Combining a Droplet-Based Microfluidic Tubing System with Gated

Letter pubs.acs.org/acssensors

Combining a Droplet-Based Microfluidic Tubing System with Gated Indicator Releasing Nanoparticles for Mercury Trace Detection Jérémy Bell, Estela Climent, Mandy Hecht,† Merwe Buurman, and Knut Rurack* Bundesanstalt für Materialforschung und−prüfung (BAM), Richard-Willstätter-Str. 11, D-12489 Berlin, Germany S Supporting Information *

ABSTRACT: A droplet-based microfluidic sensor was developed for the detection of Hg2+ traces in water. The approach uses gated mesoporous nanoparticles loaded with a fluorescent BODIPY dye. The squaraine-based gating mechanism is highly selective for Hg2+ and the indicator release mechanism ensures sensitive detection. The microfluidic system is modular and was assembled from simple PTFE/PFA tubes, while detection was realized with standard optomechanic, optic, and electronic parts. The sensor shows a stable response without memory effects and allows the detection of Hg2+ in water down to 20 ppt. KEYWORDS: microfluidic sensor, gated delivery system, fluorescence, mercury, hybrid nanoparticles

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as well as facile handling and assembly.9 Such low-cost devices can not only be used for single-liquid applications, but droplet handling with the aid of “off-the-self” components is also possible.10 In our case, droplet-based liquid handling is essential because of the signaling amplification scheme invoked to reach trace detection. As Figure 1 illustrates, a gated indicator release system consists of a porous particle that is loaded with a dye and capped with stoppers. The capping chemistry is chosen in such a way that advent of the target analyte leads to a chemical reaction that removes the stoppers, thus opening the pores and liberating the cargo. Since the amount of released indicator is directly proportional to the concentration of analyte, released indicator and indicator still contained in the closed pores have to be separated for analyte quantification. Up to now, this signaling scheme has been successfully demonstrated for lateral flow assays,11 but the step to exploit the potential of such gated sensory delivery systems for online analysis has not been made yet. To realize the latter, we conceived a liquid−liquid extraction unit as the integral part of the device (Figure 1). Such biphasic approaches are well documented for microfluidic devices and usually allow for efficient extraction because of a large exchange surface and a microreactor-like behavior of the single droplets.12,13 Here, target recognition by the functionalized nanoparticles happens in the water droplets with quantitative extraction of the released dye into the carrier fluid dichloromethane (CH2Cl2). For the chemical signaling concept, we relied on our earlier work published in ref 14 and used mesoporous silica

he demand for powerful miniaturized sensing devices is constantly increasing, basically driven by a society that wants to be ever earlier, better, and more comprehensively informed about many different aspects of life, work, and the environment.1 Whether because of environmental considerations,2 safety concerns,3 process control, 4 or medical diagnostics,5 the most promising format for liquid sample analysis in on-site applications is presumably microfluidic devices. Bringing the analytical method to the sample rather than taking the sample to the laboratory, however, implies that such miniaturized methods have to fulfill certain requirements on sensitivity and selectivity. For notorious contaminants such as heavy metal ions, for instance, this means that the sensory devices shall be able to detect very low concentrations of the analytetypically ≤10 ppb for As, Cd, Pb, or Hg ions6in a reliable and reproducible fashion, at best also quantitatively. Although miniaturized devices in general offer several advantages such as small size, low reagent consumption, and short assay times, the method usually has to reach the goal of trace detection without elaborated pretreatment or concentration steps. Powerful detection schemes are thus required, with gated indicator release systems having recently been established as a potent strategy for simple assay formats.7 In the present contribution, we report on the combination of gated dye-releasing nanoparticles with a droplet-assisted microfluidic tubing system for the ultratrace detection of Hg2+ in aqueous solution. Despite the fact that microfluidic technology holds great promise, a significant part of the nonacademic community is still hesitant to widely exploit it, mainly because of the complexity of the required infrastructure for development and testing of customized microfluidic chips.8 Alternative approaches use modular tubing systems that offer high flexibility © XXXX American Chemical Society

Received: December 22, 2015 Accepted: January 28, 2016

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DOI: 10.1021/acssensors.5b00303 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors

Figure 2. Fluorescence spectra of the organic phase after recognition of increasing Hg2+ concentrations by M1 (cM1 = 0.4 g L−1; H2O/ CH2Cl2, 1/1, v/v; PBS buffer, λexc = 505 nm). Inset zooms into the low-concentration region.

