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Letter
pubs.acs.org/acssensors

Aptamer-Modied Graphene-Based Catalytic Micromotors: OOn

Fluorescent Detection of Ricin
Berta Esteban-Fernandez de A vila, Miguel Angel Lopez-Ramirez, Daniela F. Baez, Adrian Jodra,
Virendra V. Singh, Kevin Kaufmann, and Joseph Wang*
Department of Nanoengineering, University of California, San Diego, La Jolla, California 92093, United States
S Supporting Information
*

ABSTRACT: An aptamer-based catalytic micromotor sensing

strategy for Of f-On real-time uorescent detection of the
ricin B toxin is described. This approach relies on selfpropelled reduced graphene-oxide (rGO)/platinum (Pt)
micromotors, modied with a specic ricin B aptamer tagged
with a uorescein-amidine (FAM) dye, whose uorescence is
quenched due to interactions with the rGO surface. The
continuous movement of the motor in the sample accelerates
the specic binding of the ricin B toxin to the aptamerdye
conjugate and leads to real-time uorescent On detection.
Coupling the Of f-On uorescent switching properties of the
aptamer modied-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.
KEYWORDS: ricin, aptamer, micromotors, real time-detection, graphene
ecient uid mixing capability of tubular micromotors has been
shown to be extremely eective in accelerating targetreceptor
interactions, 1820 detection, 21 and detoxication processes.2225 The new Of fOn ricin B toxin sensing system
relies on catalytic micromotors consisting of bilayer reduced
graphene oxide (rGO)/platinum (Pt) microtubes, modied
with a specic ricin B aptamer tagged with uorescein amidine
(FAM) dye (Figure 1). Aptamer-based detection methods have
gained great recent interest because of their high selectivity and
anity toward their targets.26,27 The aptamer selected here for
specic recognition of ricin B chain displays a stable folding
conformation (detailed in SI Figure S1) across a wide pH (2
7) and temperature (463 C) range,8 making it attractive for
defense applications in harsh environments. The Of f signal
(Figure 1A(a)) is attributed to the eective uorescence
quenching capability of graphene surfaces upon adsorption of
the dye-labeled aptamer through stacking interactions
between the nucleotide bases and the carbon material.28,29 The
high sensitivity and speed of the new uorescence On ricin
detection method reect the greatly accelerated ricinaptamer
binding associated with the motor-induced enhanced uid
transport. Highly specic ricin detection is demonstrated in the
presence of excess coexisting proteins and using untreated
complex samples. The present micromotor strategy thus oers

icin is considered one of the deadliest poisons worldwide

due to its extremely low lethal dose.1,2 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
receptors, respectively, leading to rapid cell death.3 The
widespread availability of the castor plant Ricinus communis
(from which ricin toxin is derived),4 ease of extraction, and
acute toxicity, make ricin a potent biological warfare agent
(BWA).5 Since there is no eective treatment for ricin
exposure, there are urgent needs to develop rapid and reliable
methods for its detection in dierent elds, such as medical
diagnostics, food or water safety testing, and biodefense.2
Current methodologies for ricin detection include biosensors,1,68 chromatographic methods,9 polymerase chain reaction (PCR) assays,10 spectroscopic methods,11 and enzymelinked immunoassays.12 However, the practical application of
these methodologies is limited by their low sensitivity and
selectivity, slow response, false positive readings, nonportability,
operational complexity, and diculties in real-time monitoring.
Developing rapid and reliable ricin detection methods is thus
crucial to facilitate timely warning and reduce the mortality
rate.13
The present work demonstrates a powerful uorescent Of f
On system for the detection of ricin based on self-propelled
aptamer-modied graphene micromotors. Chemically powered
micromotors oer tremendous sensing opportunities in diverse
elds, owing to their attractive on-the-move binding,
isolation, cargo-towing, and self-mixing capabilities.1417 The
2016 American Chemical Society

Received: December 21, 2015

Accepted: January 6, 2016
Published: January 6, 2016
217

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characteristic of the conjugated FAM dye (em 520 nm; ex

495 nm). The quenching of the uorescence intensity is largest
upon incubating the dye-aptamer with the rGO-based micromotors for 15 min (SI Figure S2). The rGO-Pt micromotors
were thus successfully modied by incubating in a 5 M FAMricin B aptamer solution for 15 min (Figure 1B (4)). Scanning
electron microscopy (SEM) images were captured 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 modied rGO/Pt microrocket. The micromotors are 10
m long and 5 m in diameter, reecting the pore size of the
PC membrane. An energy dispersive X-ray spectroscopy
analysis (EDX), shown in the right part of Figure 1C,
demonstrates the successful composition and modication 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. Modication of the microrocket with the FAMricin B aptamer is indicated from the presence of phosphorus
corresponding to the phosphate groups of the dye aptamer.
To demonstrate that the observed Of fOn uorescence
response is produced solely by the specic interaction with the
target ricin and not by nonspecic bindings, we evaluated the
specicity of the new micromotor detection system in the
presence of excess nontarget proteins. For this purpose, bovine
serum albumin (BSA) and saporin toxin were selected as a
generic soluble protein and alternative RIP toxin,31,32
respectively. Figure 2A displays the schematic illustration of
FAM-aptamer-modied-rGO/Pt micromotors in a sample
mixture containing ricin, BSA and saporin (Figure 2A(a)),
and the specic interaction of ricin with the surface-bound
aptamer, accelerated by the micromotor propulsion (Figure
2A(b)). The aptamer is initially conned on the motor surface,

