Label-Free Fluorescence Detection of Aromatic Compounds in Chip

Article pubs.acs.org/ac

Label-Free Fluorescence Detection of Aromatic Compounds in Chip Electrophoresis Applying Two-Photon Excitation and TimeCorrelated Single-Photon Counting Reinhild Beyreiss,† David Geißler,† Stefan Ohla,† Stefan Nagl,† Tjorben Nils Posch,‡ and Detlev Belder*,† †

Institut für Analytische Chemie, Universität Leipzig, Linnéstraße 3, 04103 Leipzig, Germany Forschungszentrum Jülich GmbH, Zentralinstitut für Engineering, Elektronik und Analytik (ZEA), Analytik (ZEA-3), 52425 Jülich, Germany

‡

ABSTRACT: In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by timecorrelated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying twophoton excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

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the implementation of two-photon excitation (TPE). Thereby, the fluorophore is excited by interaction with two photons of a wavelength from the visible region, allowing the use of common glass optics and the application of economic but non-UVtransparent chip materials, such as borosilicate glass (BG), cyclic olefin polymer (COP), or polydimethylsiloxane (PDMS). Further positive features of TPE are the small excitation volume, low background fluorescence, and reduced photodegradation. The simultaneous absorption of two photons was first described theoretically in 1931 by Göppert-Mayer31 and experimentally proven by Kaiser and Garett.32 A breakthrough was achieved by the development of two-photon fluorescence microscopy by Denk and co-workers.33 Two-photon induced fluorescence is widely used in microscopy34−38 and spectroscopy.39−42 It was also utilized in capillary electrophoresis (CE).43−46 However, this attractive detection technique has only rarely been applied in microfluidics. Dittrich et al. presented the determination of flow parameters in microfluidic channels via two-photon fluorescence cross-correlation spectroscopy.47 TPE microscopy was used for characterization of micromixers48 and immobilized worms.49 Microscopic investigations with two-photon excitation and fluorescence lifetime determination were performed in the context of mixing in

eaturing an enormous potential for high-speed analysis at minimal reagent consumption, microchip electrophoresis (MCE) has emerged as a widely used separation technique since its first introduction in 1992.1−5 One of the main challenges in miniaturized separation devices is the sensitive detection of minute analyte amounts. In this context, established approaches are mainly based on electrochemical,6 mass spectrometric,7,8 and, in particular, optical9−11 techniques. Absorbance detection is commonly used in capillary electrophoresis (CE); however, it is limited in sensitivity when using chip-based methods because of the short optical path lengths. In contrast, fluorescence detection offers superior sensitivity, as well as selectivity.9,12,13 Accordingly, techniques based on fluorescence are frequently applied in microfluidics. However, in most cases, additional labeling steps are necessary to mark analytes with suitable fluorophors. This procedure is often laborious and results in complex reaction mixtures if analytes possess more than one functional group.14 Moreover, the modification with a fluorophore changes the chemical properties of the molecule of interest. A feasible way to avoid labeling procedures is to use light of the deep UV spectral region for excitation, enabling label-free fluorescence detection of various analytes, ranging from small aromatics to proteins.15−17 This approach has been utilized in various formats in CE18−24 as well as in MCE.25−30 Major drawbacks of this methodology include steep requirements concerning optical transparency of chip materials and optical components. Nevertheless, it is possible to circumvent the need for fused-silica (FS) chips and lenses via © 2013 American Chemical Society