a suspension of the particles (0.4 g L−1) in PBS at pH = 7.3 (50 mM), in contact with an equivalent amount of dichloromethane, induced a release of 1 which did not occur in a control assay lacking the analyte. To find optimum conditions, assays were carried out in various buffered (MES, PBS, TRIS, acetate, phosphate; see Section I, Supporting Information) and nonbuffered solutions (HCl, NaOH) at various pH between 2.5 and 10. Even if a sensor response was measured in each case, the quickest and most stable results were obtained for PBS. Dye release was followed by an efficient phase transfer to the organic phase which can be analyzed by fluorescence without a centrifugation step as the NPs still containing dye and the released dye are in different phases. Figure 2 shows that besides a huge signal generated by 1 ppm of Hg2+, ppb concentrations of the metal ion could also be detected. A possible counterion influence was assessed with chloride and ClO4− in phosphate buffer and was found to be negligible. The kinetics of the chemical system were determined in a cuvette experiment. Upon addition of Hg2+ the response of M1 reaches a plateau of the signal after 10 min. In the microfluidic sensor, analyte recognition, uncapping, and indicator release happen in the water phase. Phase transfer of the dye, however, occurs at the interface between carrier phase and water droplets. The efficiencies of the processes scale with interaction time and surface which are determined by tube diameter and length as well as flow. In view of the kinetics of the assays in cuvette (600 s), stabilization of the droplet-based system using surfactants seems indicated.17 Unfortunately, surfactants, like phospholipids, can induce blank dye release at the high concentrations that would be required, letting us discard such a step.11,18 Because upon mixing of the analyteand M1-containing water phases exhaustive dye release occurs in ca. 100 s, an incubation step prior to droplet generation in the organic phase was thus considered. After this recognition and liberation step, the dye has to be extracted into the organic phase which requires ca. 500 s for an aqueous solution saturated with 1. Taking into account these considerations, the modular microfluidic setup was conceived entirely from micrometersized PTFE (polytetrafluoroethylene) and PFA (perfluoroalkoxyalkane) tubing connected with commercially available microconnectors, allowing us to use a flexible design that has proven advantageous, for instance, for multiple-inlet or biphasic stop-flow setups19−21 in connection with our sensory particles. Aqueous sample solution and aqueous M1 suspension were injected into an incubation loop, allowing for 100 s of dwell time, before homogeneous water droplets in CH2Cl2 carrier

Figure 1. Scheme of analyte-induced indicator release from gated nanoparticles (top; indicator = yellow balls, stopper = red balls, capping chemistry = blue strings, analyte = green balls), uncapping reaction between 2,4-bis(4-dialkylaminophenyl)-3-hydroxy-4-alkylsulfanylcyclobut-2-enone (APC) and Hg2+ (middle) and microfluidic setup (bottom).

nanoparticles (NPs) of MCM-41 type with a diameter of 120 nm. The NP format is essential to avoid precipitation and aggregation of the particles in the channels, potentially clogging the microfluidic device. The NPs were loaded with an indicator dye before grafting 3-(mercaptopropyl)trimethoxysilane (MPTS) onto the surface of the scaffold. Grafting of the loaded particles ensures that the MPTS linkers are only attached to the outer surface of the ensemble. The pores are subsequently closed with a bulky stopper unit, here squaraine 2 (for chemical and structural details of the system, see Figure S1, Supporting Information) that can be selectively removed through reaction with the target species Hg2+.14,15 The architecture guarantees that relatively few analyte-responsive units are located on the nanoparticles’ outer surface while a large amount of indicator dyes are trapped in the void pore system. Once a few analyte ions lead to an uncapping of the pores, massive release of the fluorescent cargo occurs, equipping the system with the intrinsic features of signal amplification. Adaptation of the initial hybrid system required exchange of the dye since the partitioning coefficient of safranine O used in ref 14 proved to be too low to undergo efficient phase transfer from water to CH2Cl2. Optimum performance was found for 1, a water-soluble PEGylated borondipyrromethene (BODIPY) dye recently developed by us16 with a more suitable partitioning coefficient Ko/w = 0.99 as determined here for the two-phase system. Hence, M1 contains ca. 90 μmol gparticles−1 of 1. Dye 1 presents conventional photophysical properties of BODIPYs both in water and in CH2Cl2 (Section III, Supporting Information) with absorption and emission maxima at ca. 520 and 530 nm, respectively. The fluorescence quantum yield of 1 is high, independent of the solvent employed, i.e., ca. 0.9. Moreover, in line with our previous findings,16 dimer or aggregate formation does not occur for 1 in water, enabling quantitative phase transfer. Before introducing sensor particles M1 into the microfluidic system, a study of the chemical, spectroscopic, and kinetic parameters of their response to Hg2+ was carried out in cuvettebased experiments (Figure 2). As expected, addition of Hg2+ to B

