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Functional Nanostructured Materials (including low-D carbon)
Supramolecularly Assembled Ratiometric Fluorescent Sensory Nanosystem for “Traffic Light”-type Lead Ion or pH Sensing Yuqian Liu, Qingsheng Guo, Xiaojun Qu, and Qingjiang Sun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10007 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018
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ACS Applied Materials & Interfaces
Supramolecularly Assembled Ratiometric Fluorescent Sensory Nanosystem for “Traffic Light”-type Lead Ion or pH Sensing Yuqian Liu,† Qingsheng Guo,† Xiaojun Qu and Qingjiang Sun* State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China *Fax: (86) 25-83792349. Email:
[email protected] ABSTRACT: The combination of functional nucleic acids and nanomaterials enables the continuous development of hybrid nanosystems that have found wide applications including chemo/biosensing. Herein, we report the supramolcecular assembly of a “sesame biscuit”-like superstructural nanosystem based on aptamer, quantum dot (QD) and graphene oxide (GO), and its diverse applications in Pb2+ and pH sensing. The nanosystem was assembled via adsorbing silica-encapsulated green-emitting QD onto the edge of GO by ionic interaction, followed by absorbing aptamer-modified red-emitting QD onto the GO surface via the π-stacking interaction. The nanosystem showed the characteristic of the nonquenched green fluorescence due to the silica encapsulation and quenched red fluorescence owing to nanomaterials surface energy transfer. The nanosystem responded to Pb2+/pH in ratiometric fluorescence: the red fluorescence varied upon analyte-driven conformational changes of the aptamer, whereas the green one remained constant. Under optimized conditions, the nanosystem was demonstrated to be capable of quantifying Pb2+ with a detection limit of 11.7 pM, as well as pH with a sensing resolution of 0.1 pH unit. More importantly, the ratiometric nanosystem facilitated visualization of analytes in a distinct “traffic light” manner, which was exemplified by semiquantification of exogenous Pb2+ in living cells. To demonstrate practicality, fluorescent test strips were fabricated by immobilizing the nanosystem on paper. The fluorescent test strips displayed “traffic light”-type fluorescence-color changes, with the capacity for on site, naked-eye detection of Pb2+ in real samples, as well as pointof-care pH testing in routine urinalysis. KEY WORDS: quantum dot; graphene oxide; aptamer; supramolecular assembly; ratiometric fluorescence; lead ion; pH sensing
so on have been successfully selected to specifically recognize Pb2+. The recognition predominantly utilizes the allosteric characteristics of the aptamers, and can be broadly classified into two categories. One is Pb2+induced G-quadruplex (G4) formation from a random coil,20 hairpin,21 or loosely paired duplex.22 The other is Pb2+-driven structural rearrangement of G4, for example, a change from K+-stabilized antiparallel-G4 to more compact parallel-G4.23,24 By labeling with organic dyes, different sensory systems based on chemiluminescence, fluorescence or Förster resonance energy transfer (FRET) have been developed. Chang, et al.25 reported that an aptamer FRET system can achieve the limit of detection (LOD) of 300 pM for aqueous Pb2+. However, applications of such systems for intracellular Pb2+ analysis are comprised as aptamers lack cell-membrane permeability and are susceptible to nuclease digestion. With the development of nanomaterials, the integration of aptamers with nanomaterials continuously generates robust sensory nanosystems that have found wider applications.26-29 Among others, graphene oxide (GO), a 2D carbon nanocrystal, is particularly intriguing, due to its unique surface and electronic properties.30,31 In the past, Pb2+ sensory nanosystems have been constructed by a combination of GO with Pb2+-specific aptamers. The GO avidly adsorbs a randomly coiled aptamer via the π-stacking interaction, whereas it cannot adsorb G4, thereby
1. INTRODUCTION 2+
Ionic lead (Pb ) is one of the most toxic heavy metal pollutants that can accumulate in aquatic ecosystems. The U.S. Environmental Protection Agency (EPA) has established a maximum allowable limit of 72 nM for Pb2+ in drinking water.1 Exposure to even low-level Pb2+ leads to deleterious effects for almost all the major organs and causes neurological, cardiovascular, and developmental disorders for both children and adults.2 Substantial studies of Pb2+ poisoning mechanisms at the cellular and molecular levels reveal that Pb2+ causes the generation of reactive oxygen species in cells which results in critical damage to various biomolecules such as DNA, enzymes, proteins and membrane lipids.3,4 The environmental and biological significance has motivated the development of new sensory systems that permit real time, on site or in situ Pb2+ analysis. Fluorescent sensory systems, featuring simplicity and sensitivity, have emerged as powerful tools toward achieving these goals.5-7 Functional nucleic acids including i-motifs,8,9 DNAzymes10-12 and aptamers13,14 have been widely employed as the recognition elements in fluorescent sensory systems. The aptamers, in vitro selected short DNA/RNA oligonucleotides, which can recognize a library of analytes with high specificity, have the advantages in terms of cost and simplicity.15,16 Guaninerich aptamers such as PS2.M,17 AGRO100,18 T30695,19 and 1
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Scheme 1. Illustration of the supramolecular assembly of superstructural nanosystem, and the principle for Pb2+ and pH sensing.
