Mercedes
Vázquez
ab,
Celeste
Frankenfeld
b,
Wendell K. Tomazelli
Coltro
bc,
Emanuel
Carrilho
c,
Dermot
Diamond
a and
Susan M.
Lunte
*b
aCentre for Bioanalytical Sciences, National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Dublin, Ireland
bRalph N. Adams Institute for Bioanalytical Chemistry, MRB, The University of Kansas, Lawrence, KS 66047-1620, USA. E-mail: slunte@ku.edu; Fax: +1 785 864 1916; Tel: +1 785 864 3811
cInstituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil
First published on 10th November 2009
A new approach for the integration of dual contactless conductivity and amperometric detection with an electrophoresis microchip system is presented. The PDMS layer with the embedded channels was reversibly sealed to a thin glass substrate (400 μm), on top of which a palladium electrode had been previously fabricated enabling end-channel amperometric detection. The thin glass substrate served also as a physical wall between the separation channel and the sensing copper electrodes for contactless conductivity detection. The latter were not integrated in the microfluidic device, but fabricated on an independent plastic substrate allowing a simpler and more cost-effective fabrication of the chip. PDMS/glass chips with merely contactless conductivity detection were first characterized in terms of sensitivity, efficiency and reproducibility. The separation efficiency of this system was found to be similar or slightly superior to other systems reported in the literature. The simultaneous determination of ionic and electroactive species was illustrated by the separation of peroxynitrite degradation products, i.e. NO3− (non-electroactive) and NO2− (electroactive), using hybrid PDMS/glass chips with dual contactless conductivity and amperometric detection. While both ions were detected by contactless conductivity detection with good efficiency, NO2− was also simultaneously detected amperometrically with a significant enhancement in sensitivity compared to contactless conductivity detection.
New trends in microfluidics, however, are focused on the coupling of two detection techniques in a single electrophoresis microchip rather than on the improvement of individual detection modes. Combination of the inherent properties of individual detection modes in a dual detection system has become a powerful tool to greatly enhance sample characterization and widen the range of application. However, not many publications have explored this concept to date. Obviously, the coupling of two different detection techniques in a single microfluidic device is not trivial and many parameters have to be considered, such as compatibility of both detection modes and ease of their integration with the microchip. Dual detection systems coupling fluorescence with light-scattering,6 and amperometry with LIF,7 C4D8 and electrochemiluminescence (ECL)9 have been reported so far. Additionally, a dual-electrode electrochemical detection was also introduced.10
In this work, a new approach for the integration of an electrophoresis microchip with dual C4D and amperometric detection is introduced. The coupling of C4D and amperometric detection has been reported earlier in connection with commercially available glass microchips.8 To the best of our knowledge, this is the first time that the successful integration of both detection techniques with an in-house hybrid PDMS/glass chip is reported. A description of the analytical characteristics of this new system and its advantages compared to the system presented by Wang et al.8 is presented in this report. Additionally, the hybrid PDMS/glass microchip with dual C4D and amperometric detection was used in the determination of peroxynitrite (ONOO−) degradation products. Peroxynitrite is a reactive oxidative species that forms within the body from the reaction of the free radicals nitric oxide (˙NO) and superoxide (O2˙−).11 Once formed, ONOO− rapidly decomposes to species that can indiscriminately oxidize and nitrate surrounding tissue. When the production of ONOO− is balanced by the scavenging actions of endogenous antioxidants, or when it is generated by the immune system to attack foreign microbes, the break-down of ONOO− is not harmful. However, in instances such as inflammation, excess ONOO− and its decomposition products can denature DNA,12 destroy lipid membranes13,14 and alter protein function.15,16 These destructive effects of ONOO− have been implicated in the development of atherosclerosis,17–19 diabetes,19,20 and neurodegenerative21 and autoimmune diseases.22,23
The precise degradation mechanism for ONOO− is still under debate,24–27 although it is well known that peroxynitrite degrades rapidly into its main metabolites, i.e., nitrate (NO3−) and nitrite (NO2−). A CE method for the separation and direct UV detection of peroxynitrite, nitrate and nitrite has been published elsewhere.28 However, direct detection of peroxynitrite is still very challenging due to its extremely short lifetime, and most analytical methods are based on the detection of its degradation products.26 Here, we present a new method for the separation and detection of NO3− and NO2− resulting from degradation of peroxynitrite using a hybrid PDMS/glass chip with C4D and amperometric detection. Both anions can be detected by C4D; however, only nitrite can be detected by amperometry as it is the only electrochemically active species from the two anions. We found that the coupling of C4D and amperometric detection allowed a more comprehensive analysis of peroxynitrite samples.
