0 μg, with a detection limit of 22 ng based on 3σ (signal-to-noise). The obtained detection limit was about five orders, two orders and one order of magnitude
Figure 2. UV–vis absorption spectra of LuAuNPs in the absence and presence of 1 μg MC. Inset shows the corresponding photographic images.
red to blue (inset of figure 2). The SPR peak intensity of LuAuNPs decreased, and a new absorption band at 659 nm appeared in the presence of MC (figure 2), which was ascribed to the near-field coupling that occurs when the interparticle distance decreases [22]. As protonated MC is a cation, its electrostatic interaction with the negatively charged LuAuNPs decreases the interparticle distance, resulting in the aggregation of LuAuNPs. The MC-induced aggregation of LuAuNPs was further demonstrated by TEM. TEM images of LuAuNPs in the
Figure 3. TEM images of LuAuNPs: (a) in the absence of MC; (b) in the presence of 50 ng MC; (c) 3 μg MC; (d) 7 μg MC. 4
Nanotechnology 25 (2014) 455502
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Figure 4. (a) UV–vis absorption spectra of LuAuNPs in the presence of MC with different concentrations (0, 30 ng, 50 ng, 70 ng, 90 ng, 300 ng, 700 ng, and 1000 ng). Inset: the linear calibration plot for MC detection. (b) Photographs of LuAuNPs solutions in the presence of MC with different concentrations (vial 1 to 7: the MC concentrations were 0, 30, 70, 90, 300, 700, and 1000 ng).
Figure 5. (a) CL kinetic curves for the reaction of LuAuNPs and
AgNO3 (pH = 12) in the presence of MC with different concentrations: (i) 0, (ii) 10 ng, (iii) 30 ng, (iv) 50 ng, (v) 70 ng, (vi)100 ng, (vii) 300 ng, (viii) 500 ng, (ix) 700 ng, and (x) 1000 ng. (b) The linear relationship between the relative CL response and logarithm of the concentration of MC. Relative CL intensity equals I0-I, I0 and I are the CL signals in the absence and presence of MC, respectively.
lower than that of the reported CE, FL and HPLC methods [6–9], respectively. This detection limit was also comparable to the ELISA method. The reproducibility of this detection mode was estimated by assaying three MC levels for 5 replicate measurements of the 50, 100, 500 ng MC solution which yielded reproducible UV–vis responses with coefficients of deviation of 1.5%, 2.0%, and 4.5%, respectively, suggesting the good reproducibility of this detection mode. In addition, the MC-induced aggregation of LuAuNPs would cause a color change from red to purple and then to blue (figure 4(b)), the naked eye alone can judge the presence of MC as low as 30 ng without the aid of any advanced instruments.
reaction, while the size of the aggregated LuAuNPs in the presence of MC became large, which reduced their catalytic activity, resulting in a weak CL emission. As expected, the CL signal gradually decreased with the increase in the concentration of MC as shown in figure 5(a). The calibration plot showed a good linear relationship between the relative CL intensity and the logarithm of the MC concentration in the range from 10 ng to 1000 ng with a correlation coefficient of 0.9908. The limit of detection at a signal-to noise ratio of 3 was 9.7 ng, which was about five orders, two orders, and one order of magnitude lower than that of the reported CE, FL, and HPLC methods [6–9]. This sensitivity was higher than that of the colorimetric method above (22 ng), and this CL detection modality needed less reagents consumption (the amounts of AuNPs and MC, see the experimental section). The reproducibility of this detection model was also evaluated by analyzing three MC concentration levels for five replicate measurements in the same condition. The coefficients of variation of this assay were 2.2%, 1.6%, and 2.1% at 50, 100, and 500 ng of MC, respectively, which exhibited satisfactory precision.
