Detection of concanavalin A based on attenuated fluorescence resonance energy transfer between quantum dots and mannose-stabilized gold nanoparticles

Abstract

Highly sensitive biosensing of concanavalin A (Con A) has been developed based on the attenuation of FRET efficiency between amine-terminated quantum dots and mannose-stabilized Au nanoparticles in the presence of Con A, which inhibits the H-bonding formation of the QDs–AuNPs assembly, thereby causing the fluorescence recovery.

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Lectins are proteins that specifically interact with multiple carbohydrates on cell surfaces in most biological systems, and the lectin–carbohydrate interactions play significant roles in a variety of biological processes such as cell-surface recognition, cell–cell communications, immune response, and pathogen infection.¹ Since the carbohydrate–lectin interactions are not strong, highly sensitive detection method for the carbohydrate–lectin interaction should be developed for glycomics research and clinical application. The developed detection techniques so far include NMR, isothermal titration calorimetry, surface plasmon resonance, and quartz crystal microbalance.²

In recent years, quantum dots (QDs) have been widely employed as a donor in the fluorescence resonance energy transfer (FRET)-based detection of biomolecules due to several advantageous properties, which include high quantum yield, broad absorption spectra, narrow emission spectra, size-dependent emission wavelengths, and high photochemical stability.³ Moreover, the water soluble property enables QDs to be well used in the biological system. Gold nanoparticles (AuNPs), meanwhile, have been used as a FRET acceptor because of their high extinction coefficient and a broad absorption spectrum in a visible region which is superimposed on the emission wavelength of usual FRET donors such as organic dyes and QDs.^4,5 To date, a number of studies have been directed towards the selective detection of biological substances and ions based on the attenuation of FRET efficiency in QDs–AuNPs assembly, in which the target analytes inhibit the specific interactions between QDs and AuNPs. For example, specific interactions to form the QDs–AuNPs assembly were inhibited by the biological substances such as avidin,⁵ DNA,⁶ glucose,⁷ glycoprotein,⁸ proteases,⁹ and cancer markers¹⁰ which attenuates the FRET process and thus cause the fluorescence recovery. In addition, the QDs–AuNPs assembly formed by electrostatic interaction and H-bonding were used to selectively detect lead ion¹¹ and fluoride anion¹² in the FRET-inhibition assay.

In this report, we present a novel FRET-based biosensing method for a target lectin Con A as a model analyte. Con A from Canavalia ensilformis is a tetramer with four carbohydrate binding sites which specifically binds to mannose and glucose.¹³Scheme 1 outlines our biosensing method based on the attenuation of FRET efficiency between the amine-terminated QDs and the mannose-stabilized AuNPs in the presence of Con A. When the amine-terminated QDs and the mannose-stabilized AuNPs were mixed together, the assembly of QDs and AuNPs is formed by hydrogen bonds between the amine groups of QDs surfaces and the hydroxyl groups of the mannose on the surface of the AuNPs, thereby leading to strong FRET efficiency between QDs and AuNPs (scheme 1A). However, in the presence of Con A, the specific bindings between Con A and mannose-stabilized AuNPs make Con A-induced aggregation of AuNPs, preventing the formation of hydrogen bonds between the amine-terminated QDs and the mannose-stabilized AuNPs. Therefore, the FRET efficiency between QDs and AuNPs is attenuated (Scheme 1B). Since the degree of attenuation in the FRET efficiency is strongly dependent upon the amount of added Con A, highly sensitive detection of Con A is possible.

Scheme 1 Schematic illustration of (A) FRET between amine-terminated QDs and mannose-stabilized AuNPs, and (B) Con A-induced attenuation of FRET.

