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Aptamer-Modified Graphene-Based Catalytic Micromotors: Off-On Fluorescent Detection of Ricin. Berta Esteban-Fernández de Ávila, Miguel Angel Lopez Ramirez, Daniela F. Báez, Adrian Jodra, Virendra V Singh, Kevin Kaufmann, and Joseph Wang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.5b00300 • Publication Date (Web): 06 Jan 2016 Downloaded from http://pubs.acs.org on January 7, 2016
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Aptamer-Modified Graphene-Based Catalytic Micromotors: Off-On Fluorescent Detection of Ricin. Berta Esteban-Fernández de Ávila, Miguel Angel Lopez-Ramirez, Daniela F. Báez, Adrian Jodra, Virendra V. Singh, Kevin Kaufmann, and Joseph Wang*. Department of Nanoengineering, University of California, San Diego, La Jolla, California 92093, United States. KEYWORDS: ricin, aptamer, micromotors, real time-detection, graphene
Supporting Information Placeholder ABSTRACT: An aptamer-based catalytic micromotor sensing strategy for “Off-On” real-time fluorescent detection of the ricin B toxin is described. This approach relies on selfpropelled reduced graphene-oxide (rGO)/platinum (Pt) micromotors, modified with a specific ricin B aptamer tagged with a fluorescein-amidine (FAM) dye, whose fluorescence is quenched due to π-π interactions with the rGO surface. The continuous movement of the motor in the sample accelerates the specific binding of the ricin B toxin to the aptamer-dye conjugate and leads to real-time fluorescent “On” detection. Coupling the “Off-On” fluorescent switching properties of the aptamer modified-rGO/Pt micromotors with their inherent mixing capabilities thus leads to high speed, simplicity, and sensitivity advantages, thus addressing the limitations of current ricin detection strategies. The new micromotor strategy represents an attractive route for detecting biological threats in a variety of biodefense applications.
Ricin is considered one of the deadliest poisons worldwide 1,2 due to its extremely low lethal dose. This natural toxin is composed of two polypeptide chains, A and B, which inhibit protein synthesis and act as a binding site for cell-surface 3 receptors, respectively, leading to rapid cell death. The widespread availability of the castor plant Ricinus communis 4 (from which ricin toxin is derived), ease of extraction, and acute toxicity, make ricin a potent biological warfare agent 5 (BWA). Since there is no effective treatment for ricin exposure, there are urgent needs to develop rapid and reliable methods for its detection in different fields, such as medical diagnostics, food or water safety testing, and 2 biodefense. Current methodologies for ricin detection 1,6-8 9 include biosensors, chromatographic methods, 10 polymerase chain reaction (PCR) assays, spectroscopic 11 12 methods, and enzyme-linked immunoassays. However, the practical application of these methodologies is limited by their low sensitivity and selectivity, slow response, false positive readings, non-portability, operational complexity, and difficulties in real-time monitoring. Developing rapid and reliable ricin detection methods is thus crucial to 13 facilitate timely warning and reduce the mortality rate.