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Fig. 1 Fabrication of copper electrodes for C4D detection (A) and palladium electrode for amperometric detection (B). |
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Fig. 2 Layout of the hybrid PDMS/glass chip with C4D (A) and C4D coupled in line with amperometric detection (B). Chip dimensions: (A) 70 × 30 mm and (B) 75 × 30 mm. Channel dimensions: (A) 20 mm-long injection channel, 50 mm-long separation channel, 150 μm wide and 15 μm deep; (B) 20 mm-long injection channel, 57 mm-long separation channel, 30 μm wide and 12 μm deep. WE: working electrode; RE: reference electrode; CE: counter electrode. |
The design of the PDMS/glass chip described above was then slightly modified in order to integrate both detection modes, i.e., C4D and amperometric detection, in the same chip. In this case, a glass substrate (0.4 ± 0.1 mm thickness) containing a Pd electrode (section 2.2.2) was reversibly sealed to the PDMS layer with the Pd electrode facing the PDMS layer (Fig. 2B). The alignment of the Pd electrode at 5–20 μm from the end of the separation channel was done with the help of an optical microscope. The channel dimensions of the chip (75 × 30 mm) were the following: injection and separation channels were 20 mm and 57 mm long, respectively, 30 μm wide and 12 μm deep.
Stock solutions of NO2− and NO3− at 10 mM concentration were freshly prepared in water each week from their corresponding sodium salts, and kept at 4 °C between measurements. Different run buffers containing tetradecyltrimethylammonium hydroxide (TTAOH) as electroosmotic flow (EOF) modifier were tested for the analysis of standards containing both anions dissolved in water. Successful separation of NO2− and NO3− was finally accomplished by using 30 mM lactic acid–0.75 mM TTAOH at pH 3.5 as run buffer.
Peroxynitrite (ONOO−) was freshly synthesized in-house prior to analysis following the acidified nitrite and hydrogen peroxide reaction.29 After synthesis, the concentration of peroxynitrite was calculated based on the UV absorbance at 302 nm at which maximum absorbance for peroxynitrite occurs. The resulting reaction product had a concentration in peroxynitrite of ca. 60 mM. The separation of NO2− and NO3− resulting from the degradation of peroxynitrite was achieved by using 30 mM lactic acid–0.75 mM TTAOH at pH 3.5.
A high voltage power supply (Jenway, Essex, England) with 4 channels (0 kV ± 5 kV) and controlled by a computer was used for the electrokinetic injection and flow control. Channels were preconditioned with buffer solution for 10 min prior to use. Gated injection was employed for all electrokinetic injections due to its flexibility regarding sample plug size and its reproducibility relative to unpinched injection. Thus, a high voltage was applied to the buffer reservoir in order to fill the injection channel, and a fraction of that voltage (0.67) was applied to the sample reservoir while keeping sample and buffer waste reservoirs grounded. Sample injection was then performed by floating the voltage at the buffer reservoir for 1 s, which drove the sample plug into the separation channel. The voltage at the buffer reservoir was then resumed to allow sample separation. All the experiments were performed at room temperature (23 ± 2 °C).
Mixtures of sodium, potassium and lithium cations dissolved in water at equimolar concentrations were injected by applying 0.8 kV for 1 s along the injection channel. The electrophoretic separation of the target cations was achieved by applying 1.2 kV along the separation channel using 20 mM MES/His, pH 6.1, as run buffer. As can be seen in Fig. 3A, separation of the three cations at 500 μM (each) was achieved within 40 s with very reproducible migration times (RSD < 0.96%, n = 4). Additionally, well defined and resolved peaks were obtained under these conditions. The plate number per meter (N/m) calculated for those peaks by the 5-sigma method was 4.9 × 104 for potassium, 6.6 × 104 for sodium, and 9.5 × 104 for lithium. Thus, the separation efficiency of this system is similar or slightly superior to other systems reported in the literature (4.6–5.4 × 104 plates/m for sodium in ref. 38 and 4.3 × 104 plates/m for potassium in ref. 39). Limits of detection of 31 μM, 35 μM and 50 μM were estimated (signal to noise ratio = 3) for potassium, sodium and lithium, respectively, based on the response observed for a mixture containing the target cations at 50 μM (see Fig. S-1 in the ESI†). Lower detection limits in the range of 11 μM to 25 μM were found upon increasing the injection time from 1 s to 3 s (see Fig. S-2 in the ESI†). One of the possible causes for the slightly lower sensitivity of our system compared to others reported in the literature using polyester-toner,37 PMMA38,40 or glass39 chips is related to the thickness of the layer separating the sensing electrodes (copper) from the sample/electrolyte flowing in the microchannel. Specifically, a glass substrate of ca. 400 μm was used to produce the hybrid PDMS/glass chip employed in this work (Fig. 2A). This glass substrate served also as a physical separation between the sensing electrodes and the microchannel and, thus, the distance between sensing electrodes and microchannel was larger than in those other devices (400 μm versus 100 μm in ref. 37, 125 μm in ref. 38,10–15 μm in ref. 39, and 100 μm in ref. 40). Therefore, in order to increase the sensitivity using hybrid PDMS/glass chips with C4D, the use of a glass substrate with thickness ≤ 200 μm33 and/or the application of a higher excitation voltage33,38,40 is recommended. The former can be simply attained by using commercially available microscope cover slides of the appropriate width and length.
