Rapid Prototyping of Electrochromatography Chips for Improved Two

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Rapid Prototyping of Electrochromatography Chips for Improved Two-Photon Excited Fluorescence Detection Claudia Hackl, Reinhild Beyreiss, David Geissler, Stefan Jezierski, and Detlev Belder* Institut für Analytische Chemie, Universität Leipzig, Linnéstraße 3, 04103 Leipzig, Germany S Supporting Information *

ABSTRACT: In the present study, we introduce two-photon excitation at 532 nm for label-free fluorescence detection in chip electrochromatography. Two-photon excitation at 532 nm offers a promising alternative to one-photon excitation at 266 nm, as it enables the use of economic chip materials instead of fused silica. In order to demonstrate these benefits, one-photon and two-photon induced fluorescence detection are compared in different chip layouts and materials with respect to the achievable sensitivity in the detection of polycyclic aromatic hydrocarbons (PAHs). Customized chromatography chips with cover or bottom slides of different material and thickness are produced by means of a rapid prototyping method based on liquid-phase lithography. The design of thin bottom chips (180 μm) enables the use of high-performance immersion objectives with low working distances, which allows one to exploit the full potential of two-photon excitation for a sensitive detection. The developed method is applied for label-free analysis of PAHs separated on a polymer monolith inside polymer glass sandwich chips made from fused silica or soda-lime glass. The obtained limits of detection range from 40 nM to 1.95 μM, with similar sensitivities in fused silica thin bottom chips for one-photon and two-photon excitation. In deep-UV non- or lesstransparent devices two-photon excitation is mandatory for label-free detection of aromatics with high sensitivity.

T

achieved at about the double wavelength compared to common one-photon excitation (OPE), which shifts the excitation wavelength for aromatic molecules from the deep-UV to the visible spectral region. This opens up new avenues in lab-on-achip technology as it allows label-free fluorescence detection in non-UV transparent devices such as plastic or glass chips. Furthermore, as TPE probes only a very tiny sample volume, which is defined by the tiny focal volume of about a femtoliter, it is attractive for detection in very small microchannels and cavities. Two-photon excited fluorescence9,10 is currently mainly applied in the infrared spectral region for microscopic imaging11−15 of fluorescently labeled compounds and materials with high lateral resolution, increased penetration depth in tissues and reduced background. In this spectral region TPE has also been utilized as a detection technique in separation science such as in high-performance liquid chromatography (HPLC),16,17 capillary electrophoresis (CE),18−21 and microfluidics.22−28 The discussed potential of TPE as an alternative to OPE for label-free detection of aromatic compounds in microfluidic chips has however only rarely been explored. The electrophoretically mediated microanalysis with time-resolved two-

he detection of miniature sample amounts in tiny microchannels is one of the main challenges in microfluidic lab-on-a-chip technology. The dominant technique in chip-based microfluidic separation devices is fluorescence detection utilizing fluorescence labeling to tag analytes with bright fluorescent dyes. While this approach works smoothly in electrophoretic analysis of polar or ionic biomolecules, this is more challenging in chromatographic separations of less polar compounds, lacking functional groups for simple labeling strategies. Furthermore, it has to be considered that an additional labeling step alters the chemical properties of analytes, which can be problematic (e.g., for additional downstream processes).1 In this context, fluorescence detection in the deep-ultraviolet (UV) spectral region is a valuable alternative, enabling labelfree detection of a wide variety of intrinsic fluorescent analytes such as small aromatics. This approach has been demonstrated in microchip electrophoresis (MCE)2−7 but rarely in chromatographic techniques such as in-chip electrochromatography (ChEC).8 However, label-free fluorescence detection via deep-UV excitation relies on UV-transparent optics and microchips made of fused silica, which currently limits the widespread applicability of this technique. In this context, twophoton excitation (TPE) is an interesting alternative. In TPE the promotion of the analyte into the excited state is caused by quasi simultaneous interaction with two photons. In practice this means that label-free fluorescence detection can be © 2014 American Chemical Society

