Microchim Acta (2011) 173:331335 DOI 10.

1007/s00604-011-0567-6

ORIGINAL PAPER

Integrated micro/nano-fluidic system for mixing and preconcentration of dissolved proteins

Khalid Anwar & Taeheon Han & Samuel Yu & Sun Min Kim

Received: 25 October 2010 / Accepted: 8 February 2011 / Published online: 22 February 2011 # Springer-Verlag 2011

Abstract An integrated micro/nano-fluidic system is presented for protein analysis. It is comprised of an integrated micromixer (IMM) and a preconcentrator with a separation column. The passive and planar type of IMM is based on an unbalanced split and the cross collision of the fluidic streams. The IMM can be easily fabricated and integrated to the microfluidic system. The preconcentrator has nanochannels formed by the electrical breakdown of polydimethylsiloxane (PDMS) membrane by applying a high electrical shock, but without any nano-lithography. The integrated microdevice was used for sample preparation (mixing with tagging molecules) and subsequent concentration of proteins. Proteins were electrokinetically trapped near the junction of the micro/nanochannels. We show a conceptual design and a simple microfluidic system for purposes of mixing and preconcentration. Keywords Micro/Nano-fluidic system . Micromixer . Protein preconcentration . Electrokinetic trapping

Introduction Integration of microfluidic system is a key to the micro total analysis system [14]. Tagging and preconcentration of protein in single chip is one of the most promising fields of the application of such integrated systems. For biosensing, the detection sensitivity is still limited by the highly diluted analytes in the microfluidic system. For instance, biomarker proteins related to cancer and other diseases are often
K. Anwar : T. Han : S. Yu : S. M. Kim (*) Department of Mechanical Engineering, Inha University, Incheon 402751, Republic of Korea e-mail: sunmk@inha.ac.kr

present at low concentrations, and are difficult to detect using standard immunoassays such as enzyme-linked immunosorbent assay (ELISA). It is, therefore, critical to bring the concentration within the detection limit. Numerous micromixing devices have been developed so far to overcome the mixing problem at microscale [5]. The integrated micromixer (IMM) should be simple, efficient and easily fabricated. Recently, our group designed a new IMM (passive and planar) is based on an unbalanced split, and the cross collision of the fluidic streams, which can be easily fabricated and integrated to the microfluidic system [6]. For sample preconcentration, a preconcentrator based on permselectivity of a nanochannel was introduced. Here, a simple method was employed to fabricate nanochannels working as a permselective membrane, which is an electrical breakdown of thin polydimethylsiloxane (PDMS) membrane by applying a high electrical shock [7]. In this study, the integrated micro/nano-fluidic system comprises of an IMM (passive and planar) and a nanochannel based preconcentrator. IMM was used for sample preparation and preconcentrator was subsequently used for concentration of protein sample. By applying a continuous electric field, fluorescently labeled proteins were trapped near the junction of micro/nanochannels. Device fabrication and experimentation PDMS replica molding method was used to fabricate the microfluidic device. The PDMS device consists of two microchannels (main and sub) separated by thin PDMS membrane thickness of ~25 m (Fig. 1). The widths of the main channel and the sub channel are 300 m and 50 m, respectively. Height of microchannels is 50 m and w1/w2 =2. After sealing the PDMS replica to slide glass using plasma treatment (CUTE-100LF, FEMTO Science, Hwasung,

332 Fig. 1 Optical micrograph of the microfluidic system

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Korea), nanochannels were formed by the breakdown of the PDMS membrane in-between the microchannels. Figure 2 represents the schematics of nanochannel formation process and optical image of the nanochannel. Electric shock was applied after filling both microchannels with potassium chloride (KCl, 50 mM). Nanochannels were formed between two microchannels by applying an electrical shock of 1,000 V for 1 s. The formation of nanochannel was assured by measuring current as a function of applied voltage before and after the breakdown (Keithley 6487 source/measure unit) [7]. Voltage was applied from 5 to 25 V with a 5 V step across the microchannels. Before breakdown the measured current was almost zero for all the applied voltages. But, after breakdown the V-I curve shows linear relation, which confirms the formation of nanochannel (Fig. 2d). Mixing was performed by introducing two fluids from different inlets using multi-feed syringe pump. To analyze mixing performance fluorescent solution (Rhodamine B) was used as a one fluid. Images were obtained using the inverted fluorescence microscope (Ti-U, Nikon, Tokyo, Japan) integrated with Nikon CCD camera. The fluorescence intensity in the image has been evaluated for mixing performance at inlet and exit of IMM using NIH ImageJ software.
Fig. 2 Schematic diagram of a preconcentration part of microfluidic system, b formation of nanochannel, c optical image of nanochannel formed by the electrical breakdown and d V-I curve across the microchannels

A fluorescein isothiocyanate-bovine serum albumin (FITC-BSA, Sigma-Aldrich Corp., St. Louis, MO) was mainly used to investigate the protein preconcentration. FITC-BSA samples were prepared using a 20 mM phosphate buffered saline (PBS, Sigma-Aldrich Corp., St. Louis, MO) at pH 7.4. For preconcentration, main channel (filled with protein sample) was connected to high voltage and sub channel (filled with PBS solution) was grounded and a continuous electric field was applied. After taking fluorescence images, a rectangle area was preset near the nanochannel and average fluorescence intensity was calculated for all time sequence images with in the rectangle. Electrokinetic concentration of protein Mechanism of protein concentration can be explained based on ion-selective (or permselective) nanochannels (Fig. 3) [8]. It is known that nanofluidic channels (~50 nm in thickness) can support ion-selective to ion currents such as ion-exchange membranes by surface charge. The thickness of the electrical double layer (D, EDL) is usually of nanometer order (1~10 nm for 1~100 mM ionic strengths, assuming single valence counterions), as shown in Fig. 3b,

