Magnetic Bead-Based Multiplex DNA Sequence Detection of

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Magnetic Bead-Based Multiplex DNA Sequence Detection of Genetically Modified Organisms Using Quantum Dot-Encoded Silicon Dioxide Nanoparticles Yuan Zhao,† Changlong Hao,† Wei Ma,† Qianqian Yong,† Wenjing Yan,† Hua Kuang,† Libing Wang,*,†,‡ and Chuanlai Xu† † ‡

School of Food Science & Technology, State Key Lab of Food Science & Technology, Jiangnan University, China, 214122 Hunan Entry -Exit Inspection and Quarantine Bureau, Changsha, 410001, PR China

bS Supporting Information ABSTRACT: In this study, we report the multiplex DNA detection of genetically modified organisms (GMOs) based on fluorescence and magnetic nanostructures built up with quantum dot-encoded silicon dioxide nanoparticles (QD-encoded SiO2 NPs) and magnetic nanoparticles (MNPs). Detection was achieved by fluorescence spectrophotometry without auxiliary instruments. GMOs, including corn, soybean, colza, and cotton, were simultaneously detected, and the quantitative detection of soybean with a limit of detection of low femtomolar concentrations was successfully achieved. SiO2 NPs were encoded by different colored QDs at various ratios through a reverse microemulsion. QDencoded SiO2 NP-labeled probe DNAs and MNP-labeled capture DNAs were used to hybridize with the corresponding targets at the same time. After magnetic separation, the sandwich structures were measured by fluorescence spectrophotometry. According to the overlay spectra of QDs, we estimated the presence of corresponding targets by analyzing the results via X-ray photoelectron spectroscopy peak software. Multiplex label-free DNA detection of GMOs based on QD-encoded SiO2 NPs showed high sensitivity and can be applied in other samples.

1. INTRODUCTION With the increasing development of transgenic technology, the development of genetically modified organisms (GMOs) has received significant attention for pest and weed control and to further improve production. In addition, pesticide abuse has been largely reduced, which has had a positive effect on the environment. Transgenic technology has increasingly become an important industry. Similarly, the safety of GMOs has aroused universal controversy and involves ecological risk, environmental problems, and human health. It is of critical importance to identify novel methods for the detection of inserted exogenous genes of GMOs. To date, the analysis of GMOs is mainly based on protein and DNA detection, using such methods as enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR).1,2 ELISA has high sensitivity, specificity, and simple sample preparation. However, the application of ELISA is a complex operation and may be affected by extreme conditions, such as temperature and pH. ELISA is restricted to one single protein type and cannot be used for comprehensive GMO screens encompassing several varieties at a time. PCR is a classical approach for DNA sequencing and genetic analysis but suffers from the disadvantages of false positive results and nonspecific amplification. Nowadays, multiplex suspension array assays are increasingly applied in nucleic acid detection, immunoassays, and bioanalysis.3
8 Different from conventional planar arrays, suspension array assays with fast reaction kinetics are easy to manipulate and achieve high-throughput analysis9 and the large surface areas of the r 2011 American Chemical Society

microbeads enrich target molecules. In this study, quantum dot (QD)-encoded SiO2 NPs as a support for the suspension were applied in the detection of multiplex DNA. Compared to conventional dyes, QDs play a major role in encoding nanoparticles due to their unique optical properties, such as broad absorption spectra, size-tunable emission spectra, and excellent photostability.10 The hydroxyls on the surface of SiO2 NPs can be easily modified by amino or carboxyl groups, endowing the SiO2 NPs with good biocompatibility to further immobilize biomolecules. Various types of functional SiO2 NPs have been widely synthesized. Monodisperse core
shell-structured Fe3O4
SiO2 NPs can act as a multifunctional drug carrier or an efficient absorbent.11,12 In addition, dual-function microbeads embedded within QDs and MNPs have been widely synthesized and exhibit excellent optical properties and magnetic response,13,18
20 and QDs also were used to encode mesoporous polystyrene beads.21 Mingyuan Gao et al. successfully synthesized SiO2 NPs capped by a single number of QDs or a number of one type of QD in the core using a reverse microemulsion.22
24 This approach is characterized by simple manipulation, and SiO2 NPs have a uniform structure and good dispersivity. There are many reports on dye-doped SiO2 NPs and QD-capped SiO2 NPs in bioimaging applications and DNA detection.25
30 At the same time, Gu et al. synthesized silica Received: July 7, 2011 Revised: August 23, 2011 Published: August 26, 2011 20134

