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Lighting up micromotors with quantum dots for smart chemical sensing† B. Jurado-Sa´nchez,a A. Escarpa*b and J. Wang*a
Received 8th June 2015, Accepted 29th July 2015 DOI: 10.1039/c5cc04726a www.rsc.org/chemcomm
A new ‘‘on-the-fly’’ chemical optical detection strategy based on the incorporation of fluorescence CdTe quantum dots (QDs) on the surface of self-propelled tubular micromotors is presented. The motionaccelerated binding of trace Hg to the QDs selectively quenches the fluorescence emission and leads to an effective discrimination between different mercury species and other co-existing ions.
Self-propelled nanomachines offer considerable promise for a myriad of applications.1–8 Catalytic micromotors have been shown to be extremely useful for real-time analytical measurements, enhancing binding events through movement of the recognition element and enhanced fluid transport while obviating the need for sample collection and tedious laboratory processes.9 Changes in the speed of Au–Pt nanowires or tubular micromotors have formed the basis for the motion-based detection of toxic ions and DNA.10,11 Bioreceptor-functionalized or molecularly imprinted-based micromotors have also been used for the ‘‘on-the-fly’’ sensing of biomolecules.12,13 Here we describe powerful mobile optical microsensor platforms coupling the attractive optical properties of quantum dots (QDs) with the autonomous movement of highly efficient micromotors. Such unique coupling leads to real-time optical visualization of the analyte recognition event through rapid binding-induced changes (quenching) in fluorescence intensity. Semiconductor nanocrystals have stimulated considerable research interest over the past decade due to their high luminescence efficiency, tunable size dependent emission and excellent photostability.14,15 QDs have been widely used as biological labels,16 in electronics,17 or as sensing probes.18–20 In particular, QD basednanosensors have enabled the on-site detection of toxic heavy metal ions, in connection to field remediation strategies. Chattopadhyay et al. developed a ZnS Q-dot impregnated chitosan-based
a
Department of NanoEngineering, University of California, San Diego, La Jolla, CA 92093, USA. E-mail: [email protected] b Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcala, Alcala de Henares E-28871, Madrid, Spain. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc04726a
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paper sensor for the detection of heavy metals.21a ZnSe/ZnS colloidal nanoparticles have been employed as Hg2+ sensors based on dynamic quenching.21b A review of optical QD-based sensors for the detection of heavy metals has been written by Zhu et al.21c However, such ‘nanosensing’ probes are prone to oxidation and can exhibit a non-specific response within certain samples where diffusion is complex. A very promising approach to address these limitations involves the integration of QDs into host structures with potential self-propulsion abilities. For example, Bhattacharya et al. described rolled-up InGaAs/GaAs bilayer microtubes for optical applications.17 Similarly, Steinbock et al. incorporated CdSe–ZnS nanocrystals into silica-supported zinc oxide/hydroxide macrotubes.21d Yet, the incorporation of QDs into self-propelled mobile sensing platforms has not been reported. The nanocrystal-based micromotor fluorescent sensor, described in the following sections, was fabricated by the integration of CdTe QDs on the outer surface of poly(3,4-ethylenedioxythiophene) (PEDOT)/Pt micromotors through a positively-charged poly(diallyldimethylammonium chloride) (PDDA) layer that facilitates the electrostatic interaction of the negatively-charged COOH–CdTe QDs. The new sensing concept will be illustrated towards the selective ‘on-the-fly’ Hg2+ detection (Fig. 1A) in the presence of a large excess of other co-existing ions as well as other Hg species, indicating great promise for speciation studies. QDs have been shown to be very attractive tools for the detection of heavy metals.21 The micromotor fabrication process is highly versatile, allowing for facile integration of various QDs with different emitting wavelengths for future multiplexed operations. Fig. 1A illustrates the schematic of a CdTe-based micromotor sensor platform and of the corresponding ‘on-the-fly’ detection of mercury ions based on fluorescence quenching. The new microsensors were prepared using PEDOT poly(sodium 4-styrene suphonate) (PSS)/Pt bilayer tubular microrockets as templates for the electrostatic self-assembly of highly luminescence CdTe QDs in connection to a layer-by-layer (LBL) modification protocol involving sequential incubation in PDDA and PSS (Fig. 2A). The high efficiency of the PSS/PDDA LBL modification provided the micromotor surface with a high, uniform density of positively
