Simultaneous Determination of Concanavalin A and Peanut Agglutinin

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Simultaneous Determination of Concanavalin A and Peanut Agglutinin by Dual-Color Quantum Dots Hui Zhang, Li Zhang, Ru-Ping Liang, Jing Huang, and Jian-Ding Qiu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac402496e • Publication Date (Web): 15 Oct 2013 Downloaded from http://pubs.acs.org on October 20, 2013

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Analytical Chemistry

Simultaneous Determination of Concanavalin A and Peanut Agglutinin by Dual-Color Quantum Dots Hui Zhang, Li Zhang, Ru-Ping Liang, Jing Huang, Jian-Ding Qiu* Department of Chemistry, Nanchang University, Nanchang 330031, China *Corresponding authors. (J.-D.Qiu) Tel/Fax: +86-791-83969518. E-mail: [email protected].

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ABSTRACT

In this work, we design a novel detection strategy to realize simultaneous determination of multiplex lectin by labeling glucosamine (G1) and galactosamine (G2) with different colored semiconductor quantum dots (QDs). Based on the agglutination of the aminosugar labeled QDs induced by the exclusive binding between the lectin and sugar on the QDs surfaces, the fluorescence emission of the QDs supernatant after centrifugation decreased with relevant lectin concentration. i.e. when concanavalin A (Con A) exists alone, only green color fluorescence emission from QDs-G1 supernatant decreased, so it is peanut agglutinin (PNA) and red color fluorescence emission from QDs-G2. Moreover, since QDs can be simultaneously excited with multiple fluorescence colors and have a larger Stokes shift than organic fluorophores, when both Con A and PNA are present in the sample, both of the green and red color fluorescence emission from QDs-G1 and QDs-G2 supernatant would decrease, thus realizing the simultaneous determination of Con A and PNA. The detection limits of Con A and PNA are 0.30 and 0.18 nM (3σ), respectively. Furthermore, the present detection method can determine not only the protein/lectins by fluorescence spectral method but also can realize visualization detection by UV lamp illumination. To the best of our knowledge, this is the first report of such analytical method in multiple and simultaneous lectin detection.


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INTRODUCTION As the carbohydrate recognition domains (CRD), lectins are proteins or glycoproteins of nonimmune origin that bind to mono/oligosaccharides reversibly with a high degree of stereospecificity.1,2 For example, Con A, extracted from jack bean seeds, binds exclusively to αglucose, α-mannose and their derivatives and shows no significant affinity toward other carbohydrates (such as galactose and lactose)2-4. On the other hand, PNA as a lectin isolated from peanuts binds exclusively to β-galactose, lactose and their derivatives and shows no affinity toward other carbohydrates (such as α-glucose and maltose).2-4 Since this carbohydrate recognition is very selective and often multivalent, lectins have been implicated in many important functions like cell-cell interactions, homing of leukocytes, host pathogen interactions, biosynthesis and quality control of glycoproteins, immune response, malignancy, and metastasis.5 It is important to understand and mimic carbohydrate and bacterial lectin interactions as the foundation of pathogen detection and prevention of bacterial infection. Measurement of the binding affinity of glycoconjugates toward lectins in solution is routinely realized by agglutination inhibition assays,6 enzyme linked lectin assays (ELLA),7 enzyme linked immunosorbent assay (ELISA),8 isothermal titration calorimetry (ITC),9 colorimetry,10 scattered light11 or surface plasmon resonance (SPR).12,13 But these methods are tedious, requiring extensive instrumental setup and technical expertise. Moreover, since carbohydrateprotein interactions are generally weak and their dissociation constants are in the range of Kd = 10-6 to 10-7 M for glycoproteins, the conventional approaches based on the monovalent interaction usually involve low sensitivity.14,15 Thus far, great efforts have been devoted to overcome such shortcomings, and the weak carbohydrate-protein interactions can be compensated for by the presentation of multiple ligands to their respective binding proteins.16-19


