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Surface plasmon resonance (SPR) is a total inter-nal reflection phenomenon that occurs at metal film-medium interfaces when a fraction of energy of incident light is absorbed by surface delocal-ized electrons (plasmon), resulting in a decrease of reflected light at a certain angle [1]. The sur-face plasmon has a maximum charge density at the interface and decays exponentially from the surface of metal film with a typical decay length of approximately a few hundred nanometers [2]. Within this region, SPR is very sensitive to changes in the refractive index of the medium adjacent to the Au film, for example, change in bulk solution, and adsorption of molecules onto or desorption of molecules from the metal film surface. SPR was successfully used to monitor, in real-time, a variety of biological interactions involving drugs, peptides, proteins, nucleic acids, carbohydrates, cells and viral particles in a label-free manner, providing kinetic and thermody-namic information [3–12]. SPR was also coupled to separation techniques [13–18]. For example, Du and Zhou incorporated SPR into HPLC and real-ized protein separation and detection at micro-molar levels [14]. Whelan and Zare first coupled SPR with CE [17]. To shield surface electronics from high voltages applied during the separation, a crack was made in the capillary 5 cm from the

outlet, which was connected to the flow cell of the SPR and bathed in a 1-ml volume of run buf-fer. A three-component mixture of high refractive index materials was separated and detected at the millimolar level at an untreated Au film surface. Human IgG was electrophoretically delivered to protein A, functionalized on a Au film surface and the extent of interaction observed. Recently, Ly et al. demonstrated the possibility of integrating a confined SPR image into microchip CE (MCE) [18]. In order to avoid electrochemical corrosion of Au film, a microfluidic net with a 8-mm separa-tion channel (500-µm wide and 500-nm high) was fabricated onto a 30 nm protective polymer film of high dielectric strength applied over a Au film surface that was segmented into an array of squares (200 × 200 µm). A mixture containing BSA (15 mg/ml) and glucose oxidase (15 mg/ml) was separated and detected in a 25-mM Tris-glycine run buffer containing 10% methanol. However, the existence of the protective film resulted in a 23% loss in sensitivity and the device could only withstand a maximum field strength of 50 V/cm. Field strength of several hundred V/cm are typical in MCE to achieve optimum separa-tion and peak resolution [19–21]. Moreover, high concentrations of organic solvent (methanol in this case) can denature proteins and nucleic acids.

Facile fabrication of an interface for online coupling of microchip CE to surface plasmon resonance

Background: The aim was to develop a simple route to coupling microchip CE (MCE) to surface plasmon resonance (SPR). MCE is a microfluidic technology that utilizes microfabrication techniques to connect interacting fluid reservoirs. Its advantages include rapid analysis (typically seconds), easy integration of multiple analytical steps and parallel operation. SPR detects changes in refractive index within a short distance from the surface of a thin metal film as variations in light intensity reflected from the back of the film and, thus, does not require labeling. There is a great demand for developing hyphenated techniques like MCE–SPR that are fast, sensitive and inexpensive to analyze biological materials. Materials & Methods: The separation channel and flow cell exist as overlapping regions constructed during the microchip production and buffer solution was delivered mechanically. Such a design has successfully isolated the electrical field inherent in the MCE from the SPR detector. Consequently, the potential interference to the SPR signal (or modulation of the density of surface plasmons at the gold chip) is circumvented. Results: The limits of detection for bovine serum albumin and sodium fluorescein were determined to be 7.5 µM and 3.1 mM, respectively. Conclusion: The technique described, herein, has been successfully applied in the separation of two species. The method offers the advantages of a near zero connection dead volume, electrical shielding from the separation voltage and minimization of the mass transfer effect.