Received: November 1, 2013 Accepted: March 25, 2014 Published: March 25, 2014 3773

dx.doi.org/10.1021/ac500793e | Anal. Chem. 2014, 86, 3773−3779

Analytical Chemistry

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photon excited fluorescence detection was investigated by Zugel and co-workers.29 Microchip electrophoresis (MCE) with label-free two-photon excited fluorescence detection of the separated analytes was carried out by Schulze et al.30 Beyreiss et al. reported on label-free detection in MCE using two-photon excitation and time-correlated single photon counting (TCSPC).31 These rare reports on two-photon excitation for label-free detection in microfluidics document the applicability for TPE in electrophoretic separations of predominantly ionic compounds. Even more appealing appears, however, the application to the analysis of less polar and uncharged compounds where both, potential fluorescence labeling and electrophoretic separation, are extremely challenging, if possible at all. A model application of high practical relevance is the analysis of polycyclic aromatic hydrocarbons (PAHs) as nonionic test solutes. PAHs are environmental pollutants and of particular concern because many of these substances are highly carcinogenic already at low concentrations, with maximum allowed contaminant levels in drinking water on the order of 1 nmol/L. Common methods for the analysis of PAHs employ HPLC coupled with UV or fluorescence detection and gas chromatography (GC) with mass spectrometry (MS).32−35 As there is a high demand for on-site environmental analysis of PAHs, the application of lab-on-a-chip technology for this purpose is very appealing.36−40 In the context of portable lab-on-a-chip devices, the use of electrochromatography is an attractive separation technique, as it is technically less demanding compared to HPLC on chips where high pressure pumps, valves, and pressure-tight connections are mandatory. Chip electrochromatography has been realized in substrates of various materials, such as fused silica,8,41 borosilicate glass,42 polyimide,43,44 polydimethyl siloxane,45−47 and cyclic olefin copolymer.48−52 Especially plastic devices with porous polymer monoliths41,53 (PPM) as stationary phases hold great promise for the realization of disposable high-performance separation devices. This is the starting point of our current work, where we explore the potential of two-photon excitation for label-free detection of PAHs in chip electrochromatography. A major motivation is a comparison of two-photon with common onephoton excitation in chip-based separation devices, which has so far not been investigated thoroughly. A challenge for a fair comparison of TPE versus classical OPE are, however, the differences in the individual optimal instrumental configurations. A technical hurdle in the realization of deep-UV onephoton excitation is, besides the use of cost-intensive fused silica chips, the limited availability of UV transparent optics, which is especially true for the extremely rare fused-silica objectives. On the other hand, with TPE exciting in the visible spectral region, there is a wide selection of high-performance objectives, including low distance objectives with high numerical apertures (NA). The desirable inclusion of such low distance objectives in the current study implies, however, the use of microfluidic chips with a very low bottom plate thickness, which are difficult to manufacture and not commercially available. An interesting approach for rapid prototyping is liquid phase lithography (LPL),54,55 where a microfluidic structure is formed from a photopolymer solution sandwiched in-between two slides. In the current work, we investigate LPL54,55 as a rapid and economic prototyping technique for a straightforward

manufacturing of electrochromatography chips utilizing bottom and top slides of different material and thickness for the intended fluorescence studies. This should allow us to conduct a currently missing comparison of one-photon and two-photon induced label-free fluorescence detection in chip-based separations at individually optimized parameters.

