This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT). (No. NRF-2021R1A2C1011380)

1. Microfluidic acoustophoresis chip design
NOTE: Figure 1 shows a schematic of the separation and collection of target microbeads from microchannels by acoustophoresis. The microfluidic acoustophoresis chip is designed with a CAD program.
<ol>
	<li>Design a microfluidic acoustophoresis chip that uses a mixture of aptamer-modified beads and streptavidin-coated polystyrene (PS) beads corresponding to the size of bacteria to study the separation performance of the device.</li>
	<li>Design a microfluidic acoustophoresis chip that collects PS beads at the target outlet and discards the rest through the outlet after injecting a PS sample mixture into the sample inlet (see Figure 2 and Table 1). 
	NOTE: For this purpose, the acoustofluidic chip is designed consisting of two inlets for injecting samples and buffer, a main channel with an attached piezoelectric transducer (PZT) to allow the microbeads to be aligned centrally, and two outlets through which specimens are collected and waste are discharged (Figure 3A)</li>
	<li>Design a microfluidic acoustophoresis chip in which microbeads, buffer, bacteria, and aptamers pass through the main channel, with the microbeads aligned centrally via in-chip acoustophoresis induced by the PZT (Figure 3B,C)</li>
</ol>
2. Microfluidic acoustophoresis chip fabrication
NOTE: Assemble four layers in the following order: a borosilicate glass-silicon layer, a silicon layer, a borosilicate glass layer, and a PZT layer, as shown in Figure 3A,B.
<ol>
	<li>Prepare borosilicate glass (the uppermost layer) with 2-mm-diameter holes by sandblast17 at the inlet and outlet for connecting polyetheretherketone (PEEK) tubes. The borosilicate glass measures 20 &#215; 80 &#215; 0.5 mm3.</li>
	<li>Prepare a 200-&#181;m-thick silicon channel layer with a microchannel with a cross-sectional area of 0.2 &#215; 0.2 mm2 formed using a photoresist and a silicon pattern obtained via deep reactive ion etching (RIE)18. Drill 1 mm diameter holes for the sample and buffer inlet channels and the collection and waste outlet channels during reactive ion etching. 
	NOTE: Here, ion-etching uses the RIE process to form microchannels. A photoresist was applied in the shape of a channel on a silicon wafer for the silicon channel layer. The PR-coated silicon wafer was etched with plasma generated by applying 13.56 Mhz to fluorine18.</li>
	<li>Prepare a chip with the layers above and below the silicon layer (20 &#215; 80 &#215; 0.5 mm3) prepared in step 2.2 (20 &#215; 80 &#215; 0.5 mm3) bonded to the borosilicate glass in step 2.1 and a third borosilicate glass layer using anodization at 1,000 V and 400 &#176;C19.</li>
	<li>Attach a PZT (20 &#215; 40 mm2) to the borosilicate glass layer along the microfluidic channel using a cyanoacrylate adhesive (10 &#181;L or less). 
	&#8203;NOTE: Apply the adhesive as a very thin layer in the channel using a cotton swab to minimize any change in height. Figure 3C is a picture of the device.</li>
</ol>
3. Bacterial strains and culture
NOTE: Refer to Table 2 to select and incubate GN and Gram-positive (GP) bacteria for experiments. For the culture method, refer to steps 3.1-3.4. All bacteria should be incubated under aerobic conditions until an absorbance of 0.4 at 600 nm (OD600) is obtained.
<ol>
	<li>For GN bacteria such as Escherichia coli DH5&#945;, Escherichia coli KCTC2571, Sphingomonas insulae, and Pseudomonas pictorum&#160;and GP bacteria such as&#160;Staphylococcus epidermidis&#160;and&#160;Staphylococcus pasteuri, incubate in Luria-Bertani medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>For Enterobacter (GN) and Bacillus megaterium (GP; KCTC 1021), incubate in nutrient broth medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>For Enterococcus thailandicus (GP), incubate in de Man, Rogosa, and Sharpe (MRS) medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>For Listeria grayi (GP), incubate in brain heart infusion medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>Centrifuge (9056 x g) the cultured bacteria for 1 min at room temperature (RT), then wash twice with 1x phosphate-buffered saline (PBS) buffer.</li>
