Article pubs.acs.org/ac
Simultaneous Aptasensor for Multiplex Pathogenic Bacteria Detection Based on Multicolor Upconversion Nanoparticles Labels Shijia Wu, Nuo Duan, Zhao Shi, CongCong Fang, and Zhouping Wang* State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China S Supporting Information *
ABSTRACT: A highly sensitive and specific multiplex method for the simultaneous detection of three pathogenic bacteria was fabricated using multicolor upconversion nanoparticles (UCNPs) as luminescence labels coupled with aptamers as the molecular recognition elements. Multicolor UCNPs were synthesized via doping with various rare-earth ions to obtain well-separated emission peaks. The aptamer sequences were selected using the systematic evolution of ligands by exponential enrichment (SELEX) strategy for Staphylococcus aureus, Vibrio parahemolyticus, and Salmonella typhimurium. When applied in this method, aptamers can be used for the specific recognition of the bacteria from complex mixtures, including those found in real food matrixes. Aptamers and multicolor UCNPs were employed to selectively capture and simultaneously quantify the three target bacteria on the basis of the independent peaks. Under optimal conditions, the correlation between the concentration of three bacteria and the luminescence signal was found to be linear from 50−106 cfu mL−1. Improved by the magnetic separation and concentration effect of Fe3O4 magnetic nanoparticles, the limits of detection of the developed method were found to be 25, 10, and 15 cfu mL−1 for S. aureus, V. parahemolyticus, and S. typhimurium, respectively. The capability of the bioassay in real food samples was also investigated, and the results were consistent with experimental results obtained from plate-counting methods. This proposed method for the detection of various pathogenic bacteria based on multicolor UCNPs has great potential in the application of food safety and multiplex nanosensors. fluorophore-labeled antibodies with different specificities for recognizing different targets in the samples. However, the use of conventional fluorescent dyes or nanoparticles in multicolor immunoassays is limited because of rapid photobleaching and instability. Moreover, their narrow excitation spectrum and broad emission spectrum introduce spectral cross-talk between the different detection channels. Lanthanide-doped near-infrared (NIR)-to-visible upconversion nanoparticles (UCNPs) are capable of emitting strong visible luminescence with the excitation of NIR light (typically 980 nm).11 UCNPs have shown significant advantages over the traditional organic fluorophores as fluorescent biolabels, because of their attractive optical and chemical features, their lack of autofluorescence and the fact that a light-scattering background can be induced by biological samples.12,13 As a result, the signal-to-background ratio can be greatly improved. In addition, UCNPs have also attracted increasing interest because of their tunable optical properties, which are tunable through variation of their lanthanide dopants, which include
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athogenic bacteria can survive and spread easily in the environment and pose a serious threat to human health under the appropriate conditions. For example, Escherichia coli O157:H7, Salmonella spp., and Staphyllococcus spp. are the most common causes of foodborne illnesses. In addition, multiple bacterial pathogens may coexist at different concentrations in the same food sample but typically occur at low levels.1,2 Therefore, the challenge exists to develop rapid, sensitive, and specific methods that are capable of simultaneously detecting multiple pathogens of interest. Traditional culture-based methods are preceded by an enrichment step to increase the number of bacteria to reach the detection level. For the simultaneous detection of more than one pathogen, differences in growth requirements and growth rates should be considered. As an alternative, some molecular methods such as multiplex polymerase chain reaction (PCR) or real-time PCR detection can be also considered for the simultaneous detection of five, six, or even more pathogens.3−6 However, the currently accepted bacteria detection methods require PCR amplification and/or cell culturing, which are slow, timeconsuming, and laborious and have limited suitability for onsite analyses. In recent years, several methods based on immunological theory have been developed to address this issue.7−10 An immunoassay platform usually contain an array of © 2014 American Chemical Society
Received: December 21, 2013 Accepted: February 25, 2014 Published: February 25, 2014 3100
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Er3+, Tm3+, and Ho3+.14,15 Multicolor UCNPs can simultaneously be used as labels for multiple targets.16−19 Aptamers are DNA or RNA molecules that bind to their molecular targets with high affinity and specificity as antibodies, and they provide a variety of advantages over antibodies.20 For example, aptamers are easy to synthesize, stable, inexpensive, simple to chemically modify, and minimally immunogenic.21,22 In addition, aptamers can be modified with a variety of fluorescent dyes or other tags, which provides extraordinary flexibility in the development of assays.23−25 Herein, we describe the very promising and fascinating development of a highly sensitive and specific method that combines multicolor UCNPs with aptamers. In this study, three types of multicolor upconversion nanoparticles were synthesized as signal probes, and their independent emission peaks of multicolor UCNPs were adjusted via doping with different rare earth ions. Next, the multicolor UCNPs were conjugated with the aptamers of S. aureus, V. parahemolyticus, and S. typhimurium to simultaneously detect these bacteria. This method presented here was highly sensitive and selective because of the NIR laser-induced upconversion luminescence, the absence of autofluorescence interference, and the high specificity of the aptamers. Some interesting results related to multicolor UCNPs imaging in vivo and in vitro have appeared in the literature;26,27 however, no results referring to three colors of UCNPs as labels for detection have been reported. This method may promote the application of multicolor UCNPs in analytical methods and potentially in food safety, especially for use in the detection of pathogenic bacteria, toxins, viruses, and the others.
ethanol to the bottom of the vessel, then centrifuged to obtain a powdered sample, washed with ethanol and distilled water several times, and then dried in an oven at 60 °C. We synthesized other Mn2+-doped NaYF4:Yb, Er UCNPs in a similar manner by varying the amount of Mn2+ and rare-earth ions. Briefly, aqueous solutions of 0.6 mL of 0.5 M MnCl2, 1 mL of 0.5 M Y(NO3)3, 0.9 mL of 0.2 M Yb(NO3)3, and 0.1 mL of 0.2 M Er(NO3)3 were combined under magnetic stirring. Ultimately, Mn2+-doped NaYF4:Yb, Er UCNPs were obtained. The oleic acid-capped UCNPs disperse well in nonpolar solvents including cyclohexane, chloroform, and toluene. However, for biological labeling applications, UCNPs should be compatible with biomolecules, such as nucleic acids. The hydrophobic UCNPs were consequently converted into hydrophilic UCNPs via a slightly modified version of a previously described method for ligand exchange using PAA.32 Typically, PAA (0.5 g) was added to a flask containing 10 mL of DEG. The mixture was then heated to 110 °C with vigorous stirring under nitrogen for 15 min to form a clear solution. A toluene solution containing the oleic acid-capped UCNPs (50 mg in 2 mL) was quickly injected into the hot solution and was evaporated by heating the solution for 15 min (without reflux). Next, the system was refluxed under nitrogen at 240 °C for 2 h. The resulting solution was cooled down to room temperature and treated with 0.1 M HCl with stirring for 15 min. The precipitated powder was recovered via centrifugation (3000 rpm, 10 min) and washed three times with deionized water to remove excess PAA; the products were easily dispersed in water or an aqueous buffer solution. Attachment of Amino Oligonucleotide