Received: April 15, 2013 Accepted: August 15, 2013 Published: August 15, 2013 8150

dx.doi.org/10.1021/ac4010937 | Anal. Chem. 2013, 85, 8150−8157

Analytical Chemistry

Article

microfluidic droplets50 and DNA−dye interactions.51 These publications underline the benefits of two-photon excitation using a small excitation volume and high spatial resolution; however, they necessitated the use of fluorescent labels for detection. When used at a wavelength in the lower visible range, TPE can be an attractive alternative to one-photon excitation (OPE) in the deep UV spectral region. Intrinsic fluorescence detection of serotonin in live cells using one-, two-, and three-photon excitation at various wavelengths was investigated by Balaji et al.52 and later extended to the investigation of serotonin and 5hydroxytryptophan in live cells using time-correlated singlephoton detection by Botchway and co-workers.53 Zugel et al. reported on a chip-based electrophoretically mediated microanalysis using time-resolved two-photon induced fluorescence detection.54 Label-free detection with two-photon excitation of electrophoretically separated analytes using microchip electrophoresis was presented by Schulze and co-workers.55 Therein, small aromatics as well as proteins could be detected with labelfree steady-state fluorescence detection in fused silica and borosilicate glass chips using 420 nm as the excitation wavelength. However, this approach had to use an expensive and elaborate technique to maintain the frequency-doubled Ti/ sapphire femtosecond laser as the excitation source. TPE can also be accomplished using mode-locked picosecond solid-state laser sources as already demonstrated by Bewersdorf and Hell in 1998.56 These lasers are more economic and more stable compared to femtosecond Ti/ sapphire sources and are, thus, preferred for many applications of miniaturized analytical systems. Moreover, they can be efficiently employed at 532 nm, which is the two-photon equivalent of the commonly used 266 nm excitation wavelength for many applications of intrinsic UV fluorescence in CE and MCE. Herein, we demonstrate sensitive TPE of small aromatic compounds in MCE using ∼12 ps pulses of a frequencydoubled Nd/YVO4 laser. This approach is combined with timecorrelated single-photon counting (TCSPC) detection that we have recently introduced in MCE for label-free detection with excitation at 266 nm.57 TCSPC constitutes an additional dimension in the electropherogram using fluorescence lifetimes and allows for background discrimination and peak assignment.58−60 Thus, we can demonstrate for the first time sensitive, noninvasive, and label-free detection and inherent compound assignment of small aromatic compounds in MCE with excitation in the visible spectral range. We applied the developed methods for microchip analyses of harmala alkaloids present in plant extracts. These substances belong to the class of indole alkaloids and can stimulate the central nervous system by acting as monoamine oxidase inhibitors.61,62 They are used in hallucinogenic herbal mixtures like the Ayahuasca tea.63 Posch et al. developed a method based on nonaqueous CE with mass spectrometric detection to identify these compounds in plant materials.64 Huhn et al. demonstrated that those substances are suited for fluorescence detection with excitation at 266 nm utilizing a CE system.65

phenoxy-1,2-propanediol was purchased from TCI Europe (Zwijndrecht, Belgium). Harmol hydrochloride dihydrate was from ABCR (Karlsruhe, Germany). Sodium tetraborate, sodium phosphate, and sodium hydroxide were bought from Merck (Darmstadt, Germany). Harmine and tryptamine were obtained from Alfa Aesar (Karlsruhe, Germany). Methanol was from Carl Roth (Karlsruhe, Germany). All chemicals were used as received. For the preparation of stock and buffer solutions, doubly distilled and demineralized water was used if not stated otherwise. Stock solutions of alkaloids were prepared in methanol. All biogenic drug samples were provided by the Federal Criminal Police Office of Germany, KT-34 (Wiesbaden, Germany). All solutions were filtered through a 450 nm syringe filter before introduction into the chip. Instrumentation. Label-free fluorescence lifetime detection with 532 nm as the excitation wavelength was accomplished using a setup based on the MicroTime 200 platform (PicoQuant, Berlin, Germany) on an IX71 microscope (Olympus, Hamburg, Germany). A 20 MHz Nd/YVO4 laser (Cougar, Time-Bandwidth Products, Zürich, Switzerland) with pulse widths ∼12 ps was utilized in the second harmonic and incorporated as the excitation source, as sketched out in Figure 1. The laser beam was expanded via lenses and guided by

