Free Flow Electrophoresis Device for Continuous On-Line Separation

was performed by using inverted Y-type low dead-volume mixing units and 75-μm-i.d. fused-silica capillary reaction coils (SGE, Ringwood, Victoria...
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Anal. Chem. 2000, 72, 3881-3886

Free Flow Electrophoresis Device for Continuous On-Line Separation in Analytical Systems. An Application in Biochemical Detection M. Mazereeuw, C. M. de Best, U. R. Tjaden,* H. Irth, and J. van der Greef

Division of Analytical Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands

A free flow electrophoresis (FFE) device was developed for continuous electrophoretic separation of charged compounds and implemented in a continuous flow biochemical detection (BCD) system. These continuous separation characteristics make FFE well suitable for online implementation in a chromatographic or flow injection analysis system, in which an additional separation step of charged compounds is desired. In a heterogeneous biochemical flow assay for the determination of biotin, an analyte zone reacts with an excess of an affinity protein. Subsequently, the free binding sites of the affinity protein react with an excess of fluorescein-labeled ligand. Free and affinity protein-bound label are separated on the FFE device prior to fluorescence detection of the separated fractions. Biotin and streptavidin were chosen as, respectively, model ligand and affinity protein. Since all the compounds that are involved possess different electrophoretic properties, quantitative analysis is performed after completely separating the fluorescent affinity complex and labeled biotin in the FFE device within 2 min. Since the device is optically transparent, the separated zones can be detected in the separation compartment, using laser-induced fluorescence. The applicability of the BCD-FFE system in combination with a HPLC separation is demonstrated in the bioanalysis of biotin in human urine at the micromole per liter level. In analytical chemistry, frequently different techniques are hyphenated to create sophisticated and sometimes rather complicated systems for sensitivity and selectivity enhancement. The complexity of the total system can limit the applicability, owing to the limited operational conditions that are necessary for optimal functioning of the individual processes. In such situations, an intermediate separation step is necessary. Typical examples are the combination of liquid separation systems with mass spectrometry1,2 or biochemical reaction detection systems.3,4 Such (1) Gysler, J.; Mazereeuw, M.; Tjaden, U. R. van der Greef, J. Anal. Chem., submitted. (2) Lamoree, M. H.; van der Hoeven, R. A. M.; Tjaden, U. R.; van der Greef, J. J. Mass Spectrom. 1998, 33, 453. (3) Oosterkamp, A. J.; Irth, H.; Villaverde Herraiz, M. T.; Tjaden, U. R. van der Greef, J. Anal. Chem. 1996, 68, 1201. (4) Lutz, E. S. M.; Irth, H.; Tjaden, U. R. van der Greef, J. J. Chromatogr., A 1996, 755, 179. 10.1021/ac991202k CCC: $19.00 Published on Web 07/11/2000