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charged sites for the subsequent incorporation of negativelycharged CdTe QDs via electrostatic interactions, leading thus to a strong, well-distributed fluorescence on the micromotor surface (Fig. 1B(a), C(a) and ESI,† Video 1). In order to test the performance of the modification protocol, a control experiment (vs. PEDOT/PSS/ PDDA microrockets) was performed by incubating the unmodified (PDDA-free) PEDOT/PSS/Pt microrockets with CdTe QDs in a similar fashion. The absence of fluorescence of the resulting micromotors (see ESI,† Video 1) indicates that the CdTe QDs do not interact with the unmodified outer polymeric surface either by direct absorption or by covalent bonding, compared to their electrostatic attraction to the PDDA modifier. As indicated in Fig. 2A, PEDOT/PSS/Pt micromotors offer the ability to generate the necessary electrostatic interactions with the oppositely-charged PDDA electrolyte, thus capping the outer surface with positive charge, which facilitates the electrostatic interaction and binding of the negatively-charged COOH–CdTe. A gradual increase in the fluorescence intensity was observed upon increasing the number of layers from 2 to 10 (for details of the preparation, see Fig. 2A and the experimental section),
reflecting the increased density of active charged sites for CdTe binding (see Fig. S1(a), ESI†). SEM images of the typical micromotor sensor are displayed in Fig. 2. As seen in the cross-view (Fig. 2B), the micromotor had a defined, biconical tubular geometry with inner openings of 2 mm, and a total length of 18 mm. The microsensor also exhibits a highly rough outside polymeric surface (Fig. 2C), with numerous individual ‘‘nanodots’’, reflecting the high surface density of CdTe QDs. The EDX images in Fig. 2D also reflect such uniform surface distribution of Cd and Te ions. We also evaluated the possible quenching effect of increasing H2O2 fuel concentration, and found it to be minimal up to the 7% level (see Fig. S1(b), ESI†). Since the CdTe/PEDOT microsensors are propelled efficiently over the 1–5% peroxide levels (with high speeds ranging from 42 10 to 201 52 mm s1), such fuel concentrations were used in the mercury sensing work. As expected, while this speed is B6 times lower compared with that of the unmodified PEDOT/PSS microrockets, it does not compromise practical sensing applications (see Fig. S2 and ESI,† Video 3). The resulting micromotor-based sensor offers direct mercury detection, providing the real-time optical visualization of the Hg–CdTe quenching event based on changes in the fluorescence intensity. The response time for a micromotor moving in a solution containing 3 mg L1 of target mercury is illustrated in Fig. 3. The time lapse microscopy images and the corresponding fluorescence decay plot vs. time profile (Fig. 3B) indicated that the quenching rate was low initially (0–5 s, Fig. 3A(a and b)) and then increased rapidly with time until complete fluorescence quenching of the QDs covering the micromotor surface was achieved after 12 s navigation (B10, 6–12 s, Fig. 3A(c–e)). The response time of the microsensors was 12 s for 3 mg L1 Hg2+ solutions, indicating that short navigation times offer direct rapid detection of the target analyte. To ensure adequate operation time for further experiments, the time between fuel addition and video acquisition was set to 30 s. Fig. 4A(a–d) display images of different QD mobile microsensors propelling in PBS buffer solutions containing increasing concentrations of Hg2+ ions (0–3 mg L1), along with 5% of the peroxide fuel. The corresponding plot of fluorescence intensity (calculated as corrected fluorescence) vs. Hg2+ concentration
Fig. 2 Preparation and characterization of QD-based microsensors. (A) Schematic of the layer-by-layer electrostatic self-assembly of COOH– CdTe QDs on PEDOT microrockets: micromotors are sequentially incubated with PDDA (a) and PSS (b) electrolyte solutions until 10 layers are formed (PDDA being the last layer) followed by the adsorption of the negatively charged QDs onto the outer positively charged surface of the microrockets (c). (B and C) Scanning electron microscopy (SEM) images showing the conical morphology and rough QD surface monolayer of the resulting motors. (D) Energydispersive X-ray (EDX) spectroscopy images illustrating the distribution of Pt, Cd and Te in the QD micromotors. Scale bars, 500 nm.
Fig. 3 Effect of time on the fluorescence quenching of CdTe/PEDOT micromotors in the presence of Hg2+. (A) Time-lapse images taken after the micromotor had navigated in a 3 mg L1 Hg solution for 0 (a), 2 (b), 10, 6 (c), 10, 8 (d) and 12 s (e). (B) Plot showing the fluorescence decay of CdTe/PEDOT microrockets as a function of time. The fluorescence intensity of the microrockets was estimated using ImageJ program. Conditions: 2% peroxide, 1% sodium cholate in PBS buffer. Scale bar, 5 mm.