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EXPERIMENTAL SECTION Chemicals. Butyl acrylate, 1,3-butanediol diacrylate, poly(ethylene glycol) diacrylate (Mn 250), 3-(trichlorosilyl)propyl methacrylate, 3-(trimethoxysilyl)propyl methacrylate, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2,2-dimethoxy-2-phenylacetophenone, pyrene, benzo[a]pyrene, and chloroform were purchased from Sigma-Aldrich (Steinheim, Germany). Anthracene, benzo[a]anthracene, benzo[k]fluoranthene, fluoranthene, fluorene, naphthalene and 7-amino-4-methylcoumarin were obtained from Fluka (Buchs, Switzerland). Elastosil E43 was purchased from Wacker Chemie (Munich, Germany). Acetonitrile (Rotisolv HPLC Gradient grade), ethanol and nheptane were acquired from Carl Roth (Karlsruhe, Germany). Glacial acetic acid, ammonium acetate, ammonia (25% solution), sodium dihydrogen phosphate, and sodium hydroxide were purchased from Merck (Darmstadt, Germany). All chemicals were used as received. Buffer solutions were prepared using doubly distilled and demineralized water. Stock solutions of the PAHs with a concentration of 1, 2, or 4 mg/mL were prepared in acetonitrile. All solutions were filtered through a 450 nm syringe filter before introduction into the chip. Instrumentation. For time-resolved detection in microfluidic separation systems, a MicroTime 200 platform (PicoQuant, Berlin, Germany) based on an inverted microscope (IX71, Olympus, Hamburg, Germany) was used as described previously.56 A 20 MHz Nd:YVO4 laser (Cougar, Time-Bandwidth Products AG, Zurich, Switzerland), which can be operated at 266 nm as well as 532 nm, was used as an excitation source. For excitation at 532 nm, a 40× LUCPlanFLN objective (NA = 0.6, Olympus) or a 60× UPlanSApo objective [NA = 1.2 (water), Olympus] was applied. Measurements at 266 nm excitation were performed with a 40× fused silica objective (NA = 0.8, Partec, Münster, Germany) or a 40× LMU-40×-UVB (NA = 0.5, Thorlabs, Dachau/Munich, Germany). For more details regarding optical setup and data acquisition, please see the Supporting Information. For chip electrochromatography experiments, the chips were mounted on a homemade carrier plate on the x-y-translational stage of the microscope. High voltage was applied using a bipolar four-channel high-voltage power supply (model HCV 40M-10000, FuG Elektronik GmbH, Rosenheim, Germany) providing up to ±10 kV per channel. Electrical contact to the microfluidic structure was established via a homemade poly(methyl methacrylate) plate with integrated platinum electrodes. Wet-etched fused silica microchips with a cross channel layout were purchased from iX-factory (Dortmund, Germany). The chips had outer dimensions of 90 × 15 × 1.8 mm and microchannels of 50 μm in width and 20 μm in depth. Access holes were integrated in the cover lid to contact the microfluidic structure. Borosilicate glass chips of the same microfluidic layout were from Micronit Microfluidics (Enschede, Netherlands). 3774



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Figure 1. Chromatogram of a ChEC separation of 0.6 mM 7-amino-4-methylcoumarin (1), 3.1 mM naphthalene (2), 0.2 mM fluorene (3), 0.6 mM anthracene (4), 1.0 mM fluoranthene (5), 1.0 mM pyrene (6), 0.9 mM benzo[a]anthracene (7), 0.2 mM benzo[k]fluoranthene (8), and 0.2 mM benzo[a]pyrene (9). Chip: iX-factory fused silica, 3 cm monolith; eluent: MeCN/5 mM NH4OAc, pH 8.0 (4:1, v/v); separation field strength: 408 V/cm; λex: 266 nm, 265 μW; λem: 295−385 nm and 420−480 nm; and a 40× Partec objective. The lifetimes were obtained via monoexponential tailfitting of photon histograms, which were individually constructed for each peak (exemplarily shown for benzo[a]pyrene).