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The Journal of Physical Chemistry C colloidal crystals beads deposited by QDs using a layer-by-layer approach for DNA detection.31 However, DNA detection was distinguished by the fluorescence signal of both codes and dyelabeled oligonucleotides. Multiplex label-free DNA detection based on QD-tagged hydrogel suspension arrays was accomplished by the signal of codes and the diffraction spectra of the hydrogel.32
34 Furthermore, multiplex DNA detection was traditionally achieved by flow cytometry or a microfluidic system.35,36 In our study, we prepared QD-encoded SiO2 NPs of different colors and various intensity levels using a reverse microemulsion for the multiplex label-free DNA detection of GMOs. Briefly, QD-encoded SiO2 NPs and magnetic NPs were used to label probe DNA and capture DNA. If the target DNA existed, probe DNA and capture DNA hybridized to the target DNA, forming a new sandwich structure. After magnetic separation, the nonspecific sequences were removed, and the corresponding spectra were obtained by fluorescence measurement. For multiplex detection of GMOs in our research, four kinds of QD-encoded SiO2 NPs with distinguished fluorescence signals were, respectively, used to label four kinds of probe DNAs; meanwhile, four kinds of capture DNAs were labeled by magnetic NPs. In the presence of four target DNAs without any modification, four kinds of sandwich structures were formed; multiplex label-free DNA detection was achieved according to the overlay spectra of QD-encoded SiO2 NPs without extra optical signals, illustrating the superiority of encoding. Four types of GMOs, including corn, soybean, colza, and cotton, were simultaneously detected based on QD-encoded SiO2 NPs in a short time period. The presence of targets was determined by the fluorescence spectra of QD-encoded SiO2 NPs through fluorescence spectrophotometry, and the results only needed further analysis by XPS peak software without auxiliary instruments. This approach not only allowed multiplex label-free DNA detection of GMO but also achieved quantitative detection, and the LOD was at femtomolar (fM) concentrations.

2. EXPERIMENTAL SECTION 2.1. Materials and Apparatus. Tetraethylorthosilicate (TEOS) and (3-aminopropyl)triethoxysilane (APTES) were purchased from Aldrich Chemical Co., Inc. Nucleotide sequences of target GMOs and the corresponding probes and captures were purchased from Sangon Biotech (Shanghai) Co. Ltd. Probe DNAs and capture DNAs were modified with carboxy groups at the 50 end and amine groups at the 30 end, respectively. The morphologies of QDs, MNPs, and QD-encoded SiO2 NPs were characterized using a JEOL JEM-2100 transmission electron microscope (TEM), and the fluorescent spectra of QDencoded SiO2 NPs were determined by an F-7000 fluorescence spectrophotometer. The results were analyzed by XPS peak software. 2.2. Synthesis of MPA-Capped CdTe QDs. 3-Mercaptopropionic acid (MPA)-capped CdTe QDs were synthesized according to published procedures.24,37,38 First, 390 mg of Te powder, 189 mg of NaBH4 and 4 mL of Milli-Q water were added to a 10 mL glass bottle and sealed with a syringe needle on the top of the bottle capsule for aeration, and the mixture was reacted at 4 °C for 1 day to obtain solution A. A 570 mg portion of CdCl2 3 5H2O and 148 mL of Milli-Q water were mixed together in a 250 mL three-neck flask under continuous magnetic stirring, and then 0.5 mL of MPA was added dropwise to the above solution, while the pH was adjusted to 7.5 by NaOH solution. After deoxidation by introducing nitrogen for about 30 min, this mixture was rapidly injected into