allowing the aptamer to desorb from the surface upon Pb2+-induced G4 formation.32,33 Accordingly, an interfacial electronic process between GO and a fluorophore, namely, nanomaterials surface energy transfer (NSET),34-36 is tuned, transducing the Pb2+ sensing events. The NSET-based quenching capability of GO can minimize the background noise, thereby enhancing the signal-to-noise ratios for Pb2+sensing. Moreover, as a long-range resonance energy transfer, the NSET enables the use of bigger-sized, brighter fluorophores compared to organic dyes for greater sensitivity such as upconversion nanoparticle,37 and quantum dot (QD).38 Li, et al.39 constructed a GO/aptamer-QD nanosystem for Pb2+ analysis, achieving an LOD as low as 90 pM. Despite the encouraging progress that has been made, all Pb2+ sensory systems reported so far are based on single-emission, which may suffer from interference from environmental parameters, instrumental efficiency, and sensor concentration. Ratiometric fluorescence, which is based on the measurement of changes in the ratio of fluorescence intensities at two well-resolved wavelengths, can resolve the accuracy problem.40,41 Moreover, for fluorescence ratiometry, changing the fluorescence color significantly improves the visualization capability. With inexpensive instruments such as a UV lamp or an LED, ratiometric sensory systems enable detection of analytes by the naked eye that is as easy as colorimetric systems, but with greater sensitivity. As such, the construction of ratiometric sensory systems has drawn great interest in recent years. QDs, with their characteristic narrow-band, multiplecolor emission by single-wavelength excitation, are advantageous over other materials for construction of ratiometric systems.42,43 In this work, we report the assembly of a ratiometric Pb2+ sensory nanosystem by the combination of stateof-the-art nanomaterials: dual-colored QDs as the super fluorophores, aptamer (T30695) as the recognition element, and GO as the superquencher. Two supramolecular interactions were utilized to decorate the GO with dual-colored QDs in a site-selective manner. By ionic interaction, silica-encapsulated greenemitting QD (gQD@SiO2) was adsorbed onto the ionized edge of GO, while aptamer modified redemitting QD (apt-rQD) was adsorbed onto the hydrophobic basal plane of GO via the π-stacking
interaction, producing a “sesame biscuit”-like superstructure (Scheme 1). For the nanosystem, the fluorescence of gQD cannot be quenched by GO due to silica encapsulation, and in contrast that of rQD is quenched through NSET. The nanosystem is demonstrated to be capable of sensing Pb2+ in ratiometric fluorescence: the green fluorescence remains constant, acting as the internal reference, while the Pb2+-dependent G4 formation drives the desorption of apt-rQD from GO and gradual recovery of red fluorescence. Importantly, the red-to-green ratiometry generated green-yellow-red fluorescent “traffic light”, which is conceivably sensitive to the human naked eye. In the presence of Pb2+, “traffic light”-type ratiometric fluorescence is also demonstrated for sensing physiologically important pH, operating in a competitive manner: Pb2+ drives the apt-rQD desorption from GO and intense red fluorescence, whereas the pHdependent protonation of guanines induces apt-rQD readsorption to GO and quenching of the red fluorescence. Furthermore, nanosystem-based fluorescent test strips are fabricated, and their practical use for on-site, visual Pb2+/pH testing is demonstrated.