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ACS Sensors fluid were generated within a micro T-cross. Mixing and extraction were then achieved by chaotic advection in a flexible PFA tube instead of the much more rigid PTFE to ensure small loop diameters (flexural moduli of 586 and 496 MPa, respectively).22 The extraction efficiency of the loop system was optimized by injection of a diluted aqueous solution of 1 through one of the water inlets and step-by-step increasing the flow of the aqueous phase from 10 to 30 μL min−1 each, which induced a linear increase of the water droplet size and a linear increase of the fluorescence signal in the organic phase run at 20 μL min−1. Although higher flow rates of the aqueous phase increase the ratio of water-to-organic solvent and thus dye concentration in the organic solvent, flow rates above 20 μL min−1 were found to cause fluidic instabilities resulting in pronounced signal fluctuations. Since the extraction efficiency was still close to 100% at 20 μL min−1, we opted for this parameter. We also tested a tube without loops which, however, showed inferior performance, i.e., an extraction efficiency of only ca. 20%. This optimization of the fluidic system was greatly facilitated by the modularity of the tubing system. For example, a 2-fold decrease of the flow rates required a redimensioning of both the incubation and the extraction loops, which was much simpler for the current system than it would have been for a chip-based one. Besides flexibility, PFA tubes also possess high transparency,22 facilitating direct fluorescence signal collection. Moreover, unlike conventional microfluidic chips for which only a few examples of fluorescence measurements in the preferred 90° geometry were reported,23,24 the use of tubes is not limited with regard to the angle between the exciting beam and emission collection optics, allowing us to use 90°. In the detection unit, the tube went through an optomechanic cube with perpendicularly mounted LED excitation source and fluorescence detection optics. Optical fiber bundles were used to collect the emission and the backscattered light of the LED, to account for fluctuations in real time. After signal acquisition, numerical data treatment with logic equations (see Section V.1, Supporting Information) allowed to extract the fluorescence intensity of the organic phase. This treatment along with the separation of the signals from both phases facilitated identification and removal of outliers, potentially arising, for instance, from gas bubbles or particle aggregates. This simple laboratory setup can be further miniaturized in the future by replacing the spectrometer with photodiodes and the computer-based numerical post-treatment with electronic onboard handling. Having established the optimum dye extraction and detection parameters in the modular microfluidic tubing system, we turned our attention to integration of the gated dye-releasing NPs. Using the setup of Figure 1, we performed titrations with Hg2+ solution to adjust the features for optimum system performance. In the two aqueous channels, a solution of M1 in milli-Q water buffered at pH 7.3 with PBS and an aqueous solution containing Hg2+ at concentrations between 10−12 and 10−2 M were introduced at the same flow rate of 20 μL min−1. This configuration simulates an online monitoring assay, for instance, for water pipes: one inlet contains sensor particles and buffer, while the other feeds water potentially containing the pollutant from a bypass into the analytical system. It should be noted that sensor material, buffer, and analyte are automatically diluted by a factor of 2. Moreover, we decided to keep the concentration of M1 at 0.4 g L−1 to avoid sedimentation problems; PBS buffer was introduced at 100 mM to allow for the dilution step to reach the optimum 50 mM.

The mercury concentrations reported in the following refer to the initial concentration in the analyte inlet. Independent tests analogous to our earlier work yielded acceptable recovery rates.14 In the third channel, pure dichloromethane was injected also at 20 μL min−1. First, the stability and reversibility of the fluorescence signal of the sensor were checked. The setup consisting of PTFE and PFA tubes performed well in the sense that virtually no memory effects were observed and the repeatability over time with and without additional rinsing was acceptable (see Section V.3, Supporting Information). The behavior of the system with sensor particles was virtually identical to the preliminary assays: similar droplet volumes and similar optimum flow rates guaranteed similar fluidic stability and made any readjustment of the tubing system unnecessary. Apart from moving the syringe pump for the M1 solution to a vertical position, to limit the effect of sedimentation, major adjustments only had to be made to the optical part and the spectrometer (e.g., integration time, collection window). System optimization showed us that an acquisition time of 150 s was sufficient for stable and reproducible signals with acceptable standard deviation. Furthermore, like for the cuvette-based experiments, an increasing concentration of Hg2+ led to an increase in fluorescence intensity (Figure 3).

Figure 3. (A) Recognition of Hg2+ by the M1-based microfluidic system. (B) Enlarged measurement range of (A) for low concentrations (cM1 = 0.4 g L−1; H2O/CH2Cl2, 2/1, v/v; PBS).