3.3. CL detection of MC
It was demonstrated that AuNPs could effectively catalyze the reaction between luminol and silver nitrate, producing a CL emission. The catalytic activity of AuNPs depended on their size. That is, the small AuNPs had a better catalytic activity for this CL reaction than that of the large AuNPs [20]. Therefore, the size of LuAuNPs is relatively small (∼25 nm), which should react with silver nitrate owing to the presence of luminol on the surface of AuNPs, generating a strong CL emission due to their good catalytic activity for this CL 5
Nanotechnology 25 (2014) 455502
Y He and R Peng
Therefore, most of the TC molecules form zwitterions at pH 3.0, which cannot effectively induce the aggregation of LuAuNPs through electrostatic interaction, while MC can induce the aggregation of LuAuNPs because they have a positive charge at pH 3.0.
4. Conclusion In summary, the LuAuNPs were employed to be colorimetric and chemiluminescent probes for visual, label free, sensitive, and selective detection of MC for the first time. This assay is based on the MC-induced aggregation of LuAuNPs, generating the color/UV–vis absorption change or CL quench. With colorimetry, a detection limit of 22 ng for analyzing MC was obtained, while a lower detection limit of 9.7 ng MC achieved using the CL detection modality. Besides the high sensitivity, the LuAuNPs-based probes exhibited good selectivity for the detection of MC over other antibiotics. The present assay is highlighted by its simplicity, rapidity, facility, reliability, and sensitivity. It offers a new assay for the detection of MC and extends the application field of LuAuNPs. The proposed colorimetric and chemiluminescent probes based on LuAuNPs are also expected to find other potential applications such as disease diagnosis, molecular recognition, and drug screening.
Acknowledgments
Figure 6. (a) Selectivity of LuAuNPs as a colometric probe for
detection of MC over other antibiotics. (b) CL response of LuAuNPs in the absence (blank) and presence of MC and other antibiotics.
The support of this research by the National Natural Science Foundation of P. R. China (Grant Nos. 51372211) is gratefully acknowledged.
3.4. Selectivity of LuAuNPs based detection
The selectivity of the probes based on the LuAuNPs was evaluated by challenging them against other compounds. The results are shown in figure 6. The absorption ratio (A659/A523) or relative CL intensity showed little change in the presence of 1 μg of other antibiotics including TC, CB, CZ, PN, and CP. However, the presence of the target analyte (MC) resulted in an apparent change in absorption ratio and relative CL intensity, indicating that these antibiotics did not interfere with the detection, demonstrating that the LuAuNPs-based probes can detect MC with high selectivity. The good selectivity of the proposed assay over TC can be explained as follows. TC and MC present different ionic states depending on the solution pH because they contain different numbers of tertiary amine groups as shown in figure S3. TC has three dissociable protons at pH between 2 and 10.5. The fully protonated species of TC with one positive charge that exists at pH < 3 is shown in figure S3. As the pH increases, the first deprotonation step (pH = 3.3) occurs at the hydroxyl group on C3 leading to the formation of a zwitterion with a positive charge located on the protonated dimethylamonium group and a negative charge delocalized over the A ring (figure S3) [19, 23]. However, MC has a positive charge at pH 3.0, and the pH of the BR buffer solution is chosen as 3.0 in our study.