to Magnetic Nanoparticles and Upconversion Nanoparticles. The procedure for the preparation of oligonucleotide conjugated magnetic nanoparticles (MNPs) and UCNPs were adapted from the previously reported paper.33 For example, in the case of MNPs conjugated with complementary DNA1, 5 mg of MNPs were first dispersed in 5 mL of 10 mM PBS buffer solution at pH 7.4, and then 0.4 mL of EDC (2 mg mL−1) and 0.2 mL of NHS (2 mg mL−1) were subsequently introduced into the solution. The reaction was continued for 2 h at 37 °C in a reciprocating oscillator. After incubation, the MNPs were separated with an external magnetic field and washed three times with ultrapure water. Next, the activated MNPs were dispersed in 5 mL of PBS buffer solution, and 50 μL of 100 μM amino-modified cDNA1 was subsequently added. The mixture was then incubated at 37 °C with oscillating for another 2 h. The amino group on the 5′ end of the cDNA1 covalently bonded to the carboxyl group on the MNPs surface via a catalyzed condensation reaction. After removal of the supernatant, the cDNA1 conjugated MNPs were washed twice with PBS buffer by magnetic separation and then finally redispersed in fresh STE buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 1 mM EDTA). MNPs conjugated with cDNA2 and cDNA3 were prepared using a similar protocol. In addition, substituting centrifugation for magnetic separation, we subsequently purified UCNPs modified with 1 μM amino aptamers and redispersed them in fresh STE buffer for hybridization. Procedure for the Simultaneous Detection of Three Pathogenic Bacteria. The entire procedure for this proposed method was convenient and fast. In the present work, 200 μL of aptamers-UCNPs were hybridized with the optimized cDNA-MNPs at 37 °C for 30 min to obtain three couples of UCNPs-MNPs signal probes, respectively. The UCNPs-MNPs were separated by a magnetic field and subsequently measured
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EXPERIMENTAL SECTION Reagents. The rare earth nitrates used in this work, including Y(NO3)3·6H2O, Yb(NO3)3·5H2O, Ho(NO3)3·5H2O, and Er(NO3)3·5H2O were of 99.99% purity and were purchased from Aladdin Industrial Inc. (Shanghai, China). FeCl3·6H2O, Na3C6H5O7, MnCl2, NaF, NaOH, HCl, NaBH4, toluene, cyclohexane, and ethanol were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), poly(acrylic acid) (PAA), diethylene glycol (DEG), and oleic acid were obtained from Sigma-Aldrich (U.S.A.). The bacteria aptamers28−30 and their part complementary strands (in Table S1 in the Supporting Information) were synthesized by Shanghai Sangon Biological Science & Technology Company (Shanghai, China). Synthesis and Surface Modification of Upconversion Nanoparticles. Multicolor oleic acid-capped UCNPs were prepared by a modified hydrothermal process.31 In a typical procedure for the synthesis of NaYF4: Yb, Tm/Ho UCNPs, NaOH (1.2 g, 30 mmol), water (9 mL), ethanol (10 mL), and oleic acid (20 mL) were mixed under agitation to form a homogeneous solution. Next, 0.936 mL of 0.5 M Y(NO3)3, 0.6 mL of 0.2 M Yb(NO3)3, and 0.06 mL of 0.2 M Tm(NO3)3 or Ho(NO3)3 (Y−Yb−Tm/Ho = 78 mol %−20 mol %−2 mol %) were added to the mixture and stirred thoroughly. Subsequently, a 0.168 g NaF (4 mmol) solution was added dropwise to the previously prepared solution. After being vigorously stirred at room temperature for 15 min, the colloidal solution was transferred into a 50 mL Teflon-lined autoclave, sealed, and heated at 190 °C for 12 h. After the mixture cooled to room temperature, the products were deposited by adding 3101
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Scheme 1. Schematic Illustration of the Multiplexed Luminescence Bioassay Based on Aptamers-Modified UCNPs for the Simultaneous Detection of Various Pathogenic Bacteria
using a 980 nm laser. Various concentrations (1 to 108 cfu mL−1) of bacteria were added to this mixture and further incubated at 37 °C for 30 min. The remaining UCNPs-MNPs were then separated and washed three times, and the luminescence was measured with a 980 nm excitation laser. The concentration of the three bacteria was related to the corresponding emission peak of the multicolor UCNPs.
tion in the system. Subsequently, S. aureus was added to the system, and Apt1 preferentially bound to S. aureus and caused the dissociation of some cDNA1, thereby liberating some S. aureus-Apt1-UCNPsTm. Finally, the intensity of the emission peak at 477 nm decreased as a result of the reduced concentration of UCNPsTm-MNPs signal probes. The basic principle of the strategy was that aptamers could form a defined conformation when binding to the targets and were also able to hybridize to the complementary DNA sequences to form a duplex structure. When the targets and the complementary oligonucleotides were introduced, the aptamers preferentially bound to the targets, resulting in the specific recognition of the targets. To further study the recognition process of the developed method, we used circular dichroism (CD) measurements to confirm the conformational variations of the aptamers. The CD spectra of the aptamers solution and the solution after the aptamers were incubated with their target bacteria were recorded in Figure S1 in the Supporting Information. The most important and fascinating aspect of the proposed method is that three types of UCNPs were selected as multicolor labels. These UCNPs exhibited independent single emission peaks with three types of aptamers bonded to S. aureus, V. parahemolyticus, and S. typhimurium. According to a similar method, PAA-modified NaYF4 :Yb, Ho UCNPs
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RESULTS AND DISCUSSION Principle of Simultaneous Detection of Three Bacteria Based on Multicolor UCNPs Labeling. The luminescence bioassay platform for the simultaneous detection of foodborne pathogenic bacteria based on multicolor UCNPs labeling and MNPs separation was illustrated in Scheme 1. S. aureus and aptamers of S. aureus were selected as proof-of-concept targets. Specifically, PAA-modified NaYF4: Yb, Tm UCNPs (short for UCNPsTm) were conjugated with amino-modified Apt1 via a condensation reaction, MNPs were conjugated with cDNA1, and the complex of UCNPsTm-MNPs was assembled through hybridization of Apt1 and cDNA1. Therefore, the background luminescence was gathered from UCNPsTm-MNPs as signal probes. In the absence of S. aureus, the background luminescence was at a maximum because of the abundance of UCNPsTm-MNPs signal probes collected by magnetic separa3102
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Figure 1. Luminescence spectra of NaYF4:Yb, Tm UCNPs (a), NaYF4:Yb, Ho UCNPs (b), Mn2+ ions doped NaYF4:Yb, Er UCNPs (c) and a mixture of them (d). The insets are photographs of the naked-eye visible upconversion luminescence. All solutions were excited with an external 980 nm laser.
Figure 2. Morphology and dispersibility study. High-resolution TEM images of oleic acid-capped NaYF4:Yb, Tm UCNPs (a) dispersed in cyclohexane, after ligand exchange, PAA-modified NaYF4:Yb, Tm UCNPs (b) dispersed in aqueous solution.
(UCNPsHo) were conjugated to Apt2 and PAA-modified NaYF4:Yb, Er/Mn UCNPs (UCNPsEr) were conjugated to Apt3. Three couples of aptamers-UCNPs were obtained, in addition to the assembly of three couples of complementary DNA-MNPs. Aptamers were subsequently hybridized with the corresponding complementary DNA to form three couples of UCNPs-MNPs probes. In the presence of bacteria, the aptamers could bind to the specific bacteria and caused a
decrease in the intensity of the corresponding upconversion emission peaks. In summary, we have developed a method for the simultaneous and sensitive detection method for three types of foodborne pathogenic bacteria using multicolor UCNPs as signal probes. Characterization of Upconversion Nanoparticles and Magnetic Nanoparticles. Following 980 nm excitation, visible upconversion emission was observed for all of the 3103
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Figure 3. UV−vis absorption spectra of bare NaYF4 UCNPs and aptamers-functionalized NaYF4 UCNPs (A), bare Fe3O4 MNPs and complementary oligonucleotides-modified Fe3O4 MNPs (B).