The detection limit for Hg2+ in the sample (i.e., the liquid injected through the analyte channel) was found to be ca. 10−10 M, equaling ca. 20 ppt. For this concentration, the signal including relative errors exceeded 3σ of the background signal (see Section V.2, Supporting Information). It is noteworthy that this value is 50 times lower than the maximum allowed quantity of mercury in drinking water in the U.S. and the EU.25,26 As is evident from the calibration plot in Figure 3, the system has a broad dynamic detection range from 10−10 to 10−6 M. For concentrations above 10−6 M, for which the theoretical amount of open pores is 2.4%, a massive opening of the pores occurs (23.8% for 10−5 M, and 100% for 10−4 M), inducing a dramatic increase not only of the signal but also of the measurement uncertainty. This behavior might be due to nonoptimum extraction timing when high amounts of dyes are released. Since we aimed at trace metal detection, we kept the initial optimization for the low concentration region. The sensing behavior toward possibly interfering metal ions (Na+, K+, Ca2+, Mg2+ at 1 mM; Cu2+, Ni2+, Zn2+, Ag+, Cd2+ at 0.5 mM; Pb2+, Au3+ at 0.2 mM) was also assessed by measuring the system response using otherwise identical system and assay parameters. No significant interferences were observed, in line with the excellent selectivity seen earlier by us in refs 14, 15 (see Section V.4, Supporting Information). To further validate the sensor’s performance in complex sample matrices, fish C

DOI: 10.1021/acssensors.5b00303 ACS Sens. XXXX, XXX, XXX−XXX

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tissue extracts were analyzed by our method and with AES (see Section V.5, Supporting Information). The concentrations found by both methods for the four extracts were generally in good agreement despite a ca. 15% overestimation for the present one. The latter is supposedly due to the high content of organic matter in the sample matrix.27 In conclusion, we have successfully adapted and incorporated fluorescent gated sensory nanoparticles for Hg2+ trace detection into a biphasic droplet-assisted microfluidic sensor. This sensor system is only made of simple and commercially available components such as PTFE/PFA tubes, conventional optomechanics, and an LED, without sacrificing its efficiency. Efficient mixing of analyte and sensory particles is achieved by chaotic advection in water droplets. Recognition of Hg2+ followed by extraction of the released dyes into dichloromethane occurs best at pH 7.3 (PBS buffer). Enrichment of the liberated dyes in the organic phase allowed us to obtain pronounced fluorescence signals directly linked to Hg2+ concentration in the primary phase. The fact that the microfluidic system relies on less hydrophilic dyes as the initial assay14 is also advantageous, because the number of highly emissive, highly water-soluble dyes is limited. With the redesigned hybrid material, multiplexing applications come more into reach, especially as the modular microfluidic system allows for sequential readout at different wavelength combinations by simple separation in space along the PFA tube. The microfluidic sensor developed here might become a potent tool for in-line monitoring of traces of mercury. Although the response time of 10 min is somewhat slow, the ability to detect very small traces down to 20 ppt of Hg2+ with virtually no memory effects is outstanding and comparable to or better than that of other recently published methods (see Section V.6, Supporting Information). Current work is thus directed toward further integration by replacement of the syringe pumps and spectrometer on one hand and by achieving a multiplexed assay potentially also relying on coding schemes as reported by others recently.28

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.5b00303. Full experimental details including syntheses, spectroscopic, and photophysical properties of 1, characterization, microfluidic system description, numerical signal treatment, uncertainty budget, and selectivity (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address †

Surflay Nanotec GmbH, Max-Planck-Str.3, D-12489 Berlin, Germany

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support by the AdolfMartens-Fund and Alexander-von-Humboldt Foundation. We thank K. Keil and H. Witthuhn of BAM for support (characterization of 1 and AES controls). D

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ACS Sensors (21) Hu, Y.; Wang, S.; Abbaspourrad, A.; Ardekani, A. M. Fabrication of shape controllable janus alginate/pNIPAAm microgels via microfluidics technique and off-chip ionic cross-linking. Langmuir 2015, 31, 1885−1891. (22) Polymers: A Property Database, 2nd ed.; Ellis, B., Smith, R., Eds.; CRC: Boca Raton, 2008. (23) Zhao, L.; Wu, T.; Lefevre, J. P.; Leray, I.; Delaire, J. A. Fluorimetric lead detection in a microfluidic device. Lab Chip 2009, 9, 2818−2823. (24) Hart, S. J.; Jiji, R. D. A simple, low-cost, remote fiber-optic micro volume fluorescence flowcell for capillary flow-injection analysis. Anal. Bioanal. Chem. 2002, 374, 385−389. (25) National Primary Drinking Water Regulations; U. S. Environmental Protection Agency: Washington DC, 2009. (26) Council Directive 98/83/EC on the quality of water intended for human consumption; Council of the European Union: Brussels, 1998. (27) Radulescu, M.-C.; Danet, A. F. Mercury determination in fish samples by chronopotentiometric stripping analysis using gold electrodes prepared from recordable CDs. Sensors 2008, 8, 7157− 7171. (28) Zhao, Y.; Cheng, Y.; Shang, L.; Wang, J.; Xie, Z.; Gu, Z. Microfluidic synthesis of barcode particles for multiplex assays. Small 2015, 11, 151−174.

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DOI: 10.1021/acssensors.5b00303 ACS Sens. XXXX, XXX, XXX−XXX