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Selective fluorimetric detection of cadmium in a microfluidic device
Microchemical Journal 106 (2013) 167–173
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Selective fluorimetric detection of cadmium in a microfluidic device Haitao Zhang, Djibril Faye, Jean-Pierre Lefèvre, Jacques A. Delaire, Isabelle Leray ⁎ Laboratoire de Photophysique et Photochimie Supramoléculaires et Macromoléculaires, Institut d'Alembert, Ecole Normale Supérieure de Cachan, CNRS, 61 avenue du Président Wilson 94230 CACHAN, France
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Article history: Received 21 May 2012 Received in revised form 11 June 2012 Accepted 11 June 2012 Available online 16 June 2012 Keywords: Cadmium Microfluidics Lead Solid phase extraction Rhod-5N Fluorescent sensor
a b s t r a c t A microfluidic device equipped with a fluorimetric detection has been developed for the flow injection detection of cadmium, a very toxic metal. The fluorescent sensor, Rhod-5N, is a water-soluble commercial dye whose fluorescence is increased in presence of cadmium. A detection limit of 0.45 μg L− 1 has been determined in MOPS buffer at pH 7. Special attention was paid to the interference of other metal ions. The interference of Pb2+ was circumvented by solid phase adsorption (SPE) on aminopropyl silica. Langmuir analysis of the adsorption isotherms has shown a better affinity of this functionalized silica towards Pb2+. After optimization of both pH and flow rate of the analyte through the SPE minicolumn, cadmium could be determined even in presence of lead at a concentration 10 times larger. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cadmium is a heavy metal widely used in industrial processes such as electroplating of metals, colouring agents and rechargeable Cd–Ni batteries. Cadmium is therefore found in air, water, or in plants as a result of anthropogenic pollution mainly from the smelting and refining of nonferrous metals, fossil fuel combustion and municipal waste incineration. Unfortunately, cadmium is highly toxic for all living things as it accumulates in cells, liver and kidneys with subsequent physiological disorders or carcinogenic effects [1]. For this reason, the level of cadmium in drinking water should not exceed 3 μg L− 1 according to the World Health Organization [2]. The EU has limited the amount of cadmium ions in drinking waters to 5 μg L − 1 and this value will be decreased to 0.2 μg L − 1 in 2013 [3]. Analytical methods commonly used to determine heavy metal ions in aqueous samples include sophisticated analytical techniques such as atomic absorption spectroscopy (AAS), atomic emission spectroscopy (AES) or inductively coupled plasma spectroscopy (ICP-MS). Typical detection limits are currently about 1 ng L − 1 in solution. Numerous quantitative analytical methods such as AAS [4], inductively coupled plasma atomic emission spectrometry (ICP-AES) [5] and inductively coupled plasma mass spectrometry(ICP-MS) [6] are available for the determination of cadmium(II). Single drop microextraction technique coupled with electrothermal atomic absorption spectroscopy also leads to detection of Cd2+ with a good sensitivity of 0.7 ng L− 1 [4c]. Because of the high costs of all these laboratory instrumental methods and
⁎ Corresponding author. E-mail address: [email protected] (I. Leray). 0026-265X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2012.06.005
in order to develop portable or on-line analytical devices, electrochemical methods mainly based on anodic stripping voltammetry (ASV) are developed [7]. These techniques usually employ mercury as working electrode, which produces mercury-contaminated waste. However, different attempts have been made with other electrode materials like zeolite-modified carbon [8] or silver [9]. Meanwhile, there is a rapidly growing interest in research related to the development of optical sensors based on chromogenic and fluorogenic reagents. Different review papers relate the developed optical systems for monitoring heavy metal ions in various matrices, with their limitations in terms of selectivity, limits of detection and reversibility [10,11]. Recently, different water soluble fluorophores based on acridine [12], 8-hydroxyquinoleine [13], 4,5diamino-1,8-naphthalimide/picolylamine [14], quinoline/ picolylamine [15] or Bodipy [16] derivatives have been designed and synthesized for the sensitive and selective detection of Cd2+. However, they suffer either from a lack of selectivity or result from a long synthetic step. With the goal to develop portable devices allowing delocalized assays of surface or sewage waters, we have developed a microfabricated device able to detect traces of lead in water by using a specific fluorescent sensor. The detection limit is equal to 2 ppb [17]. In this work, we propose a fluorimetric method to selectively determine the amount of cadmium ions in water in a microfluidic device. Our objective is to use a commercially available fluorescent sensor, Rhod-5N (see Scheme 1), that we already studied in a previous work as being very sensitive to cadmium and lead ions [18], and to find a procedure leading to the selective assay of cadmium ions. This fluorescent molecular sensor consists of a BAPTA chelating moiety bound to a rhodamine fluorophore. Its fluorescence, very weak in absence of metal ions, is drastically enhanced after complexation of different metal ions, as a result of the inhibition of the photoinduced
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Scheme 1. Structure of Rhod-5N and inhibition of photoelectron transfer in its complex with cadmium.