© 2000 American Chemical Society

combinations operate within a relative small window of conditions, restricting the performance of the separation method, or demand additional separation steps to allow optimal detection properties. Free flow electrophoresis (FFE) is an electrophoretic separation technique that is capable of continuously separating ionic mixtures in streams of the individual compounds.5-7 The separation chamber is a flat compartment between two parallel plates separated by a spacer through which a laminar hydrodynamic flow is generated. Perpendicularly to the flow direction, an electric field is generated in which an electrophoretic separation is feasible with separation times in the range of 1-2 min. These features make FFE suitable as an interfacing separation technique for compounds possessing different electrophoretic mobilities. The use of bioaffinity recognition in analytical chemistry as a sample pretreatment8-10 and flow injection immunoassay11-13 in CE separations14 or as a detection method15,16 is a rapidly growing area, offering unrivaled selectivity in the analytical procedures. Biochemical detection (BCD) in conjunction with liquid separations can be performed according to several strategies.14-16 Proteins with a high affinity to compounds of interest can be added to form a protein-ligand complex. The degree of complex formation is measured by adding a fluorescent-labeled ligand (label), which, owing to the short reaction time, occupies the remaining free binding sites of the affinity protein. As the free and bound label often possess equal fluorescent properties, both have to be separated prior to detection. The concentration of the analyte is related to the amount of free or bound label and can be determined after separation of the fluorescent compounds. (5) Barrolier, V. J.; Watzke, E.; Gibian, H. Z. Naturforsh. 1958, 13B, 754. (6) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem 1994, 66, 2858. (7) Hannig, K.; Heidrich, H.-G. Free Flow Electrophorsis; GIT Verlag: Darmstadt, 1990. (8) Nillson, B. J. Chromatogr. 1983, 276, 413. (9) Farjam, A.; van der Merbel, N. C.; Nieman, A. A.; Lingeman, H.; Frei, R. W.; Brinkman, U. A. Th. J. Chromatogr. 1992, 589, 141. (10) Cole, L. J.; Kennedy, R. T. Electrophoresis 1994, 16, 549. (11) Gu ¨ bitz, G.; Shellum, C. Anal. Chim. Acta 1993, 283, 421. (12) Freytag, J. W.; Lau, H. P.; Wadsley, J. Clin. Chem. 1984, 30, 1494. (13) Whelan, J. P.; Kusterbeck, A. W.; Wemhoff, G. A.; Bredehorst, R.; Ligler, F. S. Anal. Chem. 1993, 65, 3561. (14) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18. (15) Oosterkamp, A. J. Ph.D. thesis, Leiden University, Leiden, The Netherlands, 1996. (16) Lutz, E. S. M. Ph.D. thesis, Leiden University, Leiden, The Netherlands 1998.

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Several strategies are available to perform such a separation. However, it is necessary to use a technique that allows continuous separations in order to be coupled on-line to the separation and flow reaction system. Oosterkamp et al. successfully used a short column packed with restricted-access support3,17 or affinity support18,19 in an on-line postcolumn system in combination with HPLC. After the postcolumn reaction, the free fluorescent label was trapped in the restricted access material or affinity material, while the protein-analyte complex was not retained and detected. Although the use of a short column is robust and versatile, it is not a truly continuous flow separating system and demands regular regeneration. A hollow fiber module was developed by Lutz et al. and successfully applied for postcolumn separation in a flow, containing protein-analyte complexes and unbound compounds.4,20 A pressure drop was applied over the membrane, resulting in an active and diffusion-independent separation. Compounds with a molecular mass lower than the cutoff mass of the membrane can pass the membrane unhindered and are separated from larger compounds. Also the application to the separation of proteins immobilized on column material is shown with potential applications in combinatorial chemistry.20 Although this approach is of a continuous nature, contamination of the membrane or incomplete separation can be expected. In this paper, a FFE device is described, which is used as an on-line electrophoretic separation step of fluorescent compounds in a BCD system. The BCD system is based on the interaction of the affinity protein streptavidin with biotin, in which biotin acts as an analyte. During the separation, on-device detection of the fluorescent compounds is performed with laser-induced fluorescence detection. The BCD-FFE system was on-line coupled with HPLC for the bioanalysis of biotin in human urine. EXPERIMENTAL SECTION Chemicals. Polyoxyethylene sorbitan monolaurate z.s. (Tween 20) and D(+)-biotin were purchased from Merck (Schuchardt, Germany). R,R,R-Tris(hydroxymethyl)methylamine (Tris) was supplied by Aldrich-Chemie (Steinheim, Germany). Hydroxypropylmethylcellulose (HPMC), sodium fluorescein, fluoresceinbiotin, and streptavidin were obtained from Sigma (Zwijndrecht, The Netherlands). Blocking reagent was supplied by BoehringerMannheim (Mannheim, Germany). All solution were made with HPLC grade water, which was produced in a milli-Q system (Millipore, Bedford, MA). Free Flow Electrophoresis Device. A drawing of the custommade FFE device is shown in Figure 1. The device consists of a top and bottom Plexiglas plate, each 205 × 110 × 15 mm, which are separated by 100 µm with two 50-µm spacers of polyimide (Dupont, Dordrecht, The Netherlands). The plates and the spacers are sandwiched with 12 bolts and nuts. In the bottom plate, several sections of 10 mm in depth are milled to form compartments for the electrodes, sample introduction, and buffer flow. The separation compartment is 20 mm in length and 23 mm in width and (17) Oosterkamp, A. J.; Villaverde Herraiz, M. T.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1996, 68, 1201. (18) Irth, H.; Oosterkamp, A. J.; van der Welle, W.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 633, 65. (19) Oosterkamp, A. J.; Irth, H.; Beth, M.; Unger, K. K.; Tjaden, U. R.; van der Greef, J. J. Chromatogr., B 1994, 653, 55. (20) Lutz, E. S. M.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1997, 69, 4878.