NaYF4 nanoparticles under investigation (Figure 1). The UCNPs were shown to emit visible light, such as blue, green, or red light, in response to near-infrared irradiation, and their emission colors were simply tuned by alteration of the dopant species, such as Tm3+, Ho3+, and Er3+ ions. Because of their tunable colors in the visible spectral region, these species can be potentially applied to simultaneous multiplexed labeling and analysis. In Yb3+/Tm3+ and Yb3+/Ho3+ co-doped systems, Yb3+ ions act as sensitizers, whereas Tm3+ and Ho3+ ions act as activators. The single blue emission at 477 nm, which is assigned to the transition from the 4G4 level to the 3H6 ground state, and the single green emission at 542 nm, which is assigned to the transition from the 5S2 level to the 5I8 ground state, were observed in the luminescence spectrum (Figure 1a,b). In particularly, a single-band red emission at 660 nm due to the 4H9/2 → 4I15/2 transition appeared in the spectra of the Yb3+, Er3+, and Mn2+co-doped systems when the Mn2+ concentration reached 30 mol % (Figure 1c).34 The inset of Figure 1 shows naked-eye photographs of the visible blue, green, and red upconversion luminescence of the UCNPs excited with an external 980 nm laser. More importantly, all of the predominant emission peaks exhibited a large Stokes shift and a narrow peak width and were independent and clearly distinguished from one another. Thus, these three kinds of UCNPs doping with different rare-earth ions used as labels were essential for establishment of a simultaneous detection for three foodborne pathogenic bacteria (Figure 1d). In this work, NaYF4 UCNPs were synthesized in a water− ethanol−oleic acid system via a solvothermal method. These nanoparticles were dispersible in nonpolar solvents (such as cyclohexane) because of the presence of the organic ligand (oleic acid) on the surface of the nanocrystals. The size and morphology of the oleic acid-capped UCNPs were characterized by transmission electron microscopy (TEM). As shown in Figure 2a and Figure S2a,c in the Supporting Information, the monodisperse nanocubes of the as-prepared samples with sizes of 20−30 nm suggested that the long-chain oleic acid ligands on the crystal surface prevented aggregation in cyclohexane. However, the water dispersibility of the UCNPs was crucial in bio-applications; thus, the hydrophilic modifications of their surface was performed in this work. To convert the hydrophobic oleic acid-capped UCNPs into the carboxylic acid-functionalized derivatives, PAA was used as a multidentate ligand to displace the original hydrophobic ligand
on the surface of UCNPs, eventually making the UCNPs highly water dispersible. After the ligand exchange, the PAA-modified samples still consisted primarily of single nanocubes; however, the formation of nanoarrays was prevented by the carboxylic acid interactions (Figure 2b and Figure S2b,d in the Supportting Information). These observations indicated that such ligand oxidation had no obvious effects on the size and shape of the UCNPs, except for their array formation. The luminescence intensity of the modified samples was nearly unchanged and was better than that of UCNPs coated with a silica shell. More importantly, the resultant carboxyl-functionalized UCNPs could be easily bonded to amino aptamers. FTIR spectroscopy was used to characterize the functional groups present on the surface of the NaYF4 UCNPs before and after the ligand exchange (Figure S3 in the Supporting Information). The structure of these UCNPs samples was characterized by Xray powder diffraction (XRD), as shown in Figure S4 in the Supporting Information. The carboxyl-functionalized magnetic nanoparticles applied herein were obtained with Na3Cit. TEM images (Figure S5a in the Supporting Information), FT-IR spectra (Figure S5b in the Supporting Information), and XRD patterns (Figure S5c in the Supporting Information) were used to characterize the as-synthesized magnetic nanoparticles. Characterization of Nanoparticles Conjugated with Oligonucleotides. The aptamers were conjugated onto the upconversion nanoparticle surfaces by using the amino-carboxyl reaction, as described in the Experimental Section. The conjugation of UCNPs with aptamers was characterized using UV−vis absorption spectroscopy; results were shown in Figure 3A. No absorption peaks appeared in the UV−vis spectrum of carboxyl-functionalized UCNPs (three types of UCNPs) before they were conjugated with aptamers. After the UCNPs were incubated with amino-modified aptamers and the supernatant was discarded, a new absorption peak at approximately 260 nm, the ultraviolet absorption maximum of DNA, was observed in the spectrum of the suspension as a result of the amount of aptamers combined with carboxyl-functionalized UCNPs. Zeta potential experiments indicated that the PAA-modified NaYF4 UCNPs were negatively charged, and the zeta potential was positively shifted after the UCNPs were conjugated to the aptamers. For example, in the case of UCNPsTm conjugated with Apt1 (Figure S6A in the Supporting Information), the zeta potential varied from −32.5 mV to −24.7 mV. These results 3104
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appearing at approximately 90 bp was the aptamer (lane 2). Because polyacrylamide gel electrophoresis is a molecular weight-dependent method, low-molecular-weight aptamers migrated much further compared to aptamers-UCNPs conjugates (lane 3). Similarly, low-molecular-weight complementary oligonucleotides, approximately 30 bp (lane 5), migrated much further compared to cDNA−MNPs conjugates (lane 6). The differences in the mobility of DNA versus DNA conjugates indicated that the amide coupling chemistry was successful, that is, amine modified DNA was indeed attached to the surface of the nanoparticles. Alternatively, the supernatant separated after DNA incubation with nanoparticles was also measured using PAGE electrophoresis. A shallow band appeared (lane 4) in the same position as band 2, in addition to an inconspicuous band that appeared (lane 7) in accordance with band 5. These results indicated that the concentration of DNA deceased after incubation with UCNPs and MNPs. Thus, the results obtained using the above two approaches were consistent. Optimization of Dosage of Complementary DNAMNPs. At the first separation, the background luminescence of UCNPs-MNPs reached a maximum and subsequently gradually decreased with increasing cell concentration. This result indicated that the intensity of the background luminescence was closely related to the sensitivity and linear range of this proposed method. Furthermore, either the aptamers-UCNPs or the cDNA-MNPs were a variable for the amount of UCNPsMNPs signal probes. A comparative study was performed to identify the optimal dosage of cDNA-MNPs, using blank samples (without bacteria). The detection was based on the change in the luminescence intensity after the addition of various volumes of cDNA-MNPs solution to a certain volume of aptamers-UCNPs solution. As shown in Figure S7 in the Supporting Information, the luminescence intensity at 477 nm increased in proportion to the addition of cDNA1-MNPs solution, reaching a maximum intensity with the addition of 100 μL of cDNA1-MNPs solution. Thereafter, the luminescence intensity decreased with the addition of excess cDNA1MNPs solution. Initially, a small quantity of UCNPs-MNPs was separated with an external magnetic field, and the lower concentration of cDNA1-MNPs led to the abundance of Apt1UCNPsTm not having any cDNA1-MNPs to bind on the basis of base pairing. When the concentrations of cDNA1-MNPs and the concentration of Apt1-UCNPsTm were matched, the luminescence intensity reached a maximum. However, if the quantity of cDNA1-MNPs was in excess, the luminescence intensity of the UCNPsTm-MNPs was decreased because the cDNA1-MNPs were unable to combine with more Apt1UCNPsTm, and the black Fe3O4 MNPs would block some of the luminescence signal. For a similar reason, the luminescence intensities of the UCNPsHo-MNPs and the UCNPsEr-MNPs were analyzed to optimize the dosage of cDNA2-MNPs and cDNA3-MNPs. The results in Figure S7 in the Supporting Information showed that, when 120 μL of cDNA2-MNPs and 100 μL of cDNA3-MNPs were used, the corresponding emission peaks at 542 and 660 nm reached a maximum. Multiplexed Determination of Target Bacteria. In this experiment, three types of rare earth ions doped UCNPs were synthetized and modified for using as multicolor labels due to their independent single emission peaks. The emission peaks of upconversion luminescence of S. aureus, V. parahemolyticus, and S. typhimurium were monitored at wavelengths 477, 542, and 660 nm, respectively, which obviously did not overlap. As
also verified that the nanoparticles have been successfully functionalized with amino aptamers. Similar to the UV−vis spectra of the magnetic nanoparticles, no absorption peaks appeared in the spectra of the carboxylfunctionalized MNPs and a new absorption peak appeared at approximately 260 nm, which is assigned to DNA, after the carboxyl-functionalized MNPs were incubated and underwent a condensation interaction with amino complementary oligonucleotides (Figure 3B). The zeta potential of the as-prepared MNPs was also negatively charged, which was attributed to the carboxy groups on the surface. The significance of the zeta potential is that its value is related to the stability of a colloidal dispersion. Because cDNA-modified MNPs were less stable than bare MNPs due to the weak force that prevents the nanoparticles from coming together and flocculating, the zeta potential of cDNA-modified MNPs was positively shifted after the samples were incubated. For example, the zeta potential varied from −49.2 mV to −33.9 mV for MNPs before and after they were conjugated with cDNA1, respectively (Figure S6B in the Supporting Information). The results of UV spectroscopy and zeta potential clearly demonstrated that finally the three types of cDNA−MNPs complexes were successfully formed. To verify successful coupling of the amine-modified aptamers and complementary oligonucleotides to UCNPs and MNPs, a polyacrylamide gel electrophoresis (PAGE) was performed (Figure 4). This technique revealed the obvious retardation of the electrophoretic mobility of the complexes of oligonucleotides and nanoparticles compared to that of oligonucleotides alone. This result can be explained by the increased formation of a highly convoluted and branched nanoparticles surface with multiple emanating DNA strands. The bright and visible band
Figure 4. Polyacrylamide gel electrophoresis results for nanoparticles conjugated with oligonucleotides. Lanes 1−7 represent the 150 bp DNA ladder, Apt1 alone, Apt1-UCNPsTm conjugates, the supernatant after incubation of Apt1 with UCNPsTm, cDNA1 alone, cDNA1−MNPs conjugates, and the supernatant after incubation of cDNA1 with MNPs, respectively. 3105
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Specificity. Specificity of detection is one of the most important characteristics in the evaluation of an analytical method, especially for the simultaneous detection of multiplex targets. The specificity of the proposed method was investigated via the following protocol. Two requirements should be satisfied to obtain the positive relative luminescence signal, the aptamers must match with their complementary DNA and the aptamers should specifically bind with the target bacteria. As shown in Figure 6, the relative luminescence signals
previously mentioned, in the absence of target bacteria, the three couples of luminescence intensity were maximum background luminescence (I0). In the presence of target bacteria, the aptamers preferentially bound to bacteria and caused the dissociation of some bacteria-aptamers-UCNPs from UCNPs-MNPs with a gradual decrease in the luminescence intensity (I) of the unreleased UCNPs. Various intensities of luminescence spectra obtained in the presence of different concentrations of S. aureus, V. parahemolyticus, and S. typhimurium were shown in Figure 5A. Under optimal
Figure 6. Cross reaction for multiplex detection of S. aureus, V. parahemolyticus, and S. typhimurium. The concentration of all three bacteria are 104 cfu mL−1.