electron transfer occurring from one of the aromatic amine groups of the BAPTA moiety to the rhodamine fluorophore in the 1:1 complex (see Scheme 1). In our previous work, we have shown that Rhod-5N can detect Cd2+ ions (detection limit of 3.1 μg L− 1) with a high selectivity over various metal ions, except Pb2+. In this work, we describe a simple optofluidic set-up allowing the use of Rhod-5N for Cd2+ detection, and in order to circumvent the interference with Pb2+, we propose a solid phase extraction (SPE) on aminopropyl silica beads. 2. Experimental 2.1. Chemicals Rhod-5N was purchased from Invitrogen and used without any further purification. Stock solutions of the ligand were freshly prepared. Millipore water (conductivityb 6×10− 8 Ω− 1 cm− 1 at 20 °C) was used for the preparation of the buffered solutions containing 10 mM MOPS (3-(N-morpholino)propanesulfonic acid). The pH was adjusted at pH=7.0 by adding KOH (99.99%). Calcium perchlorate, zinc perchlorate, lead(II) thiocyanate, copper perchlorate and cadmium perchlorate from Aldrich or Alfa Aesar were of the highest quality available. Stock solutions of these salts were prepared at concentrations ranging from 10− 3 to 0.1 mol L− 1 in MOPS buffer, except for lead(II) which was prepared in Millipore water in order to preclude the formation of insoluble lead(II) hydroxide. For pHs between 7 and 9, a phosphate buffer (10 mM L− 1) was used. Commercial 3-aminopropyl functionalized silica gel (SiO2-APTES)) (Sigma-Aldrich) was used for lead ion extraction. The surface area of the bond silica is 550 m2.g− 1 and the particle size is 40–63 μm. The average pore size is equal to 6 nm. The aminopropyl silyl group is loaded on silica with a 1 mmol.g− 1 concentration. 2.2. Spectroscopic measurements UV/vis absorption spectra were recorded on a UVICON spectrophotometer. Corrected emission spectra were obtained on a Jobin-Yvon Spex Fluoromax spectrofluorometer.
layer of SU8-2100 resist 77 μm thick imaged through a first mask and the mixer ridges came in a second layer of SU8-2025 resist 23 μm thick imaged through a second mask perfectly aligned with the channel. The two inlet Teflon tubing allowed us to introduce via syringes mounted on two commercial syringe pumps (Harvard type PHD 2000) both analyte and fluorescent molecular sensor solutions. The excitation of the fluorescent molecules is achieved by one light emitting diode (Roitner, B5-433 B525 nm) whose power supply modulated with a sinusoidal signal (77 Hz) allowed heterodyne detection of fluorescence. The detection chamber, as described in a previous paper [17] allows both excitation and fluorescence detection in perpendicular directions. Taking into account the value of the extinction coefficient of Rhod-5N at 525 nm (ε≈60526 L mol− 1 cm− 1) and its concentration (10− 6 mol.L− 1), the optical density of the fluorescent dye at the excitation wavelength inside the irradiation chamber has been estimated to be lower than 0.06, which ensures a homogeneous excitation across the chamber. The LED was coupled with an optical fibre (silica 100/110/125 μm, with high OH concentration); this optical fibre was embedded inside the PDMS matrix, with its polished extremity at a distance 80 μm apart from the fluidic microchannel. An interference filter centred at 525 nm (Semrock FF01-525/15-25) was intercalated between the output of the LED and the entrance of the optical fibre in order to decrease the bandwidth of the LED and consequently to have a low baseline on the fluorescence signal. Fluorescent light was collected through a bundle of 7 optical fibres (400/440 μm) and focussed through a high pass filter (λ > 570 nm) on the entrance of a PM tube (Hamamatsu R928). The electrical signal from the PM tube was amplified with a current/voltage converter with gain varying from 10 4 to 109 by step. The voltage signal was routed to a lock-in amplifier (Signal Recovery 7265 DSP) and acquired from the lock-in on a PC computer via a RS232 bus; it was in the order of one hundred millivolt level and far from a signal-to-noise ratio of one. When MOPS buffer (pH 7) was circulating through the chip, there was no detectable fluorescence; the background signal varied between 10 and 30 mV, depending on the particular microdevice. In order to study the influence of the residence time on the complexation reaction occurring inside the Y-shaped channel, two microchannels with different lengths were used: a short one with a length of 2.2 cm and a long one with a length of 13 cm [20].
2.3. Microfluidic chip and fluorescence detection The device, shown in Scheme 2, is essentially made of a Y-shape microchannel moulded in PDMS fixed on a glass substrate. The details of the manufacture of this μTAS device have already been published [17]. A passive mixer with a staggered herringbone structure is used for mixing the reactant (aqueous solution of Rhod-5N) and the analyte (Cd2+ aqueous solution) analogous to the one described by Stroock et al. [19]. The Y-shape microchip shown in Scheme 2 is made of a thick layer of PDMS (RTV 615 kit from Bayer) in which is molded the microchannel, stuck on a glass plate (standard microscope slide 50 mm long and 25 mm wide). The mould is a SU8 resist (SU8‐2100 and SU8-2025 from Micro Chem) imaged on a silicon wafer by a conventional microlithographic technique. The shape of the channel was obtained in a first
2.4. Minicolumn for solid phase extraction Solid phase extraction was carried out in a home-made mini-column as displayed on Scheme 3. A capillary of polytetrafluoroethylene (PTFE) was squeezed on one side to prevent the silica gel beads out but let circulate the fluids. From the other side, the slurry of silica gel was introduced with a syringe until the column was about 10 mm long. And then the outlet of the capillary was squeezed in the same way. The weight of this 10 mm long silica gel was about 4–5 mg. The mini-column was freshly made for each use. Before each use, in order to prevent the loss of sorbent, several milliliters of water were passed through this minicolumn at the flow rate of real operation until the length of sorbent in the column
H. Zhang et al. / Microchemical Journal 106 (2013) 167–173
169
Scheme 2. Microfluidic device used for the fluorimetric detection of cadmium.
