Research Article www.acsami.org
QD-Biopolymer-TSPP Assembly as Efficient BiFRET Sensor for Ratiometric and Visual Detection of Zinc Ion Yuqian Liu,† Xiaojun Qu,† Qingsheng Guo,† Qingjiang Sun,*,† and Xuebin Huang*,‡ †
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, P. R. China ‡ School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, P. R. China S Supporting Information *
ABSTRACT: In this work, we report a new type of quantum dot (QD)-based fluorescence resonance energy transfer (FRET) assembly and its utility for sensing Zn2+ in different media. The assembly on the QD scaffold is via first coating of poly(dA) homopolymer/double-stranded DNA, followed by loading of meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP), both of which are electrostatic, offering the advantages of cost-efficiency and simplicity. More importantly, the biopolymer coating minimizes the interfacial thickness to be ≤2 nm for QD-TSPP FRET, which results in improvements of up to 60-fold for single FRET efficiency and nearly 4-fold for total FRET efficiency of the QD-biopolymer-TSPP assemblies in comparison with silica-coating-based QD-TSPP assemblies. On the basis of Zn2+-chelation-induced spectral modulation, dualemission QD-poly(dA)-TSPP assemblies are developed as a ratiometric Zn2+ sensor with increased sensitivity and specificity. The sensor either in solution or on a paper substrate displays continuous color changes from yellow to bright green toward Zn2+, exhibiting excellent visualization capability. By utilizing the competitive displacement of Zn2+, the sensor is also demonstrated to have good reversibility. Furthermore, the sensor is successfully used to visualize exogenous Zn2+ in living cells. Together the QDbiopolymer-TSPP assembly provides an inexpensive, sensitive, and reliable sensing platform not only for on-site analytical applications but also for high-resolution cellular imaging. KEYWORDS: quantum dot, biopolymer, porphyrin, electrostatic assembly, resonance energy transfer, sensor, zinc ion including bioconjugation,16 metal-affinity coordination,17 electrostatic adsorption,18 and so forth. Numerous advances have been achieved in the use of QDFRET assemblies for biological/chemical sensing applications. So far, the QD-FRET sensors have been developed for probing nucleic acids,19 peptides,20 proteins,21 proteases,22 small molecules,23 and ions.24 The response mechanisms of QDFRET sensors overwhelmingly rely on modulation of the QD− acceptor distance or spectral overlap between the QD emission and the acceptor absorption. The QD−acceptor spatial modulation depends on the choice of linker (spacer). The well-known examples include assemblies of QD−protein− dye,25 QD−peptide−dye,26 and QD−molecular beacon.27 The QD−acceptor spectral modulation involves the changes in the acceptor absorption such as cross-sectional or wavelength shifts. Snee et al. developed the QD−poly(acrylic acid)−squaraine dye FRET assembly.28 Because the dye’s absorption is pHsensitive, the FRET efficiency becomes a function of the environmental pH. Advances in maximization of the QD-FRET
1. INTRODUCTION One of the most exciting advances in nanotechnology is the development of nanomaterials with unique optical properties and nanometer-scale interfaces for analytical, imaging, and diagnostic applications.1,2 Electron3,4 and energy transfer5,6 through the nanometer-scale interfaces provide powerful tools to sense a variety of target recognition events. Primary among them is Förster resonance energy transfer (FRET), which is a process that involves nonradiative energy transfer from a photoexcited state donor to a ground-state acceptor. The quantum dot (QD) is a class of semiconducting nanocrystals with unique optical7 and electronic properties8,9 due to quantum confinement effects. The QDs have proven to be excellent donors in “donor−spacer−acceptor” configured FRET assemblies.10−13 The QD-based FRET (QD-FRET) offers several advantages over conventional organic fluorophores14 and fluorescent proteins.15 The QD can be excited at regions far from the acceptor’s absorption bands, which minimize the cross-talk between the QD and the acceptor’s emissions. The QD emission is narrow, tunable, and symmetric allowing for the maximal spectral overlap with the acceptor absorption without tailing into the acceptor emission region. The QD scaffold offers high flexibility in assembling acceptors © XXXX American Chemical Society
Received: November 22, 2016 Accepted: January 13, 2017 Published: January 13, 2017 A
DOI: 10.1021/acsami.6b14972 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