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Figure 1. Detailed presentation of the custom-made FFE device. Two plane-parallel Plexiglas plates are separated with a 100-µm spacer. In the drawing, a segment from the base plate is removed to show the three-dimensional setting of the electrode and buffer compartments: (1) sample inlet, (2) separation compartment, (3) electrode in compartment, (4) electrode membrane, (5) 100-µm spacer, and (6) inlet (7) outlet buffer compartments. Further details are given in the text.

formed by the surfaces of both plates and the spacer. The electrode compartments and the separation compartment are electrically connected through nitrocellulose membranes with a pore size of 0.8 µm (11304-47-ACN, Sartorius, Go¨ttingen, Germany). Parafilm (American National Can, Greenwich, CT) was used between the polyimide spacer and the nitrocellulose membranes to finish the connection leak-tight. The flow through the FFE device was generated with a peristaltic pump (Skalar Analytical, Breda, The Netherlands) at a flow rate of 60 or 160 µL/min, from the inlet buffer compartment through the separation compartment into the outlet buffer compartment. The carrier electrolyte for the FFE device consisted of 10 mmol/L pH 7.5 Tris-HCl buffer with 0.2% Tween 20. A 1 mol/L pH 7.5 Tris-HCl buffer was used in the electrode compartments. The sample is introduced via a Teflon tube with an inner diameter of 200 µm at various flow rates with a syringe pump (Harvard Apparatus Inc., So. Natick, MA) or a HPLC system (see below). Samples were injected using a six-port injection valve (7413, Rheodyne, Cotati, CA) with a 1-µL internal sample loop. A low-voltage power supply (model 1000/500, Bio-Rad, Veenendaal, The Netherlands) was used to apply 100-500 V over the separation compartment. During the separation, laser-induced fluorescence detection is performed using a water-cooled argon ion laser (model 2560, Spectra-Physics, Mountain View, CA) at 488 nm. To visualize the separation, a spreadout laser beam was produced with a lens to illuminate the entire separation compartment. For analytical measurements of the fluorescent signal at 530 nm, a fixed laser spot was used with a diameter similar to the width of the sample stream. An optical table was used to accurately position the FFE device. A photomultiplier tube, model TE 206RF, an air-cooling device (Products for Research Inc., Danvers, MA), and a high-voltage power supply and current amplifier (Keithly Instruments, Cleveland, OH) were used. The signals were recorded with a chart recorder (Kipp & Zonen, Delft,