Fig. 1 (A) Schematic of the QD-based microrockets and their ‘‘on-the-fly’’ selective detection of mercury ions based on fluorescence quenching. (B) Realtime optical visualization of the mercury recognition event. Fluorescence microscopy images of CdTe/PEDOT microrockets before (a) and after addition of 3 mg L1 of Hg2+ (b) (C) Time-lapse images (taken from ESI,† Videos 1 and 2), showing the fluorescence intensity of a CdTe/PEDOT microsensor before (a) and after 30 s navigation in solutions containing 3 mg L1 of Hg2+ (b), 5 mg L1 of Pb2+ and Cu2+ (c) or 5 mg L1 or methylmercury (d). Conditions: 5% peroxide and 1% sodium cholate in PBS buffer. Scale bars, 2 mm.
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Fig. 4 Dependence of the fluorescence quenching of CdTe/PEDOT micromotors on mercury concentrations. (A) Time-lapse images (see ESI,† Video 4) taken after the microrockets had navigated for 30 s in a solution containing 5% peroxide and 0 (a), 0.5 (b), 0.8 (c) and 3 mg L1 (d) of mercury. The upper part shows the magnified images corresponding to each microrocket. (B) Plot showing the fluorescence quenching (as CCRF) of CdTe/PEDOT microrockets with increasing Hg2+ concentrations in 5% peroxide solutions. Inset: the linear relationship between the quenched fluorescence intensity and Hg2+ concentration. (C) Stern–Volmer and (D) Lineweaver–Burk curves for analyzing the fluorescence quenching data. Conditions: 1% sodium cholate, medium, PBS buffer. CCRF (calculated corrected total microsensor fluorescence) was estimated using ImageJ program (see ESI†). Scale bars, 5 mm. Error bars represent the standard deviation of 5 measurements (n = 5).
(Fig. 4B) displays linear dependence up to 1 mg L1 of mercury (see also the inset) followed by a slight curvature, with nearly 100% fluorescence quenching at 2.5 mg L1 levels. Interestingly, the CdTe/PEDOT probes for Hg2+ become a powerful quantitative sample screening-tool for fast discrimination between positive (containing mercury species) and negative (no containing mercury species) samples, according to a cut-off concentration which defines the presence or absence of a target analyte in a sample. In this work, the cut-off concentration was set as 3 times the LOD (estimated as 3Sa from the calibration plots shown in Fig. 4B, inset), which correspond to 0.3 mg L1 Hg2+, allowing an extremely fast discrimination of contaminated samples above this concentration. Also, as described in Fig. S3 (ESI†), pH and ionic strength of the solutions have negligible effects on the sensitivity of Hg(II). In order to gain further insights into the mechanism describing such fluorescence quenching behavior, the previous data were analyzed by both the Stern–Volmer (eqn (1)) and the Lineweaver– Burk equations (eqn (2)), which are usually employed to describe the dynamic and static quenching behaviour, respectively.19 These two models are represented by the following equations: F0 ¼ 1 þ KSV Hg2þ F
(1)
(F0 F)1 = (F0)1 + KLB1(F0)1[Hg2+]
(2)
where F0 and F are the calculated fluorescence of the CdTe/ PEDOT microsensor before and after 30 s navigation in different Hg2+ solutions, respectively, KSV is the Stern–Volmer quenching constant and KLB denotes the binding constant of Hg2+ and the microsensor. The plot of F0/F vs. [Hg2+] (Fig. 4C) displays good linearity (r = 0.990), whereas the plot of (F0 F)1 vs. [Hg2+] (Fig. 4D)
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shows a positive deviation. Thus, the quenching mechanism obeys the Stern–Volmer equation. As expected, due to the dynamic movement of the micromotors, the quenching was not initiated by static quenching (characterized by the formation of complex or aggregate structures) but mainly by dynamic collisions. Thus, the observed quenching mechanism can be attributed to three important factors: first, based on the strong affinity of Hg2+ with carboxylic acid groups, Hg2+ absorbs onto the surface of the COOH-capped QDs, which facilitates the electron transfer from QDs to Hg2+. Second, the solubility of HgTe is approximately 20 times lower than that of CdTe, which drives the substitution of Cd2+ in CdTe QDs by Hg2+ and leads to the formation of alloyed CdxHg1xTe. To provide further evidence for the formation of such an alloy, we calculated the bandgap of CdTe/PEDOT micromotors before and after the Hg(II) addition (see ESI† for more details) to be 2.24 and 1.45, respectively. This shift to a lower bandgap is due to the quantum confinement effect as the CdTe QDs grow to a larger size due to Hg addition, and this is good evidence for the formation of CdxHg1xTe alloy on the surface of the microsensor. These ultrasmall particles quench the recombination luminescence of CdTe nanoparticles by facilitating nonradiative recombination of excited electrons (e) in the conduction band and holes (h+) in the valence band. Third, the lower bandgap