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Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation Raheel Ahmad, Ghulam Destgeer, Muhammad Afzal, Jinsoo Park, Husnain Ahmed, Jin Ho Jung, Kwangseok Park, Tae-Sung Yoon, and Hyung Jin Sung Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03474 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 17, 2017

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Analytical Chemistry

Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation Raheel Ahmad$1, Ghulam Destgeer$1, Muhammad Afzal$2, Jinsoo Park1, Husnain Ahmed1, Jin Ho Jung1, Kwangseok Park1, Tae-Sung Yoon*2, and Hyung Jin Sung*1 1

Department of Mechanical Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea 2 Department of Proteome Structural Biology, KRIBB School of Bioscience, Korea University of Science and Technology, 125 Gwahak-ro Yuseong-gu, Daejeon 34141, Korea $

These authors contributed equally in this work. To whom correspondence should be addressed: Hyung Jin Sung ([email protected]); TaeSung Yoon ([email protected]). *

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Abstract We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particleconjugated target proteins from a complex fluid. The acoustofluidic device comprised of an interdigitated transducer (IDT) that produced high-frequency surface acoustic waves (SAW) in a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid flow inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin–biotin linkage to form the PS-apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS-apt15th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After forming the PS-apt15-th complex, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS-apt15-th separation from the non-specifically bound proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.


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Introduction Proteins play a vital role in maintaining body tissues, regulating hormones, mediating metabolic processes, and regulating immune responses.1 Protein expression variations are important for disease diagnostics.2 For example, protein aggregation, forming amyloids and fibrils, is critical to the development of obesity-related type II diabetes, stroke, hypertension, Alzheimer’s disease, and Parkinson’s disease.3 The detection and analysis of low concentrations of target proteins and their selective separation and purification are clinically important.4,5 The physicochemical properties of proteins, such as their size, charge, solubility, and affinity, are important markers in the design of conventional purification processes, such as chromatography, gel electrophoresis, enzyme-linked immune-sorbent assay (ELISA), polymerase chain reaction (PCR), and mass spectrometry, which offer reasonable detection and separation; however, the limited selectivity, low sensitivity, and high instrumentation, reagent, and labor costs associated with these techniques limit their utility.6–9 To overcome these limitations, plasmonic and digital ELISA, deoxyribonucleic acid (DNA)-mediated proximity assays, and nanomaterials (particles, rods, clusters, etc.) have been used to enhance protein sensitivity up to the aM level.4,5 As a protein target cannot be chemically duplicated for target amplification, DNA–polymer conjugates and nanoparticle-based bio-bar codes that use customized aptamers (single-stranded DNA (ssDNA)) to selectively bind target proteins, along with PCR-based signal amplification, may be used for protein detection and separation.10,11 The advent of microfluidics technologies has significantly improved the performances of conventional immunoassays used for biomarker detection by addressing several previously limiting factors, such as technical complexity, high costs, and long processing times.



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Microfluidics technologies can facilitate integration, reduce reagent consumption, and speed up sample processing by miniaturizing the sample flow channels – a fundamental advancement in point of care (POC) diagnostics.12,13 The antigen–antibody binding interactions commonly used to detect target proteins in immunoassays are being replaced by promising chemical antibodies or nucleic acid aptamers that are highly stable, small, easily manufactured, and readily modified due to their flexible structure.14,15 A microfluidic hybrid chip composed of a pH-sensitive hydrogel with integrated aptamers that reversibly bind to target molecules and non-destructively separate thrombin has been demonstrated; however, that device required iterative reagent recycling to achieve a reasonable sorting efficiency.16 An inertial microfluidic platform has been utilized with size-coded micro-beads that allow for antibody–antigen affinity-based protein and cell capture for separation without the need for sample recycling.17 However, the sorting efficiency of an inertial microfluidic platform commonly used for continuous, high-throughput, size-dependent particle sorting depends significantly on the microchannel geometry, flow conditions, and fluid properties.18,19 The magnetophoresis of silica coated superparamagnetic nanoparticles has been used to separate hemoglobin-laden nanoparticle aggregates from a complex mixture of bovine serum albumin by harnessing the electrostatic interactions between the target protein and silica coated nanoparticles.20 The magnetophoretic separation techniques have the capabilities to selectively bind and isolate the target biomolecules; however, they are limited by their fundamental requirement of the carrier micro/nanoparticles to be paramagnetic or ferromagnetic which must be driven by an on-chip integrated magnetic element or an off-chip permanent magnet.21–25 The acoustophoretic manipulation of microparticles based on their size, density, compressibility or shape has the potential to overcome the limitations of other active microfluidic techniques.26