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the EDL formed in nanochannels might be overlapped since the size of nanochannel is comparable to EDL. As PDMS surface is negatively charged, the potential within the channel is negative as compared to electroneutral solution. It is therefore, anions (co-ions) are excluded from the nanochannel, and whereas cations (counterions) are enriched in the nanochannel to ensure the overall electroneutrality of the nanochannel. Hence, the nanochannel with negatively charged surfaces acts as a cation-selective membrane, and inversely for positively charged surfaces. Thus, negatively charged proteins are excluded from entering the nanochannel (at anodic side). However, the electric field applied through the whole channel network (micro- and nanochannels in series) causes electroosmotic flow (EOF) which dominates over electrophoresis (EP). Thus, protein molecules at the anodic side of nanochannel are transported by higher EOF through the microchannel toward the nanochannel. The protein molecules arriving near nanochannel are preferentially barred from passing through the nanochannel, and hence accumulate near the micro/nanochannel junction.
Fig. 3 Electrokinetic preconcentration mechanism. a Schematic of the micro/nanochannel system after the formation of nanochannel, A-A is at the position at micro/nanochannel junction. b Cross-sectional view at A-A, showing the depth of the nanochannel is comparable to the electrical double layer, so that the EDL of top and bottom surfaces can be overlapped

Results and discussion Mixing performance of the IMM was evaluated using PBS solution with and without fluorescence (Rhodamine B) as it was used to prepare protein samples. Figure 4a shows the images of the first and last (sixth) mixing segments for an

Fig. 4 a Fluorescence images represent the first and last mixing segments. b The variations of mixing index at the exit of the IMM using the value of the fluorescence intensity in gray scale image

334 Fig. 5 a Representative images for mixing process (FITC-BSA of 50 M and PBS solution). b Fluorescence intensity plot as a function of time for quantification of preconcentration

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unbalanced split, and the cross collision of the fluidic streams at Reynolds number 80. Variations of the fluorescence intensity have been evaluated by analyzing the images at inlet and exit as shown in Fig. 4a. To quantify the degree of mixing in the IMM, the mixing index (Mo) at 5.5 mm downstream of the end of the last sub-channel is defined as follows [6], s s2 M 1 s2 max 1

Mixing and subsequent preconcentration of protein sample was carried out. Mixing was accomplished by introducing two fluids (FITC-BSA sample and PBS solution) from different inlets at Re=80 using multi-feed syringe pump. Initial concentration of FITC-BSA sample was taken as low as 50 M. Low concentration sample shows weak signal so that the mixing visualization is difficult as shown in Fig. 5a. For quantification of mixing in this case, the mixing index of the IMM for fluorescent solution (Rhodamine B) was used, Fig. 4b. After mixing

where s is the variance of the mass fraction of the mixture in a cross-section that is normal to the direction of flow and s max is the maximum variance over the range of data. The variance of the mass fraction is defined as follows, r 1 XN s c cm 2 2 i1 i N where N is the number of sampling points on cross-section, ci is the mass fraction of the sampling point i and cm is the optimal mixing mass fraction. Variations of mixing index at the exit of the IMM are shown for six Reynolds numbers in Fig. 4b. To calculate mixing index, the fluorescence intensity has been evaluated on a line positioned at inlet and exit. The value of the intensity of the image was normalized by highest and lowest value. This normalized value of intensity was used to calculate the standard deviation from the mean value. Finally, mixing index was evaluated from the value of the standard deviation.

Fig. 6 Quantification of preconcentration; average fluorescence intensity was measured within a pre-defined rectangle (ROI)

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process, fluid was kept in microchannel for 2 min for homogenization using diffusion process. Mixing process decreases the concentration of FITC-BSA sample by a factor of ~0.5, predicted concentration of sample after mixing was 25 M. Preconcentration of the protein sample was performed after mixing process. The time sequence images of preconcentration process of FITC-BSA of 50 M and PBS solution are represented by Fig. 5b. For preconcentration of proteins, an electric potential of 100 V was applied across the main channels (made as anode) and the sub channel (made as cathode). Figure 5b (a) shows the image taken before electric field is applied and Fig. 5b (b~d) images are taken after applying 100 Vacross the microchannels for 2, 4 and 6 min, respectively. Figure 6 shows the protein concentration with respect to time lapse. To measure the average intensity of fluorescence, the ROI (Region of interest) is set as the rectangular window shown in Fig. 6. The fluorescence intensity of standard solutions were filled in the whole channel and measured by applying the same ROI of sample solution after the background corrections. The analyses of preconcentration results are demonstrated by comparing the average value of fluorescence intensity within the ROI. The ROI shows the concentration region within which average value of fluorescence intensity was measured to calculate the concentration of FITC-BSA at every step of time. The calculated average fluorescence intensity for protein concentration was compared with standard solution of 50 and 100 M concentrations.

protein sample, and nanochannel based preconcentrator was subsequently used to concentrate the protein sample by the electrokinetic trapping near the junction of micro/ nanochannels. Nanochannels were formed by the electrical breakdown of PDMS membrane by applying a high electrical shock, but without any nano-lithography. This research work is aimed to develop real protein analysis microdevice, which could be useful for biosensor application.
Acknowledgments This research was supported by Agency for Defense Development Research Grant (Grant number: UD090078ID) and Basic Science Research Program through the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (KRF-2007-331-D00064 and 20090065065).

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Conclusions An integrated micro/nano-fluidic system, consisting of IMM and preconcentrator, was designed and fabricated for protein analysis. A passive and planar IMM was used to prepare