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2 mL of solution A under N2 protection. After reacting for 10 min, an orange-yellow precursor without photoluminescence was successfully synthesized and required further reaction in a reaction kettle at high temperature and high pressure.28,29 QDs with an emission wavelength between 520 and 740 nm were obtained when the emission wavelength was 350 nm. 2.3. Synthesis of MNPs. A 650 mg portion of anhydrous FeCl3 and 400 mg of sodium citrate were dissolved in 20 mL of glycol under stirring for 20 min, and then 1.2 g of anhydrous sodium acetate was added to the above solution. After 10 min, the mixture was reacted in a reaction kettle at 230 °C for 10 h. Finally, the solution was cooled to room temperature under continuous stirring. The MNPs were washed several times with ethanol and water and dispersed in 10 mL of Milli-Q water.39 MNPs with carboxyl groups on the surface were successfully synthesized by this approach and exhibited high monodispersity. 2.4. Preparation of QD-Encoded SiO2 NPs. A 2.5 mL portion of Triton X-100 and 2.5 mL of hexanol were first mixed together under magnetic stirring for 10 min, and then 10 mL of cyclohexane was added. The mixture was further stirred for 20 min, and then 2 mL of an aqueous solution with multicolored QDs at various ratios was added to the above mixture, forming a new microemulsion solution. After 15 min, 300 μL of NH4OH as a catalyst was added dropwise to the above microemulsion with stirring for a further 30 min. For the synthesis of SiO2 NPs with a single QD in the core, only 40 μL of TEOS was rapidly added to hydrolyze for 12 h. For QD-encoded SiO2 NPs, 40 μL of TEOS and 40 μL of APTES were simultaneously injected and reacted for 12 h at room temperature. At the same time, the addition of APTES endowed the SiO2 NPs with amino groups for the combination of biomolecules. Finally, the addition of 2 mL of acetone was used to break the microemulsion. The particles were washed three times with ethanol and water and dispersed in 5 mL of Milli-Q water.22,24,40 In our research, the synthesized QDs solution with various emission wavelengths had a similar concentration, so we adjusted the volume of the QD solution in the microemulsion to synthesize QD-encoded SiO2 NPs. For example, the same volume of QD solution with the emission wavelength at 525 and 585 nm was added into the microemulsion to synthesize QD-encoded with the ratios of 1:1, and the volume ratio of the QD solution could be adjusted at 1:3 and so on. 2.5. Immobilization of Probe DNA onto QD-Encoded SiO2 NPs. Probe DNAs were carboxy-functionalized at the 50 end. QD-encoded SiO2 NPs with amino groups and probe DNAs were covalently reacted using the carbodiimide method. The 10 μM probe DNAs were first activated by 4.2 mg/mL of EDC and 5.0 mg/mL of sulfo-NHS overnight, and then 100 μL of QDencoded SiO2 NPs with amino groups were added. The mixture was incubated for 5 h under continuous shaking in the dark at room temperature. Finally, QD-SiO2
probe DNAs were washed by centrifugation three times and dispersed in 100 μL of PBS buffer solution. Similarly, four types of QD-SiO2
probe DNAs corresponding to four types of GMOs were obtained. 2.6. Immobilization of Capture DNA onto MNPs. Capture DNAs were amine-functionalized at the 30 end. A 2 μL portion of MNPs with carboxy groups at the surface was first activated by 4.2 mg/mL of EDC and 5.0 mg/mL of sulfo-NHS overnight, and then 2 μL of 10 μM capture DNAs with amino groups was added. The mixture was incubated for 5 h under shaking at room temperature. Finally, MNPs
capture DNAs were washed by centrifugation three times and dispersed in 100 μL of PBS buffer 20135

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The Journal of Physical Chemistry C solution. Similarly, four types of MNPs
capture DNAs corresponding to four types of GMOs were obtained. 2.7. Multiplex GMO Detection Based on QD-Encoded SiO2 NPs. Four types of QD-SiO2
probe DNAs and four types of MNPs
capture DNAs were mixed in 500 μL of PBS buffer solution, and a certain concentration of the four types of targets was added to the above solution for hybridization in an oven at 37 °C to form a new sandwich structure. After 5 h, the conjugations were separated under an external magnetic field and further detected by fluorescence measurement.

Figure 1. Schematic illustration of multiplex label-free DNA detection based on magnetic and QD-encoded SiO2 NPs.