were positive only when the aptamers matched with the complementary DNA and target bacteria coexisted. Moreover, these results showed that there was no nonspecific adsorption on the surface of the nanoparticles. These results also revealed that no cross-reactivity occurred during the simultaneous detection of S. aureus, V. parahemolyticus, and S. typhimurium. The changes in the luminescence signal induced by the binding of five other pathogenic bacteria (Shigella dysenteriae, Listeria monocytogenes, Escherichia coli, Cronobacter sakazakii, and Streptococcus pyogenes) were compared using the same assay procedure. Figure S9 in the Supporting Information showed that the luminescence signal of S. dysenteriae, L. monocytogenes, E. coli, C. sakazakii, and S. pyogenes did not exhibit any significant changes and were analogous to the luminescence signals of the blank buffer. In contrast to the incubation of the aptamer-modified MNPs and UCNPs in S. aureus, V. parahemolyticus, and S. typhimurium, the corresponding signal intensity greatly changed. As a result, the response induced by the non-specific binding was negligible compared to the response induced by the target bacteria, which demonstrated the high specificity of the multiplexed bioassay. Application of S. aureus, V. parahemolyticus and S. typhimurium Quantification in Food Samples. The accuracy of the applied bioanalysis method was evaluated using real food samples in liquid and solid matrixes, i.e., milk and shrimp samples, respectively. The eight real food samples were simply pre-treated as previously mentioned. Next, the samples were spiked with between 1 × 102 and 1 × 105 cfu mL−1 S. aureus, V. parahemolyticus, and S. typhimurium. The analysis results were given in Table S3 in the Supporting Information. The results of the proposed method were similar to those of the plate-counting method, and no significant differences between the compared methods were observed. The
Figure 5. Typical recording output for the simultaneous detection of different concentrations of three bacteria by the developed method. Standard curve of the related upconversion luminescence intensity (I0 − I) versus the concentrations of three bacteria.
conditions, the concentration of bacteria was proportional to the decreased luminescence intensity (ΔI = I0 − I), where ΔI represents the difference in the upconversion luminescence intensity excited by a 980 nm laser in the absence and in the presence of target bacteria (Figure 5B). Three foodborne pathogenic bacteria were detected in wide concentration ranges in a mixture in which all of the linear ranges for S. aureus, V. parahemolyticus, and S. typhimurium were 50 to 106 cfu mL−1, and the limits of detection (LOD) were found to be 25, 10, and 15 cfu mL−1, respectively. As shown in Table S2 in the Supporting Information, all of the calibration curves were observed over the tested range. The relative standard deviation (RSD) of this detection indicated that the developed method exhibited good precision and reproducibility. 3106
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(8) Zhao, Y.; Ye, M. Q.; Chao, Q. G.; Jia, N. Q.; Ge, Y.; Shen, H. B. J. Agric. Food Chem. 2009, 57, 517−524. (9) Wang, L.; Zhao, W. J.; O’Donoghue, M. B.; Tan, W. H. Bioconjugate Chem. 2007, 18, 297−301. (10) Gu, H.; Ho, P. L.; Tsang, K. W.; Wang, L.; Xu, B. J. Am. Chem. Soc. 2003, 125, 15702−15703. (11) Auzel, F. Chem. Rev. 2004, 104, 139−173. (12) Wang, F.; Banerjee, D.; Liu, Y. S.; Chen, X. Y.; Liu, X. G. Analyst 2010, 135, 1839−1854. (13) Cheng, L.; Wang, C.; Liu, Z. Nanoscale 2013, 5, 23−27. (14) Liu, Y. S.; Tu, D. T.; Zhu, H. M.; Chen, X. Y. Chem. Soc. Rev. 2013, 42, 6924−6958. (15) Markus, H.; Helmut, S. Angew. Chem., Int. Ed. 2011, 50, 5808− 5829. (16) Wu, S. J.; Duan, N.; Zhu, C. Q.; Ma, X. Y.; Wang, M.; Wang, Z. P. Biosens. Bioelectron. 2011, 30, 35−42. (17) Wu, S. J.; Duan, N.; Ma, X. Y.; Xia, Y.; Yu, Y.; Wang, Z. P.; Wang, H. X. Chem. Commun. 2012, 48, 4866−4868. (18) Duan, N.; Wu, S. J.; Zhu, C. Q.; Ma, X. Y.; Wang, Z. P.; Yu, Y.; Jiang, Y. Anal. Chim. Acta 2012, 723, 1−6. (19) Wu, S. J.; Duan, N.; Ma, X. Y.; Xia, Y.; Wang, H. X.; Wang, Z. P.; Zhang, Q. Anal. Chem. 2012, 84, 6263−6270. (20) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818−822. (21) Tuerk, C.; Gold, L. Science 1990, 249, 505−510. (22) Ellington, A. D.; Szostak, J. W. Nature 1992, 355, 850−852. (23) Breaker, R. R. Curr. Opin. Chem. Biol. 1997, 1, 26−31. (24) Yamamoto, R.; Kumar, P. K. R. Genes Cells 2000, 5, 389−396. (25) Hamula, C. L .A.; Guthrie, J. W.; Zhang, H.; Li, X. F.; Le, X. C. Trends Anal. Chem. 2006, 25, 681−691. (26) Wang, M.; Mi, C. C.; Zhang, Y. X.; Liu, J. L.; Feng, L.; Mao, C. B.; Xu, S. K. J. Phys. Chem. C 2009, 113, 19021−19027. (27) Li, Z. Q.; Zhang, Y. Angew. Chem., Int. Ed. 2006, 46, 7732−7735. (28) Cao, X. X.; Li, S. H.; Chen, L. C.; Ding, H. M.; Xu, H.; Huang, Y. P.; Li, J.; Liu, N. L.; Cao, W. H.; Zhu, Y. J.; Shen, B. F.; Shao, N. S. Nucleic Acids Res. 2009, 37, 4621−4628. (29) Duan, N.; Wu, S. J.; Chen, X. J.; Huang, Y. K.; Wang, Z. P. J. Agric. Food Chem. 2012, 60, 4034−4038. (30) Duan, N.; Wu, S. J.; Chen, X. J.; Huang, Y. K.; Xia, Y.; Ma, X. Y.; Wang, Z. P. J. Agric. Food Chem. 2013, 61, 3229−3234. (31) Chen, Z. G.; Chen, H. L.; Hu, H.; Yu, M. X.; Li, F. Y.; Zhang, Q.; Zhou, Z. G.; Yi, T.; Huang, C. H. J. Am. Chem. Soc. 2008, 30, 3023−3029. (32) Liu, C. H.; Wang, H.; Li, X.; Chen, D. P. J. Mater. Chem. 2009, 19, 3546−3553. (33) Wu, S. J.; Duan, N.; Ma, X. Y.; Xia, Yu.; Wang, H. X.; Wang, Z. P. Anal. Chim. Acta 2013, 782, 59−66. (34) Tian, G.; Gu, Z. J.; Zhou, L. J.; Yin, W. Y.; Liu, X. X.; Yan, L.; Jin, S.; Ren, W. L.; Xing, G. M.; Li, S. J.; Zhao, Y. L. Adv. Mater. 2012, 24, 1226−1231.
application performance clearly demonstrated that the multiplex method based on multicolor UCNPs labeling and aptamers affinity can efficiently and simultaneously detect and quantify three bacteria that coexist in real samples.