butanediol diacrylate, 685 μL of butyl acrylate and 5 μL of 3(trimethoxysilyl)propyl methacrylate were added. Finally, the polymerization mixture was purged with nitrogen for 10 min. The reservoirs of the chip were then filled with 40 μL of the polymerization mixture, and complete filling of the channels was ensured applying reduced pressure. Parts of the chip were covered with electrical tape to define the length of the monolith. The bubble-free filled chip was illuminated on a UV exposure unit (4 × 8 W, Gie-Tec GmbH, Eiterfeld, Germany) at 350−400 nm for 9 min, residual polymer in the reservoirs was removed thereafter. Finally, the chip reservoirs were filled with acetonitrile/5 mM ammonium acetate pH 8.0 (4:1 v/v) and connected to the high-voltage power supply. The monolith was flushed and conditioned with separation buffer for at least 10 min electrokinetically. Chips were stored in a fridge overnight and with vials filled with a separation buffer. Chip Electrochromatography Experiments. Prior to measurement as well as between the analyses, the reservoirs were filled with separation buffer, and the monolith was flushed electrokinetically. For ChEC experiments, the buffer in the sample inlet reservoir was replaced by analyte solution. Then the separation channel was focused at the desired detection point, which was directly behind the monolith in order to avoid damage of the monolith. A sample plug was directed into the separation channel utilizing a pinched injection program. For that purpose, the sample was focused in the cross for 30 s, followed by switching the voltage to the separation mode and injecting a sample plug into the separation channel. Voltage parameters for sample focusing and injection into the separation channel were adjusted by monitoring the injection of a fluorescent dye. Separations were performed at effective field strengths, ranging from 200 to 800 V/cm. Safety Considerations. The employed high voltage and laser sources (laser class 4) used in this work are potentially harmful. As laser radiation can cause permanent eye damage, the use of specific eye protection is obligatory. Deep-UV radiation could induce cancer upon exposure. Some of the PAHs have been identified to be carcinogenic and teratogenic.

LPL-Chip-Production. Fused silica wafers 52 × 26 mm (height: 180 μm) were purchased from Valley Design (Shirley, MA). Fused silica wafers 100 × 100 mm (height: 1.1 mm) from iX-factory (Dortmund, Germany) were cut into slides of 76 × 26 mm to fit into used chip holders. Menzel microscope slides 76 × 26 mm (height: 1.0 mm) out of soda-lime glass were purchased from Thermo Scientific (Braunschweig, Germany). Chips were produced by means of a liquid-phase lithography (LPL) initially introduced by Beebe et al.,54 which was further developed in our working group to create microfluidic free-flow devices.57,58 With the use of this procedure, it was possible to gain poly(ethylene glycol) diacrylate sandwich chips with a cross channel layout featuring a 30 mm separation channel and 6 mm long injection channels of 200 μm width and 25 μm height that were suitable for chip electrochromatography. The chips had lids of 1.0 to 1.1 mm thickness and bottom-slides with 180 μm to 1.1 mm out of fused silica or soda-lime and borosilicate glass, respectively. More details regarding the production of the LPL chips can be found in the Supporting Information. Monolith Preparation. Prior to usage, wet-etched chips were cleaned with 1 M NaOH, water, and ethanol, and the surface was functionalized to ensure covalent binding of the monolith to the channel walls. Therefore, a mixture of 50% (v/v) ethanol, 30% (v/v) glacial acetic acid, and 20% (v/v) 3(trimethoxy-silyl)propyl methacrylate was filled into the chip and left for 30 min. Afterward, the solution was removed by applying reduced pressure and the channels were subsequently washed with ethanol and dried in air. Chips prepared by liquid phase lithography were used without an additional silanizing step. Prior to polymerization glass reservoirs with a volume of ∼70 μL were mounted on the chips using silicone adhesive (Elastosil E43). Porous polymer monoliths were prepared according to Ngola et al.,59 using a modified procedure. Five milligrams of 2,2dimethoxy-2-phenylacetophenone and 5 mg of 2-acrylamido-2methyl-1-propanesulfonic acid were dissolved in 2010 μL of a mixture of 60% (v/v) acetonitrile, 20% (v/v) ethanol, and 20% (v/v) 5 mM phosphate buffer, pH 6.8. Then, 297 μL of 1,33775



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All mandated and recommended safety precautions should be followed.

