nanofluidic device

Sensors and Actuators B 178 (2013) 683–688

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Rapid detection and quantification of bacteria using an integrated micro/nanofluidic device Zhongwei Wang a,c , Taeheon Han a,c , Tae-Joon Jeon b,c , Sungjin Park d , Sun Min Kim a,c,∗ a

Department of Mechanical Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon 402-751, Republic of Korea Department of Biological Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon 402-751, Republic of Korea Biohybrid Systems Research Center, Inha University, 100 Inha-ro, Nam-gu, Incheon 402-751, Republic of Korea d Department of Mechanical and System Design Engineering, Hongik University, 94 Wausan-ro, Mapo-gu, Seoul 121-791, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 13 June 2012 Received in revised form 21 December 2012 Accepted 7 January 2013 Available online 16 January 2013 Keywords: Bacteria Escherichia coli (E. coli) Detection Preconcentration Micromixer

a b s t r a c t In this study, we present an integrated micro/nanofluidic device that integrates a micromixer and a preconcentrator for the rapid detection of bacteria. The micromixer based on the concept of unbalanced splits and cross-collisions of fluid streams is used for mixing bacterial cells with tagging molecules. The preconcentrator consists of two microchannels (main and sub) that are connected by the nanochannels, which can be easily fabricated by electric breakdown of a ∼25 ␮m-thick polydimethylsiloxane (PDMS) membrane using high electric shock. Escherichia coli (E. coli) sample is tagged with fluorescent dye and continuously preconcentrated at the target position by applying the electric field through the junction of micro- and nanochannels. The concentration of bacterial sample can be quantified by measuring the fluorescence intensity at the preconcentrated region. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The rapid detection of bacterial cells in water plays an increasingly important role in many areas such as water contamination, food safety, public health, and pharmaceuticals. Traditional detection methods usually require a culture of single bacteria into bacterial media, followed by an identification process including morphological and biochemical tests that usually require a long measurement period, large sample volumes, high cost, and significant labor [1–4]. Microfluidic systems have been widely used for bacterial analysis and have shown many advantages over larger sized analysis systems. For example, a microfluidic system can analyze a sample with smaller reagent volumes in less time, and can perform multiple sample processing on a single device. For the detection of bacteria, several integrated microfluidic systems have been developed for the detection of bacterial DNA by real-time PCR amplification [5–8]. An electrochemical microfluidic biosensor that can detect the isothermal amplification of DNA was presented for rapid detection and quantification of Escherichia coli (E. coli) [9]. The

∗ Corresponding author at: Department of Mechanical Engineering, Inha University, 100 Inha-Ro, Nam-Gu, Incheon 402-751, Republic of Korea. Tel.: +82 32 860 7328; fax: +82 32 860 7328. E-mail address: [email protected] (S.M. Kim). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.01.017

impedance based method was also employed in the microfluidic devices to detect bacteria [10–12]. However, for a highly diluted bacteria sample, the detection time and sensitivity are still limited; thus, it is important to develop a microfluidic device that can detect bacteria rapidly and sensitively. Preconcentration is a key way to enhance the detector signal and bring it within the detection limit. Recently, an arrowheadshaped structure was designed and fabricated to guide E. coli cells to swim in a desired direction to concentrate E. coli cells without external mechanical and electrical energy sources [13]. Electrokinetic techniques have been widely applied to the preconcentration and separation of bacteria. For example, a microfluidic device that implements electrokinetic techniques, zone electrophoresis (ZE), and isoelectric focusing (IEF) was developed for the continuous concentration of bacteria [14]. The dielectrophoresis technique has also been widely used in the preconcentration of bacteria [15–17]. A microfluidic chip was demonstrated for the concentration of bacteria using the free flow electrophoresis principle [18,19]. Furthermore, a nanoporebased microfluidic device was presented for electrophoretic and dielectrophoretic trapping of particles that can be applied to the concentration of bacteria [20]. In addition, physical trapping method without additional electrokinetic effect was developed for the concentration of bacterial cells [21]. In this work, we integrated microfluidic components previously developed by our group for rapid pretreatment, preconcentration

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Fig. 1. (A) Photograph of the microdevice. (B) Schematic diagram of integrated micro/nanofluidic device for consecutive analysis with a micromixer, preconcentrator, and separation column; Inset, enlargement of preconcentrator part—the main channel and subchannel are separated by a thin PDMS membrane with a thickness of ∼25 ␮m.

