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Acupuncture Sample Injection for Microchip Capillary Electrophoresis and Electrokinetic Chromatography Ji Won Ha, and Jong Hoon Hahn Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00789 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 9, 2016

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Analytical Chemistry

Acupuncture Sample Injection for Microchip Capillary Electrophoresis and Electrokinetic Chromatography Ji Won Ha1* and Jong Hoon Hahn2,* 1

Department of Chemistry, University of Ulsan, 93 Daehak-Ro, Nam-Gu, Ulsan, 44610, South Korea 2

Department of Chemistry, BioNanotechnology Center, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, 37673, South Korea

*

To whom correspondence should be addressed.

J. W. Ha Phone: +82-52-259-2347 Fax: +82-52-259-2348 E-mail: [email protected]

J. H. Hahn Phone: +82-54-279-2118 Fax: +82-54-279-5805 E-mail: [email protected]

.

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Abstract A simple nL-scale injection technique was developed for polydimethylsiloxane(PDMS) microfluidic devices to form the well-defined sample plugs in microfluidic channels. Sample injection was achieved by performing acupuncture on a channel with a needle and applying external pressure to a syringe. This technique allowed us to achieve reproducible injection of 3nL segment into a microchannel for PDMS microchip-based capillary electrophoresis (CE). Capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) with beads packing were successfully performed by applying a single potential in the most simplified straight channel. The advantages of this acupuncture injection over the electrokinetic injection in microchip CE include capability of minimizing sample loss and voltage control hardware, capability of serial injections of different sample solutions into a same microchannel, capability of injecting sample plugs into any position of a microchannel, independence on sample solutions during loading step, ease in making microchips due to the straight channel, etc.

Keywords: acupuncture injection, needle, microchip capillary electrophoresis, beads packing

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Since the introduction of the concept of micro-total analysis systems (µ-TAS) in 1989,1 intensive efforts have been devoted to the miniaturization of conventional analytical systems for increasing sample throughput, reducing the cost and time, and improving performance. Among the various conventional analytical techniques, capillary electrophoresis (CE) has proved to be the most successful one in miniaturization.2 So far, there has been much progress in the development of separation techniques for microchip-based CE.3,4 For example, numerous CE separation processes such as capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC) have been developed for the analysis of various substances such as amino acids,5 peptides,6 proteins,7 and DNA fragments.8 Despite the great progress in the microchip separation techniques, the development of simple and cost-effective methods for sample injection on microchips is still limited. The sample injection is one of the most critical parameters to achieve efficient and sensitive analysis. So far, electrokinetic sample injection has been commonly used in microchip-based CE separation. The electrokinetic injection method is based on the application of high voltages for driving the sample solution by generation of an electroosmotic flow (EOF). The first CE chip used a teeinjector,9,10 but recent devices include cross11 and double-tee injectors12 because of the difficulties in controlling the sample solution and in dispensing of well-defined sample plugs. To form a well-defined sample plug at the injector, several electrokinetic injection modes including gated,13,14 pinched,15,16 and floating17 have been employed. Among them, the pinched mode injection, which is geometrically defined by the intersection of cross or double-tee injector, has become a common practice in CE on microchips. However, all electrokinetic injection modes need cumbersome voltage programs for dispensing well-defined sample plugs and require 3



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complicated hard and software systems for chip operation. Moreover, the amounts of sample consumption are quite large compared to real injected amounts into separation channel. The electrokinetic injection is also strongly dependent on the characteristics of the channel wall surface and exhibits a bias injection towards different species,18,19 influenced by pH, ionic strength, and viscosity of sample solution during loading step, and is not ideal in the case of cell samples because of the electroporation.20 Recently, there have been efforts to overcome the aforementioned limitations in electrokinetic injection.21-30 For example, Zhang et al. have described narrow sample channel injectors for capillary electrophoresis on microchips.21 Using a tee injector with reduced sample channel width, resolution, column efficiency, and sensitivity were improved. Bingcheng Lin’s group has proposed the injection by hydrostatic pressure in conjunction with electrokinetic force on a microchip.22 In this method, hydrostatic pressure, which is generated by controlling the liquid level in different reservoirs, was used in the process of sample loading to the intersection and electrokinetic force was employed for dispensing the sample to separation channel and for CE separation. In this paper, we present a simple and cost-effective acupuncture injection technique as an alternative to electrokinetic injection in polydimethylsiloxane(PDMS) microchip-based CE. Sample injection is achieved by performing acupuncture on a microfluidic channel with a needle and applying external pressure to a syringe by turning a micrometer screw. We demonstrate that the acupuncture injection technique allows for the serial injection of reproducible 3 nL-segments into a microchannel in continuous flow. We further describe that the acupuncture injection technique can be used for microchip CE in the most simplified straight channel by applying a single potential at two reservoirs while minimizing the amounts of sample consumption. 4