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RESULTS AND DISCUSSION A central aspect of the initial work was to individually optimize the instrumental parameters for one-photon excitation at 266 nm and for two-photon excitation at 532 nm. As a starting point for employing one-photon excitation (OPE) at 266 nm in electrochromatography experiments, we utilized an optical setup that was based on an earlier work, utilizing a UVtransparent 40× fused silica objective (NA = 0.8) from Partec, a 150 μm pinhole, and a photomultiplier tube detector.56 During optimization of two-photon excitation (TPE), we observed that a common objective (40× LUCPlanFLN, NA = 0.6) for visible light gives superior results in TPE compared to the fused silica objective. We also optimized the laser intensity to gain maximum signal-to-noise ratios (SNRs) using microfluidic chips filled with the analyte solution. In these initial comparing studies, we used fused silica chips with a wet-etched channel structure as the material is UV-transparent and can be applied for both one-photon and two-photon excitation. Monolithic stationary phases for electrochromatography based on butyl acrylate were prepared in these devices by UV-induced photopolymerization referring to an approach by Fintschenko and co-workers.8,59 The length of the separation bed was 3.0 cm, and the monolithic structure was generated in the separation channel as well as in the injection cross. Thus, sample plugs of about 50 pL were injected into the separation channel applying a pinched injection program. As analytes, we chose a mixture of eight PAHs dissolved in acetonitrile which can be excited in the deep-UV spectral region.8 A typical chromatogram of an electrochromatographic separation of eight PAHs with time-resolved label-free fluorescence detection using 266 nm as excitation wavelength is shown in Figure 1. All substances are baseline-separated within 2 min, using acetonitrile and 5 mM ammonium acetate pH 8.0 (4:1, v/v) for isocratic elution at an effective field strength of 408 V/cm. For the baseline separated peaks, fluorescence lifetimes were calculated via monoexponential tailfitting of the respective photon histogram. An exemplary photon histogram of benzo[a]pyrene and the determined fluorescence lifetime is given as insert in Figure 1. Timeresolved detection was found to be very useful to facilitate the assignment of the individual peaks due to significant differences in fluorescent lifetimes of the analytes (see Table S-1 of the Supporting Information). While one-photon excitation worked quite well, we investigated thereafter the excitation at 532 nm utilizing a TPE process. A typical chromatogram is shown in Figure 2; it can be noted that background fluorescence is considerably lower compared to that of OPE. This is in good accordance to the TPE mechanism as fluorescence is only induced near the focal point. While one-photon excitation resulted in superior signal intensities for most of the analytes, as expected, TPE showed higher sensitivities for anthracene and pyrene, which can be explained by reflections that TPE is stronger for centrosymmetric molecules.60 Thereafter, we manufactured electrochromatogaphic columns in borosilicate glass chips with only limited transparency in the deep-UV spectral region and performed analogous experiments with one- and two-photon excitation. The obtained SNRs of measurements via OPE and TPE in borosilicate glass are summarized in Table 1, and corresponding

Figure 2. Chromatogram of a ChEC separation of 0.6 mM 7-amino-4methylcoumarin (1), 3.1 mM naphthalene (2), 2.4 mM fluorene (3), 0.6 mM anthracene (4), 1.0 mM fluoranthene (5), 1.0 mM pyrene (6), 0.9 mM benzo[a]anthracene (7), 0.2 mM benzo[k]fluoranthene (8), and 0.2 mM benzo[a]pyrene (9). Chip: iX-factory fused silica, 3 cm monolith; eluent: MeCN/5 mM NH4OAc pH 8 (4:1, v/v); separation field strength: 408 V/cm; λex: 532 nm, 300 mW; λem: 270− 465 nm, > 700 nm; and a 40× Olympus LUCPlanFLN objective.

Table 1. Obtained Signal-to-Noise Ratios for Separations Using Borosilicate Glass Chips (n = 2): Conditions as in Figures 1 (OPE) and 2 (TPE)a

a

analyte

OPE

7-amino-4-methylcoumarin anthracene fluoranthene pyrene benzo[a]anthracene benzo[k]fluoranthene benzo[a]pyrene

13 ± 2 n. d. 18 ± 4 5 ± 0.1 19 ± 3 18 ± 4 19 ± 5

TPE 122 151 30 78 131 151 128

± ± ± ± ± ± ±

17 13 3 15 6 13 11

OPE: 26.5 μW, TPE: 300 mW.