	<li>Prepare the selected GN and GP bacteria for analysis by resuspending in PBS buffer.</li>
</ol>
4. Microbeads and immobilization of aptamer onto microbeads
<ol>
	<li>Resuspend the streptavidin-coated microbeads (10 &#181;m) mixture (Table of Materials) before use (mix via vortexing for 20 s).</li>
	<li>Prepare aptamer by denaturing at 95 &#176;C for 3 min and then refolding at 0 &#176;C for 2 min.</li>
	<li>Transfer 250 &#181;L of the resuspended streptavidin-coated microbeads mixture to a 1.5 mL tube and wash with Tris-HCl buffer (50 mM Tris, pH 7.4, 1 mM MgCl2, 5 mM KCL, 100 mM NaCl) at RT. Then add 100 &#181;L of biotinylated DNA aptamer to the tube.</li>
	<li>Incubate the mixture at RT for 30 min while rotating (25 rpm).</li>
	<li>After centrifugation (9056 x g), wash the tube twice with 200 &#181;L of Tris-HCl buffer at RT.</li>
	<li>Add 10 &#181;L of BSA (100 mg/mL) to the washed sample tube and incubate for 30 min at RT with rotation (25 rpm).</li>
	<li>Finally, wash the aptamer-modified microbeads twice by centrifugation (9056 x g) in Tris-HCl buffer at RT.</li>
</ol>
5. Acoustophoresis setup and operation
<ol>
	<li>Connect PEEK tubes to the two inlets for injecting two samples and buffer and the two outlets for collecting and discharging waste (Figure 4).</li>
	<li>Manually fill the microfluidic acoustophoresis channel with bubble-free demineralized water using a 10 mL syringe.</li>
	<li>Prepare a precision pressure controller with two or more output channels to control the fluid flow. Then, half-fill the vials with sample and buffer with two holes in their caps, respectively, and connect to the chip inlet. 
	NOTE: A precision pressure controller with two or more output channels can be replaced with multiple precision pressure controllers. At the buffer inlet, a buffer capable of generating a laminar flow that prevents the sample from moving to the center during sample injection is injected through a flow controller.</li>
	<li>After preparing the device, inject the sample and buffer by applying a pressure of 2 kPa to the sample inlet and 4 kPa to the buffer inlet using the precision pressure control device. 
	NOTE: At this time, for smooth laminar flow, the injection pressure of the buffer should be higher than the injection pressure of the sample. The flow is controlled by a flow controller fixed to the aspirator connected to the inlet channel.</li>
	<li>Focus on a bead to move it into the center of the microfluidic channel using the PZT while checking through the microscope. 
	NOTE: The larger the bead, the greater the effect on the waveform, so it is easier to align with the node point. Function generator with amplifier applies power to PZT to generate sine wave in the microchannel. Since the upper and lower portions of the microchannel are made of glass, the generated sine wave is reflected and creates a node point7.</li>
	<li>Generate a resonance frequency of 3.66 MHz using a single-channel function generator and amplify a typical signal by 16 dB (about nine-fold) using a power amplifier (Figure 4). 
	NOTE: The resonance frequency of the actuator must match the size of the channel; because the channel is square, the PZT operates at an accurate frequency to create a single node.</li>
	<li>Observe the separation and enrichment processes on the acoustofluidic chip with a fluorescence microscope and a high-speed camera operating at 1,200 fps.</li>
	<li>Quantify and analyze the presence or absence of GN bacteria and GP bacteria by checking the images taken with the fluorescence microscope camera of the bacteria-bound beads and bacteria discharged through the collection and waste outlet samples. 
	NOTE: Microbeads with buffer, bacteria, and aptamers pass through the main channel, and the microbeads are aligned centrally via in-chip acoustophoresis induced by the PZT. Finally, microbeads that have bound GN bacteria are collected at the collection outlet, and the uncollected bacteria are discharged through the waste outlet.</li>
</ol>