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Experimental Section Fabrication of PDMS Microchips. PDMS microchips used in this paper were fabricated by photolithography replica molding method.31-34 Channel networks were designed by using a computer aided design software package (AutoCAD; Autodesk, San Rafael, CA, USA). The layout of the CZE microchips for electrokinetic injection and acupuncture injection was schematically shown in Figure S1. Figure S2 shows photographic images of PDMS microchips fabricated for electrokinetic and acupuncture injections in this study. The detailed information on CZE microchips is described in the Supporting Information.

Acupuncture Injection Method. All microchips used in this paper were composed of a glass substrate bonded with a lower PDMS plate having a thickness of 500 µm, and the upper PDMS plate containing a microfluidic channel has a thickness of 2 mm. Figure S3 shows an instrumental setup developed to realize the acupuncture injection method and a magnified view of an injector. The instrument consists of a CCD camera (Edmund Industrial Optics, Barrington, NJ, USA), chip and syringe holders, and an injector with a micrometer screw. The system is very simple, cost effective, and portable. After setting up the system, a micro-syringe containing a sample solution was fixed to the holder of injector, and we slowly turned a micrometer screw until a first drop forms at the tip of a needle which was carefully removed with a tissue paper. Figure 1 depicts a schematic of acupuncture injection. Initially, the needle position was adjusted in the horizontal plane with the assistance of a CCD camera (Figure 1A). The microchannel was then acupunctured vertically with the needle until it touches the surface of a glass substrate (Figure 1B). In this step, there is 5



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no need of fine adjustment of the needle position in the vertical direction. This is because we experimentally optimized the thickness of a lower PDMS plate bonded with a glass substrate for the needles used in this study. After performing acupuncture, sample solution was simply introduced into a channel by applying external pressure using a micrometer screw to a syringe. The injected volume could be controlled by a capacity of a syringe used and the number of graduation of a micrometer screw turned. After injecting a nL-sample plug, we carefully pulled out the needle from the PDMS microchip. The photographs of the needles employed in this study are given in Figure S4. Figure S4A shows a 30 gauge-disposable needle that has the outer and inner diameters of 300 µm and 100 µm, respectively. Figure S4B shows a cemented, fixed needle of 10-µL glass syringe, and the tip was modified and grinded into a slant face. The inner diameter of a cemented needle was same as that of a disposable needle. In microchip CE, a cemented needle of 10-µL glass syringe was used for injecting small amounts of sample due to the better accuracy and reproducibility.

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Results and Discussion Acupuncture Sample Injection of nL-Scale Volume. We first demonstrate the capability of an acupuncture injection method for generating nL-scale segments in a PDMS microfluidic channel. Figure 2 shows consecutive CCD images of injecting a sample plug using acupuncture injection from the top (A, B, C) and side (D, E) view. Initially, the needle position in the horizontal plane was adjusted with the assistance of a CCD camera as shown in Figure 2A. The needle was then inserted into the microchannel until it touches at the surface of the glass substrate following the injection of sample (Figure 2B). Finally, a nL-scale sample plug was created directly in the microchannel after the needle was pulled out carefully from the microchip (Figure 2C). Figures 2D and 2E are CCD images of injecting a sample from the side view. The channel in Figure 2 is 200 µm wide and 100 µm deep. A 25-µL syringe with a 30 gaugedisposable needle was used and the injected volumes were calculated to be about 12 nL corresponding to 3 graduations of the micrometer screw. As the biological assays require continuous flow of the sample probes, serial acupuncture injections are necessary in continuous flow. We therefore tested if the acupuncture injection technique enables us to generate serial sample plugs in continuous flow. In this experiment, aqueous sample plugs were formed by acupuncture injection in the PDMS microchannel where perfluorodecalin, which is immiscible with aqueous phase, flows at the flow rate of 0.2 µL/min. The microchannel fabricated was 200 µm wide and 100 µm deep. A 100-µL disposable syringe was used to inject sample segments, and reproducible injection of nL-scale sample segments was achieved as shown in Figure S5. The length of a sample plug was measured by using a 10 cm ruler, which is located on the chip, and then we calculated the injected volume by multiplying 7