of HgTe compared to CdTe is also responsible for this quenching behavior.19,22 The selectivity of CdTe/PEDOT probes for Hg2+ was evaluated in the presence of a large excess of other relevant metal ions and species. The time-lapse microscopic images and the plot of Fig. S5 (ESI†) (and the corresponding ESI,† Video 5) demonstrate that a large excess (3 mg L1) of Cu2+ and Pb2+ or 5 mg L1 of CH3Hg+ has a negligible effect on the fluorescence intensity of the mobile CdTe/PEDOT microsensors. Slight fluorescence quenching by Cu2+ and Pb2+ ions was observed, mainly due to binding onto the QD core, leading to a red-shift of the maximum emission wavelength.23,24 Methylmercury can quench the fluorescence of QDs through dynamic quenching, as a result of nonradiative electron/ hole recombination. The above mechanism requires an electrostatic interaction between CH3Hg+ and the functional groups surrounding the core of CdTe QDs. The inherent hydrophobic nature of this compound, compared to the hydrophilic nature of the micromotor sensors (imparted by their outer layers), prevents such interaction from occurring, thus no fluorescence changes are observed.25 Finally, the practical utility of the new mobile QD microsensors was illustrated by its ability to selectively detect the target Hg2+ ions in complex unprocessed biological media, such as saliva samples. The microscopy images in Fig. 5A clearly illustrate the effective binding of the trace Hg present in the saliva sample, which quenched the fluorescence of the microsensor (a) after 30 s navigation in the sample solution (b). An additional experiment was also performed by using a saliva sample spiked with 5 mg L1 of CH3Hg+ (Fig. 5B). No noticeable changes in the fluorescence intensity of the micromotor sensor were observed in the sample containing CH3Hg+ after 30 s navigation (Fig. 5B(b)). These data also indicate that the matrix components did not interfere with the ‘on-the-move’ QD-based assay. Overall, the data in Fig. 5 indicate considerable promise for future, fast screening of hazardous metals in complex matrices.
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Notes and references
Fig. 5 Mercury speciation and effective propulsion of the QD microsensor in a saliva sample. (A) Time-lapse images after the CdTe-based micromotor had navigated 30 s in samples spiked with 0 (a) and 3 mg L1 (b) of Hg2+ (ESI,† Video 6). (B) Time-lapse images of the microsensor in a saliva sample spiked with 5 mg L1 of CH3Hg+ after 0 (a) and 30 s (b). Untreated samples were diluted 1 : 5 in PBS buffer. Conditions: 5% peroxide, 1% sodium cholate, medium. Scale bar, 2 mm.
In conclusion, we have demonstrated here that the coupling of the optical properties of QDs and the autonomous movement of artificial nanomachines offers considerable promise as the next generation advanced mobile microsensors that offer realtime optical visualization of the analyte recognition events. Multiple CdTe QDs or ‘‘recognition sites’’ are introduced on the surface of tubular micromotors by electrostatic self-assembly, leading for the first time to mobile QD microsensors. The microsensor holds considerable promise for a simple ’real-time’ discrimination of the two most relevant mercury species, Hg2+ and CH3Hg+. The capabilities of QD-based nanomachines for the screening of complex saliva samples, with high selectivity, have been clearly illustrated. While the QD-based micromachine concept has been demonstrated here using Hg2+ as a pollutant, it could be readily extended to the selective detection of a variety of toxic organic and inorganic threats (i.e. perfluorinated compounds). Future efforts should be aimed at increasing the sensitivity of such autonomous microsensors for detecting ultratrace mercury concentrations, e.g., through increase in the initial fluorescence intensity or the QD loading. This could be accomplished by increasing the microsensor size through replacement of the 2 mm-PC membranes employed in the template preparation of the microtubes by 5 or even 10 mm-pore size membranes. In this way, an increased area is available for increased CdTe QD loading, thus increasing the initial fluorescence intensity i.e. sensitivity. The mobile QD detection platforms represent promising alternatives to conventional fluorescent dyes and may offer diverse possibilities for new future developments in lab-on-a-chip platforms, optical and electronic applications. B. J-S acknowledges support from the People Programme (Marie Curie Actions) of the EU 7th Framework Programme (PIOF-GA-2012-326476). This project received support from the Defense Threat Reduction Agency-Joint Science and Technology Office for Chemical and Biological Defense (HDTRA1-13-10002). AE acknowledges financial support from the Spanish Ministry of Economy and Competitiveness (CTQ2014-58643-R) and the NANOAVANSENS program (S2013/MIT-3029).