and quantification of bacteria in a micro/nanofluidic device. The integrated device consists of a micromixer for sample tagging and an electrokinetic preconcentrator for bacteria cells. The micromixer (passive and planar) was used for sample preparation based on the concept of unbalanced splits and cross-collisions of fluid streams [22,23]. For bacteria preconcentration, we developed a nanochannel-based preconcentrator which can be easily fabricated by the electrical breakdown of a thin polydimethylsiloxane (PDMS) membrane by applying a high electrical shock, and it has been used for the preconcentration of much smaller protein molecules using exclusion-enrichment effect (EEE) of the nanochannel combined with electrokinetic transport of the protein [23–26]. By employing this method, preconcentration of bacterial cells is easily realized in a micro/nanofluidic device by applying a continuous electric field through the nanochannel and continuously trapping the cells near the membrane.

between the main channel and subchannel. For fabricating identical size nanochannels, the electrical breakdown of the PDMS membranes were performed under the same experimental conditions: the main channel and subchannel were filled with 50 mM potassium chloride (KCl, Sigma–Aldrich Corp., US) solution after oxygen plasma treatment, and an electrical shock of 1300 V for 1 s was applied from main channel (inlet 1, 2 and outlet 3 as anode) to subchannel (as cathode). To ascertain the formation of nanochannels, the direct electrical current across the nanochannels was measured before and after the breakdown using a picoammeter/voltage source device (6487, Keithley Instruments, Inc., US). Before breakdown, the measured current was almost zero when applying a voltage of 5–25 V in 5 V steps. After breakdown, the voltage–current (V–I) curve showed a linear relation, thereby confirming the formation of nanochannels as shown in Fig. 2(C). Then, the device was thoroughly rinsed with deionized water.

2. Materials and methods

2.3. Estimation of nanochannel dimension

2.1. Device design and fabrication

The dimensions of nanochannels were estimated with fluorescent images and the equivalent electrical circuit model [28,29]. The microdevice was filled with 0.1 mM Rhodamine B dye in deionized (DI) water, as shown in Fig. 2(B), the width and the length of the nanochannels were measured using an inverted fluorescence microscope (Ti-U, Nikon, Japan) and NIS-Elements image processing tool (Nikon, Japan). The resistance (Rn ) of the nanochannels can be approximately calculated with the electrical current through microchannels and nanochannels. The depth of the nanochannels can be computed as:

Fig. 1 shows the schematics of the microfluidic device composed of a micromixer and a preconcentrator. The widths of the main and subchannel are 300 ␮m and 50 ␮m, respectively, with a height of 50 ␮m. The micromixer has two split channels with the following widths: w1 and w2 are 200 ␮m and 100 ␮m, respectively, with a width ratio of w1 /w2 = 2. The preconcentrator part contains a main and a subchannel that are separated by a 25 ␮m-thick PDMS membrane. The microfluidic device was fabricated using the polydimethylsiloxane (PDMS) replica molding method. We poured a 10:1 weight ratio mixture of silicon elastomer and curing agent (Sylgard 184 set, Dow Corning, US) onto a SU-8 photoresist (2050, MicroChem Inc., US) patterned silicon wafer that was fabricated using photolithography techniques [27] and cured at 70 ◦ C for 2 h. The cured PDMS replica was peeled from the silicon wafer and holes for the inlets and outlets were punched. The replica was bonded to microscopic slide glass substrate using oxygen plasma treatment (CUTE-100LF, FEMTO Science, Korea) and cured at 70 ◦ C for 24 h. Then reservoirs were bonded onto the device for sample injection. For removing the PDMS debris which were formed during hole punching and other unwanted particles, 0.1 M NaOH solution was continuously pumped into the microchannel for 5 min using aspirator, then the device was thoroughly rinsed using deionized water. 2.2. Fabrication of nanochannel Fig. 2(A) shows a schematic diagram of nanochannels that were formed by the electrical breakdown of the PDMS membrane in

Dn = 

Ln Wn Rn

where  is the resistivity of the KCl solution, Dn is the depth of the nanojunction, and Ln and Wn are the length and width of the nanojunction, respectively. 2.4. Sample preparation For the cultivation of E. coli, a small colony of E. coli cells (DH5␣) grown on a Luria broth (LB) solid medium plate was cultured to a late log phase in 5 ml of Luria broth (LB) medium using a rotary shaking incubator at 37 ◦ C and 180 rpm. Then, the E. coli cells were centrifuged at 2500 × g for 10 min and the supernatant was removed. To prevent the proliferation of E. coli, the pelleted E. coli cells were killed by resuspending them in 70% isopropyl alcohol (IPA) and incubating them at room temperature for 1 h with mixing every 15 min. The E. coli cells were centrifuged at 2500 × g for 15 min and resuspended in 10 mM phosphate buffered saline

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Fig. 2. (A) Schematic diagram of the nanochannel formation by electrical breakdown of PDMS membrane using high electric shock. (B) Fluorescent image of nanochannels. (C) Current–voltage curves through the main channel to the subchannel before and after PDMS breakdown (for confirming the formation of nanochannels).