chromatograms can be found in Figure S-1 of the Supporting Information. It can be seen, that TPE yields better SNRs in borosilicate glass microchips. We were surprised to detect some signals at all when exciting at 266 nm, but as expected, the sensitivity with TPE is by far superior as the borosilicate glass is perfectly transparent at 532 nm. With the described setup using TPE, we studied the chromatographic performance of the polymer monolith in the fused silica chip, varying the separation field strength between 208 and 608 V/cm. Minimum plate heights were obtained at 208 V/cm, ranging from 3.5 to 4.5 μm. This is in good accordance with literature data.8 For details, see Figure S-2 and Table S-2 of the Supporting Information. The experiments presented so far utilized fused silica or glass chips, which are, in part, commercially available. For the intended more thorough comparison of TPE and OPE, it would be interesting to utilize high NA objectives, which should be favorable for the TPE process. Such objectives (NA ≥ 1.2), which are not available for the deep-UV spectral region, have however rather low working distances and are therefore not compatible with the relatively thick bottom plates of the utilized fused silica (1.1 mm) or glass (1 mm) chips. Respective thinbottom chips are, however, not commercially available, which is 3776



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especially true for corresponding fused silica devices due to the lack of appropriately sized fused silica substrates. While such wet-etched glass chips or fused silica chips are of high optical quality with perfect microfluidic structures, they are cost intensive and accordingly less-suited for the development of novel chip layouts and set-ups where rapid and economic prototyping of several chip generations is essential. An ideal technique in this context is liquid-phase lithography (LPL), where microfluidic structures are defined within a photopolymer placed between two slides54,55 without the need for sophisticated equipment or clean room facilities. In this context, the present technique is especially attractive, as it gives straightforward access to chips with cover and bottom plates of various thicknesses and materials. In the present study, we further developed this prototyping technique for producing sandwich-chips with fused silica and soda-lime glass substrates. Microfluidic chips with different bottom plate heights (180 μm to 1.1 mm) were fabricated, and a 2 cm monolithic column could be integrated analogous to the procedure with wet-etched glass chips. The thin bottom chips allowed the use of a highperformance objective (60× UPlanSApo water-immersion objective), with small working distance and high numerical aperture. In comparison to the wet-etched chips, the chips prepared by LPL possessed channels of 200 μm in width and 25 μm in height and a separation channel of 3 cm in length. Due to the channel layout, injection volumes of about 900 pL were obtained for LPL chips. The manufactured chips with different bottom slides were investigated with regard to the achievable detection sensitivity. For these studies, we utilized a simplified test mixture containing the five PAHs from Figure 2 with the most intense fluorescence. A corresponding chromatogram of a separation on a LPL-based chip made from fused silica with a thin bottom slide (180 μm) is shown in Figure 3. At the experimental conditions, applying a field strength of 800 V/cm benzo[a]pyrene was baseline-resolved while the other four analytes were partially separated. As we could show that the LPL-technique gives straightforward access to functional electrochromatographic chips with free choice bottom slides, we further studied the detection performance of these chips, applying one- or two-photon excitation. For a better judgment of the thin bottom approach, we also included common wet-etched fused silica chips in this study. The results from various electrochromatographic runs using the two chip types and detection techniques are summarized in Table 2. For both chip types, the optical setup has been optimized at first. The laser intensity was adjusted to 400 mW for TPE and 690 μW for OPE to obtain maximum signal-tonoise-ratios. For the thin bottom LPL chip, a 40× objective (Thorlabs, NA = 0.5) was employed for OPE while a 60× immersion objective [Olympus, NA = 1.2 (water)] was utilized in the TPE experiments. The experiments with the wet-etched fused silica chips were performed with a 40× objective (Olympus, NA = 0.6) in TPE mode and a 40× objective (Partec, NA = 0.8) for OPE detection. In order to determine the limits of detection, different concentrations of the analyte mixture were prepared via serial dilution and measured in triplicate. With the LPL-chip, we obtained limits of detection in the range from 45 nM to 2.04 μM, depending on the compound. From Table 2, it is evident that with thin bottom chips made of fused silica, one can get similar sensitivities for one-photon or two-photon excitation at

Figure 3. Chromatogram obtained on a fused silica chip with 180 μm bottom plate prepared by LPL. Separation of 285 μM 7-amino-4methylcoumarin (1), 70 μM anthracene (2), 495 μM fluoranthene (3), 125 μM pyrene (4), 110 μM benzo[a]anthracene (5), and 20 μM benzo[a]pyrene (6). Two centimeter monolith, eluent: MeCN/5 mM NH4OAc, pH 8.0 (4:1, v/v); separation field strength: 800 V/cm; λex: 532 nm, 400 mW; λem: 270−465 nm, >700 nm; and a 40× Olympus LUCPlanFLN objective.