The mannoside containing a thiol functional group at the terminal position as shown in Fig. S1† has been synthesized in a multistep sequence from a mannosyl trichloroacetimidate and an alcohol, and the prepared thiolated mannose was characterized by NMR and high-resolution mass spectrometer according to our previous report.¹⁴ The polyethyleneglycol (PEG) spacer in the thiolated mannose was used to reduce nonspecific adsorption of proteins to AuNPs. The mannose-stabilized AuNPs were prepared by the displacement self-assembly, in which commercially available citrate-capped AuNPs (ca. 10 nm diameter, British Biocell International, 5.7 × 10¹² particles per mL, 1.0 mL) were reacted with 40 μM thiolated mannose solution (1.0 mL) for 17 hours. The mixture was centrifuged and filtered with Amicon ultra-4 (Millipore, USA) in order to get the mannose-stabilized AuNPs. The filtered mannose-stabilized AuNPs were resuspended in 50 mM borate buffer (pH 8.6). The final concentration of the AuNPs was determined to be 5.0 × 10¹² particles per mL by UV-visible spectrophotometry. Amine-terminated PEG QDs (CdTe) were purchased from Invitrogen (ca. 4 nm diameter, Qdot® ITK™ amino (PEG), 8.0 μM).

The absorption spectrum of mannose-stabilized AuNPs (8.3 nM) and fluorescence spectrum of amino (PEG) QDs (0.50 nM) were obtained by using a UV-vis absorption spectrophotometer (V-660, JASCO, Japan) and a spectrofluorometer (FP-6300, JASCO), respectively. The absorption spectrum of AuNPs showed maximum wavelength at around 520 nm and was well overlapped with the fluorescence spectrum of QDs exhibiting the maximum wavelength at 525 nm as shown in Fig. S2.† When the amine-terminated QDs and the mannose-stabilized AuNPs were mixed together, the fluorescence emission intensity was greatly reduced to 55% by strong FRET process from donor (QDs) to acceptor (AuNPs) (Fig. S3†). Stern–Volmer equations can be used to characterize the efficient FRET between the QDs and the AuNPs.¹⁵

F₀/F = 1 + K_SV [AuNPs]

In the Stern–Volmer equation, [AuNPs] represents the concentration of AuNPs. F₀ and F denote the steady-state fluorescence intensities in the absence and presence of AuNPs, respectively. A plot of F₀/F versus [AuNPs] gives a straight line with the slope representing the quenching constant, K_SV. In borate buffer, K_SV was determined to be 8.66 × 10⁷ M⁻¹ (F₀/F = 1.134 + 0.069[AuNPs]) with a correlation coefficient (r) of 0.995 (Fig. S4†). The experimental results clearly indicate that the fluorescence emission is quenched by AuNPs. A possible mechanism to explain the formation of the QDs–AuNPs assembly might be the hydrogen bond between the amine groups of QDs and the hydroxyl groups of the mannose stabilized on the AuNPs.

In order to confirm the H-bond formation, energy-minimized structures of mannose and aliphatic amine were generated using MacroModel 9.1 program (Schrödinger, Inc.).¹⁶ As shown in Fig. 1, the structure was found with AMBER* force field¹⁷ in the gas phase via 50000 search in Monte Carlo conformational search.¹⁸ The corresponding distances of hydrogen bonds were 1.898 Å for d(NH⋯O), 1.955 Å for d(NH⋯O), and 2.014 Å for d(NH⋯O), which are within the literature value.¹⁹ In addition, transmission electron microscopy (TEM) analysis was conducted with high-resolution TEM (JEM-2100F, UHR type, JEOL Ltd, Japan). As shown in Fig. 2, amine-terminated QDs show spherical shape with a diameter of ca. 4 nm, and mannose-stabilized AuNPs exhibit a spherical shape with a diameter of ca. 10 nm. The addition of amine-terminated QDs to mannose-stabilized AuNPs led to the formation of QDs–AuNPs clusters (Fig. 2A and C), in which QDs and AuNPs are located within a short distance (<10 nm) by hydrogen bonds between the amine groups of QDs and the hydroxyl groups of AuNPs. In contrast, when Con A-incubated mannose-stabilized AuNPs were mixed with amine-terminated QDs, the formation of QDs–AuNP clusters between QDs and AuNPs was hindered because of the Con A-induced aggregation of AuNPs (Fig. 2B and D). This experimental result clearly supports that the QDs–AuNPs assembly is formed by H-bond between amine-terminated QDs and mannose-stabilized AuNPs, and externally added Con A induces the aggregation of AuNPs, which attenuates FRET efficiency between QDs and AuNPs.