(PBS, Sigma–Aldrich Corp., US) solution for washing, and this step was repeated two more times. The E. coli was diluted into 1 × 104 , 1 × 106 , 5 × 106 , and 1 × 107 cells/ml concentrations using 10 mM PBS. 6 ␮l propidium iodide (PI, LIVE/DEAD BacLight Bacterial Viability Kit, L7012, Molecular Probes, US) for each ml of 10 mM PBS solution was prepared to mix and stain the E. coli sample. 2.5. Micromixing After treating the device with oxygen plasma, mixing experiment was performed by injecting diluted E. coli sample solution and the PI solution into inlets at a Reynolds number of 80 using multi-feed syringe pump (KD200, KD Scientific Inc., US) [22]. The mixed solution was incubated for 5 min to combine E. coli cells with PI fluorescent dyes, and was homogenized using a diffusion process.

3. Results and discussion 3.1. Micromixing and preconcentration of E. coli Mixing was evaluated by confirming the concentration distribution of fluorescent dye across the flow at the outlet of the micromixer. The fluorescent intensity at the outlet of the micromixer became uniform that demonstrated the good mixing result [22]. It is remarkable that micromixing was accomplished by introducing E. coli sample solution and PI solution into the main channel from different inlets using multi-feed syringe pump, and this process can decrease the initial concentration of the E. coli sample by a factor of 0.5. Then, we adjusted the volume of solution in the reservoirs for zero bulk flow velocity, which provided approximately 5 min for incubating E. coli cells with fluorescent dyes. When the velocity of the bulk flow drops to zero, E. coli cells become to sink down due to gravity and adsorbed on the bottom of the device.

2.6. Preconcentration Fig. 3 illustrates the mechanism of preconcentration, which is based on electrokinetic trapping of E. coli. The surfaces of the PDMS, glass, and nanochannels are negatively charged that can attract the positively charged ions in PBS solution, which forms an electrical double layer (EDL) near the interface. The electroosmotic flow (EOF) can be developed by applying an electric field to the interfacial EDL. In micro/nano sized channel, EOF is significantly amplified and strong enough to dominate over electrophoresis (EP) and drives the fluids and particles (e.g. E. coli cells) from the anodic side to the cathodic side. The nanochannels provide physical barriers to trap E. coli cells, thereby leading to a continuous preconcentration process. For preconcentration of E. coli, the main channel was connected to high voltage and the subchannel was grounded using Pt electrodes, thereby producing a continuous electric field using a voltage supply system that was controlled by LabVIEW 8.0 software (National Instrument, US) through a data acquisition (DAQ) board (PCI-6221, National Instrument, US). To observe the mixing and preconcentration of an E. coli sample in the PDMS device, we used the inverted fluorescence microscope integrated with a charge coupled device (CCD, DS-Qi1Mc, Nikon, Japan) camera and microscope software (NIS-Elements, Nikon, Japan). Throughout the preconcentration experiment, the electric current across the nanochannels was monitored using the picoammeter/voltage source. To quantify the preconcentration, fluorescence images were obtained and the average fluorescence intensity of the area of preconcentrated E. coli was analyzed using the NIS-Elements software.

Fig. 3. Preconcentration mechanism. (A) Configuration of preconcentration (C–C is the cross-section of the preconcentrator). (B) Cross-sectional view at C–C showing that the nanochannel provides a physical barrier to trap E. coli cells.

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Fig. 5. Preconcentration of E. coli samples using the same initial concentration of 107 cells/ml and the same voltage (80 V) configuration as shown in Fig. 3(A) with different average currents.

Fig. 4. (A) Time sequence images of preconcentration of 107 cells/ml of E. coli sample. (B) Quantification of preconcentration—the average fluorescence intensity was measured within the rectangle.