became constant. The loss of sorbent for each measurement was less than 2%. 3. Results and discussion 3.1. Cadmium ion sensing by fluorimetric detection in the microfluidic device When both Rhod-5N and cadmium perchlorate solutions in MOPS buffer at pH 7 are introduced in the long microfluidic channel at a flow rate of 0.25 mL h − 1, an increase of the collected fluorescence signal was observed, with a plateau value reached for one equivalent of Cd 2+ added (Fig. 1). This increase of fluorescence has already been observed during measurements in quartz cells [18] and the plateau value reached for a ratio [Ligand]/[Metal] near 1 is the result of a high complexation equilibrium constant for the complex of stoichiometry 1:1. The same experiment could be done in the short microchannel and gave similar results. However, the noise level was higher: this was attributed to a less good mixing of both analyte and sensor in the staggered herringbone structure. The influence of the kinetics of the complexation reaction was also investigated in the short microchannel in which the residence time has been determined (8.8 s for a flow rate of 0.25 mL h− 1). With this purpose, we compared the intensity of the fluorescence signals measured when mixing both reactants before introduction inside the Y-shape microchannel and after mixing inside the microchannel. The results, depicted in Fig. 2, show that, for solutions of equal concentrations, the signals have the same intensity: it means that the complexation reaction is completed at the output of the microchannel. Of course, this conclusion is also valid for the long microchannel (residence time
Scheme 3. Minicolumn for solid phase extraction.
40 s for a flow rate of 0.25 mL h− 1). The fastness of this reaction is a favourable point for this method of determination. A calibration curve for Cd 2+ determination could be drawn from the results of Fig. 1. Under our experimental conditions, the linear part of this curve ranges from 0 to 1 μm L − 1 (Fig. 3). The detection and quantification limits, estimated as three and five times the standard deviation of the blank signal respectively, were found to be 2 nmol L− 1 and 3.4 nmol L − 1 respectively. As these concentrations refer to the concentrations inside the microchannel after dilution by the probe, the real limits are 4 nmol L − 1 and 6.8 nmol L − 1 respectively, i.e. 0.45 μg L − 1 and 0.76 μg L − 1 respectively. The blank was taken as the signal of Rhod-5N (10− 6 mol L − 1) in MOPS at pH 7. Due to a better signal over noise ratio, the detection limit was found better than the one previously found by the classical method using quartz cuvettes [18] and is only twice larger than the maximal permissible concentration of 0.2 μg L − 1 than will be fixed in 2013 in the E U.
Fig. 1. Variation of measured fluorescence intensity of Rhod-5N (10− 6 M) at the output of the microfluidic circuit (long microchannel) for different concentrations of cadmium perchlorate added in one channel of the microchip (the Cd2+ concentrations inside the microchannel are given above every signal).
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Fig. 2. Comparison between fluorescence signals for reactants mixed before introduction in the microdevice with a short channel (external mixing: Mixing OUT) and inside the microdevice (internal mixing: Mixing IN).
In spite of its very good sensitivity towards cadmium ions, Rhod-5N suffers from at least one drawback: it is also sensitive to lead (Pb2+) ions, as shown in Fig. 2. Indeed, the selectivity, defined as the ratio of the apparent stability constants of the 1:1 complexes formed with the metal ions K (Cd2+)/K (Pb2+) is only 8 [18]. The selectivity of Rhod5N was found good versus calcium and zinc which are generally interfering with cadmium. Calcium is abundant in most of drinkable waters and zinc has similar properties with cadmium and is generally detected with cadmium. Furthermore, as can be seen in Fig. 2, Pb 2+ ions also induce an increase of the fluorescence of Rhod-5N with a complex rapidly formed and emitting with a fluorescence quantum yield almost three times lower than the cadmium complex [18]. As lead is also a dangerous heavy metal whose determination is as important as determination of cadmium, a method is proposed to discriminate both ions.
impossible in our case, as between pH 7 and 9, both metal ions were complexed by dithizone. Thanks to the affinity of sulphur and nitrogen atoms for several metal cations, silica functionalized by thiol or amino groups has been frequently used for extraction of mercury, lead, zinc, etc. [22,23]. After introduction in a column, this mesoporous silica can be used as a preconcentrator for heavy metal cations which is coupled with very efficient analytic methods like AAS or ICP. Very low quantities of analytes can then be detected [24,25]. Silica functionalized by aminopropyl triethoxysilane (SiO2-APTES) has been used for pre-concentration of zinc, lead, cadmium and nickel [26]. At pH 7, it has been shown that all lead ions introduced in the minicolumn are adsorbed by silica. The reversibility of the adsorption process is obtained by decomplexation at acidic pH (Scheme 4). The adsorption isotherms of both Cd(II) and Pb(II) ions on this modified silica sorbent material were determined using batch experiments at room temperature. Solutions of cadmium(II) perchlorate or lead(II) thiocyanate were shaken in a plastic vial with SiO2-APTES in Millipore water at neutral pH for 1 h. After centrifugation at 10,000 rpm during 10 min, the metal ions in the supernatant were determined by a fluorimetric titration experiment in a quartz cuvette by using Rhod-5N as the complexing molecule and the amount Nf of adsorbed heavy metal (in mol g− 1) was calculated using Eq. (1) as follows: Nf ¼
V ðC 0 −C s Þ m
where C0 is the initial concentration of heavy metal ions in mol L − 1, CS the equilibrium concentration of heavy metal ions in mol L − 1 and m the mass of the modified silica gel in g. Fig. 4 clearly shows the increase of adsorbed ions with the increase of the ion concentration in solution, towards a plateau value which is higher for Cd2+ than for Pb2+. Adsorption isotherm can be described by using the Langmuir model which leads to Eq. (2) [27]: CS CS 1 ¼ þ Nf NS b NS
3.2. Interference with lead ions: towards a fluorimetric method to discriminate lead and cadmium ions in water Methods to separate interfering cations include liquid phase extraction (LPE) or solid phase extraction (SPE). In the literature, it has been proposed to use dithizone ligand dissolved in chloroform to separate Cd2+ from Pb 2+, Cu2+,Co2+ or Ni2+ [21]. However, the optimization of pH for the extraction process of cadmium and lead appeared to be
Fig. 3. Calibration curve for the determination of Cd2+ ions by Rhod-5N 10− 6 mol L− 1 in MOPS buffer at pH 7. Inset: enlargement plot for low concentration of cadmium.