Xiaojun Liu, Ming Du, Feimeng Zhou & Frank A Gomez*Department of Chemistry & Biochemistry, California State University, 5151 State University Drive, Los Angeles, CA, 90032-8202, USA *Author for correspondence: Tel.: +1 323 343 2368 Fax: +1 323 343 6490 E-mail: fgomez2@calstatela.edu

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Herein, we describe the facile fabrication of a microfluidics-based interface for online com-bination of MCE with SPR. The interface con-sists of an overlapping section of the separation channel and flow cell. A pressure-driven mode in CE was used to ‘push’ the sample through the microchannel. Typically used in CE injections, this mode of sample manipulation was required to obtain maximal separation of two species in the MCE–SPR system. Using an electric field and SPR utilizing a dextran-functionalized Au film, the separation and detection of BSA and sodium fluorescein was realized at a field strength of 350 V/cm. The LOD and dynamic range for the two species were assessed.

Experimental Section � Materials

N-(2-hydroxyethyl)piperazine-N´-(2-ethane-sulfonic acid), cystamine dihydrochloride, 2-(N-morpholino)ethanesulfonoic acid, carboxy-methylated dextran, BSA, sodium fluorescein, N-hydroxysuuccinimide, glycidyl methacrylate, dimethylacrylamide, tetramethylethylenedi-amine and N-(3-dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride were pur-chased from Sigma (St. Louis, MO, USA). Dialysis tubing with 3500 molecular weight cut-off was purchased from Fisher (Pittsburgh, PA, USA). Negative-type photoresist SU-8 2025 and its developer were obtained form Microchem (Newton, MA, USA). Sylgard 184 poly(dimethylsiloxane) (PDMS) oligomer and its cross-linking agent were purchased from Dow Corning (Midland, MI, USA). Au films were purchased from Biosensing Instruments (Tempe, AZ, USA). A 2-nm Cr underlayer was deposited for better adhesion of the Au film to the BK7 glass slide.

� InstrumentationDetection of species separated by MCE was per-formed on a BI SPR 1000 system (Biosensing Instrument) equipped with two separated posi-tion-sensitive photodetectors. In this work, only one detector was used for the detection, but the presence of a second detector offers possibilities for more complicated chip designs and more sophisticated applications. For example, a sec-ond detector would allow for detection of spe-cies with minimal separation simultaneously. If placed further downstream from the original detector, a greater separation of a mixture of com-ponents would be realized. The flow cell on the microchip could be easily aligned to the detector

owing to the open access to the SPR prism and visualization of the reflected laser light through a viewing window on the instrument. To suppress nonspecific adsorption, an Au film surface was modified with carboxymethylated dextran fol-lowing the procedure described by Du et al. [14]. Briefly, cystamine self-assembled mono layers (SAMs) were formed by casting onto each Au film 0.8 ml of 20 mM cystamine dihydrochlo-ride solution overnight at ambient temperature. After rinsing with water, 0.8 ml of a solution con-taining 4.35 mg/ml carboxymethylated dextran, 0.1 M N-hydroxysuuccinimide and 0.4 M N-(3-dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride were spread onto the gold film and the dextran film attachment was allowed to proceed for 3 h.

Fabrication of the interface between MCE & SPRA three-step procedure was adopted to fabricate the interface consisting of PDMS replica fabrica-tion, surface coating of the PDMS chip and sealing of the chip to a dextran-functionalized Au film.

PDMS replicaThe PDMS chip was prepared by using a silicon mold created by soft photolithography [22]. The pattern (Figure 1A) was transferred to a 3” sili-con wafer through a high-resolution (10,000 dpi) photomask (CAD/Art Services, Inc., OR, USA). Sylgard 184 PDMS prepolymer was mixed thoroughly with its curing agent at 10/1 (v/v), degassed by a vacuum pump and then cured against the mold at 75°C for 2 h. After the PDMS replica was peeled off from the mold, holes were punched for the buffer and sample reservoirs. The reservoir of the flow cell (W2) was 4.0 mm away from the channel (Figure 1A). The chan-nels were approximately 30-µm high and 150-µm wide at the bottom. In reference to Figure 1A, the distances from the reservoir buffer 1 (B1) to the reservoirs buffer 2 (B2), water 1 (W1), water 2 (W2), and sample (S) were 1.0, 7.0, 6.5 and 2.0 cm, respectively. A PDMS layer of approxi-mately 30 µm was obtained through spin-coating a silicon wafer with degassed PDMS at 1400 rpm for 2 min and curing at 75°C for 2 h. A square (150 µm × 5.0 mm) of PMDS was used as the bottom layer and the corresponding gap was used as the flow cell for SPR.

