Laminar flow assisted anisotropic bacteria absorption for chemotaxis delivery of bacteria-attached microparticle
- Keon Huh†1,
- Darong Oh†1,
- Seok Young Son†1,
- Hyung Jung Yoo1, 2,
- Byeonghwa Song1, 2,
- Dong-il Dan Cho1, 2,
- Jong-Mo Seo1, 3 and
- Sung Jae Kim1, 4Email author
© Huh et al. 2016
Received: 21 October 2015
Accepted: 19 January 2016
Published: 3 February 2016
The concepts of microrobots has been drawn significant attentions recently since its unprecedented applicability in nanotechnology and biomedical field. Bacteria attached microparticles presented in this work are one of pioneering microrobot technology for self-propulsion or producing kinetic energy from ambient for their motions. Microfluidic device, especially utilizing laminar flow characteristics, were employed for anisotropic attachment of Salmonella typhimurium flagellated chemotactic bacteria to 30 um × 30 um and 50 um × 50 um microparticles that made of biodegradable polymer. Any toxic chemicals or harmful treatments were excluded during the attachment process and it finished within 100 s for the anisotropic attachment. The attachments were directly confirmed by fluorescent intensity changes and SEM visualization. Chemotaxis motions were tracked using aspartate and the maximum velocity of the bacteria-attached microrobot was measured to be ~5 um/s which is comparable to prior state of art technologies. This reusable and scalable method could play a key role in chemotaxis delivery of functional microparticles such as drug delivery system.
KeywordsLaminar flow Anisotropic Chemotaxis Microrobot S. typhimurium
Recently, microrobots, which are capable of performing microscopic motions, have been drawn significant attentions in various fields of nanotechnology and biomedical technology due to its micro-size and controllability. Especially, due to its small size, applications of microrobots including active drug delivery system for therapeutic treatments and also a tiny lens at the tip of endoscope for diagnostic care in human body have been recently proposed [1, 2]. Microrobots, however, need suitable microactuators for propulsion and one of the major challenges are sophisticated fabrication or assembly of a proper micro and nanoscale actuator for the microrobot. In the meantime, various types of actuators have been actively investigated considering their reliability, controllability and limited size for their potential applications to these microrobots, such as an electrostatic actuator [3, 4] and a piezoelectric actuator , etc. While these actuators can provide a fast and controllable movement in precise manner inside human body (low Reynolds number environment in terms of fluid dynamics [6, 7]), they either create inherent bubbles due to electrolysis, require continuous onboard power supply or are applicable only in transparent environment. Therefore, for the past few years, bio-actuator based microrobot approaches have been widely studied for resolving these problems.
Bio-actuators have many advantages over artificial actuators because of self-propulsion or producing kinetic energy from ambient for their motions . However, these bio-actuators inevitably have complex incubation procedures, and difficulty to integrate into objects to be manipulated using toxic chemical substances. Thus, various types of bio-actuator possessing a non-toxic substance and self-movement with no external power has extensively been studied . Among these bio-actuators, the flagellated bacteria has been actively researched because the motion of the bacteria have maximum speed up to 100 um/s, if required chemical resource are supplied under appropriate environment . While a number of researches were performed on applying these active bacteria to bio-actuator, their toxicity and random motions prohibit further applications so that detoxifying the bacteria and boosting up the speed of a microrobot in desired direction are highly required [11–13]. For this purpose, several researchers have developed new adhesion methods for anisotropically attaching the bacteria on the microstructures considering the directionality , which is the main issue of the research in bacteria-attached microrobot. While one can attach the bacteria during the fabrication of the target object , the bacteria can be harmed due to the toxic fabrication process and it is recommended to attach the bacteria after the objects are completely assembled. Behkam et al. demonstrated S. marcescens flagellated chemotactic bacteria as bio-actuator for the propulsion of polystyrene microbeads by oxygen plasma treatment. With the aid of external electric field, the bacteria-attached micro-bead’s motility was assessed (15–30 um/s, E k = 216.522 × 10−21 J but this number contained an electrostatic energy) where E k is kinetic energy, which is equal to the half of mass times velocity square, that the bacteria-attached microroparticle possessed so that E k can consider both the motility and mass effects simultaneously . Park et al. also demonstrated a conjugation method based on streptavidin/biotin reagents for strong attachment of the bacteria and microstructures by a high-affinity interaction. The bacteria-attached microparticle’s motility were verified using chemotactic behavior to tumor cell (0.5 um/s, E k = 0.0147 × 10−21 J) . Sahari et al. reported anisotropic attaching the bacteria by coating poly-l-lysine to surface of the polystyrene particles with different shapes (4.0 um/s, E k = 0.941 × 10−21 J) . Martel et al. researched a method by attaching magnetotactic bacteria to the polystyrene microbeads by immune response, producing the magnetic-guided auto-propelled microrobots. By applying electromagnetic field to the microrobot, the motility and direction was controlled (21 um/s, E k = 25.936 × 10−21 J) . However, these aforementioned methods harm the bacteria, has difficulty to modify the surface, and are often toxic for human body. Therefore, a new methodology for an effective directional control of bacteria-attached microrobot without less toxicity is required.