Figure 1. Schematic drawings of the instrumental setup used for MCE with laser-induced time-resolved fluorescence detection and of a utilized microfluidic chip: BS, beam splitter; C, crystal; DM, dichroic mirror; F, filter; L, lens; N, circular neutral density filter; PD, photodiode; PMT, photomultiplier tube; SPAD, single-photon avalanche diode; λ/2, half wave plate.

several optics into the microscope. The excitation light was focused by filling the back aperture of an objective onto the microfluidic channel. Emitted light was collected by the same objective. After passing the dichroic mirror, the collimated emission was sent through several lenses to a photon-counting photomultiplier tube (PMA 165-N-M, PicoQuant) or a singlephoton avalanche photodiode (SPAD, PDM series, Micro Photon Devices (MPD), Bolzano, Italy). When operating with both detectors simultaneously, the emission light was chopped by a 50/50 beam splitter. Laser intensity at 532 nm was controlled via a rotatable half wave plate before the frequencydoubling β-barium borate crystal within the laser and a variable neutral density filter wheel (Thorlabs, Dachau, Germany) within the excitation pathway. The laser beam was reflected by a Z532RDC dichroic mirror, and power was monitored via a 90/10 beam splitter and a photodiode (both from PicoQuant) that was equipped with a ND2 filter before the dichroic mirror (filter from AHF Analysentechnik, Tübingen, Germany, mounts from PicoQuant and Thorlabs).

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EXPERIMENTAL SECTION Chemicals. Serotonin hydrochloride, L-tryptophan, harmaline, harmane, norharmane, and harmalol hydrochloride dihydrate were obtained from Sigma-Aldrich (Steinheim, Germany). Propranolol hydrochloride, α- and β-cyclodextrin were from Fluka (Buchs, Switzerland). The chemical 38151



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Measurements with 532 nm excitation were performed with a 40x LUCPlanFLN objective (NA = 0.6, Olympus). For fluorescence detection, a ZT532SPRDC dichroic mirror (AHF Analysentechnik) and a DUG11 filter (band-pass 295−385 nm, Schott, Mainz, Germany) or a band-pass filter 420−480 nm (Olympus) were applied. For 266 nm excitation, the same setup with some modification along the optical path was employed, as described in detail previously.57 Briefly, we used a 40x fused-silica objective (NA = 0.8, Partec, Münster, Germany), a Z266RDC dichroic mirror (AHF Analysentechnik), a DUG11 emission filter, and a 150 μm diameter round pinhole. Optimal tube lens and z-position on the microscope stage were determined for OPE, as well as TPE, before measurements. This was achieved by filling the microfluidic channel with the analyte of interest and maximizing the detected fluorescence intensity. Chip electrophoresis was performed using a bipolar fourchannel high-voltage power supply (model HCV 40M-10000, FuG Elektronik, 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. The chips were mounted within a homemade low-conductivity carrier plate on the xy translational stage of the microscope. Fused-silica microchips with a cross injector layout were from iX-factory (Dortmund, Germany). The outer dimensions were 90 × 15 × 1.8 mm. The microchannels were 20 μm in depth and 50 μm in width at the top. At the end of each channel, access holes were implemented in the cover lid to contact the microfluidic structure. These microreservoirs are designated sample inlet (SI), sample outlet (SO), buffer inlet (BI), and buffer outlet (BO). Borosilicate glass chips with the same chip layout were from Micronit Microfluidics (Enschede, Netherlands). Microchip Electrophoresis. The microfluidic structure was treated with 0.1 M NaOH for 5 min before the measurements. Afterward, the chip was thoroughly rinsed with water and the respective separation electrolyte. Between analyses, all channels were rinsed with separation electrolyte. For MCE experiments, the channels and reservoirs were filled with separation buffer first. Then, the buffer in one reservoir was substituted with analyte solution for sample introduction. After optically focusing on the desired detection point within the chip, high voltage according to a pinched injection66 program was applied. Samples were focused for 25 s applying the following potentials in kV: BI 0.68, BO 2.00, SI 0.75, and SO 0.00. Separation parameters in kV were BI 2.40, BO 0.00, SI and SO 2.04 or BI 6.00, BO 0.00, SI and SO 5.10 for separations with an effective separation field strength of 264 and 661 V/cm, respectively. Data Acquisition and Analysis. The detected photons were recorded by a TCSPC module (PicoHarp 300, PicoQuant). The analyses were controlled and evaluated using the SymPhoTime software (version 5.3.2.2, PicoQuant). Electropherograms were created by plotting the number of counted photons versus time. The integration time interval was set to 10 ms, which corresponds to a data acquisition rate of 100 Hz. For lifetime histogram construction, a region of interest of the electropherogram was set interactively. The area where the recorded intensity had reached at least 5% of its maximal peak height within bins of 10 ms was used for evaluation of fluorescence lifetimes. Fluorescence lifetimes were calculated via monoexponential tail-fitting. The fluorescence lifetime electropherogram in Figure 2 was obtained by fitting