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CONCLUSION In this study, a multiplexed aptasensor based on multicolor upconversion nanoparticles coupled with magnetic nanoparticles was successfully developed and evaluated for the simultaneous, rapid, and specific detection of S. aureus, V. parahemolyticus, and S. typhimurium. Various rare earth ions doped UCNPs exhibited independent emission peaks were used to label the corresponding target bacteria. More importantly, any autofluorescence originating from the biomolecules possibly contained in the solution was entirely avoided through of the use of infrared 980 nm laser excitation. The use of magnetic nanoparticles provided an efficient method for the separation and concentration of targets from the interferences in the food matrix with the help of a magnetic field, without requiring multiple pretreatment processes. In addition, the aptamers were stable (unlike traditional antibodies, which were vulnerable) and highly specific to the target bacteria. Therefore, these aptamers offer us a new approach to the fabrication of a convenient, sensitive, specific, and stable platform for a bioassay. The proposed simultaneous detection provides great potential to detect various types of pathogenic bacteria coexisting in the food matrixes through the substitution of suitable aptamers.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone/fax: +86-51085917023. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly supported by NSFC (Grant 21375049), the S&T Supporting Project of Jiangsu Province (Grant BE2011621), National Science and Technology Support Program of China (Grant 2012BAK08B01), Research Fund for the Doctoral Program of Higher Education (Grant 20110093110002), Grant NCET-11-0663, and Grant JUSRP51309A.
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REFERENCES
(1) Brecher, M. E.; Hay, S. N. Clin. Microbiol. Rev. 2005, 18, 195− 204. (2) Shorten, P. R.; Pleasants, A. B.; Soboleva, T. K. Int. J. Food Microbiol. 2006, 108, 369−375. (3) Kramer, M.; Obermajer, N.; Bogovic Matijasic, B.; Rogelj, I.; Kmetec, V. Appl. Microbiol. Biotechnol. 2009, 84, 1137−1147. (4) Stokstad, E. Science 2006, 312, 523−524. (5) Deisingh, A. K.; Thompson, M. J. Appl. Microbiol. 2004, 96, 419− 429. (6) Jofre, A.; Martin, B.; Garriga, M.; Hugas, M.; Pla, M.; RodriguezLazaro, D.; Aymerich, T. Food Microbiol. 2005, 22, 109−115. (7) Yang, L. J.; Li, Y. B. Analyst 2006, 131, 394−401. 3107
dx.doi.org/10.1021/ac404205c | Anal. Chem. 2014, 86, 3100−3107
Simultaneous Aptasensor for Multiplex Pathogenic Bacteria Detection Based on Multicolor Upconversion Nanoparticles Labels Shijia Wu, Nuo Duan, Zhao Shi, CongCong Fang, and Zhouping Wang* State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China S Supporting Information *
ABSTRACT: A highly sensitive and specific multiplex method for the simultaneous detection of three pathogenic bacteria was fabricated using multicolor upconversion nanoparticles (UCNPs) as luminescence labels coupled with aptamers as the molecular recognition elements. Multicolor UCNPs were synthesized via doping with various rare-earth ions to obtain well-separated emission peaks. The aptamer sequences were selected using the systematic evolution of ligands by exponential enrichment (SELEX) strategy for Staphylococcus aureus, Vibrio parahemolyticus, and Salmonella typhimurium. When applied in this method, aptamers can be used for the specific recognition of the bacteria from complex mixtures, including those found in real food matrixes. Aptamers and multicolor UCNPs were employed to selectively capture and simultaneously quantify the three target bacteria on the basis of the independent peaks. Under optimal conditions, the correlation between the concentration of three bacteria and the luminescence signal was found to be linear from 50−106 cfu mL−1. Improved by the magnetic separation and concentration effect of Fe3O4 magnetic nanoparticles, the limits of detection of the developed method were found to be 25, 10, and 15 cfu mL−1 for S. aureus, V. parahemolyticus, and S. typhimurium, respectively. The capability of the bioassay in real food samples was also investigated, and the results were consistent with experimental results obtained from plate-counting methods. This proposed method for the detection of various pathogenic bacteria based on multicolor UCNPs has great potential in the application of food safety and multiplex nanosensors. fluorophore-labeled antibodies with different specificities for recognizing different targets in the samples. However, the use of conventional fluorescent dyes or nanoparticles in multicolor immunoassays is limited because of rapid photobleaching and instability. Moreover, their narrow excitation spectrum and broad emission spectrum introduce spectral cross-talk between the different detection channels. Lanthanide-doped near-infrared (NIR)-to-visible upconversion nanoparticles (UCNPs) are capable of emitting strong visible luminescence with the excitation of NIR light (typically 980 nm).11 UCNPs have shown significant advantages over the traditional organic fluorophores as fluorescent biolabels, because of their attractive optical and chemical features, their lack of autofluorescence and the fact that a light-scattering background can be induced by biological samples.12,13 As a result, the signal-to-background ratio can be greatly improved. In addition, UCNPs have also attracted increasing interest because of their tunable optical properties, which are tunable through variation of their lanthanide dopants, which include
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athogenic bacteria can survive and spread easily in the environment and pose a serious threat to human health under the appropriate conditions. For example, Escherichia coli O157:H7, Salmonella spp., and Staphyllococcus spp. are the most common causes of foodborne illnesses. In addition, multiple bacterial pathogens may coexist at different concentrations in the same food sample but typically occur at low levels.1,2 Therefore, the challenge exists to develop rapid, sensitive, and specific methods that are capable of simultaneously detecting multiple pathogens of interest. Traditional culture-based methods are preceded by an enrichment step to increase the number of bacteria to reach the detection level. For the simultaneous detection of more than one pathogen, differences in growth requirements and growth rates should be considered. As an alternative, some molecular methods such as multiplex polymerase chain reaction (PCR) or real-time PCR detection can be also considered for the simultaneous detection of five, six, or even more pathogens.3−6 However, the currently accepted bacteria detection methods require PCR amplification and/or cell culturing, which are slow, timeconsuming, and laborious and have limited suitability for onsite analyses. In recent years, several methods based on immunological theory have been developed to address this issue.7−10 An immunoassay platform usually contain an array of © 2014 American Chemical Society
Received: December 21, 2013 Accepted: February 25, 2014 Published: February 25, 2014 3100
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Er3+, Tm3+, and Ho3+.14,15 Multicolor UCNPs can simultaneously be used as labels for multiple targets.16−19 Aptamers are DNA or RNA molecules that bind to their molecular targets with high affinity and specificity as antibodies, and they provide a variety of advantages over antibodies.20 For example, aptamers are easy to synthesize, stable, inexpensive, simple to chemically modify, and minimally immunogenic.21,22 In addition, aptamers can be modified with a variety of fluorescent dyes or other tags, which provides extraordinary flexibility in the development of assays.23−25 Herein, we describe the very promising and fascinating development of a highly sensitive and specific method that combines multicolor UCNPs with aptamers. In this study, three types of multicolor upconversion nanoparticles were synthesized as signal probes, and their independent emission peaks of multicolor UCNPs were adjusted via doping with different rare earth ions. Next, the multicolor UCNPs were conjugated with the aptamers of S. aureus, V. parahemolyticus, and S. typhimurium to simultaneously detect these bacteria. This method presented here was highly sensitive and selective because of the NIR laser-induced upconversion luminescence, the absence of autofluorescence interference, and the high specificity of the aptamers. Some interesting results related to multicolor UCNPs imaging in vivo and in vitro have appeared in the literature;26,27 however, no results referring to three colors of UCNPs as labels for detection have been reported. This method may promote the application of multicolor UCNPs in analytical methods and potentially in food safety, especially for use in the detection of pathogenic bacteria, toxins, viruses, and the others.