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One approach is to assemble many carbohydrate units onto the surfaces of nanoparticles (NPs), in this regard, carbon nanostructures such as carbon nanotubes and graphene as well as noble metal nanoparticles including gold and silver nanoparticles have been functionalized with carbohydrates by using covalent or noncovalent methods to induce specific lectin affinity.10,20-25 The resulting polyvalent interactions between the multiple ligands and proteins can be collectively much stronger than that of the corresponding monovalent interaction. Although various NPs have been widely used for lectin detection, most of the proposed methods deal with single lectin analysis, and the simultaneous and multiplex lectin determination still remains a great challenge. Accordingly, developing sensitive, rapid, simple, reliable and cost-effective glycotechnologies and biosensors for multiple lectins determination are highly critical. For high-throughput and easy operation homogeneous determination of proteins, semiconductor quantum dots (QDs) based assays have the potential to circumvent some of the functional limitations encountered by protein arrays and suspension arrays due to their unique optical and photophysical features.26,27 The QDs have significant advantages of higher brightness and stability against photobleaching, large absorption cross sections over a broad range of excitation wavelengths, relatively high quantum yield, and a stable surface for further chemical modification.28,29 Their size-dependent emission spectra with broad absorption spectra allow their simultaneous excitation at a single wavelength.30 These properties of QDs are beneficial to their applications in simultaneous multi-protein/virus determination. For example, Chen et al. designed a detection strategy to realize multiplex viruses (EV71 and CVB3) simultaneous determination with different colored QDs.31 The fluorescence of these QDs-Ab bioconjugates is quenched by GO then the targets can break up the complex of QDs-Ab and GO and recover the fluorescence of QDs-Ab.


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Here, we designed a novel detection strategy by labeling different colored core-shell CdSe/ZnS QDs with glucosamine (G1) and galactosamine (G2) for simultaneous detection of Con A and PNA. Our detection strategy is shown in Scheme 1. QDs-G1 was prepared by covalently conjugating G1 to carboxyl functionalized green colored QDs (525 nm, 525-QDs), and QDs-G2 was prepared by covalently conjugating G2 to carboxyl functionalize red colored QDs (605 nm, 605-QDs). In the presence of Con A, the high-affinity, multivalent interaction of Con A with G1 on the surface of 525-QDs should result in agglutination of the 525-QDs due to the formation of numerous noncovalent cross-links between the 525-QDs, leading to the fluorescence decrease of the supernatant after centrifugation. On the other hand, the presence of PNA should result in the agglutination and thus the fluorescence decrease of 605-QDs supernatant due to the multivalent and selective interaction between PNA and G2. Since 525QDs and 605-QDs can be excited at a single excitation wavelength, and the two contrasting colored fluorescence signals can be monitored at the same time without spectral overlap, simultaneous detection of Con A and PNA can be realized by exciting different colored QDs. Furthermore, this detection model can determine proteins by the fluorescence spectral method, and simultaneously, realize semiquantitative determination of protein/lectins by the colored sample solution with naked eye upon UV lamp illumination.

Scheme 1. Schematic diagram of the multi-colored biosensor for determination of Con A and PNA.


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EXPERIMENTAL SECTION Reagents and Chemicals Sodium phosphate monobasic dihydrate (NaH2PO4·2H2O), sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O), manganese chloride (MnCl2), calcium chloride (CaCl2) were obtained from Beijing chemical company (China) and were used as received without further purification. D-(+)-glucosamine hydrochloride (G1), D-(+)-galactosamine hydrochloride (G2), concanavalin A (Con A), peanut agglutinin (PNA), bull serum albumin (BSA), horseradish peroxidase (HRP), glucosamine oxidase (GOx), and 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) were commercially available from Sigma (USA). Carboxyl modified QDs (525 nm and 605 nm) were bought from Wuhan Jiayuan Quantum Dots Co., Ltd. (China). All chemicals used were of analytical grade or of the highest purity available. All solutions were prepared and diluted using ultrapure water (18.2 MΩ) from the Millipore Milli-Q system. Apparatus Fluorescence spectra were collected with a F-7000 fluorescence spectrophotometer (Hitachi, Japan) and the light scattering (LS) spectra were obtained by simultaneously scanning the excitation and emission monochromators of a common spectrofluorometer. AFM images were recorded in the ScanAsyst mode with a MultiMode 8 atomic force microscope under ambient conditions (Bruker, Germany), and the fluorescent photographs were taken by a digital camera. Preparation of QDs-G1 and QDs-G2. The QDs-G1 conjugates were prepared through a standard amide coupling reaction. In brief, 10 µL of 525-QDs (0.08 µM final concentration) was added to 360 µL borate buffer solution (pH 7.4, 10 mM), followed by sequential addition of 20.4 µL of EDC (320 µM final concentration)