The present work demonstrates a powerful fluorescent “Off–On” system for detecting of ricin based on self-propelled aptamer-modified graphene micromotors. Chemically-powered micromotors offer tremendous sensing opportunities in diverse fields, owing to their attractive “onthe-move” binding, isolation, cargo-towing and self-mixing 14-17 The efficient fluid mixing capability of capabilities. tubular micromotors has shown to be extremely effective in 18-20 21 accelerating target-receptor interactions , detection and 22-25 detoxification processes. The new “Off-On” ricin B toxin sensing system relies on catalytic micromotors consisting of bilayer reduced graphene oxide (rGO)/platinum (Pt) microtubes, modified with a specific ricin B aptamer tagged with fluorescein amidine (FAM) dye (Figure 1). Aptamerbased detection methods have gained great recent interest because of their high selectivity and affinity towards their 26,27 targets. The aptamer selected here for specific recognition of ricin B chain displays a stable folding conformation (detailed in SI Fig S1) across a wide pH (2-7) 8 and temperature (4–63˚C) range, making it attractive for defense applications in harsh environments. The “Off” signal (Figure 1A(a)) is attributed to the effective fluorescence quenching capability of graphene surfaces upon adsorption of the dye-labeled aptamer through π-π stacking interactions 28,29 between the nucleotide bases and the carbon material. The high sensitivity and speed of the new fluorescence “On” ricin detection method reflect the greatly accelerated ricinaptamer binding associated with the motor-induced enhanced fluid transport. Highly specific ricin detection is demonstrated in the presence of excess co-existing proteins and using untreated complex samples. The present micromotor strategy thus offers considerable promise as a rapid, simple, and effective method for detecting ricin in diverse biodefense scenarios. As illustrated in Figure 1, the micromotor-based ricin fluorescence “On” sensing approach relies on the fast recovery of the quenched fluorescence of the dye-tagged aptamer probe due its preferential binding with the target toxin compared to the graphene-oxide surface. When these aptamer-modified peroxide-propelled micromotors (Figure 1A(b)) are exposed and bind to the ricin B toxin, the quenched fluorescence is recovered due to the displacement
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of the dye-aptamer probe from the graphene-oxide quenching motor surface. This results in an intense fluorescent signal (Figure 1A(c)) that indicates the presence of the target ricin and leads to a rapid, selective and sensitive detection. The fluorescent signal increases linearly with the ricin B concentration over the 100 pg/mL-10 µg/mL range. The motor-based signal recovery is significantly faster when compared to static microrockets.
Figure 1. In vitro “Off-On” specific fluorescent detection of ricin-B toxin by FAM-Ricin B aptamer-modified rGO/Pt microrockets, and synthesis and characterization of modified-rGO/Pt microrockets. (A) Left side: schematic illustration of the rGO/Pt micromotors modified with the dye-tagged aptamer that quenches the dye fluorescence; inset picture showing the reduced intensity of the quenched fluorescence after 15 min incubation of the motors with the dye-aptamer (a). Right side: schematic of H2O2-propelled microrocket in the presence of ricin-B toxin, producing fluorescence recovery due to release of the dye-aptamer from the motor GO-quenching surface upon binding to the toxin; inset picture showing the fluorescence intensity corresponding to the detection of 10 µg/mL ricin-B toxin (c). (A(b)) Micromotor motion in the presence of ricin B toxin in 1% H2O2 solution; scale bar, 10 µm. (B) Template preparation of the aptamer-modified microrockets: electrodeposition of rGO and Pt (1 and 2, respectively), release from the template (3) and modification with the aptamer (4). (C) SEM images of the rGO/Pt microrockets (top and side views; left), and EDX analysis of carbon (rGO), platinum, and phosphorus. Reduced GO/Pt microrockets were synthetized by a standard membrane-template electrodeposition protocol (see Methods Section of Supporting Information). Such template deposition offers highly reproducible preparation of thousands of micromotors of similar size and shape using a single membrane. Briefly, the template protocol consisted of the electrodeposition of rGO on the inner wall of a polycarbonate (PC) membrane by a cyclic voltammetry, followed by deposition of the inner Pt layer (Figure 1B; steps 1 and 2, respectively), dissolution of the membrane and release 30 of the rGO/Pt microrockets (Figure 1B (3)). The FAM-Ricin B aptamer used in this work exhibits a strong fluorescence signal, characteristic of the conjugated FAM dye (λem 520 nm; λex 495 nm). The quenching of the fluorescence intensity is largest upon incubating the dye-aptamer with the rGO-based micromotors for 15 min (SI Fig S2). The rGO-Pt micromotors were thus successfully modified by incubating in a 5 µM FAM-ricin B aptamer solution for 15 min (Figure 1B (4)).