References [1] Cherkasov A, Hilpert K, Jenssen H, Fjell C D, Waldbrook M, Mullaly S C, Volkmer R and Hancock R E W 2008 Use of artificial intelligence in the design of small peptide antibiotics effective against a broad spectrum of highly antibiotic-resistant superbugs ACS Chem. Biol. 4 65–74 [2] Davies J and Davies D 2010 Origins and evolution of antibiotic resistance Microbiol. Mol. Biol. R. 74 417–33 [3] Viswesh V, Gates K and Sun D 2009 Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells Chem. Res. Toxicol. 23 99–107 [4] Kuck N 1976 In vitro and in vivo activities of minocycline and other antibiotics against acinetobacter (Herellea-Mima) Antimicrob. Agents Chemother. 9 493–7 [5] Townsend D R, Bagshaw S M, Jacka M J, Bigam D, Cave D and Gibney R 2009 Intraoperative renal support during liver transplantation Liver Transplant. 15 73–8 [6] Orti V, Audran M, Gibert P, Bougard G and Bressolle F 2000 High-performance liquid chromatographic assay for minocycline in human plasma and parotid saliva J. Chromatogr. B 738 357–65 [7] Li Y, Van Schepdael A, Roets E and Hoogmartens J 1996 Capillary zone electrophoresis of minocycline J. Pharmaceut. Biomed. 14 1095–9 6
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[16] Chai Y, Tian D, Wang W and Cui H 2010 A novel electrochemiluminescence strategy for ultrasensitive DNA assay using luminol functionalized gold nanoparticles multilabeling and amplification of gold nanoparticles and biotin– streptavidin system Chem. Commun. 46 7560–2 [17] Jiang J, Chai Y and Cui H 2011 The electrogenerated chemiluminescence detection of IS6110 of mycobacterium tuberculosis based on a luminol functionalized gold nanoprobe RSC Adv. 1 247–54 [18] Wang X and Guo X 2009 Ultrasensitive Pb2+ detection based on fluorescence resonance energy transfer (FRET) between quantum dots and gold nanoparticl Analyst 134 1348–54 [19] Parolo M, Avena M, Pettinari G, Zajonkovsky I, Valles J and Baschini M 2010 Antimicrobial properties of tetracycline and minocycline-montmorillonites Appl. Clay Sci. 49 194–9 [20] Cui H, Guo J Z, Li N and Liu L J 2008 Gold nanoparticle triggered chemiluminescence between luminol and AgNO3 J. Phys. Chem. C 112 11319–23 [21] He Y and Cui H 2012 Fabrication of luminol and lucigenin bifunctionalized gold nnanoparticles/graphene oxide nanocomposites with dual-wavelength chemiluminescence J. Phys. Chem. C 116 12953–7 [22] Cao R and Li B 2011 A simple and sensitive method for visual detection of heparin using positively-charged gold nanoparticles as colorimetric probes Chem. Commun. 47 2865–7 [23] Leeson L, Krueger J and Nash R 1963 Structural assignment of the second and third acidity constant of tetracycline antibiotics Tetrahedron Lett. 18 1155–60
[8] Pastor-Navarro N, Morais S, Maquieira Á and Puchades R 2007 Synthesis of haptens and development of a sensitive immunoassay for tetracycline residues: application to honey samples Anal. Chim. Acta 594 211–8 [9] Chen C, Guo C, Wang L and Jiang W 2009 Rapid determination of minocycline in pharmaceutical formulations and human urine/serum samples based on the minocycline-europium-sodium dodecylbenzene sulfonate luminescence system Anal. Lett. 42 228–42 [10] Hu M, Chen J, Li Z Y, Au L, Hartland G V, Li X, Marquez M and Xia Y 2006 Gold nanostructures: engineering their plasmonic properties for biomedical applications Chem. Soc. Rev. 35 1084–94 [11] Sperling R A, Gil P R, Zhang F, Zanella M and Parak W J 2008 Biological applications of gold nanoparticles Chem. Soc. Rev. 37 1896–908 [12] Li W, Feng L, Ren J, Wu L and Qu X 2012 Visual detection of glucose using conformational switch of i-Motif DNA and non-crosslinking gold nanoparticles Chem. Eur. J. 18 12637–42 [13] Qi Y Y, Li B X and Zhang Z J 2009 Label-free and homogeneous DNA hybridization detection using gold nanoparticles-based chemiluiminescence system Biosen. Bioelectron. 24 3581–6 [14] Qi Y and Li B 2011 A sensitive, label-free, aptamer-based biosensor using a gold nanoparticle-initiated chemiluminescence system Chem. Eur. J. 17 1642–8 [15] Cui H, Wang W, Duan C F, Dong Y P and Guo J Z 2007 Synthesis, characterization, and electrochemiluminescence of luminol‐reduced gold nanoparticles and their application in a hydrogen peroxide sensor Chem. Eur. J. 13 6975–84
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Luminol functionalized gold nanoparticles as colorimetric and chemiluminescent probes for visual, label free, highly sensitive and selective detection of minocycline
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 455502 (http://iopscience.iop.org/0957-4484/25/45/455502) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 202.28.191.34 This content was downloaded on 28/02/2015 at 08:07