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and 10 µL of glucosamine G1 (8 µM final concentration). The mixture was left to gently shaking for 3.5 h in the dark at room temperature, followed by ultrafiltration (10 kD, RCF 20 000 g) for 5 min to remove excess amount of EDC and G1. Then, the mixture was diluted with PBS to a final volume of 1 mL. QDs-G2 was prepared using the similar procedure as in the case of QDs-G1 Detection of Con A and PNA. Different amounts of Con A were added to 50 µL of QDs-G1 (8 nM final concentration) and diluted with buffer solution (20 mM PBS, pH 7.4, containing 0.1 mM MnCl2, 0.1 mM MgCl2 and 0.1 mM CaCl2) to a final volume of 500 µL,32 and then the mixtures were equilibrated at room temperature (25 °C) for 60 min. Subsequently, the mixtures were centrifuged (RCF 20 000g, 20 min) and the supernatants were transferred into 1 mL quartz cuvettes to measure the fluorescence spectra at a single excitation wavelength of 335 nm. For simultaneous determination, 50 µL of QDs-G1 (8 nM final concentration), 30 µL of QDs-G2 (4.8 nM final concentration), different concentrations of Con A and PNA were incubated in buffer solution at room temperature (25 °C) for 60 min, the data were obtained with the same procedure as that of Con A or PNA. It should be noticed that centrifugation is unnecessary prior to the measurements of the LS spectra of the mixtures.

RESULTS AND DISCUSSION Optimizing Experimental Conditions. QDs–G conjugates were prepared using the coupling strategy between carboxyl coated QDs and amino labeled oligosaccharides. It is worth emphasizing here that the selection of an appropriate concentration of carboxyl coated QDs and amino oligosaccharide is critical to get detectable signals in a simultaneous detection system. In our work, two experiments were


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performed to select an appropriate concentration of amino labeled oligosaccharides. The effect of G1 concentration (0.01-6 µM final concentration) on the fluorescence response of 525-QDs to 11 nM Con A was tested with a fixed concentration of 525-QDs (8 nM final concentration). Figure 1A shows a plot of fluorescence intensity as a function of G1 concentration. It was found that the fluorescence intensity of 525-QDs supernatant decreased to a minimum value at the G1 concentration of 0.8 µM then leveled off thereafter. At the concentration lower than 0.8 µM, the G1 might be insufficient for covalent binding to the carboxyl on QDs surfaces, and the fluorescence intensity of 525-QDs supernatant drastically decreased upon addition of Con A with increasing the concentration of G1. When the G1 concentration was higher than 0.8 µM, the steric hindrance among the G1 molecules may interfere the covalent binding between G1 and the carboxyl on QDs surfaces, and the fluorescence intensity of 525-QDs supernatant was almost maintained at constant value upon addition Con A with increasing the G1 concentration. From these control experiments, we can conclude that the proper concentration of G1 is 0.8 µM. The proper concentration of G2 was determined as 0.48 µM (Figure 1B) with the same procedure as G1. The recognition time is also an important parameter for the kinetic binding between lectins and carbohydrates on the QDs surface. Figure 1C shows the time dependent response of QDs-G1 supernatant to 11 nM Con A. The fluorescence intensity at 525 nm decreased dramatically during the early stage of the reaction and reached a plateau after 60 min (red curve), indicating that Con A selectively bound the G1 on the surfaces of QDs-G1, inducing aggregation of the particles rapidly. In a control experiment (black curve), no clear decrease in fluorescence was observed without addition of Con A to the QDs-G1, which confirms that the supernatant fluorescence decrease is due to the exclusive binding between the Con A and G1 on QDs


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surfaces to generate 525-QDs aggregates. The exclusive binding between the Con A and G1 on the QDs-G1 surfaces was accomplished within 60 min. The optimum time of PNA exclusively binding to the G2 on QDs-G2 surface was obtained with the same procedure, and the optimal time was also 60 min (Figure 1D).