ð1Þ
ð2Þ
where b is the Langmuir constant in L.mol− 1 , NS is a constant representing the maximum adsorption capacity, also known as monolayer coverage of the surface (in mol g− 1). The linear plots of Cs/Nf versus Cs (Fig. 5) show that the adsorption of cadmium and lead satisfactorily obeys the Langmuir model. The values of both parameters b and Ns resulting from these plots are gathered in Table 1. The Langmuir constant, which is the ratio of the adsorption rate versus the desorption rate, is much higher for Pb 2+ than for Cd2+, which reflects a better affinity of SiO2-APTES for lead. On the opposite, the maximum adsorption capacity is twice higher for cadmium ions, which could be partly due to the smaller size of Cd2+ ion (0.194 nm) compared to Pb 2+ (0.238 nm). Considering the better affinity of the functionalized silica for lead compared to cadmium ions, we realized a SPE of both ions in buffer solutions at different pHs between 7 and 9 in order to optimize the separation. For this purpose, we used a minicolumn with packed silica beads connected to a syringe (see Experimental section). Buffered solutions of Pb2+ or Cd2+, or a mixture of both, were run through the minicolumn at a flow rate of 2 mL h− 1 and the metal content of the eluted solution was analyzed by fluorescence spectroscopy in quartz cuvettes using Rhod-5N at the same pH as a fluorescent probe. The experiments were repeated at several metal concentrations in the range 5 ×10− 8 to 5× 10− 7 mol L− 1. As shown in Fig. 6, the recovery of both Cd2+ and Pb2+ ions in the eluant is dependent on pH. For example, at pH 8.4, Pb2+ ions are completely adsorbed, compared to less than 50% adsorption for Cd2+. At pH 8.4, a mixture of both Cd2+ and Pb2+ ions was submitted to the two steps described before : i) a SPE through a minicolumn of SiO2‐ APTES at pH 8.4; ii) fluorescence detection in the microdevice described
H. Zhang et al. / Microchemical Journal 106 (2013) 167–173
H2N
H2N
NH2
NH2 H2N
Si
NH2
171
NH2
Pb2+
Adsorption
H2N
Si
Desorption
NH2
NH2
Pb2+
NH2
NH2
Scheme 4. Adsorption and desorption of Pb2+ ions on functionalized silica SiO2-APTES.
before, with Rhod-5N as the fluorescent sensor. Fig. 7 presents the calibration curves obtained after this procedure for the determination of cadmium ions in absence and in presence of two different concentrations of Pb2+ ions. The cadmium concentrations plotted on the abscissa axis are the initial cadmium solutions introduced in the minicolumn. The deviation of the calibration curves in presence of lead don't differ from the one obtained without lead by more than 15% for a Pb2+ concentration ten times higher than Cd2+.
detection with the previously described microfluidic device. Aqueous solutions of different interfering cations at different concentrations and at neutral pH were eluted through the column and the fluorescence of Rhod-5N was then measured in the microfluidic device. The values of metal ion concentrations and the obtained fluorescence intensities are gathered in Table 2. No significant effect on this intensity was observed upon addition of these interfering cations. 4. Conclusion
3.3. Effect of interfering cations With practical application in mind, the effect of different interfering cations was evaluated by SPE adsorption followed by fluorescence
Combination of an off-line SPE and a fluorimetric detection in a microfluidic device has demonstrated effective cadmium ion determination and this is the basis for an integrated SPE-microfluidic detection system.
Fig. 4. Adsorption isotherms for Cd2+(left) and Pb2+ (right) ions on SiO2-APTES in Millipore water at neutral pH.
Fig. 5. Fits of the Langmuir adsorption model (see Eq. (2) in the text) for cadmium (left) and lead (right) ions.
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Table 1 Langmuir parameters for the adsorption of Cd2+ and Pb2+ ions on SiO2-APTES.
2+
Cd Pb2+ a b
Ns (mmol/g)a
b (L/mol)b
1.11 0.57
6.3 × 102 9.7 × 104
Ns is the maximum number of metal ions covering the porous silica. b is the Langmuir constant.
Table 2 Effect of interfering cations on the fluorimetric determination of cadmium. IF is the fluorescence intensity measured in the device after column adsorption. Cation
[M2+]/μmol L− 1
IF/A.U.
Cd2+ Ca2+ Ca2+ + Cd2+ Zn2+ Zn2+ + Cd2+ Cu2+ Zn2+ + Cd2+
0 0.1 1 1 + 0.1 5 5 + 0.1 5 5 + 0.1
1 1.34 1.02 1.37 0.96 1.29 1.01 1.37
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Fig. 6. Recovery of Cd2+ and Pb2+ ions after flowing through a minicolumn of SiO2-APTES. The points correspond to a mean value for concentrations in the range of 5 × 10− 8– 5 × 10− 7 mol.L− 1.
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Fig. 7. Calibration curves for the determination of cadmium perchlorate by Rhod-5N in a phosphate buffer at pH 8.4 after solid phase extraction through a minicolumn of SiO2APTES, in the presence of different concentrations of lead(II) thiocyanate in the initial solution: ● [Pb2+]o =0 mol.L− 1; ▴ [Pb2+]o =5×10−7 mol.L− 1; ♦ [Pb2+]o =10−6 mol.L− 1.