Surface coating of the PDMS chip The surface of PDMS is hydrophobic in its natural state causing nonspecific adsorption

Key Terms

Surface plasmon resonance: Biomolecular interaction analysis instrument that provides an automated approach to measuring real-time kinetics and thermodynamics of interactions at the sub-nanomolar to nanomolar range.

Microchip CE: Microfluidic technology that applies semiconductor-like microfabrication techniques to connect interacting fluid reservoirs. It is a technique for separating chemical constituents on application of a voltage gradient.

Self-assembled monolayer: 2D film, one molecule thick, covalently assembled at an interface.

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of biomolecules and other species. To suppress nonspecific adsorption, the surface of the micro-chip was modified to make it hydrophilic with an epoxy-functionalized polymer following the method described by Wu et al. [23]. In brief, 5.0% dimethylacrylamide (w/v) and 0.1% glycidyl methacrylate (v/v) were mixed in distilled water and thoroughly degassed for 10 min. To initiate the polymerization reaction, 0.1% tetramethyle-thylenediamine (v/v) and 0.05% potassium per-sulphate (w/v) were added into the mixture and the reaction continued for 30 min at room tem-perature. To remove unpolymerized monomers and small polymer molecules, the polymer solu-tion was extensively dialyzed by 3500 molecular weight cut-off dialysis membranes for 2 days with distilled water. The PDMS replica and the sub-strate were treated with oxygen plasma for 120 s. For irreversible sealing, they were brought together immediately in alignment where the flow cell con-nected the separation channel and the reservoir W2 (Figure 1D). The epoxy polymer solution was introduced into the microchannel, and incubated for 15 min at room temperature. The solution was completely pumped out and the PDMS microchip was directly heated at 110°C for 10 min.

Coupling of the PDMS chip to the Au filmAfter surface coating, the PDMS chip (Figure 1B) was peeled off from the silicon wafer. The PDMS chip was immediately brought into contact with a clean dextran-modified Au film for reversible sealing where the flow cell was located in the Au film (Figure 1C & D). To secure a stronger sealing, the device was placed under vacuum for 10 min and the flow cell aligned with one of the two SPR position-sensitive photodetectors.

Procedures � MCE procedure

A 10 mM N-(2-hydroxyethyl)piperazine-N -́(2-ethanesulfonic acid) solution containing 0.02% BSA (pH 7.40) was used as run buffer. All samples were dissolved in the run buffer. MCE was performed with assistance of pressure. A peristaltic pump was connected to reservoir B1. To condition the microchannels, the device was rinsed with distilled water and the run buf-fer for 5 and 10 min, respectively. Hydrostatic pressure injection was used in the study. In refer-ence to Figure 1A, 8.0 µl of the run buffer was added into reservoirs W1 and W2 and 22.0 µl of the run buffer was added into reservoir B2.

W2

W1

150 µm

B2

B1

S

PDMS

Au film

Holding glass

Interface

Figure 1. Microfluidic chip and instrument setup. (A) Design of the PMDS chip; (B) microchip assembly for coupling microchip CE and surface plasmon resonance showing interface; (C) side view of the interface; (D) side view of separation channel; and, (E) current instrument design showing placement of microchip and connecting electrodes for CE.PDMS: Poly(dimethylsiloxane).