Figure 2. (a) TEM images of SiO2 NPs with a single QD in the center. (b) TEM images of QD-encoded SiO2 NPs.

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3. RESULTS AND DISCUSSION 3.1. Characterization of Nanoparticles. A system was built up based on magnetic and QD-encoded SiO2 NPs in the application multiplex detection. As schematically shown in Figure 1, magnetic NP-labeled capture DNAs and QD-encoded SiO2 NPlabeled probe DNAs hybridized with the corresponding target DNA. After magnetic separation and fluorescence measurement, we obtained the overlay spectra of QDs. QDs with an emission wavelength that ranged from 520 to 740 nm were used in this experiment. As shown in Figure S1 (Supporting Information), QDs showed excellent photoluminescence and good dispersivity. However, the synthesis of QDs with the emission wavelength at 740 nm was hard to manipulate and exhibited lower fluorescence intensity than the other three types of QDs. TEM images showed that the average diameter was bigger than 585 nm emitting QDs and the structure of QDs was inhomogeneous. The W/O microemulsion method yielded uniform QD-encoded SiO2 NPs and was easy to operate. In the microemulsion system, cyclohexane acted as a continuous phase, and Triton X-100 and hexanol acted as emulsifiers, forming a spherical micelle. The addition of QD-aqueous solution created a microwater pool. Because of Brownian movement, TEOS molecules perforated the micelle layer and were catalyzed by NH4OH to form the silica intermediate. The amount of TEOS determined the silicon dioxide shell thickness, which can enhance the photostability of QDs. The alkaline condition due to NH4OH caused a negative charge on the silica intermediate and MPA-stabilized CdTe QDs; therefore, electrostatic repulsion eliminated redundant CdTe QDs from the microwater pool, resulting in the preparation of monodispersive SiO2 NPs with a single QD located in the center. As shown in Figure 2a, a single QD was seen in the red circle. To obtain QD-encoded SiO2 NPs for multiplex DNA detection, the number of QDs in the core of SiO2 NPs could be adjusted by controlling the electrostatic repulsion between the silica intermediate and MPA-stabilized CdTe QDs. It has been reported that poly(diallydimethylammonium chloride) (PDDA) and 3-aminopropyltrimethoxysilane (APS) were applied to balance the electrostatic repulsion.14,15 In our study, APTES and TEOS were simultaneously hydrolyzed by NH4OH to obtain SiO2 NPs with a number of QDs located in the center. CH2CH2CH2NH2 groups of APTES would also partially replace OCH2CH3 groups of TEOS, decreasing the number of and the charge density of silica intermediates. Furthermore, NH2 and silanol groups would create hydrogen bonds, reducing the electrostatic repulsion. As shown in Figure 2b, the number of QDs in the core of SiO2 NPs increased; therefore, multicolored QDs with various intensity levels could be applied to encode SiO2 NPs. The images showed the excellent fluorescence characteristic of encoded SiO2 NPs with various ratios (Supporting Information, Figure S2). Interestingly, the hydrolysis of APTES synchronously achieved the modification of amino groups at the surface of SiO2 NPs, creating a platform for combining biomolecules.

Table 1. Nucleotide Sequences of Target GMOs, Probes, and Captures Used for the Experiments GMO

targets (50 -30 )

probes (50 -30 )

captures (50 -30 )

corn soybean

GAG TGG AAG TCT GTC GCG TGG TAC CA AGG GAT GAC GCA CAA TCC CAC TAT CC

TGG TAC CAC GCG AC GGA TAG TGG GAT TG

AGA CTT CCA CTC TGC GTC ATC CCT

colza

GCA GCT GCA TTC GCT GAA GGT GCT AC

GTA GCA CCT TCA GC

GAA TGC AGC TGC

cotton

CTC CAA CCC AGC GAT TTC GGT TAC TT

AAG TAA CCG AAA TC

GCT GGG TTG GAG

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Figure 3. Fluorescence spectra of QD-encoded SiO2 NPs with the ratios of 525 nm:585 nm = 1:1 (a), 525 nm:585 nm = 1:3 (b), 665 nm:740 nm = 1:1 (c), and 665 nm:740 nm = 1:3 (d). Panel (e) is the spectral overlay of mixed QD-encoded SiO2 NPs after hybridization with the ratio of 525 nm:585 nm:665 nm:740 nm = 1:2:1:2.