Fig. 1 Energy-minimized structures of H-bonds between the amine groups of QDs surfaces and the hydroxyl groups of the mannose stabilized on the AuNPs.

Fig. 2 TEM images of QDs–AuNPs assembly in the absence (A) and the presence of Con A (B). Magnified images (C and D) from A and B.

FRET-based inhibition assay has been carried out for Con A as a model lectin. Different concentrations of Con A were added to the mannose-stabilized AuNPs at a fixed concentration of 8.31 nM and were incubated for 10 min. Then, the QDs were added to the above mixture at a final concentration of 250 pM, and the fluorescence spectra were measured at different Con A concentrations. As expected, the fluorescence intensity was gradually increased as the concentration of added Con A was increased (Fig. 3A). In addition, the relative fluorescence intensity (F/F₀) increases logarithmically with increasing concentration of the added Con A (r² = 0.999) as shown in Fig. 3B, where F₀ represents the FL intensity of QDs–AuNPs assembly in the absence of Con A, and F represents the observed FL intensity of the QDs–sAuNPs assembly when Con A is present at each concentration. From these experiments, the linear dynamic range of Con A was determined to be 0.20–200 μg mL⁻¹. The present detection method provides a good detection limit (S/N = 3) of 0.0747 μg mL⁻¹, which is much lower than those obtained with colorimetric methods based on mannose-stabilized Au and Ag NPs (10.3 μg mL⁻¹ (ref. 20) and 4.1 μg mL⁻¹,²¹ respectively) and is comparable to that obtained with FRET detection based on pyrene-conjugated maltose–graphene assembly (0.082 μg mL⁻¹)²² and also to those obtained with microgravimetric and electrochemical detection methods based on NPs-induced signal amplification (0.013 μg mL⁻¹ (ref. 14) and 0.070 μg mL⁻¹,²³ respectively).

Fig. 3 (A) FL spectra of QDs in the presence of mannose-stabilized AuNPs with varying concentration of Con A, 0 μg mL⁻¹, 0.2 μg mL⁻¹, 0.5 μg mL⁻¹, 2.0 μg mL⁻¹, 5.0 μg mL⁻¹, 10 μg mL⁻¹, 50 μg mL⁻¹, 200 μg mL⁻¹, 500 μg mL⁻¹ (λ_ex = 350 nm). (B) Calibration curve of Con A.

To assess the selectivity of the present method for Con A, the effect of other proteins and lectins were examined. MALII (maackia amurensis lectin II), RCA₁₂₀ (ricinus communis agglutinin), WGA (wheat germ agglutinin) and CT (cholera toxin) are lectins not binding to the mannose. The relative FL intensities for BSA, MALII, RCA₁₂₀, WGA and CT were 1.13 ± 0.05, 1.25 ± 0.03, 1.13 ± 0.01, 1.13 ± 0.01 and 1.21 ± 0.11 (n = 3), respectively, compared to that measured from the same concentration of Con A (50 μg mL⁻¹). The result indicates that the present method presents good selectivity against other proteins. The PEG on the surface of QDs and AuNPs might alleviate the nonspecific binding.

In conclusion, we developed a novel biosensing method for the detection of a target lectin Con A based on FRET between amine-terminated QDs and mannose-stabilized AuNPs. The present method is quite simple and more sensitive than other methods, and thus can be conveniently extended to the quantitative analysis of various lectins with high sensitivity by just exchanging the carbohydrate stabilized on the surface of AuNPs. Therefore, the present biosensing method could possibly provide a versatile tool for the analysis of clinically important lectin proteins containing several binding sites, and for the bioassays of disease or cancer related glycans based on competitive binding.

Financial support for this work has been provided by Basic Science Research Program through the National Research Foundation of Korea (2012-R1A1A2006994). We acknowledge Prof. K.S. Jeong and Dr J.-M. Suk at Yonsei University for the discussion and help for the H-bond modelling.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Chemical structure of thiolated mannose, Absorption and fluorescence spectrum of AuNPs and QDs, and Stern–Volmer plot. See DOI: 10.1039/c2ay26128f

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