In this paper, we proposed a simple, but effective microfluidic structure for anisotropical absorption of bacteria to microparticles for demonstrating bacteria integrated microrobot. By flowing attenuated Salmonella typhimurium bacteria into the microfluidic channel, hydrophobicity of microparticles made of biodegradable polycaprolacton (PCL) enabled a quick bacterial absorption (<100 s) without any toxic adhesive substance or chemical modification. Moreover, the bacteria were attached only at the surfaces of microcubics facing against the flow direction and the rest of the surfaces were free from the absorption, since Laminar flow can eliminate any backflow or vortical flow. The maximum motility of the bacteria-attached microparticle was measured to be ~ 5 um/s (E k = 58.952 × 10−21 J) by chemotaxis which is comparable to prior state of art technologies.
A silicon master for poly-dimethyl siloxane (PDMS) mold was prepared as following. A 4-inch wafer was cleaned in piranha solution (H2SO4: H2O2 = 4:1) to remove residual organics. SU-8 2050 (MicroChem, USA) photoresist was spun (JSP4A, JD Tech, Korea). The coater parameters were set depending on the target height, for example, 500 rpm of 15 s for spreading and 4000 rpm of 40 s for conformal coating in case of 35 um, etc. After soft bake for 3 min at 95 °C, the layer was exposed by ultraviolet (UV) of 365 nm wavelength for 6.4 s (160 mW) using MA-6 Aligner (Karl-suss, Germany). After post baking at 95 °C for 6 min, the wafer was cooled down slowly to room temperature. Then the pattern was developed in PEGMA developer for 10 min. After rinsing with iso-propyl alcohol, hard bake was performed at 120 °C for 5 min. For a PDMS cast, we followed the general soft-lithographical fabrication processes . Briefly, PDMS (Sylgard 184 Silicone elastomer kit, Dow Corning) base was mixed with a curing agent at 10:1 and bubbles were removed by vacuum jar for 1 h. After pouring the solution onto the master, it was cured in an oven at 75 °C for 4 h. Cured PDMS piece was peeled off from the master and cut into each device. Punching out the inlet and outlet holes was followed. Then PDMS piece and slide glass were bonded by oxygen plasma treatment (Femto Science, Korea).
The biodegradable PCL micro chamber was prepared using the x-ray-lithography-based microfabrication method . Briefly, a 10 % PCL solution in di-chloro-methane (DCM) was poured on a silicon wafer and coated at 1400 rpm to obtain PCL film with the thickness of 15 um. A lamination process was used to form the double layered PCL solid film with the thickness of 30 um . X-ray synchrotron irradiation was performed at Pohang Accelerator Laboratory (PAL), Korea to pattern the PCL film with 30 um × 30 um cubic microparticles. A 45 % (w/v) potassium hydroxide (KOH) solution was used to separate the pattered PCL microparticles from the substrate. The microparticles were dispensed in deionized water so that the number density of the microparticles can be 104 ea./mL.
The random speed and motions of bacteria are controlled by the taxis characteristics . Among various taxis mechanisms, chemotactic characteristics have been recently studied  because a target site (e.g. cancer site) often released a specific chemical for bacteria to be followed. S. marcescens and S. typhimurium especially shows the directional movement toward chemo-attractant by flagella motor [24, 25]. Bacteria used in this work were attenuated S. typhimurium (SHJ2037) since it has chemotactic behavior toward a breast cancer site and also has shown a reasonable safety level by several clinical trials . In addition, the attenuated S. typhimurium is characterized by fluorescence expression for easy tracking of their movements. S. typhimurium were inoculated in solid Luria–Bertani broth medium (LB medium) consisted of 500 mL DI water, 7.5 g agarose powder (DUKSAN, Korea), 5 g sodium chloride (NaCl, DUKSAN, Korea), 5 g tryptone (Becton, Dickinson and Company, USA) and 2.5 g yeast extract (Becton, Dickinson and Company, USA). The solid LB medium was incubated at 37 °C for overnight since it is easier to get highly active bacteria in solid LB than in liquid LB. From the inoculated solid LB medium, one bacterial colony was picked and cultivated in the 10 mL of liquid LB medium that contained 50 ug/mL of Kanamycin and Ampicillin. Then the bacteria was incubated at 37 °C on shacking incubator at 120 rpm for 3 h. The engineered S. typhimurium at an optical density (OD600) of 1.0–1.5 (UV/VUIS Spectrophotometer-Optizen 2120 UV, Mechasys, Korea) were used in this experiment.