ethanol to the bottom of the vessel, then centrifuged to obtain a powdered sample, washed with ethanol and distilled water several times, and then dried in an oven at 60 °C. We synthesized other Mn2+-doped NaYF4:Yb, Er UCNPs in a similar manner by varying the amount of Mn2+ and rare-earth ions. Briefly, aqueous solutions of 0.6 mL of 0.5 M MnCl2, 1 mL of 0.5 M Y(NO3)3, 0.9 mL of 0.2 M Yb(NO3)3, and 0.1 mL of 0.2 M Er(NO3)3 were combined under magnetic stirring. Ultimately, Mn2+-doped NaYF4:Yb, Er UCNPs were obtained. The oleic acid-capped UCNPs disperse well in nonpolar solvents including cyclohexane, chloroform, and toluene. However, for biological labeling applications, UCNPs should be compatible with biomolecules, such as nucleic acids. The hydrophobic UCNPs were consequently converted into hydrophilic UCNPs via a slightly modified version of a previously described method for ligand exchange using PAA.32 Typically, PAA (0.5 g) was added to a flask containing 10 mL of DEG. The mixture was then heated to 110 °C with vigorous stirring under nitrogen for 15 min to form a clear solution. A toluene solution containing the oleic acid-capped UCNPs (50 mg in 2 mL) was quickly injected into the hot solution and was evaporated by heating the solution for 15 min (without reflux). Next, the system was refluxed under nitrogen at 240 °C for 2 h. The resulting solution was cooled down to room temperature and treated with 0.1 M HCl with stirring for 15 min. The precipitated powder was recovered via centrifugation (3000 rpm, 10 min) and washed three times with deionized water to remove excess PAA; the products were easily dispersed in water or an aqueous buffer solution. Attachment of Amino Oligonucleotide to Magnetic Nanoparticles and Upconversion Nanoparticles. The procedure for the preparation of oligonucleotide conjugated magnetic nanoparticles (MNPs) and UCNPs were adapted from the previously reported paper.33 For example, in the case of MNPs conjugated with complementary DNA1, 5 mg of MNPs were first dispersed in 5 mL of 10 mM PBS buffer solution at pH 7.4, and then 0.4 mL of EDC (2 mg mL−1) and 0.2 mL of NHS (2 mg mL−1) were subsequently introduced into the solution. The reaction was continued for 2 h at 37 °C in a reciprocating oscillator. After incubation, the MNPs were separated with an external magnetic field and washed three times with ultrapure water. Next, the activated MNPs were dispersed in 5 mL of PBS buffer solution, and 50 μL of 100 μM amino-modified cDNA1 was subsequently added. The mixture was then incubated at 37 °C with oscillating for another 2 h. The amino group on the 5′ end of the cDNA1 covalently bonded to the carboxyl group on the MNPs surface via a catalyzed condensation reaction. After removal of the supernatant, the cDNA1 conjugated MNPs were washed twice with PBS buffer by magnetic separation and then finally redispersed in fresh STE buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 1 mM EDTA). MNPs conjugated with cDNA2 and cDNA3 were prepared using a similar protocol. In addition, substituting centrifugation for magnetic separation, we subsequently purified UCNPs modified with 1 μM amino aptamers and redispersed them in fresh STE buffer for hybridization. Procedure for the Simultaneous Detection of Three Pathogenic Bacteria. The entire procedure for this proposed method was convenient and fast. In the present work, 200 μL of aptamers-UCNPs were hybridized with the optimized cDNA-MNPs at 37 °C for 30 min to obtain three couples of UCNPs-MNPs signal probes, respectively. The UCNPs-MNPs were separated by a magnetic field and subsequently measured
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EXPERIMENTAL SECTION Reagents. The rare earth nitrates used in this work, including Y(NO3)3·6H2O, Yb(NO3)3·5H2O, Ho(NO3)3·5H2O, and Er(NO3)3·5H2O were of 99.99% purity and were purchased from Aladdin Industrial Inc. (Shanghai, China). FeCl3·6H2O, Na3C6H5O7, MnCl2, NaF, NaOH, HCl, NaBH4, toluene, cyclohexane, and ethanol were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), poly(acrylic acid) (PAA), diethylene glycol (DEG), and oleic acid were obtained from Sigma-Aldrich (U.S.A.). The bacteria aptamers28−30 and their part complementary strands (in Table S1 in the Supporting Information) were synthesized by Shanghai Sangon Biological Science & Technology Company (Shanghai, China). Synthesis and Surface Modification of Upconversion Nanoparticles. Multicolor oleic acid-capped UCNPs were prepared by a modified hydrothermal process.31 In a typical procedure for the synthesis of NaYF4: Yb, Tm/Ho UCNPs, NaOH (1.2 g, 30 mmol), water (9 mL), ethanol (10 mL), and oleic acid (20 mL) were mixed under agitation to form a homogeneous solution. Next, 0.936 mL of 0.5 M Y(NO3)3, 0.6 mL of 0.2 M Yb(NO3)3, and 0.06 mL of 0.2 M Tm(NO3)3 or Ho(NO3)3 (Y−Yb−Tm/Ho = 78 mol %−20 mol %−2 mol %) were added to the mixture and stirred thoroughly. Subsequently, a 0.168 g NaF (4 mmol) solution was added dropwise to the previously prepared solution. After being vigorously stirred at room temperature for 15 min, the colloidal solution was transferred into a 50 mL Teflon-lined autoclave, sealed, and heated at 190 °C for 12 h. After the mixture cooled to room temperature, the products were deposited by adding 3101
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Scheme 1. Schematic Illustration of the Multiplexed Luminescence Bioassay Based on Aptamers-Modified UCNPs for the Simultaneous Detection of Various Pathogenic Bacteria
using a 980 nm laser. Various concentrations (1 to 108 cfu mL−1) of bacteria were added to this mixture and further incubated at 37 °C for 30 min. The remaining UCNPs-MNPs were then separated and washed three times, and the luminescence was measured with a 980 nm excitation laser. The concentration of the three bacteria was related to the corresponding emission peak of the multicolor UCNPs.
tion in the system. Subsequently, S. aureus was added to the system, and Apt1 preferentially bound to S. aureus and caused the dissociation of some cDNA1, thereby liberating some S. aureus-Apt1-UCNPsTm. Finally, the intensity of the emission peak at 477 nm decreased as a result of the reduced concentration of UCNPsTm-MNPs signal probes. The basic principle of the strategy was that aptamers could form a defined conformation when binding to the targets and were also able to hybridize to the complementary DNA sequences to form a duplex structure. When the targets and the complementary oligonucleotides were introduced, the aptamers preferentially bound to the targets, resulting in the specific recognition of the targets. To further study the recognition process of the developed method, we used circular dichroism (CD) measurements to confirm the conformational variations of the aptamers. The CD spectra of the aptamers solution and the solution after the aptamers were incubated with their target bacteria were recorded in Figure S1 in the Supporting Information. The most important and fascinating aspect of the proposed method is that three types of UCNPs were selected as multicolor labels. These UCNPs exhibited independent single emission peaks with three types of aptamers bonded to S. aureus, V. parahemolyticus, and S. typhimurium. According to a similar method, PAA-modified NaYF4 :Yb, Ho UCNPs
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RESULTS AND DISCUSSION Principle of Simultaneous Detection of Three Bacteria Based on Multicolor UCNPs Labeling. The luminescence bioassay platform for the simultaneous detection of foodborne pathogenic bacteria based on multicolor UCNPs labeling and MNPs separation was illustrated in Scheme 1. S. aureus and aptamers of S. aureus were selected as proof-of-concept targets. Specifically, PAA-modified NaYF4: Yb, Tm UCNPs (short for UCNPsTm) were conjugated with amino-modified Apt1 via a condensation reaction, MNPs were conjugated with cDNA1, and the complex of UCNPsTm-MNPs was assembled through hybridization of Apt1 and cDNA1. Therefore, the background luminescence was gathered from UCNPsTm-MNPs as signal probes. In the absence of S. aureus, the background luminescence was at a maximum because of the abundance of UCNPsTm-MNPs signal probes collected by magnetic separa3102
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Figure 1. Luminescence spectra of NaYF4:Yb, Tm UCNPs (a), NaYF4:Yb, Ho UCNPs (b), Mn2+ ions doped NaYF4:Yb, Er UCNPs (c) and a mixture of them (d). The insets are photographs of the naked-eye visible upconversion luminescence. All solutions were excited with an external 980 nm laser.