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width, depth, and the length. The injected volumes were calculated to be about 56 nL and were reproducible (Figure S5). We further tried to reduce the amounts of sample injected into a microchannel. In this study, the microchannel fabricated was 100 µm wide and 100 µm deep. A 10-µL glass syringe with a cemented needle (Figure S4B) was used due to the better accuracy and reproducibility. Figure 3 shows consecutive CCD images of introducing sample plugs into a microchannel where perfluorodecalin flows at the flow rate of 0.2 µL/min. Notably, we could achieve serial, reproducible 3 nL-injections of a sample solution in continuous flow of perfluorodecalin (Figure 3D). We further checked if leakage at the injection point occurs on a PDMS microchip. A sample was continuously introduced at the injection point by the acupuncture injection method, while perfluorodecalin was flowed in the PDMS microchannel at the flow rate of 5 µL/min. As a result of that, the first leakage at the injection point was observed after injecting a sample segment of 147 times under the condition (Table S1). This result indicates that the acupuncture injection method can be applied to PDMS microchip-based CE without the leakage problem.

Microchip Capillary Zone Electrophoresis. We tested if the acupuncture injection technique capable of nL-scale sample injections can be directly used as an alternative to electrokinetic injection in microchip CE. In the present study, we performed CZE separations of FITC and fluorescein by using gated, pinched, and acupuncture injection modes, and the experimental conditions were same except for the sample injection mode. The experimental setup for CZE is shown in Figure S6, and Figure S7 shows a CCD image of fluorescent sample plug (~3 nL) injected into a straight channel by the acupuncture injection method. Figure 4 shows the result of CZE separations obtained from electrokinetic (gated, pinched) and 8


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acupuncture mode injections. It is clear from the electropherograms that the mixture of 10 µM FITC and 10 µM fluorescein was separated completely by all the three injection modes. We then compared the separation efficiency for all the three injection modes in terms of the number of theoretical plates calculated using equation 1.

 t   N = 5.54 ×  W  1/ 2 

2

(1)

In the above equation, N is the number of theoretical plates, t is the migration time, and W1/2 is the peak width at half height. As shown in Table S2, the number of theoretical plates of acupuncture injection was larger than those of electrokinetic injections for both FITC and fluorescein. This indicates the improved resolution achieved by using acupuncture injection mode as compared to electrokinetic injection. This can be ascribed to a well-defined sample plug formed by the acupuncture injection method. Therefore, we confirmed that the acupuncture injection method developed in this study can be employed for microchip CE separations. A great advantage of acupuncture injection over electrokinetic injection is the capability of serial injections of different sample solutions into a same microchannel. As demonstrated in Figure S5, another sample solution with a different color (blue) can be introduced into the same microchannel by the acupuncture injection method. To further demonstrate this, we performed a CZE separation by changing the composition of analyte solution. In this experiment, 15% glycerol was added to sample solution (v/v). As shown in Figure 4D, the mixture of 10 µM FITC and 10 µM fluorescein was separated completely. More notably, we found an enhanced resolution indicated by the increase in the number of theoretical plates in the presence of glycerol (Table S2). This may be attributed to the decreased diffusion of the sample plugs to be 9



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introduced into the microchannel because of glycerol added to sample solution. Besides the aforementioned advantage of the acupuncture injection over the electrokinetic injection, the other advantages in microchip CZE include capability of minimizing the sample loss and voltage control hardware, a decrease of injection bias effects, capability of injecting a sample segment into any position of a microchannel, ease in making microchips due to the straight channel, etc. Therefore, we believe that the acupuncture injection mode can be conveniently used as an alternative to electrokinetic injection in microchip CZE.