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Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Selective fluorimetric detection of cadmium in a microfluidic device Haitao Zhang, Djibril Faye, Jean-Pierre Lefèvre, Jacques A. Delaire, Isabelle Leray ⁎ Laboratoire de Photophysique et Photochimie Supramoléculaires et Macromoléculaires, Institut d'Alembert, Ecole Normale Supérieure de Cachan, CNRS, 61 avenue du Président Wilson 94230 CACHAN, France
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Article history: Received 21 May 2012 Received in revised form 11 June 2012 Accepted 11 June 2012 Available online 16 June 2012 Keywords: Cadmium Microfluidics Lead Solid phase extraction Rhod-5N Fluorescent sensor
a b s t r a c t A microfluidic device equipped with a fluorimetric detection has been developed for the flow injection detection of cadmium, a very toxic metal. The fluorescent sensor, Rhod-5N, is a water-soluble commercial dye whose fluorescence is increased in presence of cadmium. A detection limit of 0.45 μg L− 1 has been determined in MOPS buffer at pH 7. Special attention was paid to the interference of other metal ions. The interference of Pb2+ was circumvented by solid phase adsorption (SPE) on aminopropyl silica. Langmuir analysis of the adsorption isotherms has shown a better affinity of this functionalized silica towards Pb2+. After optimization of both pH and flow rate of the analyte through the SPE minicolumn, cadmium could be determined even in presence of lead at a concentration 10 times larger. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cadmium is a heavy metal widely used in industrial processes such as electroplating of metals, colouring agents and rechargeable Cd–Ni batteries. Cadmium is therefore found in air, water, or in plants as a result of anthropogenic pollution mainly from the smelting and refining of nonferrous metals, fossil fuel combustion and municipal waste incineration. Unfortunately, cadmium is highly toxic for all living things as it accumulates in cells, liver and kidneys with subsequent physiological disorders or carcinogenic effects [1]. For this reason, the level of cadmium in drinking water should not exceed 3 μg L− 1 according to the World Health Organization [2]. The EU has limited the amount of cadmium ions in drinking waters to 5 μg L − 1 and this value will be decreased to 0.2 μg L − 1 in 2013 [3]. Analytical methods commonly used to determine heavy metal ions in aqueous samples include sophisticated analytical techniques such as atomic absorption spectroscopy (AAS), atomic emission spectroscopy (AES) or inductively coupled plasma spectroscopy (ICP-MS). Typical detection limits are currently about 1 ng L − 1 in solution. Numerous quantitative analytical methods such as AAS [4], inductively coupled plasma atomic emission spectrometry (ICP-AES) [5] and inductively coupled plasma mass spectrometry(ICP-MS) [6] are available for the determination of cadmium(II). Single drop microextraction technique coupled with electrothermal atomic absorption spectroscopy also leads to detection of Cd2+ with a good sensitivity of 0.7 ng L− 1 [4c]. Because of the high costs of all these laboratory instrumental methods and
⁎ Corresponding author. E-mail address: [email protected] (I. Leray). 0026-265X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2012.06.005
in order to develop portable or on-line analytical devices, electrochemical methods mainly based on anodic stripping voltammetry (ASV) are developed [7]. These techniques usually employ mercury as working electrode, which produces mercury-contaminated waste. However, different attempts have been made with other electrode materials like zeolite-modified carbon [8] or silver [9]. Meanwhile, there is a rapidly growing interest in research related to the development of optical sensors based on chromogenic and fluorogenic reagents. Different review papers relate the developed optical systems for monitoring heavy metal ions in various matrices, with their limitations in terms of selectivity, limits of detection and reversibility [10,11]. Recently, different water soluble fluorophores based on acridine [12], 8-hydroxyquinoleine [13], 4,5diamino-1,8-naphthalimide/picolylamine [14], quinoline/ picolylamine [15] or Bodipy [16] derivatives have been designed and synthesized for the sensitive and selective detection of Cd2+. However, they suffer either from a lack of selectivity or result from a long synthetic step. With the goal to develop portable devices allowing delocalized assays of surface or sewage waters, we have developed a microfabricated device able to detect traces of lead in water by using a specific fluorescent sensor. The detection limit is equal to 2 ppb [17]. In this work, we propose a fluorimetric method to selectively determine the amount of cadmium ions in water in a microfluidic device. Our objective is to use a commercially available fluorescent sensor, Rhod-5N (see Scheme 1), that we already studied in a previous work as being very sensitive to cadmium and lead ions [18], and to find a procedure leading to the selective assay of cadmium ions. This fluorescent molecular sensor consists of a BAPTA chelating moiety bound to a rhodamine fluorophore. Its fluorescence, very weak in absence of metal ions, is drastically enhanced after complexation of different metal ions, as a result of the inhibition of the photoinduced
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Scheme 1. Structure of Rhod-5N and inhibition of photoelectron transfer in its complex with cadmium.