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Lighting up micromotors with quantum dots for smart chemical sensing† B. Jurado-Sa´nchez,a A. Escarpa*b and J. Wang*a
Received 8th June 2015, Accepted 29th July 2015 DOI: 10.1039/c5cc04726a www.rsc.org/chemcomm
A new ‘‘on-the-fly’’ chemical optical detection strategy based on the incorporation of fluorescence CdTe quantum dots (QDs) on the surface of self-propelled tubular micromotors is presented. The motionaccelerated binding of trace Hg to the QDs selectively quenches the fluorescence emission and leads to an effective discrimination between different mercury species and other co-existing ions.
Self-propelled nanomachines offer considerable promise for a myriad of applications.1–8 Catalytic micromotors have been shown to be extremely useful for real-time analytical measurements, enhancing binding events through movement of the recognition element and enhanced fluid transport while obviating the need for sample collection and tedious laboratory processes.9 Changes in the speed of Au–Pt nanowires or tubular micromotors have formed the basis for the motion-based detection of toxic ions and DNA.10,11 Bioreceptor-functionalized or molecularly imprinted-based micromotors have also been used for the ‘‘on-the-fly’’ sensing of biomolecules.12,13 Here we describe powerful mobile optical microsensor platforms coupling the attractive optical properties of quantum dots (QDs) with the autonomous movement of highly efficient micromotors. Such unique coupling leads to real-time optical visualization of the analyte recognition event through rapid binding-induced changes (quenching) in fluorescence intensity. Semiconductor nanocrystals have stimulated considerable research interest over the past decade due to their high luminescence efficiency, tunable size dependent emission and excellent photostability.14,15 QDs have been widely used as biological labels,16 in electronics,17 or as sensing probes.18–20 In particular, QD basednanosensors have enabled the on-site detection of toxic heavy metal ions, in connection to field remediation strategies. Chattopadhyay et al. developed a ZnS Q-dot impregnated chitosan-based
a
Department of NanoEngineering, University of California, San Diego, La Jolla, CA 92093, USA. E-mail: [email protected] b Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcala, Alcala de Henares E-28871, Madrid, Spain. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc04726a
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paper sensor for the detection of heavy metals.21a ZnSe/ZnS colloidal nanoparticles have been employed as Hg2+ sensors based on dynamic quenching.21b A review of optical QD-based sensors for the detection of heavy metals has been written by Zhu et al.21c However, such ‘nanosensing’ probes are prone to oxidation and can exhibit a non-specific response within certain samples where diffusion is complex. A very promising approach to address these limitations involves the integration of QDs into host structures with potential self-propulsion abilities. For example, Bhattacharya et al. described rolled-up InGaAs/GaAs bilayer microtubes for optical applications.17 Similarly, Steinbock et al. incorporated CdSe–ZnS nanocrystals into silica-supported zinc oxide/hydroxide macrotubes.21d Yet, the incorporation of QDs into self-propelled mobile sensing platforms has not been reported. The nanocrystal-based micromotor fluorescent sensor, described in the following sections, was fabricated by the integration of CdTe QDs on the outer surface of poly(3,4-ethylenedioxythiophene) (PEDOT)/Pt micromotors through a positively-charged poly(diallyldimethylammonium chloride) (PDDA) layer that facilitates the electrostatic interaction of the negatively-charged COOH–CdTe QDs. The new sensing concept will be illustrated towards the selective ‘on-the-fly’ Hg2+ detection (Fig. 1A) in the presence of a large excess of other co-existing ions as well as other Hg species, indicating great promise for speciation studies. QDs have been shown to be very attractive tools for the detection of heavy metals.21 The micromotor fabrication process is highly versatile, allowing for facile integration of various QDs with different emitting wavelengths for future multiplexed operations. Fig. 1A illustrates the schematic of a CdTe-based micromotor sensor platform and of the corresponding ‘on-the-fly’ detection of mercury ions based on fluorescence quenching. The new microsensors were prepared using PEDOT poly(sodium 4-styrene suphonate) (PSS)/Pt bilayer tubular microrockets as templates for the electrostatic self-assembly of highly luminescence CdTe QDs in connection to a layer-by-layer (LBL) modification protocol involving sequential incubation in PDDA and PSS (Fig. 2A). The high efficiency of the PSS/PDDA LBL modification provided the micromotor surface with a high, uniform density of positively