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 25 (2014) 455502 (7pp)
doi:10.1088/0957-4484/25/45/455502
Luminol functionalized gold nanoparticles as colorimetric and chemiluminescent probes for visual, label free, highly sensitive and selective detection of minocycline Yi He1 and Rufang Peng2 1
School of National Defence Science & Technology, Southwest University of Science and Technology, Mianyang, 621010, People’s Republic of China 2 School of Materials Science and Engineering & State Key Laboratory Cultivation, Base for Nonmetal Composite and Functional Materials, Southwest University of Science and Technology, Mianyang, 621010, People’s Republic of China E-mail: [email protected] Received 2 June 2014, revised 8 September 2014 Accepted for publication 9 September 2014 Published 20 October 2014 Abstract
In this work, luminol functionalized gold nanoparticles (LuAuNPs) were used as colorimetric and chemiluminescent probes for visual, label free, sensitive and selective detection of minocycline (MC). The LuAuNPs were prepared by simple one-pot reduction of HAuCl4 with luminol, which exhibited a good chemiluminescence (CL) activity owing to the presence of luminol molecules on their surface and surface plasmon resonance absorption. In the absence of MC, the color of LuAuNPs was wine red and their size was relatively small (∼25 nm), which could react with silver nitrate, producing a strong CL emission. Upon the addition of MC at acidic buffer solutions, the electrostatic interaction between positively charged MC and negatively charged LuAuNPs caused the aggregation of LuAuNPs, generating a purple or blue color. Simultaneously, the aggregated LuAuNPs did not effectively react with silver nitrate, producing a weak CL emission. The signal change was linearly dependent on the logarithm of MC concentration in the range from 30 ng to 1.0 μg for colorimetric detection and from 10 ng to 1.0 μg for CL detection. With colorimetry, a detection limit of 22 ng was achieved, while the detection limit for CL detection modality was 9.7 ng. S Online supplementary data available from stacks.iop.org/NANO/25/455502/mmedia Keywords: minocycline, gold nanoparticles, colorimetry, chemiluminescence (Some figures may appear in colour only in the online journal) 1. Introduction
(MC) was synthesized semi-synthetically from natural TC antibiotics by Lederle laboratories in 1972, and has a broader spectrum than the other members of the group [4]. It is always used to treat many different bacterial infections such as urinary tract infections, respiratory infections, skin infections, chlamydia, and others. However, the dose-limiting side effect of MC has the possibility for renal and liver toxicity, which may cause autoimmune disorders such as drug related lupus and auto-immune hepatitis [5]. In order to acquire the best
Antibiotic pollution has attracted more and more attention because it is considered to be a serious threat to human health. Misuse or overuse of antibiotics does not just lead to drugresistant superbugs, it may also permanently eliminate the body’s good bacteria, which could contribute to the rise of chronic diseases such as obesity, asthma, and cancer [1–3]. As a broad-spectrum tetracycline (TC) antibiotic, minocycline 0957-4484/14/455502+07$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Nanotechnology 25 (2014) 455502
Y He and R Peng
2. Experimental section
therapeutic effect and weaken the toxicity of MC, some reliable and sensitive analytical techniques have been developed to detect MC, including high performance liquid chromatography (HPLC), capillary electrophoresis (CE), enzymelinked immunosorbent assay (ELISA), and fluorescence (FL) [6–9]. Nevertheless, these approaches are usually challenged with some drawbacks, such as expensive instruments for HPLC and CE, time-consuming and high cost for ELISA, and low sensitivity for FL. Thus, simple and convenient assays for the detection of MC are urgently needed. Gold nanoparticles (AuNPs) are emerging as potential probes in biomedical applications due to their size and distance-dependent surface plasmon resonance (SPR) absorption, high extinction coefficient, and good biocompatibility [10]. The sensors based on the self-assembly or aggregation of AuNPs have been proven to achieve high sensitivity in visual and colorimetric detection of DNA, protein, ions and small molecules [11]. For example, Qu’s group developed a colorimetric assay for label-free detection of glucose based on glucose-fueled conformational switch of i-motif DNA and non-crosslinking AuNPs aggregation [12]. This assay was simple in operation and the sensitivity was better