Figure 1. In vitro Of fOn specic uorescent detection of ricin-B

toxin by FAM-Ricin B aptamer-modied rGO/Pt microrockets, and
synthesis and characterization of modied-rGO/Pt microrockets. (A)
Left side: schematic illustration of the rGO/Pt micromotors modied
with the dye-tagged aptamer that quenches the dye uorescence; inset
picture showing the reduced intensity of the quenched uorescence
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 uorescence recovery due to release of the
dye-aptamer from the motor GO-quenching surface upon binding to
the toxin; inset picture showing the uorescence intensity corresponding to the detection of 10 g/mL ricin-B toxin (c). (A(b))
Micromotor motion in the presence of ricin B toxin in 1% H2O2
solution; scale bar, 10 m. (B) Template preparation of the aptamermodied microrockets: electrodeposition of rGO and Pt (1 and 2,
respectively), release from the template (3) and modication 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.

considerable promise as a rapid, simple, and eective method

for detecting ricin in diverse biodefense scenarios.
As illustrated in Figure 1, the micromotor-based ricin
uorescence On sensing approach relies on the fast recovery
of the quenched uorescence of the dye-tagged aptamer probe
due its preferential binding with the target toxin compared to
the graphene-oxide surface. When these aptamer-modied
peroxide-propelled micromotors (Figure 1A(b)) are exposed
and bind to the ricin B toxin, the quenched uorescence is
recovered due to the displacement of the dye-aptamer probe
from the graphene-oxide quenching motor surface. This results
in an intense uorescent signal (Figure 1A(c)) that indicates
the presence of the target ricin and leads to a rapid, selective,
and sensitive detection. The uorescent signal increases linearly
with the ricin B concentration over the 100 pg/mL to 10 g/
mL range. The motor-based signal recovery is signicantly
faster when compared to static microrockets.
Reduced GO/Pt microrockets were synthesized by a
standard membrane-template electrodeposition protocol (see
Methods Section of Supporting Information). Such template
deposition oers highly reproducible preparation of thousands
of micromotors of similar size and shape using a single
membrane. Briey, the template protocol consisted of the
electrodeposition of rGO on the inner wall of a polycarbonate
(PC) membrane by cyclic voltammetry, followed by deposition
of the inner Pt layer (Figure 1B; steps 1 and 2, respectively),
dissolution of the membrane and release of the rGO/Pt
microrockets (Figure 1B (3)).30 The FAM-Ricin B aptamer
used in this work exhibits a strong uorescence signal,

Figure 2. Specicity of FAM-Ricin B aptamer-rGO/Pt micromotors

toward the ricin B target. (A) Schematic illustration of dye-aptamermodied-rGO/Pt micromotors in the presence of coexisting ricin,
saporin, and BSA (a), and specic ricin B-aptamer interaction
accelerated by the motor propulsion (b). Fluorescence images in the
presence of 10 g/mL ricin B toxin (B), and in the presence of BSA
(C) and saporin (D), both at the 100 g/mL level. Images for the
detection of 10 g/mL ricin B toxin in the presence of 100 g/mL (E)
BSA and (F) saporin. (G) Corresponding uorescence intensities
expressed as percentage recovery of the prequenching signal.
Experimental conditions: 3 min treatment; 1% H2O2; 1.5% NaCh;
PBS pH = 7.4.
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resulting in a loss of the dye uorescence, which is recovered
upon the specic binding with the target ricin.
As shown in Figure 2, an excess of both nontarget proteins
(100 g/mL vs 10 g/mL ricin) displays a negligible
uorescence recovery (Figure 2C,D,G), compared to the
strong recovery observed in the presence of the target ricin B
(Figure 2B and G(a)). Similarly, the uorescent signal is
recovered fully upon adding the ricin toxin to solutions
containing an excess of BSA and saporin (Figure 2E and F,
respectively, and bars in G), demonstrating the high specicity
of the surface-bound aptamer toward 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 uorescence
recovery (due to the toxin binding) and the speed of the
motors (in the presence of dierent fuel levels) was tested
(Figure 3). No uorescence quenching eect was observed at
the dierent H2O2 fuel concentrations used (SI Figure S3).

recovery relates directly to the micromotor speed at each fuel

level (0, 155, 380, and 860 m/s, respectively), reecting the
increased likelihood of ricin contacts with the surface-conned
aptamer due to faster movement of the receptor and enhanced
uid transport. As can be observed in Figure 3B, the fast
movement of the aptamer-modied micromotors in the 2%
peroxide solution resulted in nearly 100% recovery of the
quenched-uorescence, reecting the highest aptamerricin
contact rate. Overall, the data of Figure 3B clearly indicate that
the speed of the aptamer-modied micromotors plays a crucial
role in the accelerated detection of the toxin.
To investigate the sensitivity and detection limit of the dyeaptamer-modied micromotors for ricin detection and
demonstrate the quantitative nature of the uorescent On
response, the modied micromotors were exposed to dierent
ricin B concentrations. As illustrated from the images of Figure
4A, the recovery of the uorescent signal (i.e., fraction of the