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charged sites for the subsequent incorporation of negativelycharged CdTe QDs via electrostatic interactions, leading thus to a strong, well-distributed fluorescence on the micromotor surface (Fig. 1B(a), C(a) and ESI,† Video 1). In order to test the performance of the modification protocol, a control experiment (vs. PEDOT/PSS/ PDDA microrockets) was performed by incubating the unmodified (PDDA-free) PEDOT/PSS/Pt microrockets with CdTe QDs in a similar fashion. The absence of fluorescence of the resulting micromotors (see ESI,† Video 1) indicates that the CdTe QDs do not interact with the unmodified outer polymeric surface either by direct absorption or by covalent bonding, compared to their electrostatic attraction to the PDDA modifier. As indicated in Fig. 2A, PEDOT/PSS/Pt micromotors offer the ability to generate the necessary electrostatic interactions with the oppositely-charged PDDA electrolyte, thus capping the outer surface with positive charge, which facilitates the electrostatic interaction and binding of the negatively-charged COOH–CdTe. A gradual increase in the fluorescence intensity was observed upon increasing the number of layers from 2 to 10 (for details of the preparation, see Fig. 2A and the experimental section),
reflecting the increased density of active charged sites for CdTe binding (see Fig. S1(a), ESI†). SEM images of the typical micromotor sensor are displayed in Fig. 2. As seen in the cross-view (Fig. 2B), the micromotor had a defined, biconical tubular geometry with inner openings of 2 mm, and a total length of 18 mm. The microsensor also exhibits a highly rough outside polymeric surface (Fig. 2C), with numerous individual ‘‘nanodots’’, reflecting the high surface density of CdTe QDs. The EDX images in Fig. 2D also reflect such uniform surface distribution of Cd and Te ions. We also evaluated the possible quenching effect of increasing H2O2 fuel concentration, and found it to be minimal up to the 7% level (see Fig. S1(b), ESI†). Since the CdTe/PEDOT microsensors are propelled efficiently over the 1–5% peroxide levels (with high speeds ranging from 42 10 to 201 52 mm s1), such fuel concentrations were used in the mercury sensing work. As expected, while this speed is B6 times lower compared with that of the unmodified PEDOT/PSS microrockets, it does not compromise practical sensing applications (see Fig. S2 and ESI,† Video 3). The resulting micromotor-based sensor offers direct mercury detection, providing the real-time optical visualization of the Hg–CdTe quenching event based on changes in the fluorescence intensity. The response time for a micromotor moving in a solution containing 3 mg L1 of target mercury is illustrated in Fig. 3. The time lapse microscopy images and the corresponding fluorescence decay plot vs. time profile (Fig. 3B) indicated that the quenching rate was low initially (0–5 s, Fig. 3A(a and b)) and then increased rapidly with time until complete fluorescence quenching of the QDs covering the micromotor surface was achieved after 12 s navigation (B10, 6–12 s, Fig. 3A(c–e)). The response time of the microsensors was 12 s for 3 mg L1 Hg2+ solutions, indicating that short navigation times offer direct rapid detection of the target analyte. To ensure adequate operation time for further experiments, the time between fuel addition and video acquisition was set to 30 s. Fig. 4A(a–d) display images of different QD mobile microsensors propelling in PBS buffer solutions containing increasing concentrations of Hg2+ ions (0–3 mg L1), along with 5% of the peroxide fuel. The corresponding plot of fluorescence intensity (calculated as corrected fluorescence) vs. Hg2+ concentration
Fig. 2 Preparation and characterization of QD-based microsensors. (A) Schematic of the layer-by-layer electrostatic self-assembly of COOH– CdTe QDs on PEDOT microrockets: micromotors are sequentially incubated with PDDA (a) and PSS (b) electrolyte solutions until 10 layers are formed (PDDA being the last layer) followed by the adsorption of the negatively charged QDs onto the outer positively charged surface of the microrockets (c). (B and C) Scanning electron microscopy (SEM) images showing the conical morphology and rough QD surface monolayer of the resulting motors. (D) Energydispersive X-ray (EDX) spectroscopy images illustrating the distribution of Pt, Cd and Te in the QD micromotors. Scale bars, 500 nm.
Fig. 3 Effect of time on the fluorescence quenching of CdTe/PEDOT micromotors in the presence of Hg2+. (A) Time-lapse images taken after the micromotor had navigated in a 3 mg L1 Hg solution for 0 (a), 2 (b), 10, 6 (c), 10, 8 (d) and 12 s (e). (B) Plot showing the fluorescence decay of CdTe/PEDOT microrockets as a function of time. The fluorescence intensity of the microrockets was estimated using ImageJ program. Conditions: 2% peroxide, 1% sodium cholate in PBS buffer. Scale bar, 5 mm.