Figure 1. Optimizing the monosaccharide concentration and incubation time. (A) Plot of fluorescence intensity of 525-QDs supernatant conjugated with different concentrations of G1 in the presence (red curve) and absence (black curve) of 11 nM Con A. (B) Plot of fluorescence intensity of 605-QDs supernatant conjugated with different concentrations of G2 in the presence (red curve) and absence (black curve) of 8 nM PNA. (C) Time-dependent fluorescence responses of the QDs-G1 supernatant in the presence (red curve) and absence (black curve) of 11 nM Con A. (D) Time-dependent fluorescence responses of the QDs-G2 supernatant in the presence (red curve) and absence (black curve) of 8 nM PNA. Final concentrations: 525-QDs, 8 nM; 605-QDs, 4.8 nM; Con A, 11 nM; PNA, 8 nM. Ex = 335 nm.


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Quantitative Determination of Con A and PNA.

Figure 2. (A) Fluorescence spectra of QDs-G1 supernatant upon increasing the concentration of Con A; (B) Plot of fluorescence intensity of QDs-G1 supernatant as a function of Con A concentration (inset: calibration curve of quantitative determination Con A in logarithmic scales). (C) Fluorescence spectra of QDs-G2 supernatant upon increasing the concentration of PNA; (D) Plot of fluorescence intensity of QDs-G2 supernatant as a function of PNA concentration (inset: calibration curve of quantitative determination PNA in logarithmic scales). Final concentrations: QDs-G1, 8 nM, QDs-G2, 4.8 nM. Ex = 335 nm. Under the optimum conditions, Con A and PNA were determined by this fluorescence detection platform, respectively. For Con A detection, once Con A was added into the QDs-G1 solution, the interaction of Con A with G1 on the surfaces of QDs-G1 resulted in aggregation of the QDs-G1, leading to the fluorescence decrease of 525-QDs supernatant after centrifugation. It was found that the fluorescence intensities of the supernatant were inversely proportional to the


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concentration of lectins, and the results were shown in Figure 2A-B. The fluorescence intensity of 525-QDs supernatant decreased rapidly when the concentration of Con A was low, and then leveled off thereafter due to the steric hindrance among the Con A molecules. The inset of Figure 2B reveals that the fluorescence intensity of 525-QDs supernatant decreased with a linear relationship against the logarithm of the Con A concentration over the range of 0.4-46 nM with a detection limit (LOD) of about 0.3 nM (3σ), which is comparable to the limits obtained by other sensors.21,33 Similarly, for PNA determination, as shown in Figure 2C-D, the fluorescence intensity of 605-QDs supernatant decreased with a linear relationship against the logarithm of the PNA concentration over the range of 2.2-22 nM, and the LOD was calculated as 0.18 nM (3σ), which is much lower than the limits obtained by other sensor.2 Such data suggests that the fluorescent sensing platform is of excellent sensitivity for Con A and PNA detection, which has potential applications in protein-carbohydrate studies, pathogen diagnosis and cell biology. This detection approach relies on target lectin-induced aggregation of QDs-G1 and QDs-G2, thus, it is supposed that enhanced light scattering (LS) signals owing to the distance changes between QDs can be collected as a supplemental strategy for monitoring the concentration of lectin. The LS signals of aggregated QDs-G1 and QDs-G2 were measured by scanning synchronously

both

the

excitation

and

emission

monochromators

of

a

common

spectrofluorometer from 220 to 700 nm with a slit width for excitation and emission of 5 nm. It is worth noting that the maximum LS peak was located at 284 nm rather than the absorption region at about 600 nm, indicating that the recorded LS signals should be attributed to the conventional Rayleigh scattering rather than resonance light scattering.34,35 Figures 3 displays the LS responses for Con A and PNA with varying concentrations. As expected, the LS signals of QDs increased in the presence of the target lectin. It was found that the enhanced LS signals


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linearly increased with the increasing Con A concentration in the range of 0.4 to 46 nM with the LOD around 0.3 nM (3σ, in logarithmic scales) (Figure 3A-B). Similarly, for PNA detection, a linear relationship between the increased LS signals of QDs-G2 and the concentration of PNA in the range of 0.6-22 nM was observed (Figure 3C-D). The LOD was calculated to be 0.18 nM (3σ, in logarithmic scales). The enhanced LS intensity as a function of lectin confirms our speculation that the fluorescence decrease of QDs supernatant is the result of its agglutination. It should be noticed that the maximum scattering peaks of both the red and green QDs were located at 284 nm, the LS signals raised by different target protein could not be distinguished, and thus the LS platform could be applied to the multiple rather than simultaneous detection of Con A and PNA.