To prevent this problem, the preconcentration process should be immediately carried out after adjusting bulk flow. We compared the influence of incubation time on tagging efficiency of E. coli with PI and found that there was no significant difference between the standard 15-min incubation and on-chip incubation of E. coli with PI. It is also previously reported that the incubation time does not significantly influence on the tagging efficiency of bacteria with this LIVE/DEAD BacLight Bacterial Viability Kit [30]. After incubating the E. coli with PI fluorescent dyes for 5 min, preconcentration was carried out with a voltage of 80 V applied across the nanochannels. The image of the nanochannels are shown in Fig. 2 with the dimensions of 25 ␮m length (Ln ), 24 ␮m width (Wn ), and the depth (Dn ) is approximately calculated to be ∼205 nm using an electrical circuit model. Fig. 4(A) shows time sequence images of the preconcentration of the E. coli sample with an initial concentration of 1 × 107 cells/ml in 10 mM PBS solution. As shown in Fig. 4(A), the preconcentration process images were taken before the electric field was applied (at 0 min) after applying a voltage of 80 V across the main channel (anode) and the subchannel (cathode) for 3 and 6 min respectively, with a current of ∼44 ␮A, which was monitored using the picoammeter/voltage source throughout the preconcentration experiment. To quantify the preconcentration process, the average fluorescence intensity of the preconcentrated E. coli within the red rectangular window was measured after a background correction. The fluorescence intensity increased rapidly and showed good preconcentration performance within 6 min, as shown in Fig. 4(B). 3.2. Optimization of the preconcentration process When the same voltage is applied across the nanochannels, the current depends on the dimensions (or electrical resistance) of the nanochannels which are generated by the electric breakdown of the PDMS membrane. In some cases, the resistances of the nanochannels are irregularly formed even under the same experimental conditions, which may affect the preconcentration rate and cause experimental errors. For investigating this problem, additional preconcentration experiments were performed using

different devices with different resistances of nanochannels, which resulted in different currents during preconcentration experiments. The same initial concentration of E. coli samples (1 × 107 cells/ml) and the same voltage (80 V) were used, as shown in Fig. 4. The high current led to high rate of fluorescence intensity growth, which resulted in high preconcentration rate (Fig. 5). However, when the improper currents were applied, the fluorescence intensity profile shows an irregular pattern (40 ␮A) or slow increase (25 ␮A and 31 ␮A). These phenomena can be explained by examining Fig. 3(B). When the depth of the nanochannel is comparable to the electrical double layer thickness (EDL, D ) of the charged surfaces, a perm-selective nanochannel is formed. Negatively charged ions are excluded from this nanochannel, which leads to a continuous enrichment of negatively charged ions on the anodic side of the nanochannel [31]. The continuous enrichment of negatively charged ions could initiate strong concentration polarization near the nanochannel, even at moderate buffer concentrations. This strong concentration polarization generates a nonequilibrium electroosmotic flow (EOF) and fast fluid vortices that affect the preconcentration process [32,33]. As shown in Fig. 5, the proposed device exhibited good preconcentration performance within 6 min. The value of the fluorescence intensity at 6 min is proportional to the current when the same voltage was fixed at 80 V. This result well corresponds with the previous study [32]. However, there is no significant difference in fluorescence signals when the currents are 40, 44 and 45 ␮A. Thus, monitoring current during preconcentration plays an important role and the experimental results can be optimized by performing the experiment under the same experimental configurations: same dimensions of nanochannels and the same V–I curves after PDMS breakdown. 3.3. Quantification of E. coli preconcentration For quasi-quantification of E. coli sample with our method, we proposed to draw a standard curve using the preconcentration results of standard samples. In micro-sized channel, the sample volume is extremely small (down to several microliters) and the detection limits of E. coli are often down to single cell per microliter which is equivalent to 1 × 103 cells/ml. Thus, E. coli preconcentration experiments were performed with four different initial concentrations of E. coli samples (1 × 107 , 5 × 106 , 1 × 106 , and 1 × 104 cells/ml) and the devices with almost the same dimensions of nanochannels (Ln = 25 ␮m, Wn = 24 ± 2 ␮m, and Dn = 205 ± 10 nm). All the preconcentration experiments were under the same electrical configuration in Fig. 3(A), which had the same currents (42 ± 2 ␮A) when a voltage of 80 V was applied. Each