Fig. 1 (A) Schematic of the QD-based microrockets and their ‘‘on-the-fly’’ selective detection of mercury ions based on fluorescence quenching. (B) Realtime optical visualization of the mercury recognition event. Fluorescence microscopy images of CdTe/PEDOT microrockets before (a) and after addition of 3 mg L1 of Hg2+ (b) (C) Time-lapse images (taken from ESI,† Videos 1 and 2), showing the fluorescence intensity of a CdTe/PEDOT microsensor before (a) and after 30 s navigation in solutions containing 3 mg L1 of Hg2+ (b), 5 mg L1 of Pb2+ and Cu2+ (c) or 5 mg L1 or methylmercury (d). Conditions: 5% peroxide and 1% sodium cholate in PBS buffer. Scale bars, 2 mm.
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Fig. 4 Dependence of the fluorescence quenching of CdTe/PEDOT micromotors on mercury concentrations. (A) Time-lapse images (see ESI,† Video 4) taken after the microrockets had navigated for 30 s in a solution containing 5% peroxide and 0 (a), 0.5 (b), 0.8 (c) and 3 mg L1 (d) of mercury. The upper part shows the magnified images corresponding to each microrocket. (B) Plot showing the fluorescence quenching (as CCRF) of CdTe/PEDOT microrockets with increasing Hg2+ concentrations in 5% peroxide solutions. Inset: the linear relationship between the quenched fluorescence intensity and Hg2+ concentration. (C) Stern–Volmer and (D) Lineweaver–Burk curves for analyzing the fluorescence quenching data. Conditions: 1% sodium cholate, medium, PBS buffer. CCRF (calculated corrected total microsensor fluorescence) was estimated using ImageJ program (see ESI†). Scale bars, 5 mm. Error bars represent the standard deviation of 5 measurements (n = 5).
(Fig. 4B) displays linear dependence up to 1 mg L1 of mercury (see also the inset) followed by a slight curvature, with nearly 100% fluorescence quenching at 2.5 mg L1 levels. Interestingly, the CdTe/PEDOT probes for Hg2+ become a powerful quantitative sample screening-tool for fast discrimination between positive (containing mercury species) and negative (no containing mercury species) samples, according to a cut-off concentration which defines the presence or absence of a target analyte in a sample. In this work, the cut-off concentration was set as 3 times the LOD (estimated as 3Sa from the calibration plots shown in Fig. 4B, inset), which correspond to 0.3 mg L1 Hg2+, allowing an extremely fast discrimination of contaminated samples above this concentration. Also, as described in Fig. S3 (ESI†), pH and ionic strength of the solutions have negligible effects on the sensitivity of Hg(II). In order to gain further insights into the mechanism describing such fluorescence quenching behavior, the previous data were analyzed by both the Stern–Volmer (eqn (1)) and the Lineweaver– Burk equations (eqn (2)), which are usually employed to describe the dynamic and static quenching behaviour, respectively.19 These two models are represented by the following equations: F0 ¼ 1 þ KSV Hg2þ F
(1)
(F0 F)1 = (F0)1 + KLB1(F0)1[Hg2+]
(2)
where F0 and F are the calculated fluorescence of the CdTe/ PEDOT microsensor before and after 30 s navigation in different Hg2+ solutions, respectively, KSV is the Stern–Volmer quenching constant and KLB denotes the binding constant of Hg2+ and the microsensor. The plot of F0/F vs. [Hg2+] (Fig. 4C) displays good linearity (r = 0.990), whereas the plot of (F0 F)1 vs. [Hg2+] (Fig. 4D)
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shows a positive deviation. Thus, the quenching mechanism obeys the Stern–Volmer equation. As expected, due to the dynamic movement of the micromotors, the quenching was not initiated by static quenching (characterized by the formation of complex or aggregate structures) but mainly by dynamic collisions. Thus, the observed quenching mechanism can be attributed to three important factors: first, based on the strong affinity of Hg2+ with carboxylic acid groups, Hg2+ absorbs onto the surface of the COOH-capped QDs, which facilitates the electron transfer from QDs to Hg2+. Second, the solubility of HgTe is approximately 20 times lower than that of CdTe, which drives the substitution of Cd2+ in CdTe QDs by Hg2+ and leads to the formation of alloyed CdxHg1xTe. To provide further evidence for the formation of such an alloy, we calculated the bandgap of CdTe/PEDOT micromotors before and after the Hg(II) addition (see ESI† for more details) to be 2.24 and 1.45, respectively. This shift to a lower bandgap is due to the quantum confinement effect as the CdTe QDs grow to a larger size due to Hg addition, and this is good evidence for the formation of CdxHg1xTe alloy on the surface of the microsensor. These ultrasmall particles quench the recombination luminescence of CdTe nanoparticles by facilitating nonradiative recombination of excited electrons (e) in the conduction band and holes (h+) in the valence band. Third, the lower bandgap of HgTe compared to CdTe