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Initially, 32 µl of sample solution was added to reservoir S. This hydrostatic pressure injection continued for 20 s (Figure 2A). For the separa-tion, 2.5 kV was applied to reservoirs B2 and W1 accompanied with a flow rate of 0.5 µl/s from res-µl/s from res-/s from res-ervoir B (Figure 2B). After 100 s, the flow rate was increased to 3.2 µl/s. After each run, the channels were washed with the run buffer for 2 min.

Results & discussion � Interface

Connection dead volume of the interface Connection volume of the interface directly affects the separation resolution and LOD. In our study, the overlapped region consisting of a segment of the separation channel and the flow cell formed the interface between MCE and SPR. The connection volume of the interface was deter-mined by the overlapped length of the two chan-nels since the height and width of the channels are the same (Figure 1C & D). In an ideal case, the length of the overlapping channels is the same as the height of the channels and the connection dead volume can be considered as zero. Early prototype microchips containing large dead vol-umes affected separation efficiencies and yielded diminished LOD.

Linkage PDMS is elastomeric and its surface free energy is low (~20 erg/cm2) [24–26]. The characteristics

endow PDMS microchip’s ability to reversibly seal onto Au film without distorting the microchannel when the PDMS chip is brought into contact with the Au film surface. In our study, the interface was placed under vacuum to strengthen the seal. To test how high a flow rate the sealing could withstand, a food dye was pumped through the device at an increasing flow rate. The sealing was still leakage-free even at a flow rate of 5.1 µl/s (the maximum flow rate the pump provided). Thus, the device could be fixed onto SPR only with adhesive tape.

Mass transfer effectThe analyte molecules must be delivered to the vicinity of the Au film surface before they are detected. Transportation of the analyte molecules from the run buffer to the Au film is referred to as mass transfer. To obtain kinetic information on the interested interaction, there is the require-ment that the rate constant of mass transfer (K

m)

be larger than that of the association reaction [1]. The rate constant of mass transfer is dependent on the characteristics of the analyte molecules and the solution, immobilization capacity of Au film and the instrument factors, and is expressed as in equAtion 1 [27]:

equAtion 1

Where D, h, f, b, and x are the diffusion coeffi-cient of analyte molecules, the height of the flow cell, the flow rate, the width of the flow cell and the thickness of diffusion layer, respectively. In other SPR experiments, the height of the flow cell is fixed. In our device, the height of the flow cell can be easily adjusted by changing the spin-coating speed and time. For example, when the spin-coated speed varied from 800–1200 rpm for 30 s, the height of the flow cell changed from 65–45 µm. Accordingly, the rate con-stant of mass transfer theoretically increased by one-third.

Electric field effectIn MCE, analyte molecules are separated according to different charge-to-mass ratios at certain field strengths; the higher the field strength, the higher the separation resolution. Meanwhile, application of a positive poten-tial (1.5 V) to a Au film improved the SPR measurement’s sensitivity [28]. Initially, we attempted to take advantage of this charac-teristic by directly connecting the Au film to

+-

ElectrodeElectrode

Injection

Pump

Pump

Fluid flow

Species B

Species A

Figure 2. Microchip and electrophoresis separation configurations. (A) Chip configuration and injection and (B) microchip electrophoresis separation assisted with pressure.

. / /K D h f bx0 98m 32

31

= ^ ^h h

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a power supply and using it as one electrode for MCE. The strategy was not successful as bubbles were produced and gathered at the interface between the MCE and SPR compo-nents of the system. We attributed this to the electrochemical reaction 2H+ + 2e = H

2. We

also found that the Au film gradually peeled off and that the peeling began from the area near-est to the Pt electrode if the Au film was con-nected to the power supply through the buffer solution. We believe that this peeling results from the electrochemical corrosion of the Cr underlayer and the Au film of the SPR chip. In subsequent experiments, the pressure-assisted MCE strategy was used as shown in Figure 2B. Pressure was incorporated into conventional MCE to introduce the separated compound into the flow cell over the Au film to which no voltage was applied.