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Fig. 3 Electrophoretic separation of a 500 μM equimolar mixture of potassium, sodium and lithium chloride dissolved in water (A) and a 500 μM equimolar mixture of chloride, nitrate and perchlorate dissolved in run buffer (B). Separation parameters: run buffer, 20 mM MES/His, pH 6.1; injection voltage, 0.8 kV (A) and −0.8 kV (B); separation voltage, 1.2 kV (A) and −1.2 kV (B); injection time, 1 s; separation channel effective length, ca. 35 mm. C4D parameters: frequency, 500 kHz; excitation voltage, 10 Vpeak-to-peak. |
Additionally, equimolar mixtures of chloride, nitrate and perchlorate anions dissolved in run buffer (20 mM MES/His, pH 6.1) were injected at −0.8 kV (injection time, 1 s), and subsequently separated by applying −1.2 kV along the separation channel. As can be seen in Fig. 3B, separation of the three anions at 500 μM (each) was achieved within 65 s (RSD < 0.7% for three consecutive injections). Once more, the sensitivity of this system for the anions tested was found to be lower than in other systems reported earlier.40 Limits of detection of 79 μM, 67 μM and 77 μM were estimated (signal to noise ratio = 3) for chloride, nitrate and perchlorate, respectively, based on the response observed for a mixture containing the target anions at 100 μM (see Fig. S-3 in the ESI†). Wider peaks were observed for the anions compared to the cations tested (Fig. 3A), which could be caused by a larger contribution of diffusional band broadening as a result of longer migration times.40 However, the resolution was still good enough (Rs = 1.2) to allow a satisfactory separation of the target anions with rather good efficiencies (5.5 × 104 plates/m for chloride, 7.9 × 104 plates/m for nitrate and 13.0 × 104 plates/m for perchlorate).
The fabrication of the palladium electrode for amperometric detection (section 2.2.2) and its integration in the hybrid PDMS/glass chip was rather straightforward, and made the construction of a special chip/electrode holder unnecessary. The latter represents a clear advantage as compared to other systems using screen-printing electrodes as working electrodes for amperometric detection.8 Moreover, replacement of the PDMS/glass chips in the event of any channel fouling was easily done by peeling off the used PDMS layer from the glass containing the working electrode and reversibly sealing a new PDMS layer.
The selection of the background electrolyte (BGE) for this application was not trivial. BGEs of low conductivity are typically used for conductivity detection because they enhance sensitivity by maximizing the difference in conductivity between the analyte zone and the carrier electrolyte.41 However, buffers commonly used for amperometric detection are highly conducting and, thus, generate significant baseline noise when used for conductivity detection, with a consequent loss of sensitivity. The separation of NO2− and NO3− further complicates buffer selection due to their similar electrophoretic mobilities.42
Optimization of the separation conditions for the determination of ONOO− degradation products was carried out by using standards. A mixture of nitrate and nitrite anions dissolved in water was injected at −1.0 kV (injection time, 1 s) and then separated by applying −1.5 kV along the separation channel. In order to reverse the EOF, a cationic surfactant was added to the run buffer. Considering that TTAOH had been previously used for the separation of nitrate and nitrite by CE with contact conductivity detection,43 that was the EOF modifier of choice. As shown in Fig. 4, the separation of nitrate (1 mM) and nitrite (5 mM) anions was finally achieved in less than 50 s (RSD < 1.2% for three consecutive injections) by using 30 mM lactic acid–0.75 mM TTAOH, pH 3.5, as run buffer. This BGE exhibited the appropriate conductivity and ionic strength to allow a good separation by C4D (Fig. 4A). The effect of pH on the separation was proved to be very significant. No satisfactory resolution was achieved by using the same buffer composition at lower pH (pH 2.5 vs. pH 3.5). Therefore, the pH, near the pKa of NO2−, was essential for the separation of both ions by microchip electrophoresis with C4D. However, a better signal-to-noise ratio was observed for the NO2− peak by amperometric detection (Fig. 4B), together with flat baselines and low noise levels. Limits of detection were estimated (signal