The Netherlands) and a laboratory-developed integration package on a PC. The integration software and the analog-to-digital transformer were laboratory-build. HPLC-BCD-FFE. A 10-cm HPLC column with a 3-mm inner diameter was laboratory packed with 5-µm C18 material (Nucleosil, Macherey-Nagel, Du¨ren, Germany). The mobile phase was delivered with a HPLC pump (Gilson, Villiers le Bel, France) at a constant flow of 400 µL/min. The mobile phase consisted of 10 mmol/L pH 7.5 Tris-HCl buffer. Samples were injected using a six-port injection valve (Rheodyne) with a 20-µL sample loop. A UV absorbance detector (Spectroflow, Kratos, Ramsey, NJ) was used to monitor the column effluent. Downstream to the detector, a two-stage splitter was used to reduce the effluent flow from 400 to 1 µL/min. In the first stage, an adjustable restriction was used, whereas the second split contained a T-piece with a (16 cm) 150µm-i.d. fused-silica capillary as a restriction. Mixing of the effluent with the affinity protein, streptavidin, and fluorescein-labeled biotin was performed by using inverted Y-type low dead-volume mixing units and 75-µm-i.d. fused-silica capillary reaction coils (SGE, Ringwood, Victoria, Australia). The reagents were delivered using a syringe pump (Harvard Apparatus), and the reaction took place at room temperature. Urine Sample Pretreatment. Human urine was spiked with biotin at a concentration of 4 µmol/L. Blank and spiked urine samples were diluted 1:1 and filtered through a 0.2-µm membrane filter. Subsequently, the filtrate was injected into the HPLC system and analyzed with the BCD-FFE system. RESULTS AND DISCUSSION Design of the FFE Device. The developed FFE device consists of two parallel Plexiglas plates that are separated with a spacer of 100-µm thickness (see Figure 1). Through the separation compartment of 20 × 23 × 0.1 mm, a laminar flow is generated and perpendicular to the flow direction an electric field for zone electrophoretic separations. The space between the parallel plates was kept as small as possible to allow maximum electric field strength, with minimal Joule heating effects, such as gas bubble formation and protein degradation. Also, the FFE device was constructed from Plexiglas in which a low electroosmotic flow and minor protein adsorption was expected in combination with the used buffer system.21 The electrical connection between the electrodes and the separation compartment is of crucial importance and needs a large pressure resistance in combination with a high electrical conductivity. Ion-exchange membranes were tested. However, the material was too rigid and was pressed into the Plexiglas plates, causing leakage after renewal. Wet paper membranes of ∼100-µm thickness performed well. The paper is squashed without leaving any imprints on the Plexiglas and adapts to the correct proportions. Finally, nitrocellulose membranes with a 0.8-µm pore size were tested and used because of their superior conductive properties compared to paper. Moreover, due to the good conducting properties, a homogeneous electric field through the separation compartment can be generated. When high voltages are applied (.250 V), the liquid in the membranes is removed electrokinetically, which is visible as areas in the membranes with a slightly different color. This results in loss of electrical field strength in (21) Shao, X.; Shen, Y.; Lee, M. L. J. Microcolumn Sep. 1999, 11, 325.

the separation compartment and an unstable and not homogeneous electrical conduction. However, it was demonstrated that by applying voltages up to 250 V, no irregularities during the FFE separations appeared. As the FFE device is transparent, optical on-device detection of fluorescent compounds during the electrophoretic separation is feasible by illuminating either the entire separation compartment or a local spot. Initial experiments in order to visualize the performance of the FFE separation were performed using sodium fluorescein. The separation compartment was entirely illuminated with a diverged 30-mW laser beam of an argon ion laser at 488 nm. Sodium fluorescein was continuously infused and deflected toward the anode in an electric field of ∼100 V/cm. The carrier electrolyte consisted of 10 mmol/L pH 7.5 Tris-HCl buffer and 0.05% Tween 20, which is a surfactant to reduce the interaction of biomolecules with the surface of the separation compartment. The maintenance of a stable pH in the separation compartment during the FFE separation is of crucial importance as electrophoretic mobilities and the detection properties of the involved compounds are strongly pH dependent. Fluorescein, for example, occurs in several charged states of which only the anionic state, at high pH, has a high fluorescence yield.22 As protons and hydroxylic ions will be generated in the electrode compartments during the electrophoretic separations, a pH gradient is present in the separation compartment affecting the fluorescence signal of fluorescein. The pH gradient was visualized using a solution of bromphenol blue, which changed to yellow, indicating a pH lower than 3. However, formation of the pH gradient was effectively suppressed by using and regular renewal of 1 mol/L pH 7.5 TrisHCl buffer in the electrode compartments. Continuous Flow Biochemical Detection. In BCD systems, analytes are recognized and bound by a protein with a high affinity. An extensively studied and widely applied model for BCD studies is the biotin-streptavidin interaction23 in which biotin (B) or a biotin-labeled compound can be used as an analyte. Streptavidin (S) is a 60 kDa (pI ∼7) affinity protein with four biotin binding sites, forming strong complexes with a dissociation constant of ∼10-14 mol-1 and a half-life of ∼3 days (pH 7).24 Below, a typical BCD reaction scheme is given:

B + S T S-B + Sfree

(1)

Sfree + FB T S-FB + FBfree

(2)

S-B + FB f no product

(3)

The affinity protein streptavidin is added in excess to biotin (eq 1) to form strong streptavidin-biotin complexes (S-B) subsequently, an excess of fluorescein-labeled biotin, in the following denoted as fluorescein-biotin (FB), is added to react with all the free binding sites of the streptavidin (Sfree, eq 2) to form streptavidin-fluorescein-biotin complexes. Due to the short reaction time that is available (e.g., 1-2 min), fluorescein-biotin does not react with the saturated biotin-streptavidin complex (eq 3). (22) Leonhardt, H.; Gordon, L.; Livingston, R. J. Phys. Chem. 1971, 75, 245. (23) Oosterkamp, A. J.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1994, 66, 4295. (24) Haugland, R. P. Molecular Probes Catalog, Handbook of Fluorescent Probes and Research Chemicals; 1992-1994, pp 69-70.

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Figure 2. Graphical representation of the visually observed separation of a mixture of free fluorescein-biotin (FB) and the streptavidinbound fluorescein-biotin (S-FB). The strongly deflected line toward the anode originates from fluorescein (F), which is present as a pollution in the fluorescein-biotin stock solutions.

The concentration of biotin is determined by measuring the concentration of either the free or the streptavidin-bound fluorescein-biotin (eq 2). In general, the free and bound fractions have similar fluorescent features, making a separation step prior to the detection inevitable. All compounds that are involved in the biotin-streptavidin reaction have a net negative charge at pH 7.5 with different electrophoretic mobilities and can therefore be electrophoretically separated. Separation of Biotin and Streptavidin-Biotin. The separation of free FB and streptavidin-bound FB was demonstrated by mixing FB and streptavidin at a molar ratio of streptavidin binding sites to biotin of 1:8. The concentration of FB was 6 µmol/L. The mixture of free FB and streptavidin-FB was continuously infused into the device with a flow of 1 µL/min. The compound lines are visualized by illuminating the entire separation compartment. A drawing of the separated compound lines is shown in Figure 2. The free and the streptavidin bound FB are well separated within 60 s. As the sample solution is relatively low conducting, sharpening of the compound lines, which is due to electrophoretic stacking, was observed during the separation. The strongly deflected line (F) is caused by fluorescein, which is present as pollution in the fluorescein-biotin stock. The streptavidin-FB line is only slightly deflected toward the cathode and relatively broad, which could indicate a mixture of charged streptavidin compounds present in the sample. Band broadening due to adsorption was minimized by the addition of Tween 20 to the carrier electrolyte and by the properties of Plexiglas. Furthermore, the low angle of deflection of the streptavidin-FB line indicates a minor or no electroosmotic flow in the separation compartment. When streptavidin is mixed with a large excess of biotin and subsequently incubated with FB with the molar ratios of S:B:FB ) 1:100:8, only two lines are visible which are caused by free FB and the fluorescein impurity (F). The streptavidin-FB complex is not visible, indicating that streptavidin and fluorescein-biotin do not show significant nonspecific binding; hence. this system is in principle suitable for quantitative determinations. Flow Injection Analysis. An overview of a flow injection system for the reaction of biotin and streptavidin is shown in Figure 3 and is based on the reactions in eqs 1-3. In the present 3884

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Figure 3. Schematic overview of the flow injection system with a FFE separation step for the determination of biotin with streptavidin. The two-step biochemical reaction is performed in two reaction coils, I and II (see text). A laser beam (488 nm) is focused on the fluorescein-biotin line through the FFE device and the fluorescence signal is collected and detected with a photomultiplier (PMT). V is the injection valve, P a peristaltic pump, HV the power supply, and W waste of the FFE buffer.