Figure 4. Four barcodes, including QDs-525-585-11-SiO2 NP-labeled corn, QDs-525-585-13-SiO2 NP-labeled soybean, QDs-665-740-11-SiO2 NPlabeled colza, and QDs-665-740-13-SiO2 NP-labeled cotton, were applied in the multiplex detection. (a) The spectral overlay of mixed QD-encoded SiO2 NPs after hybridization with the ratio of 525 nm:585 nm:665 nm:740 nm = 1:1:2:4 in the presence of target corn, colza, and cotton. (b) The spectral overlay of mixed QD-encoded SiO2 NPs after hybridization with the ratio of 525 nm:585 nm:665 nm:740 nm = 1:3:1:1 in the presence of target soybean and colza. Panels (c) and (d) are cross-reactivity analysis. The abscissa 1
4 represent target corn, soybean, colza, and cotton, respectively. (c) No target soybean presented in solution, which corresponds to (a). (d) Both target corn and cotton did not exist, which corresponds to (b). All targets were present and detected (e). No targets were detected in the control experiment (f). 20137

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Figure 5. (a) Fluorescence spectra of QDs-585-665-11-SiO2 NPs after being hybridized with different concentrations of GMO soybean. (b) The linear relation between concentration of target and fluorescence signal of QDs-585-665-11-SiO2 NPs. (c) Fluorescence spectra of QDs-585-665-12-SiO2 NPs after being hybridized with different concentrations of GMO soybean. (d) The linear relation between concentration of target and fluorescence signal of QDs-585-665-12-SiO2 NPs.

3.2. Procedure for Bead Encoding. The principle of multiplexed optical encoding based on multicolored QDs was first reported by Shuming Nie.3 It was demonstrated that x intensity levels with y colors generated (xy
1) unique codes. For example, two-colored QDs at two intensity levels could be applied to generate (22
1) types of encoded SiO2 NPs, and three-colored QDs at three intensity levels can generate (33
1) codes. In our study, QDs with an emission wavelength at 525, 585, 665, and 740 nm were utilized. Two-colored and three-colored QDs were used to encode SiO2 NPs at various ratios and are illustrated in Figure S3 in the Supporting Information. 3.3. Multiplex GMO Detection Based on QD-Encoded SiO2 NPs. QD-encoded SiO2 NP-labeled probe DNAs and MNPlabeled capture DNAs simultaneously hybridized with the corresponding targets. The probe DNAs and capture DNAs were complementary to the corresponding targets and formed a sandwich structure. The magnetic NPs with the diameter at 300 nm had excellent monodispersity and a unique magnetic response in Figure S4 (Supporting Information); thus, the sandwich structures were easily separated from the mixtures under extra magnetic field after hybridization in an oven at 37 °C for 5 h. As shown in Table 1, we designed four types of specific oligonucleotides as the detection targets from GMO, including GMO corn NK 603, GMO soybean GTS 40-3-2, GMO colza RT73, and GMO cotton MON531. QDs with an emission wavelength at 525, 585, and 665 nm were used to encode SiO2 NPs. The ratios