Capillary Electrochromatography with Packing of Chromatographic Beads. To take a step further after the microchip CZE, we attempted to use the acupuncture injection method for PDMS microchip-based CEC. CEC is another CE separation technique in which the flow of the mobile phase or buffer is driven through a chromatographic column by an electric field. For the microchip-based CEC separation, we need an integration of a chamber densely packed with chromatographic beads onto a PDMS microchip. There have been a few methods used for packing of chromatographic beads into the bead chamber in microchip CEC.35-37 However, there has been large consumption of beads compared to real packed beads into the chamber.35 Moreover, the previous methods required a complicated procedure for making a column packed with silica beads.35-37 Therefore, it is highly desired to develop simpler and more cost-effective method that can overcome the drawbacks in the previous methods. To overcome the aforementioned limitations, we used the acupuncture injection technique for packing chromatographic beads into a chamber in microchip CEC. We made frit structures in the inlet and outlet of a bead chamber to construct a cavity in which beads could be trapped. The 30 gauge-disposable needle of 1 cc syringe containing the suspension of ODS10


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coated beads was directly inserted into a side channel shown in Figure S1D, and then beads were packed into a chamber by applying pressure to a syringe. The beads were unable to pass through the frit structures because the space between the frit structures (3 µm) is narrower than the diameter of the packing material (5 µm). Figures 5A and 5B show CCD images of bead-packed chamber and inlet and outlet frit structures. It is clear from the CCD images that ODS-coated beads were trapped and packed in the chamber (Figure S8). We further confirmed that the beads are packed densely into the chamber by looking at the packed chamber from the side view (Figures 5C and 5D). Therefore, the acupuncture injection allowed us to introduce chromatographic beads into the chamber, to minimize consumption of the bead, and to simplify a packing process. As a next step, reversed-phase CEC was performed on the chamber packed with ODScoated silica beads equilibrated with buffer. In this experiment, fluorogenic neutral compounds, coumarin 440 and coumarin 450, were used for the CEC test. Figure 5E demonstrates the CEC separation of coumarins using a mobile phase with a buffer composition of 65% acetonitrile/35% 50 mM tricine buffer (v/v, pH 9.0). The successful CEC separation of coumarin 440 and coumarin 450 within 9 min was achieved using the acupuncture injection method.

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Conclusions We have developed an acupuncture injection technique as an alternative to conventional electrokinetic injection in PDMS microchip-based CE. This technique allowed the formation of reproducible 3 nL-scale sample plugs in microfluidic channel. CZE separation was performed in the most simplified straight channel with a single potential. The advantages of using the acupuncture injection technique for beads packing on PDMS microchips and CEC separation of coumarins were also demonstrated. The coupling of the acupuncture injection mode with microchip CE provides several great advantages including a decrease of injection bias effects, capability of minimizing the sample loss and voltage control hardware, capability of serial injections of different samples into the same microchannel, capability of injecting samples into any position of a channel, ease in making chips, etc. Therefore, the acupuncture injection method would be more conveniently used not only in the microchip CE but also in the various research areas related to lab-on-a-chips.

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Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2015R1C1A1A01052995). J. H. Hahn gratefully acknowledges the financial support from the Green Science Research Program (2015Y060) of POSCO, South Korea.

Supporting Information Available: Detailed experimental methods including materials, apparatus and instrumentation. Supporting tables and additional supporting figures as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figures and Captions

Figure 1. Schematic diagram of an acupuncture injection method. (A) A needle position is adjusted in the horizontal plane with the assistance of a CCD camera. (B) nL-scale sample segments are introduced into a PDMS microchannel by the acupuncture injection method.

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Figure 2. Consecutive CCD images recorded at the different stages of sample injection using acupuncture injection method; top view (A, B, C) and side view (E, F). (A), (D): initial state, (B), (E): injection point, (C): after injection. Black dotted-line shows a microchannel while a pink line in E shows a boundary between a glass substrate and a lower PDMS plate.

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Figure 3. (A, B, C) Consecutive CCD images recorded at the different stages of sample injection using an acupuncture injection method from the top view; (A): initial state, (B): injection point, (C): after injection. (D) CCD image of serial injections of 3 nL-segments to a channel leading to the formation of sample plugs in continuous flow.

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Figure 4. CZE separations of 10 µM FITC (peak 1) and 10 µM fluorescein (peak 2) using three sample injection modes; (A) gated mode injection, (b) pinched mode injection, and (C, D) acupuncture mode injection.

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Figure 5. CCD images of a bead-packed chamber with inlet and outlet frit structures; the top view (A, B) and side view (C, D). (E) Electropherogram obtained from CEC separation of 20 µM coumarin 440 (peak 1) and 40 µM coumarin 450 (peak 2) using a mobile phase with a buffer composition of 65% acetonitrile/35% 50 mM tricine buffer (v/v, pH 9.0).

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Graphical Abstract

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Acupuncture Sample Injection for Microchip ... - ACS Publications

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