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Fig. 6. (A) Preconcentration of E. coli samples with four initial concentrations using the same electrical configuration as shown in Fig. 3(A) (the average current is 42 ± 2 ␮A when a voltage of 80 V is applied). (B) Standard curve (orange) of fluorescence intensity ratio to initial concentration ratio, and a linear fitting curve (black) for the standard curve. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

experimental data point shown in Fig. 6(A) is in proportion to the initial concentration of the E. coli sample. For calculating the preconcentration factor, E. coli was cultured to late log phase with a stationary concentration of ∼1 × 108 cells/ml and its fluorescence intensity was measured and shown in Fig. 6(A) as a standard sample. The fluorescence intensity of 1 × 104 cells/ml E. coli sample after 1-min preconcentration is approximately equal to the intensity value of the standard sample solution, which means about 104 -fold concentration factor was achieved within 1 min. This result shows that our device achieves much faster and higher concentration than other methods [13–21]. As far as we know, our approach shows the fastest concentration especially for bacteria up to now. Fig. 6(B) shows the fluorescence intensity ratio to the initial concentration ratio. In this plot, the lowest concentration of the E. coli samples (1 × 104 cells/ml) is defined as 1, and its fluorescence intensity after 6 min of preconcentration is also defined as 1. This plot provides a standard calibration to determine the initial concentration of the E. coli sample and compare the sample to the standard curve for quasi-quantification of E. coli sample. Also an R-Square value of a linear fit curve is 0.9067 shown in Fig. 6(B), so about 9% variance may be explained by the differences in currents (42 ± 2 ␮A) during preconcentration.

4. Conclusions A micro/nanofluidic device that integrates a passive micromixer and a preconcentrator was developed for the rapid analysis of bacteria. The proposed device can be used to mix a bacteria sample with tagging molecules and then preconcentrate the bacteria, sequentially. The nanochannel-based preconcentrator can rapidly and highly concentrate a bacteria sample within 6 min by the electrokinetic trapping of bacteria, and the preconcentration can be optimized by applying a same voltage and monitoring the electrical current through nanochannels. The nanochannel plays an important role in trapping bacteria cells and can be easily fabricated using the electric breakdown of a PDMS membrane using high electric shock. In this study, we demonstrate a novel method for rapid detection and quasi-quantification of bacteria sample using microfluidic device. The unique features of this device include the following: The device (i) uniquely integrates tagging, preconcentration and quantification of bacteria in a single device; (ii) easily realizes rapid and high preconcentration of bacteria using a nanochannelbased preconcentrator, which is the fastest method for bacterial

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Biographies Zhongwei Wang received BE degree in Mechanical Engineering from Shenyang Aerospace University, Shenyang, China in 2009. He is now taking the course of Master Degree in Thermodynamics and Fluid Mechanics at Inha University, Incheon, Republic of Korea. His research interests are Micro Total Analysis Systems (␮TAS), Bio-MEMS, Micromixer, Electrokinetic protein preconcentration, Bacteria detection, and Caenorhabditis elegans analysis. Taeheon Han received his BS (2003) and MS (2008) degrees in mechanical engineering from Inha University, Incheon, Republic of Korea. Now he is a PhD candidate in the same institute and his research interests are microfluidic system for biochemical analysis and cell based biosensors. Tae-Joon Jeon received his BS (2001) degree in the Department of Chemical Engineering from Seoul National University, Korea, and his PhD degree in the Chemical and Biomolecular Engineering from University of California, Los Angeles (UCLA) in 2008, followed by his postdoctoral work in the Department of Bioengineering at UCLA in 2008. He joined a faculty member in the Department of Biological Engineering at Inha University, Incheon, Korea, and is now an Assistant Professor in the same department. His research focuses on ion channels, lipid bilayer membranes, biomimetic membranes/cells, ion channel based biosensors, and immunobiosensors. Sungjin Park is an assistant professor in the department of mechanical and system design engineering at Hongik University at Seoul. He received PhD in the department of mechanical engineering at the University of Michigan at Ann Arbor. He received MS and BS in the Seoul National University. His areas of research are thermodynamic and fluid dynamic analysis, heat and mass transfer. Sun Min Kim received his BS (1997) and MS (1999) degrees in mechanical engineering from the Seoul National University, Korea, and an MS degree in biomedical engineering and his PhD degree in mechanical engineering from the University of Michigan, Ann Arbor in 2005 and 2006, respectively. In 2007, he joined the faculty of the Department of Mechanical Engineering, Inha University, following post-doctoral work at the Brigham and Women’s Hospital, Harvard Medical School, MA. He is interested in the fundamental understanding and development of micro/nanofluidic systems for biochemical sample analysis, cell-based biosensor, and cell analysis.