than colorimetric glucose sensors. On the other hand, AuNPs have been employed as catalysts for various chemiluminescence (CL) systems. The catalytic activity of AuNPs is always related to their size. For instance, the catalytic activity of aggregated AuNPs for the luminol and hydrogen peroxide CL reaction is better than that of dispersed AuNPs. On this basis, two assays for the detection of DNA and proteins were designed by Li’s group [13, 14]. However, the assays needed a high hybridization temperature and unstable reagent (hydrogen peroxide), which may reduce the stability of AuNPs and generate false signals, leading to bad accuracy. Therefore, the construction of CL assays on the basis of AuNPs and stable CL systems under mild experimental conditions still remains a great challenge. To further enlarge the application fields of AuNPs, it is necessary to functionalize AuNPs with suitable functional molecules such as luminol, an important chemiluminescent reagent. In previous work, luminol functionalized AuNPs (LuAuNPs) were synthesized, and exhibited a unique CL and electrochemiluminescence (ECL) activity. They have been labeled with biomolecules as bioprobes for ECL detection of various targets, including protein and DNA [15–17]. Herein, we use LuAuNPs as colorimetric and chemiluminescent probes for visual, label free, sensitive and selective detection of MC for the first time. As MC bears two tertiary amine groups, it can be protonated in acidic solutions. The protonated MC was demonstrated to be able to directly cause the aggregation of LuAuNPs through electrostatic interaction between positively charged MC and negatively charged LuAuNPs. The status of aggregated LuAuNPs could be reflected by either colorimetric change or CL change. The signal changes were employed to detect MC. This strategy combines the advantages of the high sensitivity of CL assays and the convenience of colorimetric assays.
2.1. Chemicals and materials
Luminol was obtained from Sigma-Aldrich. MC, TC, clenbuterol (CB), cefazolin (CZ), penicillin (PN), chloromycetin (CP), glucose, and arginine were supplied by Sangon Biotech (Shanghai, China). HAuCl4, NaCl, NaOH, MgCl2, KCl, AgNO3, (NH4)2SO4, H3PO4, HAc, H3BO3, and cetrimonium bromide were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Other chemicals were analytical grade and used without purification. Mill-Q water (18.2 MΩ) was used throughout. 2.2. Apparatus and characterizations
The morphology of LuAuNPs was observed by a JEM-2100 field emission transmission electron microscope (TEM) operating at 200 KV (Japan Electronic Company, Japan). The UV–vis extinction spectra were recorded on a UV–vis spectrophotometer (Shimadzu UV-1800, Japan). CL measurements were carried out on a microplate luminometer (Centro LB 960, Berthold, Germany). 2.3. Preparation of LuAuNPs
LuAuNPs were synthesized according to the literature with little modification [15]. Briefly, a 100 mL portion of HAuCl4 solution (0.01%, weight/weight) was heated to boiling point. While stirring vigorously, 2 mL of luminol solution (0.01 mol L−1) was added rapidly to the solution above. The mixture solution was maintained at boiling point for 27 h, during which time a color change from yellow to black to purple was observed before a wine red or purple color was reached. After cooling down to room temperature, the asprepared LuAuNPs were centrifuged at 12 500 rpm for 5 min and re-dispersed in ultrapure water (18.2 MΩ) and stored at 4 °C for further use. The concentration of LuAuNPs (ca. 4.78 nM) was calculated by Beer’s law, using an extinction coefficient of ca. 6 × 108 M−1 cm−1 at 523 nm [18]. 2.4. UV–vis spectroscopic analysis
The LuAuNPs-based colormetric detection was performed at room temperature. In a typical experiment, 300 μL of LuAuNPs was added into 300 μL of Britton-Robinson (BR) buffer solution (pH 3.0) containing different concentrations of MC and kept for 15 mins before the UV–vis spectra were recorded. The presence of MC caused the absorption peak of LuAuNPs redshift from 523 to 659 nm, thus the quantitative determination was based on the absorption ratio (A659/A523). 2.5. CL analysis
CL determination of MC was performed in the AuNPsluminol-AgNO3 reaction system at room temperature. Typically, 50 μL of LuAuNPs solution, and 50 μL of BR buffer solution (pH 3.0) containing different concentrations of MC were sequentially added to a well in a polystyrene 96-well 2
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Figure 1. (a) The molecular structures of MC, luminol, and 3-aminophthalate. (b) Schematic of LuAuNPs utilized as colorimetric and
chemiluminescent probes for visual and label free detection of MC.