<ol>
	<li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustofluidic+separation+of+cells+and+particles.">Acoustofluidic separation of cells and particles.</a> Microsystem &amp; Nanoengineering. 5 (1), 1-18 (2019).</li><li>Lee, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer+affinity-bead+mediated+capture+and+displacement+of+Gram-negative+bacteria+using+acoustophoresis.">Aptamer affinity-bead mediated capture and displacement of Gram-negative bacteria using acoustophoresis.</a> Micromachines. 10 (11), 770 (2019).</li><li>Hirvonen, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+sample+preparation+of+positive+blood+cultures+for+the+direct+detection+of+methicillin-sensitive+and+-resistant+Staphylococcus+aureus+and+methicillin-resistant+coagulase-negative+staphylococci+within+one+hour+using+the+automated+GenomEra+CDXTM+PCR+system.">One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDXTM PCR system.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 31 (10), 2835-2842 (2012).</li><li>Swaminathan, B., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+food-borne+pathogenic+bacteria.">Rapid detection of food-borne pathogenic bacteria.</a> Annual Review of Microbiology. 48 (1), 401-426 (1994).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+manipulation+of+single+microparticles,+cells,+and+organisms+using+surface+acoustic+waves.">On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves.</a> Proceeding of the National Academy of Sciences of the United States of America. 109 (28), 11105-11109 (2012).</li><li>Karthick, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+impedance-based+size+independent+isolation+of+circulating+tumor+cells+from+blood+using+acoustophoresis.">Acoustic impedance-based size independent isolation of circulating tumor cells from blood using acoustophoresis.</a> Lab on a Chip. 18 (24), 2802 (2018).</li><li>Lenshof, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustofluidics+8:+Applications+of+acoustophoresis+in+continuous+flow+microsystems.">Acoustofluidics 8: Applications of acoustophoresis in continuous flow microsystems.</a> Lab on a Chip. 12 (7), 1210-1223 (2012).</li><li>Persson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+microfluidic+chip+technology+to+facilitate+automation+of+phage+display+selection.">Acoustic microfluidic chip technology to facilitate automation of phage display selection.</a> The FEBS journal. 275 (22), 5657-5666 (2008).</li><li>Klussmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The aptamer handbook: Functional oligonucleotides and their applications. , (2006).</li><li>Ellington, A., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346 (6287), 818-822 (1990).</li><li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligands+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249 (4968), 505-510 (1990).</li><li>Namnabat, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+numerical+simulation+of+acoustophoretic+motion+induced+by+boundary-driven+acoustic+streaming+in+standing+surface+acoustic+wave+microfluidics.">3D numerical simulation of acoustophoretic motion induced by boundary-driven acoustic streaming in standing surface acoustic wave microfluidics.</a> Scientific Reports. 11 (1), 11326 (2021).</li><li>Nimjee, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer+as+therapeutics.">Aptamer as therapeutics.</a> Annual Review of Pharmacology and Toxicology. 57, 61-79 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+aptamer+discovery+and+application.">Recent advances in aptamer discovery and application.</a> Molecules. 24 (5), 941 (2019).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acousto-microfluidics+for+screening+of+ssDNA+aptamer.">Acousto-microfluidics for screening of ssDNA aptamer.</a> Scientific Reports. 6 (1), 1-9 (2016).</li><li>Persson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+microfluidic+chip+technology+to+facilitate+automation+of+phage+display+selection.">Acoustic microfluidic chip technology to facilitate automation of phage display selection.</a> The FEBS Journal. 275 (22), 5657-5666 (2008).</li><li>Van Toan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+processes+for+glass+micromachining.">An investigation of processes for glass micromachining.</a> Micromachines. 7 (3), 51 (2016).</li><li>Jansen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+on+the+reactive+ion+etching+of+silicon+in+microtechnology.">A survey on the reactive ion etching of silicon in microtechnology.</a> Journal of Micromechanics and Microengineering. 6 (1), 14 (1996).</li><li>Hanneborg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicon-to-silicon+anodic+bonding+with+a+borosilicate+glass+layer.">Silicon-to-silicon anodic bonding with a borosilicate glass layer.</a> Journal of Micromechanics and Microengineering. 1 (3), 139 (1991).</li><li>Mach, A. J., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+scalable+blood+filtration+device+using+inertial+microfluidics.">Continuous scalable blood filtration device using inertial microfluidics.</a> Biotechnology and bioengineering. 107 (2), 302-311 (2010).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+filter+microchip+for+rapid+separation+of+plasma+and+viruses+from+whole+blood.">Simple filter microchip for rapid separation of plasma and viruses from whole blood.</a> International Journal of Nanomedicine. 7, 5019-5028 (2012).</li><li>Ai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+Escherichia+coli+bacteria+from+peripheral+blood+mononuclear+cells+using+standing+surface+acoustic+waves.">Separation of Escherichia coli bacteria from peripheral blood mononuclear cells using standing surface acoustic waves.</a> Analytical Chemistry. 85 (19), 9126-9134 (2013).</li><li>Ohlsson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+impedance+matched+buffers+enable+separation+of+bacteria+from+blood+cells+at+high+cell+concentrations.">Acoustic impedance matched buffers enable separation of bacteria from blood cells at high cell concentrations.</a> Scientific Reports. 8 (1), 1-11 (2018).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+dielectrophoretic+bacterial+separation+and+concentration+from+physiological+media+of+high+conductivity.">Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity.</a> Lab on a Chip. 11 (17), 2893-2900 (2011).</li><li>Kim, U., Soh, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+sorting+of+multiple+bacterial+targets+using+integrated+Dielectrophoretic-Magnetic+Activated+Cell+Sorter.">Simultaneous sorting of multiple bacterial targets using integrated Dielectrophoretic-Magnetic Activated Cell Sorter.</a> Lab on a Chip. 9 (16), 2313-2318 (2009).</li><li>Cai, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluidic+device+for+immunomagnetic+separation+of+foodborne+bacteria+using+self-assembled+magnetic+nanoparticle+chains.">A fluidic device for immunomagnetic separation of foodborne bacteria using self-assembled magnetic nanoparticle chains.</a> Micromachines. 9 (12), 624 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