2. EXPERIMENTAL SECTION 2.1 Materials and Reagents. GO (4 mg/mL, dispersed in water), and lead nitrate (Pb(NO3)2, 99.99%) were obtained from Sigma-Aldrich. T30695 ((GGGT)4) modified with 5’-thiol or 3’-FAM were synthesized by Invitrogen (Shanghai, China) and purified with HPLC. Glucose (Glu), cysteine (Cys), histidine (His), glutathione (GSH), bovine serum albumin (BSA), fetal bovine serum were purchased from Fcmacs (Nanjing, China). Ammonium chloride (NH4Cl), urea, uric acid (UA) and ascorbic acid (AA) were purchased from Aladdin (Shanghai, China). Exonuclease I (20 U/μL) was supplied by Abcam (Shanghai, China). Cellulose papers and standard pH test strips (pH 5.5-9.0) were purchased from Wanqing Chemical (Nanjing, China). Pure water (18.2MΩ cm) was obtained from a Pall Cascade AN system. 2.2 Assembly of the Nanosystem. The aminomodified gQD@SiO2, wherein one gQD (CdSe/ZnS: EM: 520 nm) was embedded within 18-nm-thick silica, was prepared by a modified microemulsion method.44 The apt-rQD was prepared by tethering the rQD (InP/ZnS, 2
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ACS Applied Materials & Interfaces EM: 590 nm) with thiol-modified aptamers.45 To assemble the nanosystems, 0.4 mg/mL of GO was first mixed with 1 equiv. of gQD@SiO2 (0-160 nM) in PBS buffer (10 mM, pH 7.4) for 10 min, followed by centrifugation (15000 rpm, 20 min). Next, varying concentrations (0-1.2 mg/mL) of formed gQD@SiO2/GO were, respectively, mixed with 1 equiv. of apt-rQD (100 nM) for 30 min, followed by centrifugation. 2.3 Stability of the Nanosystem. The stability experiments for the nanosystem were conducted by adjusting the solution pH (4.0-10.0) with 1 M of HCl/NaOH, varying the ionic strength (0-1000 mM, NaCl), and challenging with various urine components (30 mM for urea, 1 mM for UA, 8 mM for AA and NH4Cl) and biomolecules (11 mM for Glu, 5 mM for GSH, 3 mM for Cys and His, 0.2 mM for BSA, and 50 U/mL for exonuclease I), respectively. Samples were left to stand for 12 h prior to each fluorescence measurement. 2.4 Pb2+ Sensing. The nanosensor solutions were prepared by dispersing the nanosystems in PBS buffers with varying pH (6.0, 6.5, 7.0, 7.4, 8.0, 9.0, 10.0), respectively, and 0.4 mg/mL was kept for each solution. Pb2+ solutions were prepared by dissolving Pb(NO3)2 into PBS buffers with varying pH, and diluted to different concentrations, respectively. The sensitivity experiments were conducted by mixing varying concentrations of Pb2+ solutions, respectively, with 1 equiv. of the nanosensor solution at identical pH for 30 min, followed by the fluorescence measurements. The selectivity experiments were conducted by equally mixing the nanosensor solution (pH 8.0) with solutions of various metal ions spiked with/without 2 μM of Pb2+ for 30 min, respectively, followed by fluorescence measurements. For the anti-interference experiments, varying concentration of Pb2+ solutions were, respectively, mixed with 1 equiv. of 5 mM of GSH-spiked nanosensor solution at pH 7.4 for 30 min, followed by fluorescence measurements. 2.5 Intracellular Pb2+ Imaging. HeLa cells were cultured in 10% FBS supplemented Dulbecco’s Modified Eagle Medium at 37 °C in a 5% CO2 humidified atmosphere. By spiking 10 nM and 1 μM of Pb2+, respectively, into in the culture medium for 4 h, the Pb2+-treated cells were obtained. Prior to Pb2+ imaging, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) assay was performed by incubating the native cells with varying concentrations of the nanosystem for 16 h, and subsequent staining with MTT for 4 h, followed by absorption measurements. Kinetics of cellular uptake of the nanosystems was conducted by incubating the native cells with 0.2 mg/mL of the nanosystem for different time, followed by fluorescence imaging. Specific to track the endocytosis, lysosomes of the cells were stained with LysoTracker blue (EX: 375 nm; EM: 410-430 nm). For fluorescence imaging, the native and Pb2+-treated HeLa cells were, respectively, incubated with 0.2 mg/mL of nanosensor for 12 h, followed by 3 times washing with PBS buffer. Excitation wavelength of laser was 405 nm, and the emissions at 505-525 nm and 570-610 nm were collected. The fluorescent images were analyzed by ImageJ (NIH, http://rsb.info.nih.gov/nihimage/index.html).