Figure 4. (A) Correlation between the ricin B concentration and the

uorescence intensity: images corresponding to each ricin B
concentration level over the 100 pg/mL to 10 g/mL range (a, f,
respectively); the corresponding calibration plot (g). (B) Fluorescence
recovery (top) and micromotor movement (bottom) in dierent ricin
B toxin-spiked samples (from a to e: PBS buer, 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-modied micromotors at 1% H2O2; (B: a-e, bottom part)
microscope images showing the movement of the micromotor in each
sample. (B, f) Percentages of uorescence recovery and micromotor
speeds in the dierent samples. Experimental conditions: as in Figure 2
using 10 g/mL ricin B toxin. Scale bar, 10 m.

Figure 3. Ricin-triggered uorescence recovery: inuence of the

reaction time and motor speed upon the uorescence recovery due to
binding of the toxin to the surface-conned aptamer. (A) Dependence
of the ricin-triggered uorescence recovery upon the incubation time
under static conditions (a) and with H2O2-propelled micromotors (b).
(B) Dependence of the uorescence recovery upon the concentration
of the peroxide fuel. (CF) Fluorescence images (top part) of the
ricin B toxin solution after 3 min incubation with the dye-aptamermodied micromotors using dierent 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
g/mL; 1% H2O2 (A); 1.5% NaCh; 3 min treatment (BF). Error
bars estimated as a triple of the standard deviation (n = 3).

displaced aptamerdye conjugate) increases gradually upon

increasing the ricin B concentration from 100 pg/mL to 10 g/
mL (af). 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
modied micromotors at low (ng/mL) ricin concentrations and
that the uorescent intensity can provide a quantitative
estimation of the target concentration.
The Centers for Disease Control and Prevention have
classied ricin as a category B agent, which means that it could
potentially aect our food or water supply.7 In order to evaluate
the practical applicability of the new micromotor ricin detection
method, dierent complex matrices were spiked with a known
amount of ricin (10 g/mL). Figure 4B displays the
dependence of the micromotors speed on the uorescence
recovery using six dierent untreated real samples (ae: PBS
buer, tap water, saliva, serum, and urine, respectively) along
with 1% H2O2 fuel. While the aptamer-modied micromotors
display ecient movement in these dierent biological media,
their speed varies based on the nature of the medium. We
observed a more ecient uorescence recovery in PBS buer
(up to 90%) compared to these real samples after 3 min

The eect of the ricin-micromotor incubation time on the

uorescence ricin response was studied under motor movement
and static conditions (Figure 3A: b and a, respectively).
Optimal detection of 10 g/mL ricin B was obtained using a 3
min incubation. Such a period resulted in recovery of 90% of
the uorescence signal in the presence of the self-propelled
modied motors compared to 12% recovery under static
conditions (Figure 3A). This dramatic enhancement reects the
uid convection induced by the rapid micromotor motion and
the corresponding bubble trail.
Figure 3B shows the values of the uorescence recovery
(corresponding uorescence images in Figure 3CF, top part)
obtained after 3 min incubation of the dye-aptamer-micromotors in ricin solutions containing dierent H2O2 levels (0%,
0.5%, 1%, and 2%, respectively). As indicated from the optical
images of Figure 3CF (bottom part), the rate of uorescence
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incubation with the micromotors, reecting the observed speed

change. For example, due to the higher viscosity of saliva, the
micromotor speed is slow, which leads to slow uorescence
recovery. Nevertheless, uorescence recoveries higher than
65% have been observed in all samples within 3 min,
illustrating the applicability of the micromotor method for the
rapid and specic detection of ricin in real samples without
preparatory and washing steps.
In summary, we developed an eective micromotor-based
protocol for detecting the ricin B toxin based on specic dyelabeled aptamer-modied microengines. The new ricin
detection method oers rapid on-the-y binding of the
toxin along with a visual Of fOn uorescent 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

S Supporting Information
*

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acssensors.5b00300.
Supporting videos description, experimental section,
supporting gures and table (PDF)
Speed of FAM-Ricin B aptamer-modied rGO/Pt
micromotors at dierent fuel concentrations (AVI)
Motion of FAM-Ricin B aptamer-modied rGO/Pt
micromotors in dierent Ricin B toxin-spiked samples
(AVI)

AUTHOR INFORMATION

Corresponding Author

*E-mail: josephwang@ucsd.edu.
Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the nal version of
the manuscript.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
This work was supported by the Defense Threat Reduction
Agency (grants nos. HDTRA1-13-1-0002 and HDTRA1-14-10064). D. F. Baez and A. Jodra acknowledge fellowships from
CONICYT-CHILE and the Spanish Ministry of Economy and
Competitiveness, respectively. The authors thank A da Mart n
for her advice.

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