Main microfluidic channel of device was connected with syringe pump (Harvard PHD 2000, USA) using four-port switching valve (IDEX, USA) for sequential injection and the outlet channel was linked with another syringe pump. First, the mixture of microparticles (100 ea./mL) was diluted with dimethyl sulfoxide (DMSO, DAEJUNG, Korea) and injected into the device at a flow rate of 0.1 mL/min for 10 min. DMSO reduces friction between microparticles and the surface of channel. Since DMSO has toxicity to bacteria, DI water was injected to wash out DMSO. After DMSO was completely removed, S. typhimurium was pumped into main channel at 3 uL/s. The hydrophobicity of microcubics made of biodegradable PCL enabled a bacterial absorption without any adhesive substance or chemical modification. Moreover, the bacteria can be attached only to the surfaces of microparticles facing against the flow direction and the rest of the surfaces were free from the absorption. Lastly, back-pressure from the outlet channel was applied into the main channel for releasing and collecting the bacteria-attached microparticles. All of experiments were monitored with IX51 inverted fluorescent microscope (Olympus, Japan) and the images were post processed using CellSens software (Olympus, Japan).
Chemotaxis of S. typhimurium
Aforementioned S. typhimurium was attracted by aspartate acid . For evaluating the chemotactic motility, we used 10 mM of α-methyl-DL-aspartic acid (Sigma-Aldrich, USA) solution as a chemo-attractant in the flow-free microfluidic channel. The concentration of attractant was chosen as previous studies [16, 27]. Before the demonstration using the microparticles, the chemotaxis of S. typhimurium itself was tested in Y-shaped microchannel. The bacteria were injected from the left reservoir and 10 mM of aspartic acid was dropped only at bottom right reservoir. See Additional file 1. While capturing this movie, bacteria were able to be moved only by diffusion since there was no net flow from anywhere. However, the bacteria tended to move toward the bottom right reservoir and there was no reason for this movement except the chemotaxis. This video clearly showed that S. typhimurium has the chemotaxis properties.
Results and discussions
Microfluidic trapper for anisotropic bacterial absorption
Bacteria also followed the laminar streamline to approach to the microparticle. Since excess amount of bacteria were supplied from inlet reservoir, there was a higher stochastic chance for the bacteria to contact the microparticle surface than no flow condition in previous literature. However, the flow rate should be carefully determined since the bacteria should stay around the cube for the residence time (i.e. the time requires for the absorption.)
Microparticle trapping efficiency
Anisotropic bacteria absorption
Chemotaxis test of bacteria-attached microparticle
While this velocity value was considerably faster than previous bacteria-attached microparticle, one needs to have set up a proper measure, since previous studies had employed different mass (or size) of microparticles. Kinetic energy (E k = 0.5mv2) would be the candidate to investigate the efficiency of bacteria-attached microparticle. Larger mass could reduce the velocity, but could have higher E k value. Our presenting S. typhimurium attached microparticle has E k of 58.952 × 10−21 J which is more efficient to other studies mentioned in introduction section.
Laminar flow assisted anisotropic attachment of S. typhimurium flagellated bacteria to the microparticles were demonstrated for a chemotactic delivery. A low cost and scalable microfluidic device was designed to trap microparticles and controlling flows upwardly/downwardly enables an anisotropic attachment of the bacteria. Direct visualizations using fluorescent intensity and SEM imaging were performed the attachment and it was measured to be less than 100 s to complete the absorption. Chemotaxis test revealed that the maximum velocity of the bacteria-attached microparticle was ~5 um/s which is comparable to the state of art researches. Besides, this method excludes any toxic fabrication steps or chemicals so that the devices is reusable. Therefore, the presenting method is highly useful for a chemotaxis delivery of microparticles such as drug delivery system if the microparticles contains a drug. While the size of microparticle (30–50 um) are far too large to pass through the circulatory system, they are still useful when injected right near a lesion, not through a circulatory system.
KH and DO participated in design, fabrication and carried out testing and drafted the manuscript. SS reviewed the test results and wrote the manuscript. HY and BS fabricated the micro-particles that were needed on the experiment. DC and JS participated in editing the manuscript. SK guided design development, reviewed all test results, and supervised this study. All authors read and approved the final manuscript.
This work was supported by Future based Technology Development Program (Nano Fields) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2012-0001033 and 2012-0009563), Basic Science Research Program (2013R1A1A1008125), the Center for Integrated Smart Sensor (CISS-2011-0031870) and Korean Health Technology RND project, Ministry of Health and Welfare, Republic of Korea (HI13C1468, HI14C0559). The author gratefully acknowledged the financial support from BK21 plus program of Creative Research Engineer Development IT, Seoul National University.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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