2.6 pH Sensing. For the pH sensing experiments, the nanosystem was mixed with varying concentrations (10-2000 nM) of Pb2+, followed by titration with 1 M of HCl/NaOH to adjust the solution pH from pH 5.0 to pH 8.5, respectively. After 30 min standing, the solutions were subjected to fluorescence measurements. 2.7 Fluorescent Test Strips. The cellulose paperbased strip (2 cm × 20 cm) was hydrophobically processed with wax to obtain a line of spots (diameter: 0.8 cm). Then, 20 μL of nanosensor solution (0.8 mg/mL) was dropped onto each spot and naturally dried. The strip was stuck onto a piece of A4 paper for further testing. For the Pb2+ testing, varying concentrations of Pb2+-spiked samples (PBS buffer (pH 7.0), tap water, and 10-fold diluted serum) were dropped onto the test strips (80 μL for each spot) and incubated for 30 min. For the pH testing, varying pH of buffer and urine samples spiked with 2 μM of Pb2+ were dropped onto the test strips. The urine samples (stored at 4 oC) were pretreated with centrifugation at 12000 rpm for 40 min before the use. Under the illumination of a hand-held UV lamp, fluorescent images were taken with a digital camera placed 30 cm from the top of all the test strips, and analyzed by ImageJ. 2.8 Characterization. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. All the fluorescence measurements were conducted with a 0.2-cm-thick quartz cuvette, an excitation wavelength of 405 nm, and a 5 nm slit. X-ray photoelectron spectroscopy (XPS) was obtained on a PHI 5000 VersaProbe spectrometer (ULVAC-PHI, Inc.). Fourier transform-infrared (FT-IR) spectra were acquired with Nicolet-5700 instrument (Nikon, Japan). The ζ-potential measurement was conducted with a Malvern Zetasizer Nano-ZS particle analyzer (Malvern, U.K.). Atomic absorption spectroscopy (AAS) analyses were conducted with a Hitachi 180-80 atomic absorption spectrometer. Circular dichroism (CD) spectra were obtained with a Chirascan circular dichroism spectrophotometer (Applied Photophysics, UK). Cellular fluorescence images were obtained from a TCS SP8 confocal fluorescence microscope (Leica, Germany). Tunneling electron microscopy (TEM) images were acquired with a JEOL JEM-2100 instrument.