We developed a sonic levitation microfluidic device for capturing and transferring GN bacteria from culture samples at high speed based on a continuous running method according to their size and type, and aptamer-modified microbeads. The long, square microchannel enables a simpler design and greater cost-efficiency for 2D acoustophoresis than previously reported20,21,22,23,<sup class...

Microfluidic platforms are being developed to isolate bacteria from medical and environmental samples, in addition to methods based on dielectric transfer, magnetophoresis, bead extraction, filtering, centrifugal microfluidics and inertial effects, and surface acoustic waves1,2.&#160;The detection of pathogenic bacteria is continued using polymerase chain reaction (PCR), but it is usually laborious, complex, and time-consuming3,4. Microfluidic acoustophoresis systems are an alternative to address this through reasonable throughput and non-contact cell isolation5,6,7. Acoustophoresis is a technology that separates or concentrates beads using the phenomenon of material movement through a sound wave. When sound waves enter the microchannel, they are sorted according to the size, density, etc., of the beads, and cells can be separated according to the biochemical and electrical properties of the suspension medium7,8. Accordingly, many acoustophoretic studies have been actively pursued9,10,11, and recently, 3D numerical simulations of acoustophoretic motion induced by boundary-driven acoustic streaming in standing surface acoustic wave microfluidics have been introduced12.
Studies in various fields are examining how to replace antibodies2,3. Aptamer is a target material having high selectivity and specificity, and many studies are being conducted2,9,10,13. Aptamers have advantages of small size, excellent biological stability, low cost, and high reproducibility compared to antibodies and are being studied in diagnostic and therapeutic applications2,3,14.
Here, this article describes a microfluidic acoustophoresis technology protocol that can be used for the rapid, efficient separation of Gram-negative (GN) bacteria from a medium using aptamer-modified microbeads. This system generates a two-dimensional (2D) acoustic standing wave through single piezoelectric actuation by simultaneously stimulating two orthogonal resonances within a long rectangular microchannel to align and focus aptamer-attached microbeads at the node and anti-node points for separation efficiency2,11,15,16.&#160;There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration.
This protocol can be helpful in the field of early diagnosis of bacterial infectious diseases, as well as a rapid, selective, and sensitive response to pathogenic bacterial infections through real-time water monitoring.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 &#181;m polystyrene microbeads</td>
			<td>Bang Laboratories</td>
			<td>PS04001</td>
			<td>Cell size beads</td>
		</tr>
		<tr>
			<td>10 &#181;m Streptavidin-coated microbeads</td>
			<td>Bang Laboratories</td>
			<td>CP01007</td>
			<td>Aptamer affinity beads</td>
		</tr>
		<tr>
			<td>4-inch Silicon Wafer/SU-8 mold</td>
			<td>4science</td>
			<td>29-03573-01</td>
			<td>Components of chip</td>
		</tr>
		<tr>
			<td>Aptamer</td>
			<td>Integrated DNA Technologies</td>
			<td>GN3-6&#39;</td>
			<td>RNA for bacteria conjugation</td>