electron transfer occurring from one of the aromatic amine groups of the BAPTA moiety to the rhodamine fluorophore in the 1:1 complex (see Scheme 1). In our previous work, we have shown that Rhod-5N can detect Cd2+ ions (detection limit of 3.1 μg L− 1) with a high selectivity over various metal ions, except Pb2+. In this work, we describe a simple optofluidic set-up allowing the use of Rhod-5N for Cd2+ detection, and in order to circumvent the interference with Pb2+, we propose a solid phase extraction (SPE) on aminopropyl silica beads. 2. Experimental 2.1. Chemicals Rhod-5N was purchased from Invitrogen and used without any further purification. Stock solutions of the ligand were freshly prepared. Millipore water (conductivityb 6×10− 8 Ω− 1 cm− 1 at 20 °C) was used for the preparation of the buffered solutions containing 10 mM MOPS (3-(N-morpholino)propanesulfonic acid). The pH was adjusted at pH=7.0 by adding KOH (99.99%). Calcium perchlorate, zinc perchlorate, lead(II) thiocyanate, copper perchlorate and cadmium perchlorate from Aldrich or Alfa Aesar were of the highest quality available. Stock solutions of these salts were prepared at concentrations ranging from 10− 3 to 0.1 mol L− 1 in MOPS buffer, except for lead(II) which was prepared in Millipore water in order to preclude the formation of insoluble lead(II) hydroxide. For pHs between 7 and 9, a phosphate buffer (10 mM L− 1) was used. Commercial 3-aminopropyl functionalized silica gel (SiO2-APTES)) (Sigma-Aldrich) was used for lead ion extraction. The surface area of the bond silica is 550 m2.g− 1 and the particle size is 40–63 μm. The average pore size is equal to 6 nm. The aminopropyl silyl group is loaded on silica with a 1 mmol.g− 1 concentration. 2.2. Spectroscopic measurements UV/vis absorption spectra were recorded on a UVICON spectrophotometer. Corrected emission spectra were obtained on a Jobin-Yvon Spex Fluoromax spectrofluorometer.
layer of SU8-2100 resist 77 μm thick imaged through a first mask and the mixer ridges came in a second layer of SU8-2025 resist 23 μm thick imaged through a second mask perfectly aligned with the channel. The two inlet Teflon tubing allowed us to introduce via syringes mounted on two commercial syringe pumps (Harvard type PHD 2000) both analyte and fluorescent molecular sensor solutions. The excitation of the fluorescent molecules is achieved by one light emitting diode (Roitner, B5-433 B525 nm) whose power supply modulated with a sinusoidal signal (77 Hz) allowed heterodyne detection of fluorescence. The detection chamber, as described in a previous paper [17] allows both excitation and fluorescence detection in perpendicular directions. Taking into account the value of the extinction coefficient of Rhod-5N at 525 nm (ε≈60526 L mol− 1 cm− 1) and its concentration (10− 6 mol.L− 1), the optical density of the fluorescent dye at the excitation wavelength inside the irradiation chamber has been estimated to be lower than 0.06, which ensures a homogeneous excitation across the chamber. The LED was coupled with an optical fibre (silica 100/110/125 μm, with high OH concentration); this optical fibre was embedded inside the PDMS matrix, with its polished extremity at a distance 80 μm apart from the fluidic microchannel. An interference filter centred at 525 nm (Semrock FF01-525/15-25) was intercalated between the output of the LED and the entrance of the optical fibre in order to decrease the bandwidth of the LED and consequently to have a low baseline on the fluorescence signal. Fluorescent light was collected through a bundle of 7 optical fibres (400/440 μm) and focussed through a high pass filter (λ > 570 nm) on the entrance of a PM tube (Hamamatsu R928). The electrical signal from the PM tube was amplified with a current/voltage converter with gain varying from 10 4 to 109 by step. The voltage signal was routed to a lock-in amplifier (Signal Recovery 7265 DSP) and acquired from the lock-in on a PC computer via a RS232 bus; it was in the order of one hundred millivolt level and far from a signal-to-noise ratio of one. When MOPS buffer (pH 7) was circulating through the chip, there was no detectable fluorescence; the background signal varied between 10 and 30 mV, depending on the particular microdevice. In order to study the influence of the residence time on the complexation reaction occurring inside the Y-shaped channel, two microchannels with different lengths were used: a short one with a length of 2.2 cm and a long one with a length of 13 cm [20].
2.3. Microfluidic chip and fluorescence detection The device, shown in Scheme 2, is essentially made of a Y-shape microchannel moulded in PDMS fixed on a glass substrate. The details of the manufacture of this μTAS device have already been published [17]. A passive mixer with a staggered herringbone structure is used for mixing the reactant (aqueous solution of Rhod-5N) and the analyte (Cd2+ aqueous solution) analogous to the one described by Stroock et al. [19]. The Y-shape microchip shown in Scheme 2 is made of a thick layer of PDMS (RTV 615 kit from Bayer) in which is molded the microchannel, stuck on a glass plate (standard microscope slide 50 mm long and 25 mm wide). The mould is a SU8 resist (SU8‐2100 and SU8-2025 from Micro Chem) imaged on a silicon wafer by a conventional microlithographic technique. The shape of the channel was obtained in a first
2.4. Minicolumn for solid phase extraction Solid phase extraction was carried out in a home-made mini-column as displayed on Scheme 3. A capillary of polytetrafluoroethylene (PTFE) was squeezed on one side to prevent the silica gel beads out but let circulate the fluids. From the other side, the slurry of silica gel was introduced with a syringe until the column was about 10 mm long. And then the outlet of the capillary was squeezed in the same way. The weight of this 10 mm long silica gel was about 4–5 mg. The mini-column was freshly made for each use. Before each use, in order to prevent the loss of sorbent, several milliliters of water were passed through this minicolumn at the flow rate of real operation until the length of sorbent in the column
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169
Scheme 2. Microfluidic device used for the fluorimetric detection of cadmium.
became constant. The loss of sorbent for each measurement was less than 2%. 3. Results and discussion 3.1. Cadmium ion sensing by fluorimetric detection in the microfluidic device When both Rhod-5N and cadmium perchlorate solutions in MOPS buffer at pH 7 are introduced in the long microfluidic channel at a flow rate of 0.25 mL h − 1, an increase of the collected fluorescence signal was observed, with a plateau value reached for one equivalent of Cd 2+ added (Fig. 1). This increase of fluorescence has already been observed during measurements in quartz cells [18] and the plateau value reached for a ratio [Ligand]/[Metal] near 1 is the result of a high complexation equilibrium constant for the complex of stoichiometry 1:1. The same experiment could be done in the short microchannel and gave similar results. However, the noise level was higher: this was attributed to a less good mixing of both analyte and sensor in the staggered herringbone structure. The influence of the kinetics of the complexation reaction was also investigated in the short microchannel in which the residence time has been determined (8.8 s for a flow rate of 0.25 mL h− 1). With this purpose, we compared the intensity of the fluorescence signals measured when mixing both reactants before introduction inside the Y-shape microchannel and after mixing inside the microchannel. The results, depicted in Fig. 2, show that, for solutions of equal concentrations, the signals have the same intensity: it means that the complexation reaction is completed at the output of the microchannel. Of course, this conclusion is also valid for the long microchannel (residence time
Scheme 3. Minicolumn for solid phase extraction.