3. RESULTS AND DISCUSSIONS 3.1 The Assembly of the Nanosystem: Siteselective Decoration of GO by Two Supramolecular Interactions. The GO-based nanosystem assembly depends on its surface properties, which was characterized by XPS (Figure S1 in the SI). The spectra showed four types of carbon bonds, i.e. C-C (286 eV), COH (286.5 eV), C=O (288 eV), and O=C-OH (289.5 eV), clearly revealing that the GO was characteristic of hydrophobic basal plane with charged groups.46 Two supramolecular (noncovalent) interactions were therefore utilized to decorate the GO with dual-colored QDs. First, ionic interaction was chosen to adsorb amino-modified gQD@SiO2 onto the GO. Figure 1A shows TEM images of the gQD@SiO2/GO prepared with varying concentrations of gQD@SiO2. It was found that the ionic interaction drove an “edge-to-basal plane” adsorption process: for concentrations of ≤60 nM, 3
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gQD@SiO2 were preferentially adsorbed onto the GO edge, owing to the high density of carboxyl/hydroxyl groups situated there; further increasing the concentration, the gQD@SiO2 expanded onto the basal plane, due to the presence of ionized defects (with a lower density of carboxyl/hydroxyl groups compared to the edge). This result reveals that the ionic interactiondriven decoration can be controlled in a site-selective manner by optimizing the nanoparticle-to-GO ratio. In our case, 60 nM of gQD@SiO2 was chosen to decorate the GO edge, retaining the intact basal plane to adsorb a high density of apt-rQD. Upon the formation of gQD@SiO2/GO, the green fluorescence at 520 nm was found to be nearly proportional to the increased decoration, without being quenched by GO due to the silica encapsulation (Figure 1B). Second, the π-stacking interaction between the hexagonal cells of GO and the aromatic bases of the aptamer was utilized to further decorate the gQD@SiO2/GO with apt-rQD. Figure 1C shows the fluorescent spectra of gQD@SiO2/GO/aptQD nanosystems prepared with varying concentrations of gQD@SiO2/GO. With increased gQD@SiO2/GO, the green fluorescence increased, and the red fluorescence at 590 nm gradually quenched due to NSET between GO and rQD. With 0.4 mg/mL of gQD@SiO2/GO, the red-to-green intensity ratio reached 1:2; the red fluorescence could not be further quenched upon further increasing the concentration, indicative of a threshold of saturation for the apt-rQDs on GO. Under the optimized condition (0.4 mg/mL of gQD@SiO2/GO mixed with 100 nM of apt-rQD), the nanosystem featured a “sesame biscuit”-like superstructure, with a few of big-sized gQD@SiO2 “peanuts” situated at the edge, and a high density of small-sized apt-rQD “sesames” spreading across the surface (Figure 1D). The supramolecular assembly of the nanosystem was also confirmed by ζ-potential and FT-IR measurements (Figure S2 in the SI). Upon adsorbing amino-modified gQD@SiO2 (60 nM) onto GO, the ζ-potential positively shifted from -40.3 mV to -18.7 mV; further adsorbing apt-rQD to the gQD@SiO2/GO, the ζ-potential negatively shifted to -27.6 mV, due to the negatively charged property of aptamer and rQD. In the FT-IR spectra, characteristic bands of deformation vibration (1560.2 cm-1) and bending vibration (788.7 cm-1) of amino groups on gQD@SiO2 appeared upon the ionic assembly of gQD@SiO2/GO; characteristic bands of deformation vibration of glycosidic bond (1400.2 cm-1) and stretching vibration of phosphate group (1089.7 cm-1) on the aptamer appeared upon the assembly of the superstructural nanosystem. 3.2 Use of the Nanosystem-Based Fluorescent “Traffic Light” to Visualize Pb2+. The superstructural nanosystem with a red-to-green intensity ratio of 1:2 was used as the nanosensor for Pb2+ analysis. The final concentration of nanosensor was kept as 0.2 mg/mL. The response time was defined as 30 min, identical to that of a GO/apt-FAM system (Figure S3 in the SI). As shown in Figure 2A, the nanosensor exhibited a ratiometric fluorescent response toward Pb2+. With increased Pb2+ concentration, the red fluorescence
Figure 1. (A) TEM images of gQD@SiO2/GO prepared with varying gQD@SiO2 concentrations: a) 20 nM, b) 40 nM, c) 60 nM, d) 80 nM. (B) Fluorescent spectra of the gQD@SiO2/GO in (A). (C) Fluorescent spectra of aptrQD (100 nM) incubated with varying concentrations of gQD@SiO2/GO. (D) TEM image of the nanosystem. The insets show microscopic images of gQD@SiO2 and aptrQD. Scale bar: 20 nm.