Figure 2. Morphology and dispersibility study. High-resolution TEM images of oleic acid-capped NaYF4:Yb, Tm UCNPs (a) dispersed in cyclohexane, after ligand exchange, PAA-modified NaYF4:Yb, Tm UCNPs (b) dispersed in aqueous solution.
(UCNPsHo) were conjugated to Apt2 and PAA-modified NaYF4:Yb, Er/Mn UCNPs (UCNPsEr) were conjugated to Apt3. Three couples of aptamers-UCNPs were obtained, in addition to the assembly of three couples of complementary DNA-MNPs. Aptamers were subsequently hybridized with the corresponding complementary DNA to form three couples of UCNPs-MNPs probes. In the presence of bacteria, the aptamers could bind to the specific bacteria and caused a
decrease in the intensity of the corresponding upconversion emission peaks. In summary, we have developed a method for the simultaneous and sensitive detection method for three types of foodborne pathogenic bacteria using multicolor UCNPs as signal probes. Characterization of Upconversion Nanoparticles and Magnetic Nanoparticles. Following 980 nm excitation, visible upconversion emission was observed for all of the 3103
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Figure 3. UV−vis absorption spectra of bare NaYF4 UCNPs and aptamers-functionalized NaYF4 UCNPs (A), bare Fe3O4 MNPs and complementary oligonucleotides-modified Fe3O4 MNPs (B).
NaYF4 nanoparticles under investigation (Figure 1). The UCNPs were shown to emit visible light, such as blue, green, or red light, in response to near-infrared irradiation, and their emission colors were simply tuned by alteration of the dopant species, such as Tm3+, Ho3+, and Er3+ ions. Because of their tunable colors in the visible spectral region, these species can be potentially applied to simultaneous multiplexed labeling and analysis. In Yb3+/Tm3+ and Yb3+/Ho3+ co-doped systems, Yb3+ ions act as sensitizers, whereas Tm3+ and Ho3+ ions act as activators. The single blue emission at 477 nm, which is assigned to the transition from the 4G4 level to the 3H6 ground state, and the single green emission at 542 nm, which is assigned to the transition from the 5S2 level to the 5I8 ground state, were observed in the luminescence spectrum (Figure 1a,b). In particularly, a single-band red emission at 660 nm due to the 4H9/2 → 4I15/2 transition appeared in the spectra of the Yb3+, Er3+, and Mn2+co-doped systems when the Mn2+ concentration reached 30 mol % (Figure 1c).34 The inset of Figure 1 shows naked-eye photographs of the visible blue, green, and red upconversion luminescence of the UCNPs excited with an external 980 nm laser. More importantly, all of the predominant emission peaks exhibited a large Stokes shift and a narrow peak width and were independent and clearly distinguished from one another. Thus, these three kinds of UCNPs doping with different rare-earth ions used as labels were essential for establishment of a simultaneous detection for three foodborne pathogenic bacteria (Figure 1d). In this work, NaYF4 UCNPs were synthesized in a water− ethanol−oleic acid system via a solvothermal method. These nanoparticles were dispersible in nonpolar solvents (such as cyclohexane) because of the presence of the organic ligand (oleic acid) on the surface of the nanocrystals. The size and morphology of the oleic acid-capped UCNPs were characterized by transmission electron microscopy (TEM). As shown in Figure 2a and Figure S2a,c in the Supporting Information, the monodisperse nanocubes of the as-prepared samples with sizes of 20−30 nm suggested that the long-chain oleic acid ligands on the crystal surface prevented aggregation in cyclohexane. However, the water dispersibility of the UCNPs was crucial in bio-applications; thus, the hydrophilic modifications of their surface was performed in this work. To convert the hydrophobic oleic acid-capped UCNPs into the carboxylic acid-functionalized derivatives, PAA was used as a multidentate ligand to displace the original hydrophobic ligand
on the surface of UCNPs, eventually making the UCNPs highly water dispersible. After the ligand exchange, the PAA-modified samples still consisted primarily of single nanocubes; however, the formation of nanoarrays was prevented by the carboxylic acid interactions (Figure 2b and Figure S2b,d in the Supportting Information). These observations indicated that such ligand oxidation had no obvious effects on the size and shape of the UCNPs, except for their array formation. The luminescence intensity of the modified samples was nearly unchanged and was better than that of UCNPs coated with a silica shell. More importantly, the resultant carboxyl-functionalized UCNPs could be easily bonded to amino aptamers. FTIR spectroscopy was used to characterize the functional groups present on the surface of the NaYF4 UCNPs before and after the ligand exchange (Figure S3 in the Supporting Information). The structure of these UCNPs samples was characterized by Xray powder diffraction (XRD), as shown in Figure S4 in the Supporting Information. The carboxyl-functionalized magnetic nanoparticles applied herein were obtained with Na3Cit. TEM images (Figure S5a in the Supporting Information), FT-IR spectra (Figure S5b in the Supporting Information), and XRD patterns (Figure S5c in the Supporting Information) were used to characterize the as-synthesized magnetic nanoparticles. Characterization of Nanoparticles Conjugated with Oligonucleotides. The aptamers were conjugated onto the upconversion nanoparticle surfaces by using the amino-carboxyl reaction, as described in the Experimental Section. The conjugation of UCNPs with aptamers was characterized using UV−vis absorption spectroscopy; results were shown in Figure 3A. No absorption peaks appeared in the UV−vis spectrum of carboxyl-functionalized UCNPs (three types of UCNPs) before they were conjugated with aptamers. After the UCNPs were incubated with amino-modified aptamers and the supernatant was discarded, a new absorption peak at approximately 260 nm, the ultraviolet absorption maximum of DNA, was observed in the spectrum of the suspension as a result of the amount of aptamers combined with carboxyl-functionalized UCNPs. Zeta potential experiments indicated that the PAA-modified NaYF4 UCNPs were negatively charged, and the zeta potential was positively shifted after the UCNPs were conjugated to the aptamers. For example, in the case of UCNPsTm conjugated with Apt1 (Figure S6A in the Supporting Information), the zeta potential varied from −32.5 mV to −24.7 mV. These results 3104
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appearing at approximately 90 bp was the aptamer (lane 2). Because polyacrylamide gel electrophoresis is a molecular weight-dependent method, low-molecular-weight aptamers migrated much further compared to aptamers-UCNPs conjugates (lane 3). Similarly, low-molecular-weight complementary oligonucleotides, approximately 30 bp (lane 5), migrated much further compared to cDNA−MNPs conjugates (lane 6). The differences in the mobility of DNA versus DNA conjugates indicated that the amide coupling chemistry was successful, that is, amine modified DNA was indeed attached to the surface of the nanoparticles. Alternatively, the supernatant separated after DNA incubation with nanoparticles was also measured using PAGE electrophoresis. A shallow band appeared (lane 4) in the same position as band 2, in addition to an inconspicuous band that appeared (lane 7) in accordance with band 5. These results indicated that the concentration of DNA deceased after incubation with UCNPs and MNPs. Thus, the results obtained using the above two approaches were consistent. Optimization of Dosage of Complementary DNAMNPs. At the first separation, the background luminescence of UCNPs-MNPs reached a maximum and subsequently gradually decreased with increasing cell concentration. This result indicated that the intensity of the background luminescence was closely related to the sensitivity and linear range of this proposed method. Furthermore, either the aptamers-UCNPs or the cDNA-MNPs were a variable for the amount of UCNPsMNPs signal probes. A comparative study was performed to identify the optimal dosage of cDNA-MNPs, using blank samples (without bacteria). The detection was based on the change in the luminescence intensity after the addition of various volumes of cDNA-MNPs solution to a certain volume of aptamers-UCNPs solution. As shown in Figure S7 in the Supporting Information, the luminescence intensity at 477 nm increased in proportion to the addition of cDNA1-MNPs solution, reaching a maximum intensity with the addition of 100 μL of cDNA1-MNPs solution. Thereafter, the luminescence intensity decreased with the addition of excess cDNA1MNPs solution. Initially, a small quantity of UCNPs-MNPs was separated with an external magnetic field, and the lower concentration of cDNA1-MNPs led to the abundance of Apt1UCNPsTm not having any cDNA1-MNPs to bind on the basis of base pairing. When the concentrations of cDNA1-MNPs and the concentration of Apt1-UCNPsTm were matched, the luminescence intensity reached a maximum. However, if the quantity of cDNA1-MNPs was in excess, the luminescence intensity of the UCNPsTm-MNPs was decreased because the cDNA1-MNPs were unable to combine with more Apt1UCNPsTm, and the black Fe3O4 MNPs would block some of the luminescence signal. For a similar reason, the luminescence intensities of the UCNPsHo-MNPs and the UCNPsEr-MNPs were analyzed to optimize the dosage of cDNA2-MNPs and cDNA3-MNPs. The results in Figure S7 in the Supporting Information showed that, when 120 μL of cDNA2-MNPs and 100 μL of cDNA3-MNPs were used, the corresponding emission peaks at 542 and 660 nm reached a maximum. Multiplexed Determination of Target Bacteria. In this experiment, three types of rare earth ions doped UCNPs were synthetized and modified for using as multicolor labels due to their independent single emission peaks. The emission peaks of upconversion luminescence of S. aureus, V. parahemolyticus, and S. typhimurium were monitored at wavelengths 477, 542, and 660 nm, respectively, which obviously did not overlap. As