Figure 2. (a) Electropherogram with respective fluorescence lifetime histograms of a mixture of serotonin (1), propranolol (2), and tryptophan (3) obtained at a concentration of 1 mM each; separation buffer: borate buffer (20 mM, pH 9.2); effective separation length: 13 mm; effective field strength: 264 V/cm; λex: 532 nm, 400 mW; λem: 295−385 nm. (b) Fluorescence lifetime electropherograms with bins of 100 ms; threshold for lifetime determination: 5 kcounts/s.

the photon counts in ranges of 100 ms using a threshold of 500 photons per 100 ms. For determination of migration times and electrophoretic resolutions, the software Clarity (version 3.0.4.444, DataApex, Prague, Czech Republic) was used. Limits of detection were extrapolated by linear fitting with OriginPro 8G SR4 (v8.0951, Origin Lab, Northampton, USA). Plant Sample Extraction. Ten milligrams of the dried and ground plant material was dissolved in 100 μL of methanol. After 20 min of ultrasonication, the samples were centrifuged at 13 000 rpm for 10 min. The supernatant was removed and diluted with separation electrolyte prior to electrophoretic analyses.64 Safety Considerations. The employed high voltage and laser sources (laser class 4) used in this work are potentially harmful. The laser radiation can cause permanent eye damage, and the use of dedicated protection devices is obligatory. Radiation in the deep UV spectral region can induce cancer upon exposure. Sodium tetraborate and its respirable dusts are toxic. All mandated and recommended safety precautions should be followed.

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RESULTS AND DISCUSSION The stimulation of intrinsic fluorescence by 266 nm excitation allows label-free detection of a wide variety of analytes. In the context of microfluidics, the requirements imposed on optics and chip materials concerning the optical quality and especially the optical transparency in the UV region are quite high. The materials of choice for this wavelength region are usually fused 8152



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Table 1. Migration Times (tm) in s, Electrophoretic Resolutions (R), and Fluorescence Lifetimes (τ) in ns with Standard Deviations (n = 3) of Small Aromatics Obtained in MCE Experiments. Conditions As Shown in Figure 2 for TPE: FS, FusedSilica Chip; BG, Borosilicate Glass Chip FS

BG

analyte

tm (s)

R

τ (ns)

tm (s)

R

τ (ns)