		</tr>
		<tr>
			<td>Borosilicate glass</td>
			<td>Schott</td>
			<td>BOROFLOAT 33</td>
			<td>Components of chip</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Daihan</td>
			<td>CF-10</td>
			<td>Wasing particles</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>3M</td>
			<td>AD100</td>
			<td>Attach PZT to microchip</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>KCTC</td>
			<td>KCTC2571</td>
			<td>Target bacteria</td>
		</tr>
		<tr>
			<td>Functional generator</td>
			<td>GW Instek</td>
			<td>AFG-2225</td>
			<td>Generate frequency</td>
		</tr>
		<tr>
			<td>High-speed camera</td>
			<td>Photron</td>
			<td>FASTCAM Mini</td>
			<td>Observation of separation</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>As one</td>
			<td>HI-1000</td>
			<td>Heating plate for curing of liquid PDMS</td>
		</tr>
		<tr>
			<td>KOVAX-SYRINGE 10 mL Syringe</td>
			<td>Koreavaccine</td>
			<td>22G-10ML</td>
			<td>Fill the microfluidic acoustophoresis channel with bubble-free demineralized water.</td>
		</tr>
		<tr>
			<td>Liquid polydimethylsiloxane, PDMS</td>
			<td>Dow Corning Inc.</td>
			<td>Sylgard 184</td>
			<td>Components of chip</td>
		</tr>
		<tr>
			<td>LB Broth Miller</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>Cell culture (Luria-Bertani medium)</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Corp.</td>
			<td>IX-81</td>
			<td>Observation of separation</td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Capricorn scientific</td>
			<td>PBS-1A</td>
			<td>Wasing bacteria</td>
		</tr>
		<tr>
			<td>PEEK Tubes</td>
			<td>Saint-Gobain Ppl Corp.</td>
			<td>AAD04103</td>
			<td>Inject or collect particles</td>
		</tr>
		<tr>
			<td>Piezoelectric transducer</td>
			<td>Fuji Ceramics</td>
			<td>C-213</td>
			<td>Generate specific wave in channel</td>
		</tr>
		<tr>
			<td>Power amplifier</td>
			<td>Amplifier Research</td>
			<td>75A250A</td>
			<td>Amplify frequency</td>
		</tr>
		<tr>
			<td>Pressure controller/&#956;flucon</td>
			<td>AMED</td>
			<td>AMED-&#956;flucon</td>
			<td>Control of air pressure/flow controller</td>
		</tr>
		<tr>
			<td>Tris-HCl buffer</td>
			<td>invitrogen</td>
			<td>15567027</td>
			<td>Wasing particles</td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>SeouLin Bioscience</td>
			<td>SLRM-3</td>
			<td>Modifiying aptamer and bead</td>
		</tr>
	</tbody>
</table>

microfluidic acoustophoresis for flowthrough separation of gram-negative bacteria using aptamer affinity beads

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for the fast, efficient isolation of Gram-negative bacteria from a medium. This method enhances the separation efficiency using a mix of long, square microchannels. In this system, the sample and buffer are injected into the inlet port through a flow controller. For bead centering and sample separation, AC power is applied to the piezoelectric transducer via a function generator with a power amplifier to generate acoustic radiation force in the microchannel. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration. The device has a recovery rate of &gt;98% and purity of 97.8% up to a 10x dose concentration. This study has demonstrated a recovery rate and purity higher than the existing methods for separating bacteria, suggesting that the device can separate bacteria efficiently.