also verified that the nanoparticles have been successfully functionalized with amino aptamers. Similar to the UV−vis spectra of the magnetic nanoparticles, no absorption peaks appeared in the spectra of the carboxylfunctionalized MNPs and a new absorption peak appeared at approximately 260 nm, which is assigned to DNA, after the carboxyl-functionalized MNPs were incubated and underwent a condensation interaction with amino complementary oligonucleotides (Figure 3B). The zeta potential of the as-prepared MNPs was also negatively charged, which was attributed to the carboxy groups on the surface. The significance of the zeta potential is that its value is related to the stability of a colloidal dispersion. Because cDNA-modified MNPs were less stable than bare MNPs due to the weak force that prevents the nanoparticles from coming together and flocculating, the zeta potential of cDNA-modified MNPs was positively shifted after the samples were incubated. For example, the zeta potential varied from −49.2 mV to −33.9 mV for MNPs before and after they were conjugated with cDNA1, respectively (Figure S6B in the Supporting Information). The results of UV spectroscopy and zeta potential clearly demonstrated that finally the three types of cDNA−MNPs complexes were successfully formed. To verify successful coupling of the amine-modified aptamers and complementary oligonucleotides to UCNPs and MNPs, a polyacrylamide gel electrophoresis (PAGE) was performed (Figure 4). This technique revealed the obvious retardation of the electrophoretic mobility of the complexes of oligonucleotides and nanoparticles compared to that of oligonucleotides alone. This result can be explained by the increased formation of a highly convoluted and branched nanoparticles surface with multiple emanating DNA strands. The bright and visible band
Figure 4. Polyacrylamide gel electrophoresis results for nanoparticles conjugated with oligonucleotides. Lanes 1−7 represent the 150 bp DNA ladder, Apt1 alone, Apt1-UCNPsTm conjugates, the supernatant after incubation of Apt1 with UCNPsTm, cDNA1 alone, cDNA1−MNPs conjugates, and the supernatant after incubation of cDNA1 with MNPs, respectively. 3105
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Analytical Chemistry
Article
Specificity. Specificity of detection is one of the most important characteristics in the evaluation of an analytical method, especially for the simultaneous detection of multiplex targets. The specificity of the proposed method was investigated via the following protocol. Two requirements should be satisfied to obtain the positive relative luminescence signal, the aptamers must match with their complementary DNA and the aptamers should specifically bind with the target bacteria. As shown in Figure 6, the relative luminescence signals
previously mentioned, in the absence of target bacteria, the three couples of luminescence intensity were maximum background luminescence (I0). In the presence of target bacteria, the aptamers preferentially bound to bacteria and caused the dissociation of some bacteria-aptamers-UCNPs from UCNPs-MNPs with a gradual decrease in the luminescence intensity (I) of the unreleased UCNPs. Various intensities of luminescence spectra obtained in the presence of different concentrations of S. aureus, V. parahemolyticus, and S. typhimurium were shown in Figure 5A. Under optimal
Figure 6. Cross reaction for multiplex detection of S. aureus, V. parahemolyticus, and S. typhimurium. The concentration of all three bacteria are 104 cfu mL−1.
were positive only when the aptamers matched with the complementary DNA and target bacteria coexisted. Moreover, these results showed that there was no nonspecific adsorption on the surface of the nanoparticles. These results also revealed that no cross-reactivity occurred during the simultaneous detection of S. aureus, V. parahemolyticus, and S. typhimurium. The changes in the luminescence signal induced by the binding of five other pathogenic bacteria (Shigella dysenteriae, Listeria monocytogenes, Escherichia coli, Cronobacter sakazakii, and Streptococcus pyogenes) were compared using the same assay procedure. Figure S9 in the Supporting Information showed that the luminescence signal of S. dysenteriae, L. monocytogenes, E. coli, C. sakazakii, and S. pyogenes did not exhibit any significant changes and were analogous to the luminescence signals of the blank buffer. In contrast to the incubation of the aptamer-modified MNPs and UCNPs in S. aureus, V. parahemolyticus, and S. typhimurium, the corresponding signal intensity greatly changed. As a result, the response induced by the non-specific binding was negligible compared to the response induced by the target bacteria, which demonstrated the high specificity of the multiplexed bioassay. Application of S. aureus, V. parahemolyticus and S. typhimurium Quantification in Food Samples. The accuracy of the applied bioanalysis method was evaluated using real food samples in liquid and solid matrixes, i.e., milk and shrimp samples, respectively. The eight real food samples were simply pre-treated as previously mentioned. Next, the samples were spiked with between 1 × 102 and 1 × 105 cfu mL−1 S. aureus, V. parahemolyticus, and S. typhimurium. The analysis results were given in Table S3 in the Supporting Information. The results of the proposed method were similar to those of the plate-counting method, and no significant differences between the compared methods were observed. The
Figure 5. Typical recording output for the simultaneous detection of different concentrations of three bacteria by the developed method. Standard curve of the related upconversion luminescence intensity (I0 − I) versus the concentrations of three bacteria.
conditions, the concentration of bacteria was proportional to the decreased luminescence intensity (ΔI = I0 − I), where ΔI represents the difference in the upconversion luminescence intensity excited by a 980 nm laser in the absence and in the presence of target bacteria (Figure 5B). Three foodborne pathogenic bacteria were detected in wide concentration ranges in a mixture in which all of the linear ranges for S. aureus, V. parahemolyticus, and S. typhimurium were 50 to 106 cfu mL−1, and the limits of detection (LOD) were found to be 25, 10, and 15 cfu mL−1, respectively. As shown in Table S2 in the Supporting Information, all of the calibration curves were observed over the tested range. The relative standard deviation (RSD) of this detection indicated that the developed method exhibited good precision and reproducibility. 3106
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application performance clearly demonstrated that the multiplex method based on multicolor UCNPs labeling and aptamers affinity can efficiently and simultaneously detect and quantify three bacteria that coexist in real samples.
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CONCLUSION In this study, a multiplexed aptasensor based on multicolor upconversion nanoparticles coupled with magnetic nanoparticles was successfully developed and evaluated for the simultaneous, rapid, and specific detection of S. aureus, V. parahemolyticus, and S. typhimurium. Various rare earth ions doped UCNPs exhibited independent emission peaks were used to label the corresponding target bacteria. More importantly, any autofluorescence originating from the biomolecules possibly contained in the solution was entirely avoided through of the use of infrared 980 nm laser excitation. The use of magnetic nanoparticles provided an efficient method for the separation and concentration of targets from the interferences in the food matrix with the help of a magnetic field, without requiring multiple pretreatment processes. In addition, the aptamers were stable (unlike traditional antibodies, which were vulnerable) and highly specific to the target bacteria. Therefore, these aptamers offer us a new approach to the fabrication of a convenient, sensitive, specific, and stable platform for a bioassay. The proposed simultaneous detection provides great potential to detect various types of pathogenic bacteria coexisting in the food matrixes through the substitution of suitable aptamers.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone/fax: +86-51085917023. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly supported by NSFC (Grant 21375049), the S&T Supporting Project of Jiangsu Province (Grant BE2011621), National Science and Technology Support Program of China (Grant 2012BAK08B01), Research Fund for the Doctoral Program of Higher Education (Grant 20110093110002), Grant NCET-11-0663, and Grant JUSRP51309A.
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