Figure 3. (A) LS spectra of QDs-G1 treated by various amount of Con A. (B) Plot of LS intensity at 284 nm vs. Con A concentration (inset: calibration curve of detection Con A in logarithmic scales). (C) LS spectra of QDs-G2 treated by various amount of PNA. (D) Plot of LS


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intensity at 284 nm vs. PNA concentration (inset: calibration curve of detection PNA in logarithmic scales). Final concentrations: QDs-G1, 8 nM, QDs-G2, 4.8 nM. Atomic force microscopic (AFM) imaging was performed to monitor the formation of QDsG1 aggregates in the presence of target lectin. As shown in Figure 4A and 4C, they clearly show that QDs-G1 were mainly small dotlike nanoparticle with cross-sectional heights about 2 nm. Upon interacting with the corresponding lectin, i.e. Con A, the aggregation of QDs-G1 was monitored, as indicated by the formation of wirelike chains (the cross-sectional height of 9-11 nm) of nanoparticles in Figure 4B and 4D. These observations support our suggestion that the fluorescence decrease after centrifugation should be ascribed to the formation of condensed and compact aggregates as a result of the intense and exclusive combination of glucosamine molecules on QDs surfaces and Con A.

Figure 4. (A) AFM image of the dispersive QDs-G1 deposited on freshly cleaved mica and the inset shows the statistical analysis of the QDs-G1. Size: 5.0 × 5.0 µm. (B) AFM image of the QDs-G1 aggregates induced by Con A. size: 5.0 × 5.0 µm. (C) and (D) are the height distribution


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of the QDs-G1 as shown in (A) and (B), respectively. Final concentrations: QDs-G1, 8 nM, Con A, 11 nM. Selectivity of the Dual-Colored QDs-oligosaccharide Probe. Since both 525-QDs and 605-QDs could be excited to multiple fluorescence colors by a single excitation light source at 335 nm, we can realize simultaneous detection of Con A and PNA by the above described methods by exciting different colored QDs simultaneously. Before simultaneous detection of Con A and PNA, it is necessary to investigate whether the detection system can differentiate the two lectins by QDs with different colored fluorescence emission. As shown in Figure 5A, when Con A alone was existed in the sample solution, the fluorescence emission of 525-QDs supernatant decreased while the fluorescence emission of 605-QDs supernatant almost remained constant. This demonstrates a vital point: agglutination of 525-QDs was the result of a high density of glucosamine G1 on the 525-QDs surfaces that specifically bound with Con A. Similarly, fluorescence measurements (Figure 5B) show that when PNA was alone existed in the analysis sample, only the fluorescence intensity of 605-QDs supernatant decreased and the fluorescence emission of 525-QDs supernatant remained constant. Thus, the two different colored QDs assay can be easily and effectively used to differentiate the two lectins.


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Figure 5. (A) Fluorescence spectra of QDs-G1 and QDs-G2 supernatant upon increasing the concentration of Con A. (B) Fluorescence spectra of QDs-G1 and QDs-G2 supernatant upon increasing the concentration of PNA. Final concentrations: QDs-G1, 8 nM; QDs-G2, 4.8 nM. Ex = 335 nm. To testify the selectivity of this detection system, some possible interfering proteins, BSA, HRP, and GOx were chosen as controls to investigate the selectivity of the method. As indicated in Figure 6, at a concentration equal to that of target lectin, we incubated aliquots of the QDs-G1 and QDs-G2 solutions separately with Con A, PNA, BSA, HRP, and GOx, the results demonstrate that these proteins could not induce the fluorescence intensity of the QDs supernatant to decrease except for Con A and PNA. These data again demonstrate that the interactions between lectin and GDs-1 and GQs-2 are selectively, and possible interferences from other proteins are neglectable. Thus, this highly specific sensor has great potential for use in detecting Con A and PNA simultaneously in complex real samples.

Figure 6. Selectivity of the dual-colored QDs-oligosaccharide probe, the concentration of these proteins is equal to that of target lectin (136 nM). Final concentrations: QDs-G1, 8 nM; QDs-G2, 4.8 nM. Ex = 335 nm. Simultaneous Determination of Con A and PNA.