is also responsible for this quenching behavior.19,22 The selectivity of CdTe/PEDOT probes for Hg2+ was evaluated in the presence of a large excess of other relevant metal ions and species. The time-lapse microscopic images and the plot of Fig. S5 (ESI†) (and the corresponding ESI,† Video 5) demonstrate that a large excess (3 mg L1) of Cu2+ and Pb2+ or 5 mg L1 of CH3Hg+ has a negligible effect on the fluorescence intensity of the mobile CdTe/PEDOT microsensors. Slight fluorescence quenching by Cu2+ and Pb2+ ions was observed, mainly due to binding onto the QD core, leading to a red-shift of the maximum emission wavelength.23,24 Methylmercury can quench the fluorescence of QDs through dynamic quenching, as a result of nonradiative electron/ hole recombination. The above mechanism requires an electrostatic interaction between CH3Hg+ and the functional groups surrounding the core of CdTe QDs. The inherent hydrophobic nature of this compound, compared to the hydrophilic nature of the micromotor sensors (imparted by their outer layers), prevents such interaction from occurring, thus no fluorescence changes are observed.25 Finally, the practical utility of the new mobile QD microsensors was illustrated by its ability to selectively detect the target Hg2+ ions in complex unprocessed biological media, such as saliva samples. The microscopy images in Fig. 5A clearly illustrate the effective binding of the trace Hg present in the saliva sample, which quenched the fluorescence of the microsensor (a) after 30 s navigation in the sample solution (b). An additional experiment was also performed by using a saliva sample spiked with 5 mg L1 of CH3Hg+ (Fig. 5B). No noticeable changes in the fluorescence intensity of the micromotor sensor were observed in the sample containing CH3Hg+ after 30 s navigation (Fig. 5B(b)). These data also indicate that the matrix components did not interfere with the ‘on-the-move’ QD-based assay. Overall, the data in Fig. 5 indicate considerable promise for future, fast screening of hazardous metals in complex matrices.
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Notes and references
Fig. 5 Mercury speciation and effective propulsion of the QD microsensor in a saliva sample. (A) Time-lapse images after the CdTe-based micromotor had navigated 30 s in samples spiked with 0 (a) and 3 mg L1 (b) of Hg2+ (ESI,† Video 6). (B) Time-lapse images of the microsensor in a saliva sample spiked with 5 mg L1 of CH3Hg+ after 0 (a) and 30 s (b). Untreated samples were diluted 1 : 5 in PBS buffer. Conditions: 5% peroxide, 1% sodium cholate, medium. Scale bar, 2 mm.
In conclusion, we have demonstrated here that the coupling of the optical properties of QDs and the autonomous movement of artificial nanomachines offers considerable promise as the next generation advanced mobile microsensors that offer realtime optical visualization of the analyte recognition events. Multiple CdTe QDs or ‘‘recognition sites’’ are introduced on the surface of tubular micromotors by electrostatic self-assembly, leading for the first time to mobile QD microsensors. The microsensor holds considerable promise for a simple ’real-time’ discrimination of the two most relevant mercury species, Hg2+ and CH3Hg+. The capabilities of QD-based nanomachines for the screening of complex saliva samples, with high selectivity, have been clearly illustrated. While the QD-based micromachine concept has been demonstrated here using Hg2+ as a pollutant, it could be readily extended to the selective detection of a variety of toxic organic and inorganic threats (i.e. perfluorinated compounds). Future efforts should be aimed at increasing the sensitivity of such autonomous microsensors for detecting ultratrace mercury concentrations, e.g., through increase in the initial fluorescence intensity or the QD loading. This could be accomplished by increasing the microsensor size through replacement of the 2 mm-PC membranes employed in the template preparation of the microtubes by 5 or even 10 mm-pore size membranes. In this way, an increased area is available for increased CdTe QD loading, thus increasing the initial fluorescence intensity i.e. sensitivity. The mobile QD detection platforms represent promising alternatives to conventional fluorescent dyes and may offer diverse possibilities for new future developments in lab-on-a-chip platforms, optical and electronic applications. B. J-S acknowledges support from the People Programme (Marie Curie Actions) of the EU 7th Framework Programme (PIOF-GA-2012-326476). This project received support from the Defense Threat Reduction Agency-Joint Science and Technology Office for Chemical and Biological Defense (HDTRA1-13-10002). AE acknowledges financial support from the Spanish Ministry of Economy and Competitiveness (CTQ2014-58643-R) and the NANOAVANSENS program (S2013/MIT-3029).
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