provided by Invitrogen (Shanghai, China). The pure water (18.2 MΩ cm) was obtained from a Pall Cascade AN system. UV−vis absorption experiments were performed on a Hitachi U-4100 spectrophotometer (Hitachi, Japan). Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. Transmission electron microscopy (TEM) images were acquired with a JEOL JEM-2100 instrument. Fourier transform-infrared (FTIR) spectra were acquired with Nicolet-5700 instrument (Nikon, Japan). The ζ-potential and hydrodynamic diameter measurements were conducted with a Malvern Zetasizer Nano-ZS particle analyzer (Malvern, U.K.). Fluorescence images of living cells were obtained from TCS SP8 confocal fluorescence microscope (Leica, Germany). 2.2. Assembly of QD-Poly(dA)-TSPP, QD-DNA-TSPP, and QDSiO2-TSPP. The synthesis of oil soluble green/yellow-emission CdSe@ZnS QDs (517QD and 560QD) was according to the singlestep method.41 Water-soluble AET-capped QDs were achieved via typical ligand exchange.42 The QD-poly(dA) and QD-DNA were prepared by mixing an equal volume of poly(dA)/DNA (0−32 mg L−1) with 2 μM AET-QDs (517QD/560QD), respectively, under stirring for 10 min. The amino-capped QD-SiO2 were prepared according to a microemulsion method described previously.35 The fluorescence quantum yields of as-prepared QD-poly(dA)/DNA/SiO2 were estimated by using rhodmine 6G (with a quantum yield of 0.95 in ethanol) as the reference. Afterward, QD-poly(dA)-TSPP, QD-DNATSPP and QD-SiO2-TSPP were assembled, respectively, by mixing equal volume of 0−24 μM TSPP with 1 μM QD-poly(dA)/DNA/ SiO2, under stirring. All the assemblies were subjected to ultracentrifugation at 40 000 rpm for 10 min, and the precipitates were redispersed in PBS buffer (pH 7.4) for further use. 2.3. Characterization of QD-Based Assemblies. ζ-potential measurements were conducted for aqueous solutions of AET-QD, QD-poly(dA)/DNA with varying poly(dA)/DNA coatings, or QDpoly(dA)/DNA-TSPP with varying TSPP densities at room temperature and pH 7.4. Hydrodynamic diameters of AET-QD, and QDpoly(dA)/DNA/SiO2 were estimated, respectively, by the dynamic light scattering (DLS) measurements. FTIR measurements were conducted for dry samples of AET-QD, QD-poly(dA) and QDpoly(dA)-TSPP, with the spectral resolution of 1.928 cm−1. Morphologies of QD-poly(dA)/DNA/SiO2 and QD-poly(dA)-TSPP were determined by TEM measurements, which were conducted by dropping diluted solutions onto carbon films supported by a Cu grid, respectively. 2.4. Fluorescence Measurements. To optimize the biopolymer spacer thickness, different amounts of poly(dA)/DNA were mixed with an equal volume of 2 μM 517QD, respectively, followed by the addition of 8.0 μM TSPP, for fluorescence measurements. To discern the FRET efficiencies of different QD-TSPP assemblies, varying densities of TSPP were assembled on 517QD-poly(dA)/DNA/SiO2, for fluorescence measurements. For experiments of sensing Zn2+, 1 μM of 517QD-poly(dA)-TSPP, 517 QD-DNA-TSPP, 517QD-SiO2 (4 nm)-TSPP, and 517QD-SiO2 (7 nm)-TSPP with optimized TSPP densities were prepared, respectively, as single-emission sensors. The dual-emission QD-BiFRET sensor was prepared by mixing 517QD-poly(dA)-TSPP with 560QD-poly(dA)TSPP at a molar ratio of 1:1. Zn2+ solutions were prepared by spiking varying amounts of Zn2+ into FBS, which was diluted by PBS buffer (pH 7.4) with a volume-to-volume (v/v) ratio of 10%. After the Zn2+ solutions were incubated with 1 equiv of the sensor solutions, respectively, for 30 min under the catalysis of imidazole (60 mM), fluorescence measurements were conducted. All fluorescence measurements were performed under ambient condition, with solutions placed in a 0.2 cm quartz cuvette. The excitation wavelength was fixed at 460 nm with a slit width of 5 nm. 2.5. Calculation of FRET Efficiencies. For different assemblies, the FRET efficiencies were calculated using the following equations:9
efficiency are also found, including increasing the acceptor-toQD ratios,29 the use of Au nanopaticles as the acceptors,30,31 and the development of multiplex FRET systems.32 Zinc ions (Zn2+), which rank second in abundance among transition metals in the human body, is believed to play vital roles in many biological processes, influencing the brain function, gene transcription, immune function, and mammalian reproduction.33 Alterations in Zn2+ homeostasis is confirmed to be closely correlated with several diseases such as Alzheimer’s disease, diabetes, epilepsy, and cerebral ischemia.34 Owing to the biological significances, the development of sensors for detecting Zn2+ in biological settings is of particular interest. Previously, we assembled a QD-based binary FRET (QDBiFRET) sensor to detect Zn2+ with a spectral modulation mechanism.35 In this sensor, distinctly emissive QDs were used as dual donors; ultrathin silica shell and meso-tetra(4sulfonatophenyl)porphine dihydrochloride (TSPP) were used as the spacer and the only acceptor, respectively. Binary FRET processes (FRET-1 and FRET-2) took place on the basis of spectral overlaps between dual QD emissions and two absorption bands of TSPP. Upon chelating with Zn2+, the two absorption bands of TSPP changed oppositely. Accordingly, binary FRET efficiencies were altered: FRET-1 was restricted and FRET-2 was enhanced. This led to enhancement of one QD emission but further quenching of another QD emission, producing net ratiometric response as a function of the Zn2+ concentration. However, from a FRET prospective, FRET efficiency of the QD-SiO2-TSPP assembly is far from maximal, and the sensitivity of QD-BiFRET sensor is not optimal. This is because that the thickness of silica spacer is difficult to reach