microtiter plate and incubated at room temperature for 15 mins. Subsequently, 50 μL of 0.01 M NaOH and 50 μL of 1 mM AgNO3 were injected into each well successively to initiate the CL reaction. The CL emission was collected by a microplate luminometer. The voltage of the photomultiplier tube was set at 1000 V. The quantitative determination was based on the peak height of the total CL emission.
effectively catalyze the reaction between luminol and silver nitrate, generating a strong CL emission [20]. The CL mechanism of this system involves the following process. Luminol is oxidized by AgNO3 by virtue of catalysis of AuNPs to produce Ag and luminol radical, which react with the dissolved oxygen resulting in CL emission. The small AuNPs show better catalytic activity than their counterparts because they have a large surface and high particle concentration leading to higher CL intensity [20, 21]. In the presence of protonated MC with positive charge, the surface charge of LuAuNPs would be neutralized, which contributed to increased van der Waals attractive force among MC-coated LuAuNPs, producing a rapid aggregation and a purple color. Meanwhile, the size of the aggregated LuAuNPs increased significantly and no longer effectively catalyzed the corresponding CL reaction, leading to a weak CL emission, thus enabling LuAuNPs to serve as colorimetric and chemiluminescent probes for visual and label free detection of MC.
3. Results and discussion 3.1. Principle of LuAuNPs based sensing MC
LuAuNPs were synthesized by one pot reduction of HAuCl4 with luminol in aqueous solution at reflux temperature. It was demonstrated that luminol and its oxidation product 3-aminophthalate (AP2−) (figure 1(a)) were capped on the surface of AuNPs as stabilizers via Au-N covalent interaction [15]. The as-prepared LuAuNPs were well-dispersed because the electrostatic repulsion between the surface-bound AP2− ions prevented the strong van der Waals attraction between AuNPs from causing them to aggregate. Additionally, as previously reported, the tertiary amine groups of the MC molecule can be protonated in an acidic solution [19], leading to the formation of a species with one positive charge (MC+, 2.8 < pH < 5) or two positive charges (MC2+, pH < 2.8). As shown in figure 1(b), the well-dispersed LuAuNPs are wine red in color, and the size of LuANPs is relatively small, which can
3.2. Colorimetric and visual detection of MC
Initially, the LuAuNPs were centrifuged at 12 500 rpm for 5 mins and re-dispersed in ultrapure water (18.2 MΩ) to remove the free luminol molecules, and the color of the well dispersed colloid was wine red due to their SPR absorption at 523 nm. Upon exposure of the LuAuNPs to 1 μg MC dispersed in BR buffer solution (0.04 M H3PO4−0.04 M HAc −0.04 M H3BO3), the color of the gold colloid changed from 3
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absence of MC revealed uniform monodisperse particles with an average size of ∼25 nm in diameter (figure 3(a)), while obvious aggregation of LuAuNPs occurred in the presence of 3 μg and 7 μg MC, with a few hundred nanometers to micrometers in diameter of the aggregates (figures 3(c) and (d)). The TEM results gave direct evidence for MC-induced aggregation of LuAuNPs. To optimize the conditions for the detection of MC, some important experimental factors, including pH of BR buffer solution and the concentration of LuAuNPs, were evaluated. The effect of pH of BR buffer solution was examined in the range of 2.0–8.0. At