system, biotin acts as analyte (B), which reacts in coil I with streptavidin to form an undetectable complex (S-B, eq 1). In a second step (eq 2) in coil II, fluorescein-biotin (FB) is added to react with the remaining free streptavidin binding sites (Sfree). After a reaction time of ∼200 s, a fluorescence signal is generated by free and streptavidin-FB complex. The samples were introduced in the FIA system via a six-port valve with a 1-µL internal loop. Quantitative measurements of the fluorescence intensity are performed with a focused laser beam, which is fixed at a fluorescent compound stream through the FFE device. The fluorescence signal was collected and filtered using a broad-band filter to transmit in the range of 512 nm. The light was transmitted via an optical fiber and detected with a photomultiplier tube. The optical setup was fixed permanently and only readjusted after changing electrophoretic parameters or for maintenance reasons. To reduce interinjection variations in the signal intensity, caused by small migration differences of the measured compound, the laser spot was slightly larger than the width of the sample stream. CZE analysis demonstrated that the fluorescein-biotin stock solution contained a fluorescent impurity with an electrophoretic mobility identical to that of the streptavidin-FB complex. To avoid signal overlap, all quantitative measurements in the FFE device were performed on the fluorescence-biotin signal. In Figure 4, three repetitive injections of 0.5 µmol/L biotin are shown. The biotin sample was introduced and reacted in the flow injection system. Free FB and streptavidin-bound FB are separated in the FFE device. The detector response (R) is determined by the concentration of free FB, which is proportional to the biotin concentration (eq 4):

R ) r(FBfree)

(4)

in which r is the slope of the calibration curve. A calibration curve was measured in the concentration range of 10-7-2.4 × 10-6 mol/ L, with a regression correlation coefficient of 0.993. For 0.5 µmol/ L, a relative standard deviation of the peak height of ∼6% (n ) 3)

Figure 4. Flow injection analysis of three repetitive injections of 0.5 µmol/L biotin. The carrier electrolyte consisted of 10 mmol/L pH 7.5 Tris-HCl buffer with 0.2% Tween 20. A 1 mol/L pH 7.5 Tris-HCl buffer was used in the electrode compartments.

Figure 6. HLPC-BCD-FFE analysis of biotin. A 4 µmol/L solution of biotin was injected into the HPLC system, equipped with a C18 analytical column. (A) UV absorbance signal prior to the BCD-FFE system. (B) Fluorescence signal of free fluorescein-biotin recorded on-device with laser-induced fluorescence detection. Figure 5. Experimental setup of the HPLC-BCD-FFE system. P is the HPLC pump, I the injection valve, C the C18 column, UV the UV absorbance detector, SP the 100:1 split, BCD the biochemical detection system, and FFE the free flow electrophoresis device with laser-induced fluorescence detection.

was found. The absolute detection limit was determined at 10 pmol. At concentrations higher than 2.4 × 10-6 mol/L, the calibration curve deviates from linearity and reaches a plateau, which is due to the saturation of streptavidin binding sites (eq 1). The detection limit is determined by the performance of the optical system and the generated background noise, which was relatively high in the setup used and thus compromising the detection limit. Also, nonspecific binding of biotin to the wall of the sample vials resulted in a severe loss of signal at low concentrations. Therefore, preincubation of the sample vials with a blocking agent to reduce adsorption to the vial surfaces was essential and resulted in a extension of the linear range of the calibration curve toward the detection limit. Liquid Chromatography with BCD-FFE Detection. A large-bore HPLC system was coupled to the FIA-BCD-FFE setup for a feasibility study of the LC analysis of biotin (Figure 5). After the HPLC separation, UV absorbance detection at 210 nm is performed. The column effluent was reduced from 400 to 1 µL/min with a two-stage split system to make the flows compatible with the FFE flow. The mobile phase consisted of 10 mmol/L pH 7.5 Tris-HCl buffer, without an organic modifier. However, the performance of the FFE separation of streptavidin and an excess of fluorescein-labeled biotin was not affected when 30% (v/v) methanol or ethanol was added to the FFE buffer. In Figure 6 the UV absorbance signal of a 4 µmol/L biotin sample and the fluorescence signal after biochemical reaction detection is shown. The mixture of free FB and streptavidin-bound FB flows from the postcolumn BCD system into the FFE device and is subsequently separated. Finally, detection was performed