serotonin propranolol tryptophan

17.0 ± 0.3 17.4 ± 0.2 18.9 ± 0.3

3.2 ± 0.1 6.9 ± 0.2

3.37 ± 0.05 8.83 ± 0.39 3.36 ± 0.06

16.8 ± 0.3 17.2 ± 0.3 18.7 ± 0.3

2.8 ± 0.0 6.4 ± 0.1

3.39 ± 0.06 9.00 ± 1.07 3.34 ± 0.03

silica for microfluidic chips and fused silica or calcium fluoride for refractive optical components, along with dedicated coatings. However, these material requirements can be circumvented when employing the corresponding two-photon excitation process in the visible range. In order to develop methods for microchip electrophoresis with time-resolved fluorescence detection after two-photon excitation at 532 nm, we first investigated a mixture consisting of the neurotransmitter serotonin, the β-blocker propranolol, and the amino acid tryptophan. Those compounds are moderately fluorescent when excited at 266 nm. In initial electrophoretic experiments, we utilized experimental conditions based on earlier work with one-photon excitation at 266 nm.57 This included a UV-transparent 40x objective (NA = 0.8), a fusedsilica chip, a pinhole of 150 μm, and a photomultiplier tube detector. One advantage of TPE in this context is the much wider selection range of suitable objectives in the visible spectral region. Additionally, they often possess superior imaging properties compared to rare UV-transparent objectives needed for OPE at 266 nm. Furthermore, due to the small excitation volume in TPE and the negligible background fluorescence, a pinhole is superfluous. By using a common achromatically corrected objective (40x, NA = 0.6) and omitting the pinhole, we observed improved signal-to-noise ratios (SNR) by a factor of about 10 (data not shown). Thereafter, the laser intensity was optimized for further improving the detection sensitivity. A typical electropherogram of the test mixture obtained after method optimization is shown in Figure 2. A noticeable characteristic is the very low background fluorescence. That is in accordance to the mechanism of TPE where fluorescence is induced only at and near the focal volume. All three substances were baseline-separated within 15 s using a borate buffer (20 mM, pH 9.2), an effective separation field strength of 264 V/ cm, and a separation length of only 13 mm. The applied laser power was approximately 400 mW. The obtained migration times and calculated electrophoretic resolution for peak pairs are given in Table 1. Due to the utilized time-correlated singlephoton counting mechanism, additional information about fluorescence decay characteristics were gathered on-the-fly. The respective fluorescence lifetime histograms are given as insets in Figure 2. With these data, the corresponding fluorescence lifetime for each signal in the electropherogram was easily accessible by monoexponential tail-fitting. The determined fluorescence lifetimes given in Table 1 are in good agreement with those determined previously utilizing one-photon excitation at 266 nm.57 For propranolol, the determined value after TPE was within the error range of the value obtained using OPE. For serotonin and tryptophan, the differences of the mean fluorescence lifetimes with respect to the value obtained with OPE were 6 and 18%, respectively. Fluorescence lifetimes could be determined reliably by TPE at all investigated concentrations from 6.25 to 100 μM with

standard deviations typically on the order of 0.2 ns or less. Fluorescence lifetime determinations at lower concentrations close to the limits of detection (LOD) were not attempted within this study. However, it is expected that with few available photons the significance of the fluorescence decay curve for compound identification will decrease rapidly close to the LOD. A direct comparison of electropherograms obtained with OPE and TPE is difficult because the respective optimal experimental conditions are different. On the one hand, the laser intensity has to be much higher for TPE in order to achieve the necessary high photon densities. On the other hand, objectives with superior imaging performance can be used for two-photon excitation. Although a fair comparison is difficult, Figure 3 shows electropherograms obtained with OPE and

Figure 3. Separations of serotonin (1), propranolol (2), and tryptophan (3) obtained with OPE (black) and TPE (red); separation buffer: borate buffer (20 mM, pH 9.2); effective separation length: 13 mm; effective field strength: 264 V/cm; λem: 295−385 nm; OPE (black): 10 μM each; λex: 266 nm, 100 μW; pinhole: 300 μm; TPE (red): 100 μM each; λex: 532 nm, 400 mW, no pinhole.