40 s for a flow rate of 0.25 mL h− 1). The fastness of this reaction is a favourable point for this method of determination. A calibration curve for Cd 2+ determination could be drawn from the results of Fig. 1. Under our experimental conditions, the linear part of this curve ranges from 0 to 1 μm L − 1 (Fig. 3). The detection and quantification limits, estimated as three and five times the standard deviation of the blank signal respectively, were found to be 2 nmol L− 1 and 3.4 nmol L − 1 respectively. As these concentrations refer to the concentrations inside the microchannel after dilution by the probe, the real limits are 4 nmol L − 1 and 6.8 nmol L − 1 respectively, i.e. 0.45 μg L − 1 and 0.76 μg L − 1 respectively. The blank was taken as the signal of Rhod-5N (10− 6 mol L − 1) in MOPS at pH 7. Due to a better signal over noise ratio, the detection limit was found better than the one previously found by the classical method using quartz cuvettes [18] and is only twice larger than the maximal permissible concentration of 0.2 μg L − 1 than will be fixed in 2013 in the E U.
Fig. 1. Variation of measured fluorescence intensity of Rhod-5N (10− 6 M) at the output of the microfluidic circuit (long microchannel) for different concentrations of cadmium perchlorate added in one channel of the microchip (the Cd2+ concentrations inside the microchannel are given above every signal).
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Fig. 2. Comparison between fluorescence signals for reactants mixed before introduction in the microdevice with a short channel (external mixing: Mixing OUT) and inside the microdevice (internal mixing: Mixing IN).
In spite of its very good sensitivity towards cadmium ions, Rhod-5N suffers from at least one drawback: it is also sensitive to lead (Pb2+) ions, as shown in Fig. 2. Indeed, the selectivity, defined as the ratio of the apparent stability constants of the 1:1 complexes formed with the metal ions K (Cd2+)/K (Pb2+) is only 8 [18]. The selectivity of Rhod5N was found good versus calcium and zinc which are generally interfering with cadmium. Calcium is abundant in most of drinkable waters and zinc has similar properties with cadmium and is generally detected with cadmium. Furthermore, as can be seen in Fig. 2, Pb 2+ ions also induce an increase of the fluorescence of Rhod-5N with a complex rapidly formed and emitting with a fluorescence quantum yield almost three times lower than the cadmium complex [18]. As lead is also a dangerous heavy metal whose determination is as important as determination of cadmium, a method is proposed to discriminate both ions.
impossible in our case, as between pH 7 and 9, both metal ions were complexed by dithizone. Thanks to the affinity of sulphur and nitrogen atoms for several metal cations, silica functionalized by thiol or amino groups has been frequently used for extraction of mercury, lead, zinc, etc. [22,23]. After introduction in a column, this mesoporous silica can be used as a preconcentrator for heavy metal cations which is coupled with very efficient analytic methods like AAS or ICP. Very low quantities of analytes can then be detected [24,25]. Silica functionalized by aminopropyl triethoxysilane (SiO2-APTES) has been used for pre-concentration of zinc, lead, cadmium and nickel [26]. At pH 7, it has been shown that all lead ions introduced in the minicolumn are adsorbed by silica. The reversibility of the adsorption process is obtained by decomplexation at acidic pH (Scheme 4). The adsorption isotherms of both Cd(II) and Pb(II) ions on this modified silica sorbent material were determined using batch experiments at room temperature. Solutions of cadmium(II) perchlorate or lead(II) thiocyanate were shaken in a plastic vial with SiO2-APTES in Millipore water at neutral pH for 1 h. After centrifugation at 10,000 rpm during 10 min, the metal ions in the supernatant were determined by a fluorimetric titration experiment in a quartz cuvette by using Rhod-5N as the complexing molecule and the amount Nf of adsorbed heavy metal (in mol g− 1) was calculated using Eq. (1) as follows: Nf ¼
V ðC 0 −C s Þ m
where C0 is the initial concentration of heavy metal ions in mol L − 1, CS the equilibrium concentration of heavy metal ions in mol L − 1 and m the mass of the modified silica gel in g. Fig. 4 clearly shows the increase of adsorbed ions with the increase of the ion concentration in solution, towards a plateau value which is higher for Cd2+ than for Pb2+. Adsorption isotherm can be described by using the Langmuir model which leads to Eq. (2) [27]: CS CS 1 ¼ þ Nf NS b NS
3.2. Interference with lead ions: towards a fluorimetric method to discriminate lead and cadmium ions in water Methods to separate interfering cations include liquid phase extraction (LPE) or solid phase extraction (SPE). In the literature, it has been proposed to use dithizone ligand dissolved in chloroform to separate Cd2+ from Pb 2+, Cu2+,Co2+ or Ni2+ [21]. However, the optimization of pH for the extraction process of cadmium and lead appeared to be
Fig. 3. Calibration curve for the determination of Cd2+ ions by Rhod-5N 10− 6 mol L− 1 in MOPS buffer at pH 7. Inset: enlargement plot for low concentration of cadmium.