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ACS Applied Materials & Interfaces fluorescence-color changes, which could be easily distinguished by the naked eye (Figure 2C). The “green light” switched on as the Pb2+ concentration was no more than 1 nM; the “yellow light” twinkled in the range of 5-50 nM of Pb2+; the “red light” warned for a Pb2+ concentration greater than 50 nM. Therefore, with the fluorescent “traffic light”, the nanosensor facilitated the realization of naked-eye detection of Pb2+. Notably, the fluorescent “traffic light” could not be achieved under acidic conditions.
3.3 Cellular Fluorescent “Traffic Light” for Semiquantification of Exogenous Pb2+. The visualization of intracellular Pb2+ is of particular importance for both fundamental study of the Pb2+poisoning mechanism and clinical diagnosis. The nanosensor was used to visualize exogenous Pb2+ in HeLa cells. Prior to cellular imaging, the photostability, biostability and biocompatibility of the nanosensor were evaluated under different conditions. The fluorescence of the nanosensor was found to be insensitive to a wide range of pH (4.0-10.0), and ionic strength (0-1000 mM NaCl), and tolerant of the presence of various biomolecules, such as glucose, cysteine, histidine, BSA, and even exonuclease I, indicative of high stability (Figure S6 in the SI). The MTT assay revealed that the nanosensor had negligible effect on the cell viability in 16 h, featuring low toxicity (Figure S7 in the SI). Cellular uptake of the nanosensor was investigated by fluorescence imaging (Figure S8 in the SI). By fluorescent colocalization of LysoTracker blue and the nanosensor at the lysosomes, the nanosensor was observed to enter the cells via endocytosis. By monitoring the reference green fluorescence in cytoplasm, the nanosensor was found to heavily enter the cells within 10 h. The intracellular environment is complex, and biothiols, such as GSH, may combine with Pb2+, interfering with the sensing reliability. To evaluate the anti-interference capability, a competitive assay was conducted by exposing the nanosensor and GSH together to Pb2+. The presence of 5 mM of GSH was found not to interfere with the fluorescence ratiometry induced by Pb2+ in the concentration range of subnanomolar to a few tens of nanomolar, and only slightly reduced the fluorescence ratiometry at higher Pb2+ concentrations (Figure S9 in the SI). This result indicated that the nanosensor could be reliably used for sensing intracellular Pb2+, which is ascribed to the higher affinity of the aptamer toward Pb2+ compared to GSH.47 Figure 3 shows microscopic fluorescent images of the native and Pb2+-treated HeLa cells with the nanosensor. The native cells exhibited a red-to-green fluorescence intensity ratio of 1:2, identical to that for the nanosensor in solution. For the Pb2+-treated cells, the green fluorescence remained stable, while the red fluorescence increased with respect to that of the native cells. Quantitatively, the red-to-green intensity ratio increased to 1:1 and 1.9:1, respectively, for 10 nM and 1 μM of Pb2+-treated cells. As a result, the overlaid cell images displayed distinct “traffic light”-type fluorescence-colors: the “green light” to indicate the absence of Pb2+; the “yellow light” for an allowable
Figure 2. (A) Fluorescent spectra of the nanosensor as a function of Pb2+ concentration at pH 8.0. Evolution of (B) the relative red-to-green intensity ratio and (C) fluorescence-colors of the nanosensor with respect to the Pb2+ concentration for varying pH. increased gradually, whilst the green fluorescenceremained constant. The fluorescent response was found to be pH dependent (Figure S4 in the SI). Under neutral and basic conditions, the ratiometric fluorescence responded to Pb2+ in a dosedependent manner, while for acidic conditions, the fluorescent response became less sensitive. At a Pb2+ concentration of 2 μM, approximately 3.0-fold increases in red fluorescence were achieved at pH ≥7.0, and less than 1.6-fold increases were observed at pH