were 525 nm:585 nm = 1:2 (QDs-525-585-12-SiO2 NPs) and 585 nm:665 nm = 1:2 (QDs-585-665-12-SiO2 NPs), which possessed similar fluorescence intensity. Oligonucleotides from corn and soybean were simultaneously detected based on QDs525-585-12-SiO2 NP-labeled corn probe DNA and QDs-585665-12-SiO2 NP-labeled soybean probe DNA. Because of the overlay of 585 nm in the two codes, the fluorescence intensity at 585 nm was enhanced in the presence of corn and soybean. In theory, the code would change to 525 nm:585 nm:665 nm = 1:3:2 after fluorescence measurement in Figure S5 (Supporting Information). If there were no corn DNAs or soybean DNAs in the sample, MNPs only captured QDs-585-665-12-SiO2 NPs or QDs-525585-12-SiO2 NPs, respectively. Not only could two targets be simultaneously detected but also four targets could be further detected. Oligonucleotides from corn, soybean, colza, and cotton were successfully detected in one step. In this study, QDs with an emission wavelength at 525, 585, 665, and 740 nm were used to encode SiO2 NPs. As shown in Figure 3a
d, the ratios were 525 nm:585 nm = 1:1 (QDs-525-585-11-SiO2 NPs), 525 nm:585 nm = 1:3 (QDs-525585-13-SiO2 NPs), 665 nm:740 nm = 1:1 (QDs-665-740-11SiO2 NPs), and 665 nm:740 nm = 1:3 (QDs-665-740-13-SiO2 NPs), which possessed similar fluorescence intensity. The codes were applied to label corresponding probe DNAs of corn, soybean, colza, and cotton, respectively. After hybridization and magnetic separation, the spectral overlay was changed to 525 nm:585 20138

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The Journal of Physical Chemistry C nm:665 nm:740 nm = 2:4:2:4 (1:2:1:2) in the presence of four types of targets. As shown in Figure 3e, the spectral overlay indicated the presence of targets, achieving the synchronous detection of multiplex targets in a short time period. The specificity of multiplex detection was assessed. Four barcodes were simultaneously applied in the experiment. In the absence of target soybean, the final spectra became 525 nm:585 nm:665 nm: 740nm = 1:1:2:4 overlaid by QDs-525-585-11-SiO2 NP-labeled corn, QDs-665-740-11-SiO2 NP-labeled colza, and QDs-665740-13-SiO2 NP-labeled cotton (Figure 4a,c). If there were no target corn and cotton, we obtained the spectra with the ratio of 525 nm:585 nm:665 nm:740 nm = 1:3:1:1 overlaid by QDs-525585-13-SiO2 NP-labeled soybean and QDs-665-740-11-SiO2 NP-labeled colza (Figure 4b,d). If all targets were present or no targets exist in the control experiment, we obtained the spectra with highest and lowest fluorescence intensity, respectively (Figure 4e,f). The cross-reactivity analysis indicated that quantum dot barcodes could specifically hybridize with the corresponding targets, achieving simultaneously multiplex detection. 3.4. Quantitative Detection. In our research, the sensitivity of DNA detection based on QD-encoded SiO2 NPs was evaluated. Because of the high fluorescence intensity of QD-encoded SiO2 NPs, this method showed an excellent signal in the quantitative DNA detection of soybean. To a certain extent, the fluorescence intensity of QD-encoded SiO2 NPs was proportional to the amount of target DNA. A 100 μL portion of QDs-585-665-11-SiO2 NPs was used to label the soybean probe and 1 μL of MNPs was used to label the soybean capture. As shown in Figure 5a,b, the detection range was just from 10 μM to 1 nM. Not only was the optical property of QD-encoded SiO2 NPs an important factor but also the amount of MNPs played a critical role in detection sensitivity. Therefore, the amount of MNPs was adjusted to 2 μL, but it was worthy to note that QDs-585-665-12-SiO2 NPs were used to label the soybean probe. In Figure 5c, with the increased number of MNPs, the number of QD-encoded SiO2 NPs captured by MNPs increased, resulting in high fluorescence intensity after dilution. As demonstrated in Figure 5d, a standard curve was established. The equation was log Y = 3.6382 + 0.1925 log X, where Y was the fluorescence intensity of QD-encoded SiO2 NPs after hybridization and X was the concentration of the target. The standard deviation (SD) was 0.07589. For the calculation of LOD, Y = ycontrol + 3  SD, where y was the fluorescence intensity of the control groups, which was caused by nonspecific adsorption of QD-encoded SiO2 NPs and MNPs. The corresponding X (LOD) was calculated to be 4.48 fM. The large amount of MNPs also led to nonspecific adsorption of QDencoded SiO2 NPs. An appropriate concentration of MNPs was necessary for quantitative detection. DNA hybridization based on QD-encoded SiO2 NPs achieved quantitative detection and exhibited excellent fluorescence signals.