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Scanning electron microscopy (SEM) images were carried out to examine the structural morphology of rGO/Pt microrockets. The left part of Figure 1C shows SEM images of the top and side views of a modified rGO/Pt microrocket. The micromotors are ~10 μm long and 5 µm in diameter, reflecting the pore size of the PC membrane. An energy dispersive X-ray spectroscopy analysis (EDX), shown in right part of Figure 1C, demonstrates the successful composition and modification of the functionalized rGO/Pt microrocket (right part of Figure 1C). These EDX images clearly show the presence of carbon and platinum, corresponding to the outer and inner layers, respectively. Modification of the microrocket with the FAM-ricin B aptamer is indicated from the presence of phosphorous corresponding to the phosphate groups of the dye aptamer. To demonstrate that the observed “Off-On” fluorescence response is produced solely by the specific interaction with the target ricin and not by non-specific bindings, we evaluated the specificity of the new micromotor detection system in the presence of excess non-target proteins. For this purpose, bovine serum albumin (BSA) and saporin toxin were selected as a generic soluble protein and 31,32 alternative RIP toxin, respectively. Figure 2A displays the schematic illustration of FAM-aptamer-modified-rGO/Pt micromotors in a sample mixture containing ricin, BSA and saporin (Figure 2A(a)), and the specific interaction of ricin with the surface-bound aptamer, accelerated by the micromotors propulsion (Figure 2A(b)). The aptamer is initially confined on the motor surface, resulting in a loss of the dye fluorescence, which is recovered upon the specific binding with the target ricin.
Figure 2. Specificity of FAM-Ricin B aptamer-rGO/Pt micromotors towards the ricin B target. (A) Schematic illustration of dye-aptamer-modified-rGO/Pt micromotors in the presence of coexisting ricin, saporin and BSA (a), and specific ricin B-aptamer interaction accelerated by the motor propulsion (b). Fluorescence images in the presence of 10 µg/mL ricin B toxin (B), and in presence of BSA (C) and saporin (D), both at 100 µg/mL level. Images for the detection of 10 µg/mL ricin B toxin in the presence of 100 µg/mL (E) BSA and (F) saporin. (G) Corresponding fluorescence intensities expressed as percentage recovery of the pre-quenching signal. Experimental conditions: 3 min treatment; 1% H2O2; 1.5% NaCh; PBS pH= 7.4.
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As shown in Figure 2, an excess of both non-target proteins (100 µg/mL vs 10 µg/mL ricin) displays a negligible fluorescence recovery (Figure 2C, 2D, and 2G), compared to the strong recovery observed in the presence of the target ricin B (Figure 2B and 2G(a)). Similarly, the fluorescent signal is recovered fully upon adding the ricin toxin to solutions containing an excess of BSA and saporin (Figure 2E and 2F, respectively, and bars in 2G), demonstrating the high specificity of the surface-bound aptamer towards the ricin B toxin. To prove the important role of the self-propelled micromotors and their self-mixing capability in the dramatically accelerated of ricin detection, the dependence between the fluorescence recovery (due to the toxin binding) and the speed of the motors (in presence of different fuel levels) was tested (Figure 3). No fluorescence quenching effect was observed at the different H2O2 fuel concentrations used (SI Figure S3). The effect of the ricin-micromotor incubation time on the fluorescence ricin response was studied under motor movement and static conditions (Figure 3A: b and a, respectively). Optimal detection of 10 µg/mL ricin B was obtained using a 3 min incubation. Such period resulted in recovery of 90% of the fluorescence signal in the presence of the self-propelled modified motors compared to 12% recovery under static conditions (Figure 3A). This dramatic enhancement reflects the fluid convection induced by the rapid micromotor motion and the corresponding bubble trail.