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Under the optimized conditions, the multi-colored QDs-G probe was used for quantitative detection of Con A and PNA simultaneously. As shown in Figure 7, once the green and red colored QDs-oligosaccharide probes were exposed to the target lectins, i.e. Con A and PNA, protein specifically combined with certain carbohydrate groups on the surface of QDs, resulting in agglutination and thus fluorescence decrease of the relevant QDs supernatant due to the formation of numerous noncovalent cross-links between the QDs. With the increased concentration of Con A and PNA, the fluorescence intensity of the green and red colored QDs supernatant decreased, respectively (Figure 7A). It can be seen that the fluorescence intensity decreased linearly with the concentration of Con A from 0.4 to 46 nM with the LOD around 0.3 nM (3σ, in logarithmic scales) (Figure 7B). For PNA detection, the fluorescence intensity decreased with a linear relationship against the concentration of PNA over the range 2.2-22 nM and the LOD was about 0.18 nM (3σ, in logarithmic scales) (Figure 7C).

Figure 7. (A) Fluorescence spectra of QDs-G1, QDs-G2 supernatant with increasing concentration of Con A and PNA. (B) Plot of the fluorescence intensity at 525 nm vs. Con A concentration, inset: calibration curve of detection Con A in logarithmic scales. (C) Plot of the fluorescence intensity at 605 nm vs. PNA concentration, inset: calibration curve of detection PNA in logarithmic scales. Final concentrations: QDs-G1, 8 nM; QDs-G2, 4.8 nM. Ex = 335 nm.


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As a simple homogeneous methodology for lectin analysis, visualization detection can also be evidently realized by UV lamp illumination. Figure 8 shows the photograph of semiquantitative determination Con A and PNA respectively and simultaneously (upon UV lamp illumination). When 525-QDs and Con A existed in solution, the color of green QDs supernatant shallowed rapidly with increasing the concentration of Con A due to the exclusive binding between con A and glucosamine G1 on the surface of 525-QDs (Figure 8A). Similarly, when 605-QDs and PNA existed in solution, the color of red QDs supernatant shallowed with increasing the concentration of PNA due to the high-affinity of PNA to galactosamine G2 coupled on 605-QDs. If both Con A and PNA existed in the sample solution, the fluorescence emission of the two distinguishing colored QDs (525-QDs and 605-QDs) supernatant decreased at the same time and the solution exhibited its mixed color (Figure 8C).

Figure 8. Photograph of semiquantitative determination Con A and PNA respectively and simultaneously upon UV lamp illumination (365 nm). (A) QDs-G1 supernatant with various amounts of Con A (from left to right): 0, 0.4, 1, 8, 11, 46, and 136 nM; (B) QDs-G2 supernatant with various amounts of PNA (from left to right): 0, 0.6, 2.2, 4, 8, 22, and 46 nM; (C) QDs-G1 and QDs-G2 supernatant with various amounts of Con A and PNA. The concentrations of Con A


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were 0, 1, 8, 46, and 136 nM and the concentrations of PNA were 46, 22, 8, 2.2, and 0 nM (from left to right).

CONCLUSION In summary, a simple, sensitive, highly selective, and rapid QDs-based sensing platform has been developed for simultaneous detection of Con A and PNA with dual-color fluorescence output signals in homogeneous solution. The high-affinity, multivalent interaction of lectins with carbohydrate groups on the surface of QDs results in agglutination and thus the fluorescence decrease of the QDs supernatant due to the formation of numerous noncovalent cross-links between the QDs. Since QDs can be excited at a single excitation wavelength, two contrasting colored fluorescence signals can be monitored at the same time without spectra overlap, leading to the simultaneous detection of Con A and PNA. Compared with previous multiplex protein/virus determination strategy,31 our present assay proposed a different novel sensing mechanism and a simple experimental methodology. Moreover, this approach allows rapid and precise quantitative determination of lectins by evaluating changes in fluorescent intensity and semiquantitative determination of lectin by digital visualization upon UV lamp illumination as well.

ACKNOWLEDGEMENTS We greatly appreciate the supports of the National Natural Science Foundation of China (21163014, 21105044, 21265012 and 21265017) and the Program for New Century Excellent Talents in University (NCET-11-1002).

REFERENCES (1) Lis, H.; Sharon, N. Chem. Rev., 1998, 98, 637-674.


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