with laser-induced fluorescence, with a focused laser spot on the free FB trace. During the transfer from the UV detector, through the BCD system into the FFE device, band broadening occurs, as can be seen from Figure 6. In this BCD system, the concentration of binding sites of streptavidin and biotin is 2 and 2.26 µmol/L, respectively. A calibration curve was constructed with a linear range between 2 and 8 µmol/L biotin. The selectivity of the immunochemical reaction detection and the applicability of an added FFE separation was tested with a bioanalysis. Human urine was spiked with 4 µmol/L biotin, diluted with water at a ratio of 1:1, and separated on the C18 analytical HPLC column. In Figure 7 , an on-line UV absorbance signal of biotin and the fluorescence signal of free FB are shown and compared with the blank urine signal. The selectivity of the biochemical reaction with laser-induced fluorescence detection, compared to UV absorbance detection, is clearly demonstrated. In the analysis of the urine sample, no interfering matrix compounds are visible, and despite the high ion concentration of the urine sample, no adverse effects were observed in the FFE separation. CONCLUSIONS AND PERSPECTIVES A FFE device was developed for continuous electrophoretic separation of compounds in a bulk flow and was shown to be an effective tool to enhance the compatibility of hyphenated systems when compounds with different electrophoretic mobilities are involved. The electrophoretic separation is directly influenced by the buffer composition, the velocity of the hydrodynamic flow, and the electric field strength. High electric field strengths are necessary when a high flow rate or small differences in the electrophoretic mobilities are present; however, as a consequence, gas bubble formation in the separation compartment and electrode membrane breakdown can appear. Also, pH changes in the Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

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Figure 7. Urine spiked with biotin at a concentration of 4 µmol/L biotin and a blank signal. UV absorbance and laser-induced fluorescence signal (LIF) of (A) blank urine and (B) spiked urine. In the UV signal, biotin is visible, while the fluorescein-biotin signal was detected on-device with LIF.

separation compartment toward a low pH can strongly affect the fluorescence yield and hence influence the signal of the fluorescein labeled compounds. The FFE system was successfully applied in a BCD system for biotin with streptavidin as affinity protein and fluorescein(25) Fintschenko, Y.; van den Berg, A. J. Chromatogr., A 1998, 819, 3.

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biotin as reporter molecule. All compounds are in an anionic state in the pH 7 background electrolyte and possess different electrophoretic mobilities. In the device, the streams of fluoresceinbiotin and the streptavidin-bound fluorescein-biotin were zone electrophoretically separated in a time span of ∼3 min. Quantitative on-device detection was performed with laser-induced fluorescence by illuminating a spot on the device through which the separated not-reacted fluorescein-biotin flows. The combination of a HPLC separation with the BCD-FFE system was demonstrated with a bioanalysis of 4 µmol/L biotin in human urine. FFE is a continuous and relatively fast electrophoretic separation technique and can be used in a wide variety of regular and miniaturized25 analytical systems. Besides the demonstrated use in biochemical detection, potential applications can be found in related areas, such as enzyme amplification detection and screening of combinatorial libraries, provided that the involved compounds possess different electrophoretic mobilities. Also, FFE can be a powerful tool in the continuous removal of unwanted species in a liquid flow, such as involatile compounds in the effluent of a liquid separation system prior to mass spectrometric detection. ACKNOWLEDGMENT Henk Verpoorten from the Fine Mechanical Workshop at Leiden University is kindly acknowledged for constructive discussions and the manufacturing of the free flow electrophoresis device. Received for review October 18, 1999. Accepted May 31, 2000. AC991202K