Figure 3. Ricin-triggered fluorescence recovery: influence of the reaction time and motor speed upon the fluorescence recovery due to binding of the toxin to the surface-confined aptamer. (A) Dependence of the ricin-triggered fluorescence recovery upon the incubation time under static conditions (a) and with H2O2-propelled micromotors (b). (B) Dependence of the fluorescence recovery upon the concentration of the peroxide fuel. (C-F) Fluorescence images (top part) of the ricin B toxin solution after 3 min incubation with the dye-aptamer-modified micromotors using different H2O2 levels (0, 0.5, 1 and 2%, respectively). Bottom part, images corresponding to the micromotor motion at each fuel level. Experimental conditions: Ricin B toxin, 10 µg/mL; 1% H2O2 (A); 1.5% NaCh; 3 min treatment (B-F). Error bars estimated as a triple of the standard deviation (n = 3). Figure 3B shows the values of the fluorescence recovery (corresponding fluorescence images in Figure 3: C-F, top part) obtained after 3 min incubation of the dye-aptamer-
micromotors in ricin solutions containing different H2O2 levels (0, 0.5, 1 and 2%, respectively). As indicated from the optical images of Figure 3 C-F (bottom part), the rate of fluorescence recovery relates directly to the micromotor speed at each fuel level (0, 155, 380 and 860 µm/s, respectively), reflecting the increased likelihood of ricin contacts with the surface-confined aptamer due to faster movement of the receptor and enhanced fluid transport. As can be observed in Figure 3B, the fast movement of the aptamer-modified micromotors in the 2% peroxide solution resulted in nearly 100% recovery of the quenchedfluorescence, reflecting the highest aptamer-ricin contact rate. Overall, the data of Figure 3B clearly indicate that the speed of the aptamer-modified micromotors plays a crucial role in the accelerated detection of the toxin. To investigate the sensitivity and detection limit of the dye-aptamer-modified micromotors for ricin detection and demonstrate the quantitative nature of the fluorescent “On” response, the modified micromotors were exposed to different ricin B concentrations. As illustrated from the images of Figure 4A, the recovery of the fluorescent signal (i.e., fraction of the displaced aptamer-dye conjugate) increases gradually upon increasing the ricin B concentration between 100 pg/mL to 10 µg/mL (a-f). The corresponding calibration plot (shown in Figure 4A(g)) is highly linear over the entire range. These data indicate an attractive detection performance of the dye-aptamer modified micromotors at low (ng/mL) ricin concentrations and that the fluorescent intensity can provide a quantitative estimation of the target concentration.
Figure 4. (A) Correlation between the ricin B concentration and the fluorescence intensity: images corresponding to each ricin B concentration level over the 100 pg/mL to 10 µg/mL range (a, f, respectively); the corresponding calibration plot (g). (B) Fluorescence recovery (top) and micromotor movement (bottom) in different ricin B toxin-spiked samples (from a to e: PBS buffer, tap water, saliva, serum, and urine, respectively). Fluorescence pictures (B: a-e, top part) of each spiked sample after 3 min incubation with the FAM-ricin B aptamer-modified micromotors at 1% H2O2; (B: a-e, bottom part) microscope images showing the movement of the micromotor in each sample. (B, f) Percentages of fluorescence recovery and micromotor speeds in the different samples. Experimental conditions: as in Figure 2 using 10 µg/mL ricin B toxin. Scale bar, 10 µm. The Centers for Disease Control and Prevention have classified ricin as a category B agent, which means that 7 it could potentially affects our food or water supply. In order to evaluate the practical applicability of the new micromotor ricin detection method, different complex matrices were spiked with a known amount of ricin (10 µg/mL). Figure 4B displays the dependence of the micromotors’ speed on the
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fluorescence recovery using six different untreated real samples (a-e: PBS buffer, tap water, saliva, serum and urine, respectively) along with 1% H2O2 fuel. While the aptamermodified micromotors display efficient movement in these different biological media, their speed varies based on the nature of the medium. We observed a more efficient fluorescence recovery in PBS buffer (up to 90%) compared to these real samples after 3 min incubation with the micromotors, reflecting the observed speed change. For example, due to the higher viscosity of saliva, the micromotor speed is slow, which leads to slow fluorescence recovery. Nevertheless, fluorescence recoveries higher than ~65% have been observed in all samples within 3 min, illustrating the applicability of the micromotor method for the rapid and specific detection of ricin in real samples without preparatory and washing steps. In summary, we developed an effective micromotor-based protocol for detecting the ricin B toxin based on specific dye-labeled aptamer-modified microengines. The new ricin detection method offers rapid ‘on-the-fly’ binding of the toxin along with a visual “Off-On” fluorescent response that enables to fast, sensitive and selective real-time measurements in diverse untreated media. Trace amounts of ricin B toxin can thus be detected within a few minutes in complex biological and environmental samples. The aptamer-micromotor concept could be expanded for detecting multiple biothreats (via judicious design of the aptamer) and holds considerable promise for variety of biodefense applications.
ASSOCIATED CONTENT Supporting Information Supporting videos description, experimental section, supporting figures and table. The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was supported by the Defense Threat Reduction Agency (grants nos. HDTRA1-13-1-0002 and HDTRA1-14-10064). D. F. Báez and A. Jodra acknowledge fellowships from CONICYT-CHILE and the Spanish Ministry of Economy and Competitiveness, respectively. The authors thank Aída Martín for her advice.
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