Figure 5 shows the image of bead flow as a function of PZT voltage (OFF, 0.1 V, 0.5 V, 5 V). In the case of the acoustophoretic chip introduced in this study, it was confirmed that as the voltage of the PZT increased, the central concentration of the 10 &#181;m-sized beads increased. Most of the 10 &#181;m-sized beads were concentrated in the center at 5 V of the PZT voltage. Through this result, a resonant frequency of 3.66 MHz was generated in a single channel function generator, and a gen...

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Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

This paper describesthe fabrication and operation of microfluidic acoustophoretic chips using the microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for fast, efficient isolation of Gram-negative bacteria from a medium.

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and ...

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2.4K Views.  Inje University. This paper describesthe fabrication and operation of microfluidic acoustophoretic chips using the microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for fast, efficient isolation of Gram-negative bacteria from a medium.

Video: Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

Terahertz Microfluidic Sensing Using a Parallel-plate Waveguide Sensor

Refractive index (RI) sensing is a powerful noninvasive and label-free sensing technique for the identification, detection and monitoring of microfluidic samples with a wide range of possible sensor designs such as interferometers and resonators 1,2. Most of the existing RI sensing applications focus on biological materials in aqueous solutions in visible and IR frequencies, such as DNA hybridization and genome sequencing. At terahertz frequencies, applications include quality control, monitoring of industrial processes and sensing and detection applications involving nonpolar materials.
Several potential designs for refractive index sensors in the terahertz regime exist, including photonic crystal waveguides 3, asymmetric split-ring resonators 4, and photonic band gap structures integrated into parallel-plate waveguides 5. Many of these designs are based on optical resonators such as rings or cavities. The resonant frequencies of these structures are dependent on the refractive index of the material in or around the resonator. By monitoring the shifts in resonant frequency the refractive index of a sample can be accurately measured and this in turn can be used to identify a material, monitor contamination or dilution, etc.
The sensor design we use here is based on a simple parallel-plate waveguide 6,7. A rectangular groove machined into one face acts as a resonant cavity (Figures 1 and 2). When terahertz radiation is coupled into the waveguide and propagates in the lowest-order transverse-electric (TE1) mode, the result is a single strong resonant feature with a tunable resonant frequency that is dependent on the geometry of the groove 6,8. This groove can be filled with nonpolar liquid microfluidic samples which cause a shift in the observed resonant frequency that depends on the amount of liquid in the groove and its refractive index 9.
Our technique has an advantage over other terahertz techniques in its simplicity, both in fabrication and implementation, since the procedure can be accomplished with standard laboratory equipment without the need for a clean room or any special fabrication or experimental techniques. It can also be easily expanded to multichannel operation by the incorporation of multiple grooves 10. In this video we will describe our complete experimental procedure, from the design of the sensor to the data analysis and determination of the sample refractive index.

The procedure for implementing a refractive index sensor for terahertz frequencies based on a grooved parallel-plate waveguide geometry is described here. The method yields a measurement of the refractive index of a small volume of liquid through monitoring of the shift in the resonant frequency of the waveguide structure

Refractive index (RI) sensing is a powerful noninvasive and label-free sensing technique for the identification, detection and monitoring of microfluidic ...

Picoinjection of Microfluidic Drops Without Metal Electrodes

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.

We have developed a technique for picoinjecting microfluidic drops that does not require metal electrodes. As such, devices incorporating our technique are simpler to fabricate and to use.

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of ...

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.

We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including ...