to noise ratio = 3) based on the response observed for a mixture containing 1 mM nitrite and 0.2 mM nitrate. Limits of detection found for nitrite with C4D (Fig. 4A) and amperometric detection (Fig. 4B) were 308 μM and 20 μM, respectively. The detection limit estimated for nitrate based only on C4D was 67 μM, in good agreement with the value previously calculated in section 3.1. The increased sensitivity shown by amperometric detection as compared to C4D is consistent with the LOD values reported in the literature for electrophoresis microchips.2 Good separation efficiency was found for C4D with 3.8 × 104 plates/m for nitrite and 4.5 × 104 plates/m for nitrate. It should be noted that the effective length of the separation channel for amperometric detection (ca. 47 mm) was longer than for C4D (ca. 42 mm) what resulted in a shift of the nitrite peak towards longer migration times for amperometric detection (Fig. 4). This peak shift can be prevented by transforming the time-based x-axis electropherograms obtained for both detectors into the corresponding effective mobility-scaled x-axis,44 as shown in the ESI†.
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Fig. 4 Electrophoretic separation of a mixture of 5 mM NO2− and 1 mM NO3− in water detected simultaneously by: A) C4D (frequency, 450 kHz; excitation voltage, 15 Vpeak-to-peak; effective length, ca. 42 mm); B) amperometric detection (voltage applied to the working electrode, 850 mV; effective length, ca. 47 mm). Separation parameters: run buffer, 30 mM lactic acid–0.75 mM TTAOH, pH 3.5; injection voltage, −1.0 kV; separation voltage, −1.5 kV; injection time, 1 s. |
Finally, the peroxynitrite sample synthesized in-house (ca. 60 mM) was analyzed by using the separation conditions optimized for nitrate and nitrite standards. Peroxynitrite is very unstable at pH lower than 12.28 Therefore, at pH 3.5, peroxynitrite degrades rapidly into its main metabolites, i.e., NO2− and NO3−. As can be seen in Fig. 5, the concentrations of NO2− and NO3− generated from the peroxynitrite degradation differed significantly. While a well-defined peak was observed for NO3−, both detectors showed distorted peaks for NO2− indicating nitrite overloading. However, further dilution of the peroxynitrite sample was not attempted as that could have prevented the satisfactory determination of nitrate by C4D. As shown in Fig. 4, a shift of the nitrite peak towards longer migration times is also observed in Fig. 5 for amperometric detection (effective length, ca. 47 mm) compared to C4D (effective length, ca. 42 mm). See the corresponding effective mobility-scaled electropherograms in the ESI†.
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Fig. 5 Electrophoretic separation of NO2− and NO3− resulting from the degradation of peroxynitrite (ca. 60 mM) detected simultaneously by: A) C4D (frequency, 450 kHz; excitation voltage, 15 Vpeak-to-peak; effective length, ca. 42 mm); B) amperometric detection (voltage applied to the working electrode, 850 mV; effective length, ca. 47 mm). Separation parameters: run buffer, 30 mM lactic acid–0.75 mM TTAOH, pH 3.5; injection voltage, −1.0 kV; separation voltage, −1.5 kV; injection time, 1 s. |
The overall aim of this research was to demonstrate the suitability of the proposed microchip-based electrophoretic system for the detection of analytes with very different electrochemical properties in real samples, and thus, no attempt was made to accurately quantify the concentrations of NO2− and NO3− resulting from the degradation of peroxynitrite. However, it was still possible to obtain estimations of those by extrapolation of the calibration curves calculated for the nitrate and nitrite standards with the C4D detector. These were 1.1 mM for NO3− (calibration curve correlation coefficient, R = 0.9299) and 22.2 mM for NO2− (R = 0.9393), respectively.
Footnote |
† Electronic supplementary information (ESI) available: electropherograms showing the separation of cations at 50 μM (and 25 μM) and anions at 100 μM equimolar concentrations by C4D; electropherogram showing the separation of nitrite and nitrate at 500 μM and 100 μM, respectively, by simultaneous C4D and amperometric detection; effective mobility-scaled electropherograms showing the separation of nitrate and nitrite in a standard mixture and in a peroxynitrite sample. See DOI: 10.1039/b908985c |
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