MCE separation & SPR detection facilitated by the interfaceSPR is sensitive to a change in the refractive index of a solution adjacent to the Au film. We chose BSA (refractive index: 1.445) and fluorescein sodium (refractive index: 1.33) [29] as model analytes to verify the interface. Figure 3A shows a representative separation and detection of BSA (80 s) and fluorescein sodium (118 s) at a field strength of 350 V/cm in posi-tive polarity. A lower voltage may not have realized separation between the two species. We found that fluorescein sodium migrated back into reservoir S if the flow rate was <0.3 µl/s. We assumed this was because the back-/s. We assumed this was because the back-ward electrophoretic mobility was larger than the forward rate due to the peristaltic pump

(“electro-osmotic” flow was near zero due to the modification of the PDMS chip). To decrease the migration time of fluorescein sodium, the flow rate was changed from 0.5 to 3.2 µl/s after the BSA migrated out. This increase in flow rate was also reason that the peak width of fluorescein sodium was smaller than that of BSA. We also assessed the dynamic range and LOD. Figure 3B shows the linear portion of the calibration curve. The dynamic range for both species was over two orders of magnitude. The LOD was 7.5 µM (~3.3 pmol) and 3.1 mM (~1.6 nmol) for BSA and fluorescein sodium, respectively. Recent work in another labora-tory [18] used BSA as an exemplar and was able to detect it at the 30 pmol level. The differ-ence in LOD of BSA and fluorescein sodium is believed to result from their difference in refractive index.

ConclusionWe have described a facile fabrication of a sim-ple microfluidic PDMS chip for coupling MCE to SPR. This interface offers the advantages of a near zero connection dead volume, electri-cal shielding from the separation voltage and minimization of the mass transfer effect. This configuration allows a high separation voltage to be used. The dynamic range for both species is over two orders of magnitude. The LOD is 7.5 µM and 3.1 mM for BSA and sodium fluo-rescein, respectively. The present study is not an exhaustive one and to further broaden the viability of the MCE–SPR hyphenated tech-nique, separations involving mixtures of pro-teins and more than two biomolecules should be examined.

Concentration (µM)

Fluorescein sodium

Fluorescein sodium

BSABSA

RU R

U

14

12

10

Time (s)

8

6

4

2

00 20 40 60 80 100 120 140 160 180

180

160

140

120

100

80

60

40

20

0

-200 100 200 300 400 500 600 700 800

Figure 3. Electropherogram and dynamic range plot. (A) A representative electropherogram of the separation of BSA and sodium fluorescein and (B) dynamic range.

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Future perspectiveMCE coupled to SPR has great potential as a hybrid analytical technique as it provides both separation and detection in efficient times cales coupled to small sample volume and reagent size requirements. A further advantage of the tech nique is that there is little need for sample modifi cation since SPR detects minor differ-ences in refractive indices. Potential applications of the system include their use in genetic analysis, clinical analysis and immuno assays. Modifying the current design to an SPR imager would increase the sensitivity of the current MCE–SPR design. Further work will focus on demonstrat-ing MCE–SPR in a high-throughput platform setting.

AcknowledgementsThe authors gratefully acknowledge Y Li and D Jiang for helpful discussions.

Financial & competing interests disclosureThe authors gratefully acknowledge financial support for this research by grants from the National Science Foundation (DMR-0351848, CHE-0515363 and CBET-0723271 to FG and CHE-0555244 to FZ). The authors have no other relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Executive summary

� Simple fabrication of a microfluidic chip as an interface for integrating microchip capillary electrophoresis with surface plasmon resonance has been described.

� The unique interface design allows for isolation of the electrical field minimizing any potential interference to the surface plasmon resonance signal at a field strength of 350 V/cm.

� The LOD for BSA and sodium fluorescein were determined to be 7.5 and 3.1 mM, respectively.

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