individually optimized parameters, including the chip. Taking into account that in TPE only an extremely small volume portion of the microfluidic channel is probed, this is remarkable and indicates that TPE exhibits a high potential for sensitive detection in submicrometer channels. Compared to standard methods for the PAH analysis, we do not reach the limit of detection (LOD) obtained by HPLC with fluorescence detection, which are in the middle picomolar range34,35 but can keep up with those from GC/MS.32,33 Nevertheless, the LOD obtained with TPE in the LPL chip are in the range of those reported by Benhabib et al., using the portable McMOA.36 The experiments in wet-etched FS chips revealed lower sensitivities with LOD ranging from 1.82 to 150 μM. Due to the difference in channel dimensions and, by that, injection and detection volumes, a quantitative comparison of the LPL and wet-etched chips with regard to sensitivity data is difficult. Furthermore, we used our method to prototype LPL-chips with common soda-lime glass slides. We were able to detect our analytes in this nondeep-UV-transparent material by applying TPE with SNRs, ranging from 39 to 52 with concentrations and parameters as in Figure 3. In summary, we developed a technique for fast prototyping of electrochromatography chips, offering a free selection of cover and bottom slides, which allows one to optimize the ray path for optical detection in microfluidic chips in a fast and economic way. It shows that the combination of thin bottom chips with high-quality objectives, which are not available in the deep-UV spectral region, makes TPE a very interesting and sensitive technique for label-free detection in microfluidics.

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CONCLUSION We demonstrated the application of label-free time-resolved fluorescence detection in microchip electrochromatography, together with a rapid prototyping process for chip manufacturing. We developed a rapid prototyping process for electrochromatography chips with varying bottom slide materials 3777



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Table 2. Determined Limits of Detection (SNR = 3) for Investigated PAHs in Wet-Etched and Homemade Fused Silica Chipsa wet-etched chip FS LOD (n = 3) anthracene fluoranthene pyrene benzo[a]anthracene benzo[a]pyrene

OPE (266 nm) 90.3 25.2 21.8 8.70 2.12

± ± ± ± ±

7.0 μM 1.7 μM 1.1 μM 0.49 μM 0.07 μM

LPL chip FS, thin bottom

TPE (532 nm) 14.5 150 29.0 18.8 1.82

± ± ± ± ±

0.8 μM 15 μM 1.9 μM 1.0 μM 0.13 μM

OPE (266 nm) 1.86 617 406 170 65

± ± ± ± ±

0.22 μM 44 nM 22 nM 10 nM 3 nM

TPE (532 nm) 262 2.04 400 358 45

± ± ± ± ±

4 nM 0.10 μM 47 nM 39 nM 6 nM

Two centimeter monolith; eluent: MeCN/5 mM NH4OAc, pH 8.0 (4:1, v/v); separation field strength: 800 V/cm; OPE: λex = 266 nm, 690 μW, and λem: >266 nm; TPE: λex = 532 nm, 400 mW, λem: 270−465 nm, and >700 nm.

a

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based on liquid phase lithography. Polymer glass sandwich chips made of soda-lime glass as well as fused silica were successfully utilized for optimizing two-photon excited fluorescence detection. Applying a thin bottom fused silica polymer sandwich chip, sensitivities in the nanomolar range were achieved for the detection of polycyclic aromatic hydrocarbons. We could show that at individually optimized conditions label-free two-photon excitation at 532 nm can reach similar sensitivities as one-photon excitation at 266 nm. While labelfree OPE is restricted to fused silica substrates, two-photon excitation can be applied to detect UV fluorophores in quite diverse materials (e.g. soda-lime glass) and potentially also polymer chips.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +49-341-9736115. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Deutsche Forschungsgemeinschaf t DFG (Project BE 1992/8−1). C.H. thanks the Studienstiftung des deutschen Volkes for the scholarship.

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Rapid Prototyping of Electrochromatography Chips for Improved Two

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