4. CONCLUSION In this study, we aimed to establish the multiplex label-free DNA detection of GMOs based on QD-encoded SiO2 NPs. We obtained the overlay spectra of QDs using a fluorescence spectrophotometer and further determined the presence of corresponding targets by analyzing the overlay spectra of QDs through XPS peak software. When soybean was used as an example, we successfully achieved quantitative detection with an LOD of 4.48 fM. Multiplex DNA detection based on QD-encoded SiO2 NPs showed high sensitivity and a low LOD; the analysis of overlay spectra

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without auxiliary instruments identified a new platform in multiplex detection.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional details on the characterization of QDs and magnetic clusters and QD-encoded SiO2 NPs. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work is financially supported by the National Natural Science Foundation of China (21071066, 20835006, 91027038, 21101079, 21175034), the Key Programs from MOST (2010AA06Z302, 2010DFB3047, 2008BAK41B03, 2009BAK61B04, 2008ZX08012-001, 2010GB2C100167), and grants from Natural Science Foundation of Jiangsu Province, MOF and MOE (BK2010001, BK2010141, JUSRP10921, JUSRP11019, 201110060, 201110016, 201110061, 201010078, 201010216). ’ REFERENCES (1) Mavropoulou, A. K.; Koraki, T.; Ioannou, P. C.; Christopoulos, T. K. Anal. Chem. 2005, 77, 4785–4791. (2) Petit, L.; Baraige, F.; Balois, A. M.; Bertheau, Y.; Fach, P. Eur. Food Res. Technol. 2003, 217, 83–89. (3) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631–635. (4) Li, Y.; Liu, E. C. Y.; Pickett, N.; Skabara, P. J.; Cummins, S. S.; Ryley, S.; Sutherland, A. J.; O’Brien, P. J. Mater. Chem. 2005, 15, 1238–1243. (5) Zhao, Y.; Chen, W.; Peng, C. F.; Liu, L. Q.; Xue, F.; Zhu, S. F.; Kuang, H.; Xu, C. L. J. Colloid Interface Sci. 2010, 352, 337–342. (6) Yang, Y. H.; Wen, Z. K.; Dong, Y. P.; Gao, M. Y. Small 2006, 2, 898–901. (7) Wang, D. Y.; Rogach, A. L.; Caruso, F. Nano Lett. 2002, 2, 857–861. (8) Thaxton, C. S.; Elghanian, R.; Thomas, A. D.; Stoeva, S. I.; Lee, J. S.; Smith, N. D.; Schaeffer, A. J.; Klocker, H.; Horninger, W.; Bartsch, G.; Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18437–18442. (9) Zhao, Y. J.; Zhao, X. W.; Hu, J.; Li, J.; Xu, W. Y.; Gu, Z. Z. Angew. Chem., Int. Ed. 2009, 48, 7350–7352. (10) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602–7625. (11) Gai, S. L.; Yang, P. P.; Li, C. X.; Wang, W. X.; Dai, Y. L.; Niu, N.; Lin, J. Adv. Funct. Mater. 2010, 20, 1166–1172. (12) Deng, Y.; Qi, D.; Deng, C.; Zhang, X.; Zhao, D. J. Am. Chem. Soc. 2008, 130, 28–29. (13) Sathe, T. R.; Agrawal, A.; Nie, S. Anal. Chem. 2006, 78, 5627–5632. (14) Schroedter, A.; Weller, H. Nano Lett. 2002, 2, 1363–1367. (15) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11–18. (16) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Sto1lzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5, 331–338. (17) Mandal, A.; Nakayama, J.; Tamai, N.; Biju, V.; Isikawa, M. J. Phys. Chem. B 2007, 111, 12765. (18) Mandal, A.; Tamai, N. J. Phys. Chem. C 2008, 112, 8244–8250. (19) Yi, D. K.; Selvan, S. T.; Lee, S. S.; Papaefthymiou, G. C.; Kundaliya, D.; Ying, J. Y. J. Am. Chem. Soc. 2005, 127, 4990–4991. 20139

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dx.doi.org/10.1021/jp206443p |J. Phys. Chem. C 2011, 115, 20134–20140