pH 3.0, the highest absorption ratio (A659/A523) was obtained (figure S1). MC was protonated at this pH so that the MC was positively charged, which led to the efficient electrostatic interaction with the negatively charged LuAuNPs. Thus, the pH of the BR buffer solution was chosen as 3.0. The effect of the concentration of LuAuNPs was also studied. The results showed that the highest sensitivity was obtained when using 2.4 nM LuAuNPs (figure S2). Therefore, 2.4 nM was selected as the final concentration of LuAuNPs. Under the optimized conditions above, the UV–vis spectra of LuAuNPs in the presence of MC with different concentrations were recorded. As shown in figure 4(a), the absorption of LuAuNPs at 523 nm decreased, and the absorption at 659 nm gradually increased by increasing the MC concentration. The absorption ratio (A659/A523) exhibited a linear correlation to the logarithm of MC concentration in the range from 30 ng to 1.0 μg, with a detection limit of 22 ng based on 3σ (signal-to-noise). The obtained detection limit was about five orders, two orders and one order of magnitude
Figure 2. UV–vis absorption spectra of LuAuNPs in the absence and presence of 1 μg MC. Inset shows the corresponding photographic images.
red to blue (inset of figure 2). The SPR peak intensity of LuAuNPs decreased, and a new absorption band at 659 nm appeared in the presence of MC (figure 2), which was ascribed to the near-field coupling that occurs when the interparticle distance decreases [22]. As protonated MC is a cation, its electrostatic interaction with the negatively charged LuAuNPs decreases the interparticle distance, resulting in the aggregation of LuAuNPs. The MC-induced aggregation of LuAuNPs was further demonstrated by TEM. TEM images of LuAuNPs in the
Figure 3. TEM images of LuAuNPs: (a) in the absence of MC; (b) in the presence of 50 ng MC; (c) 3 μg MC; (d) 7 μg MC. 4
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Figure 4. (a) UV–vis absorption spectra of LuAuNPs in the presence of MC with different concentrations (0, 30 ng, 50 ng, 70 ng, 90 ng, 300 ng, 700 ng, and 1000 ng). Inset: the linear calibration plot for MC detection. (b) Photographs of LuAuNPs solutions in the presence of MC with different concentrations (vial 1 to 7: the MC concentrations were 0, 30, 70, 90, 300, 700, and 1000 ng).
Figure 5. (a) CL kinetic curves for the reaction of LuAuNPs and
AgNO3 (pH = 12) in the presence of MC with different concentrations: (i) 0, (ii) 10 ng, (iii) 30 ng, (iv) 50 ng, (v) 70 ng, (vi)100 ng, (vii) 300 ng, (viii) 500 ng, (ix) 700 ng, and (x) 1000 ng. (b) The linear relationship between the relative CL response and logarithm of the concentration of MC. Relative CL intensity equals I0-I, I0 and I are the CL signals in the absence and presence of MC, respectively.
lower than that of the reported CE, FL and HPLC methods [6–9], respectively. This detection limit was also comparable to the ELISA method. The reproducibility of this detection mode was estimated by assaying three MC levels for 5 replicate measurements of the 50, 100, 500 ng MC solution which yielded reproducible UV–vis responses with coefficients of deviation of 1.5%, 2.0%, and 4.5%, respectively, suggesting the good reproducibility of this detection mode. In addition, the MC-induced aggregation of LuAuNPs would cause a color change from red to purple and then to blue (figure 4(b)), the naked eye alone can judge the presence of MC as low as 30 ng without the aid of any advanced instruments.