Thermal Measurement Techniques in Analytical Microfluidic Devices

Thermal measurement techniques have been used for many applications such as thermal characterization of materials and chemical reaction detection. Micromachining techniques allow reduction of the thermal mass of fabricated structures and introduce the possibility to perform high sensitivity thermal measurements in the micro-scale and nano-scale devices. Combining thermal measurement techniques with microfluidic devices allows performing different analytical measurements with low sample consumption and reduced measurement time by integrating the miniaturized system on a single chip. The procedures of thermal measurement techniques for particle detection, material characterization, and chemical detection are introduced in this paper.

Here, we present three protocols for thermal measurements in microfluidic devices.

Thermal measurement techniques have been used for many applications such as thermal characterization of materials and chemical reaction detection. ...

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for the purpose of investigating phase change of those liquid segments. The magnetic beads were externally controlled using a magnet, allowing for the beads to bridge the air valve between the adjacent liquid segments. A hydrophobic coating was applied to the inner surface of the tube to enhance the separation between two liquid segments. The applied magnetic field formed an aggregate cluster of magnetic beads, capturing a certain liquid amount within the cluster that is referred to as carry-over volume. A fluorescent dye was added to one liquid segment, followed by a series of liquid transfers, which then changed the fluorescence intensity in the neighboring liquid segment. Based on the numerical analysis of the measured fluorescence intensity change, the carry-over volume per mass of magnetic beads has been found to be ~2 to 3 &#181;l/mg. This small amount of liquid allowed for the use of comparatively small liquid segments of a couple hundred microliters, enhancing the feasibility of the device for a lab-in-tube approach. This technique of applying small compositional variation in a liquid volume was applied to analyzing the binary phase diagram between water and the surfactant C12E5 (pentaethylene glycol monododecyl ether), leading to quicker analysis with smaller sample volumes than conventional methods.

Here, we present a protocol to investigate multi-component phase diagrams using externally controlled magnetic beads as liquid carriers in a lab-in-tube approach. This approach can aid in applications that seek to gather further information on phase change in complex liquid systems.

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each thermoplastic of interest. Solvent bonding is a simple and versatile method that can be used to fabricate devices from a variety of plastics. An appropriate solvent is added between two device layers to be bonded, and heat and pressure are applied to the device to facilitate the bonding. By using an appropriate combination of solvent, plastic, heat, and pressure, the device can be sealed with a high quality bond, characterized as having high bond coverage, bond strength, optical clarity, durability over time, and low deformation or damage to microfeature geometry. We describe the procedure for bonding devices made from two popular thermoplastics, poly(methyl-methacrylate) (PMMA), and cyclo-olefin polymer (COP), as well as a variety of methods to characterize the quality of the resulting bonds, and strategies to troubleshoot low quality bonds. These methods can be used to develop new solvent bonding protocols for other plastic-solvent systems.

Solvent bonding is a simple and versatile method for fabricating thermoplastic microfluidic devices with high quality bonds. We describe a protocol to achieve strong, optically clear bonds in PMMA and COP microfluidic devices that preserve microfeature details, by a judicious combination of pressure, temperature, an appropriate solvent, and device geometry.

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each ...

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

A Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and analyzing cells at the single-cell level by eliminating the influence of external factors on the cellular microenvironment. Encapsulation of cells in droplets is dictated by the Poisson distribution as a function of the number of cells present in each droplet and the average number of cells per volume of droplet. Primary cells, especially immune cells, or clinical specimens can be scarce and loss-less encapsulation of cells remains challenging. In this paper, we present a new methodology that uses pipette-tips to load cells to droplet-based microfluidic devices without the significant loss of cells. With various cell types , we demonstrate efficient cell encapsulation in droplets that closely corresponds to the encapsulation efficiency predicted by the Poisson distribution. Our method ensures loss-less loading of cells to microfluidic platforms and can be easily adapted for downstream single cell analysis, e.g., to decode cellular interactions between different cell types.

This article presents a protocol for seeding scarce population of cells using pipette-tips to droplet microfluidic devices in order to provide higher encapsulation efficiency of cells in droplets.

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and ...