ð1Þ
ð2Þ
where b is the Langmuir constant in L.mol− 1 , NS is a constant representing the maximum adsorption capacity, also known as monolayer coverage of the surface (in mol g− 1). The linear plots of Cs/Nf versus Cs (Fig. 5) show that the adsorption of cadmium and lead satisfactorily obeys the Langmuir model. The values of both parameters b and Ns resulting from these plots are gathered in Table 1. The Langmuir constant, which is the ratio of the adsorption rate versus the desorption rate, is much higher for Pb 2+ than for Cd2+, which reflects a better affinity of SiO2-APTES for lead. On the opposite, the maximum adsorption capacity is twice higher for cadmium ions, which could be partly due to the smaller size of Cd2+ ion (0.194 nm) compared to Pb 2+ (0.238 nm). Considering the better affinity of the functionalized silica for lead compared to cadmium ions, we realized a SPE of both ions in buffer solutions at different pHs between 7 and 9 in order to optimize the separation. For this purpose, we used a minicolumn with packed silica beads connected to a syringe (see Experimental section). Buffered solutions of Pb2+ or Cd2+, or a mixture of both, were run through the minicolumn at a flow rate of 2 mL h− 1 and the metal content of the eluted solution was analyzed by fluorescence spectroscopy in quartz cuvettes using Rhod-5N at the same pH as a fluorescent probe. The experiments were repeated at several metal concentrations in the range 5 ×10− 8 to 5× 10− 7 mol L− 1. As shown in Fig. 6, the recovery of both Cd2+ and Pb2+ ions in the eluant is dependent on pH. For example, at pH 8.4, Pb2+ ions are completely adsorbed, compared to less than 50% adsorption for Cd2+. At pH 8.4, a mixture of both Cd2+ and Pb2+ ions was submitted to the two steps described before : i) a SPE through a minicolumn of SiO2‐ APTES at pH 8.4; ii) fluorescence detection in the microdevice described
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H2N
H2N
NH2
NH2 H2N
Si
NH2
171
NH2
Pb2+
Adsorption
H2N
Si
Desorption
NH2
NH2
Pb2+
NH2
NH2
Scheme 4. Adsorption and desorption of Pb2+ ions on functionalized silica SiO2-APTES.
before, with Rhod-5N as the fluorescent sensor. Fig. 7 presents the calibration curves obtained after this procedure for the determination of cadmium ions in absence and in presence of two different concentrations of Pb2+ ions. The cadmium concentrations plotted on the abscissa axis are the initial cadmium solutions introduced in the minicolumn. The deviation of the calibration curves in presence of lead don't differ from the one obtained without lead by more than 15% for a Pb2+ concentration ten times higher than Cd2+.
detection with the previously described microfluidic device. Aqueous solutions of different interfering cations at different concentrations and at neutral pH were eluted through the column and the fluorescence of Rhod-5N was then measured in the microfluidic device. The values of metal ion concentrations and the obtained fluorescence intensities are gathered in Table 2. No significant effect on this intensity was observed upon addition of these interfering cations. 4. Conclusion
3.3. Effect of interfering cations With practical application in mind, the effect of different interfering cations was evaluated by SPE adsorption followed by fluorescence
Combination of an off-line SPE and a fluorimetric detection in a microfluidic device has demonstrated effective cadmium ion determination and this is the basis for an integrated SPE-microfluidic detection system.
Fig. 4. Adsorption isotherms for Cd2+(left) and Pb2+ (right) ions on SiO2-APTES in Millipore water at neutral pH.
Fig. 5. Fits of the Langmuir adsorption model (see Eq. (2) in the text) for cadmium (left) and lead (right) ions.
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Table 1 Langmuir parameters for the adsorption of Cd2+ and Pb2+ ions on SiO2-APTES.
2+
Cd Pb2+ a b
Ns (mmol/g)a
b (L/mol)b
1.11 0.57
6.3 × 102 9.7 × 104
Ns is the maximum number of metal ions covering the porous silica. b is the Langmuir constant.
Table 2 Effect of interfering cations on the fluorimetric determination of cadmium. IF is the fluorescence intensity measured in the device after column adsorption. Cation
[M2+]/μmol L− 1
IF/A.U.
Cd2+ Ca2+ Ca2+ + Cd2+ Zn2+ Zn2+ + Cd2+ Cu2+ Zn2+ + Cd2+
0 0.1 1 1 + 0.1 5 5 + 0.1 5 5 + 0.1
1 1.34 1.02 1.37 0.96 1.29 1.01 1.37
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Fig. 6. Recovery of Cd2+ and Pb2+ ions after flowing through a minicolumn of SiO2-APTES. The points correspond to a mean value for concentrations in the range of 5 × 10− 8– 5 × 10− 7 mol.L− 1.
The reached detection limit (4 nM, 0.45 μg.L − 1) is below the present European maximum admissible value in drinking water and only twice larger than the limit for 2013. As it does not use heavy, fragile or voluminous components, our setup including the minicolumn filled with silica beads can be integrated in a portable apparatus. In conclusion, several advantages emerged in this microanalysis system: a high sensitivity has been obtained with a commercial fluorescent molecular sensor, and a method using a commercial sorbent material has been developed to obtain a good selectivity.
Fig. 7. Calibration curves for the determination of cadmium perchlorate by Rhod-5N in a phosphate buffer at pH 8.4 after solid phase extraction through a minicolumn of SiO2APTES, in the presence of different concentrations of lead(II) thiocyanate in the initial solution: ● [Pb2+]o =0 mol.L− 1; ▴ [Pb2+]o =5×10−7 mol.L− 1; ♦ [Pb2+]o =10−6 mol.L− 1.
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