reaction, while the size of the aggregated LuAuNPs in the presence of MC became large, which reduced their catalytic activity, resulting in a weak CL emission. As expected, the CL signal gradually decreased with the increase in the concentration of MC as shown in figure 5(a). The calibration plot showed a good linear relationship between the relative CL intensity and the logarithm of the MC concentration in the range from 10 ng to 1000 ng with a correlation coefficient of 0.9908. The limit of detection at a signal-to noise ratio of 3 was 9.7 ng, which was about five orders, two orders, and one order of magnitude lower than that of the reported CE, FL, and HPLC methods [6–9]. This sensitivity was higher than that of the colorimetric method above (22 ng), and this CL detection modality needed less reagents consumption (the amounts of AuNPs and MC, see the experimental section). The reproducibility of this detection model was also evaluated by analyzing three MC concentration levels for five replicate measurements in the same condition. The coefficients of variation of this assay were 2.2%, 1.6%, and 2.1% at 50, 100, and 500 ng of MC, respectively, which exhibited satisfactory precision.
3.3. CL detection of MC
It was demonstrated that AuNPs could effectively catalyze the reaction between luminol and silver nitrate, producing a CL emission. The catalytic activity of AuNPs depended on their size. That is, the small AuNPs had a better catalytic activity for this CL reaction than that of the large AuNPs [20]. Therefore, the size of LuAuNPs is relatively small (∼25 nm), which should react with silver nitrate owing to the presence of luminol on the surface of AuNPs, generating a strong CL emission due to their good catalytic activity for this CL 5
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Therefore, most of the TC molecules form zwitterions at pH 3.0, which cannot effectively induce the aggregation of LuAuNPs through electrostatic interaction, while MC can induce the aggregation of LuAuNPs because they have a positive charge at pH 3.0.
4. Conclusion In summary, the LuAuNPs were employed to be colorimetric and chemiluminescent probes for visual, label free, sensitive, and selective detection of MC for the first time. This assay is based on the MC-induced aggregation of LuAuNPs, generating the color/UV–vis absorption change or CL quench. With colorimetry, a detection limit of 22 ng for analyzing MC was obtained, while a lower detection limit of 9.7 ng MC achieved using the CL detection modality. Besides the high sensitivity, the LuAuNPs-based probes exhibited good selectivity for the detection of MC over other antibiotics. The present assay is highlighted by its simplicity, rapidity, facility, reliability, and sensitivity. It offers a new assay for the detection of MC and extends the application field of LuAuNPs. The proposed colorimetric and chemiluminescent probes based on LuAuNPs are also expected to find other potential applications such as disease diagnosis, molecular recognition, and drug screening.
Acknowledgments
Figure 6. (a) Selectivity of LuAuNPs as a colometric probe for
detection of MC over other antibiotics. (b) CL response of LuAuNPs in the absence (blank) and presence of MC and other antibiotics.
The support of this research by the National Natural Science Foundation of P. R. China (Grant Nos. 51372211) is gratefully acknowledged.
3.4. Selectivity of LuAuNPs based detection
The selectivity of the probes based on the LuAuNPs was evaluated by challenging them against other compounds. The results are shown in figure 6. The absorption ratio (A659/A523) or relative CL intensity showed little change in the presence of 1 μg of other antibiotics including TC, CB, CZ, PN, and CP. However, the presence of the target analyte (MC) resulted in an apparent change in absorption ratio and relative CL intensity, indicating that these antibiotics did not interfere with the detection, demonstrating that the LuAuNPs-based probes can detect MC with high selectivity. The good selectivity of the proposed assay over TC can be explained as follows. TC and MC present different ionic states depending on the solution pH because they contain different numbers of tertiary amine groups as shown in figure S3. TC has three dissociable protons at pH between 2 and 10.5. The fully protonated species of TC with one positive charge that exists at pH < 3 is shown in figure S3. As the pH increases, the first deprotonation step (pH = 3.3) occurs at the hydroxyl group on C3 leading to the formation of a zwitterion with a positive charge located on the protonated dimethylamonium group and a negative charge delocalized over the A ring (figure S3) [19, 23]. However, MC has a positive charge at pH 3.0, and the pH of the BR buffer solution is chosen as 3.0 in our study.
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