TPE, utilizing the same fused-silica chip but different sample concentrations, resulting in similar signal-to-noise ratios. Although the electropherograms are of similar appearance, with respect to the separation performance, it is obvious that the obtained signal intensities are considerably higher for one OPE, and the background fluorescence is significantly lower in TPE at 532 nm. The resulting signal-to-noise ratios for both excitation modes were 92 ± 13, 140 ± 6, and 53 ± 6 for serotonin, propranolol, and tryptophan using OPE and 93 ± 12, 129 ± 37, and 44 ± 7 after TPE, respectively (n = 3). One of the most attractive features of UV fluorescence detection using TPE is the possibility of using chips and optics made of non-UV-transparent materials like borosilicate glass or organic polymers. In order to examine this aspect, we performed respective electrophoretic separations utilizing 8153



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Table 2. Calculated LODs (SNR = 3) in mol/L for OPE and TPE Using a FS or BG Microchip, (A) Tight Sample Focusing Conditions (As Used in Shown Separations), (B) Optimized Sample Focusing Parameters To Increase Sensitivity A

B

excitation

microchip

TPE TPE OPE TPE TPE OPE

BG FS FS BG FS FS

serotonin (3.83 (3.32 (2.79 (3.14 (5.05 (7.29

± ± ± ± ± ±

0.37) 0.10) 0.20) 0.15) 0.09) 0.61)

propranolol

× 10−6 × 10−6 × 10−7 × 10−8 ×10−8 ×10−10

borosilicate glass chips. The geometry and dimensions of the microfluidic channels, as well as the separation and detection conditions, were identical to previous studies using fused-silica devices. We obtained a similar separation performance on both chip types. The obtained plate numbers using fused silica as chip material were approximately 283 000, 169 000, and 79 000 for serotonin, propranolol, and tryptophan, respectively. Plate numbers using a borosilicate glass chip were 26 7000, 182 000, and 55 000, respectively. A comparison of migration times and obtained peak resolutions is given in Table 1. There was no indication of a significant influence of the chip material on the electrophoretic separation, as well as on the determined fluorescence lifetimes. While the separation performance is quite similar in both chips, one interesting aspect in comparing borosilicate glass with fused-silica chips for TPE at 532 nm is the achievable detection sensitivity. For this purpose, we analyzed serial dilutions of the mixture in triplicate with both chip types. The resulting signal-to-noise ratios were calculated, and the limits of detection (LOD) at a SNR of 3 were determined via extrapolation. As listed in Table 2, we obtained detection limits in the low micromolar range for TPE in both chip types. A comparison of results obtained with TPE and OPE utilizing fused-silica chips reveals that the sensitivity is about 10 times better in the case of OPE at 266 nm. The LODs for serotonin and propranolol determined in these experiments were about a factor of 20 better compared to previous work by Schulze et al. using TPE at 420 nm.55 A direct comparison of sensitivity values with those from the literature is difficult, however, because the experimental parameters, as for example the injected sample volume, have a great impact. In order to evaluate the achievable sensitivity in MCE separations of the model compounds, we optimized the injection parameters in order to gain maximum sensitivity at sufficient electrophoretic resolution (R > 1). For that purpose, the focusing parameters of the pinched injection program were altered in order to inject larger sample plugs. This affected the separation performance, resulting in reduced plate numbers of 3000, 2000, and 21 000. With these sensitivity optimized focusing parameters, the limits of detection for serotonin and propranolol using two-photon excitation could be improved by 2 orders of magnitude from 3.32 and 2.37 μM to 51 and 37 nM, respectively. For tryptophan, the enhancement was from 6.80 μM to 277 nM. Although these analytes do only feature relatively small twophoton absorption cross sections, they can be detected in MCE with good sensitivities down to the nanomolar range. Realistically, some analytes that are detectable via 266 nm excitation could feature too small two-photon cross sections to be amenable for this methodology with reasonable sensitivities. However, these initial results are quite encouraging with respect

(3.79 (2.37 (2.10 (6.50 (3.68 (8.96

± ± ± ± ± ±

0.37) 0.07) 0.03) 0.11) 0.10) 0.31)

×10−6 ×10−6 ×10−7 ×10−8 ×10−8 ×10−10

tryptophan (9.96 ± 0.75) (6.80 ± 0.12) (6.13 ± 0.31) (2.83 ± 0.33) (2.77 ± 0.14)

Label-Free Fluorescence Detection of Aromatic Compounds in Chip

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