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In the present work, we developed an active microfluidic platform that combines the aptamer affinity-based selective capture of target biomolecules using functionalized microparticles27,28 and acoustic waves to sort protein carrier particles through separate outlets to achieve separation. The on-demand electronic control of acoustic waves on an acoustofluidic platform29–32 and the use of a single aptamer-based assay circumvent the limitations of passive microfluidic devices and complex sample protocols.16,17 The acoustofluidic device is based on surface acoustic waves (SAW) technologies that are amenable to miniaturization,32– 35

and the microfluidic platform offers continuous, size-based, rapid target microparticle

separation.36–38 The bulk acoustic waves (BAW)-based acoustofluidic devices, using direct acoustic radiation forces by the standing acoustic waves, have been combined with functionalized beads using antibody-antigen interactions to realize agglutination and fluorescence assays,39,40 decomplexing of biofluids,41,42 enhancement of phage display,43 and affinity-bead-mediated cytometry.44 However, despite the advantages associated with BAWbased handling of functionalized microparticles, the dependence on antibody-antigen affinity has its limitations. The antibody’s larger size, longer developmental time, limited stability at high temperature and changing pH levels, and less flexibility in their design encourage researchers to find conjugation alternatives in aptamer-based affinity assays which can circumvent most of the limitations described above.45,46 To the best of our knowledge, active microfluidic separation techniques, and in particular acoustofluidic techniques, have not yet been used to capture and isolate targeted biomolecules from a complex fluid using aptamerbased carrier microparticles. In the present work, we conjugated customized aptamer (ssDNA) to streptavidin-functionalized polystyrene (PS) microparticles to capture target proteins from a mixed solution. The validity of this strategy was demonstrated using thrombin as a model target protein, which plays an important role in monitoring and assessing atherosclerosis, angiogenesis, hemostasis, and thrombosis.27,47 The sample protein mixture containing the



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target protein attached to the microparticles was pumped through the microchannel as two buffer sheath flows focused the sample prior to exposure to a SAW generated by an interdigitated transducer (IDT). The input AC signal frequency fed into the IDT was optimized with respect to the microparticle diameter, and a significant acoustic radiation force (ARF) was exerted on the carrier particles48,49 to deflect them from their laminar streamlines and segregate the target protein from the mixture and collect the target protein through a separate outlet port. Experimental Section Chemicals Streptavidin-coated microspheres were purchased from Polysciences, Inc. (USA). Tris (hydroxymethyl) aminomethane, sodium chloride (NaCl), potassium chloride (KCl) magnesium chloride (MgCl2), calcium chloride (CaCl2), ethylenediaminetetracetic acid (EDTA), human serum (from human male AB plasma), triton® X-100, tween® 20, and ultrapure DNase/RNase-free distilled water were purchased from Sigma-Aldrich (USA). DNase (TURBO DNA-freeTM kit) was procured from Ambion® Life Technologies (Lithuania). Human alpha thrombin was purchased from Sekisui Diagnostics, LLC. (USA). The DNA construct of mCardinal258 was synthesized and obtained in pBHA vector from Bioneer. Inc (Daejeon, Korea) and sub-cloned into pET21a vector using NdeI and XhoI restriction sites. The red fluorescent protein with polyhistidine tag was purified on an AKTA prime express system by Nickel affinity chromatography and further purified by Sephacryl S-100 (16/60) gel filtration chromatography, GE Healthcare Life Sciences (USA). The oligonucleotide (aptamer15: biotin-A15-GGT TGG TGT GGT TGG) used in this work was synthesized and purified using high-performance liquid chromatography (HPLC), GENOTECH (Daejeon, South Korea). All other chemicals were of analytical grade and were used without further


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purification. The binding buffer A consisted of 20 mM Tris-HCl, M NaCl, 1 mM EDTA, and 0.0005% Triton® X-100 with a final pH of 7.5. The binding buffer B consisted of Tris-HCl (20 mM), KCl (5 mM), MgCl2 (1 mM), NaCl (140 mM), CaCl2 (1 mM), and Tween 20 (0.1%) with a final pH of 7.4. Device Fabrication In this study, lithium niobate (LiNbO3, 128° Y–X cut, 4"dia. × 0.5 mm, 2sp, MTI Korea) was used as the piezoelectric substrates to fabricate an IDT for SAW generation. The process used to fabricate the microdevices was similar to that used previously.59–62 The IDT, comprising a bimetallic Au/Cr layer (1000 Å/300 Å thick) of comb-shaped uniformly spaced electrodes 7.5 µm in width and spacing (for a 130 MHz device), was deposited via e-beam evaporation, followed by a lift-off process.63 The polydimethylsiloxane (PDMS) channel was fabricated using conventional soft lithography. A thin layer of SU-8 2100 photoresist (MicroChem, Newton, MA) was spin-coated onto a Si wafer and patterned via ultraviolet exposure through a chrome mask. PDMS and the curing agent (10:1) were thoroughly mixed and poured onto the patterned SU-8 mold, followed by degassing to remove bubbles from the PDMS under vacuum pressure. After removing the bubbles and baking at 65°C for two hours, the PDMS was peeled from the Si substrate, and the microchannels were cut to the appropriate sizes. After punching the inlet and outlet ports, the PDMS microchannel was oxygen plasmabonded to the LiNbO3 substrate covered with a SiO2 layer (plasma exposure for 3 min at 200 W and 750 mTorr). After bonding, the device was cured in a 95°C oven for at least 15 min to strengthen the bond. The height and width of the PDMS microfluidic channel at the particle deflection or separation zone were 40 µm and 100 µm, respectively. Instruments



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The morphologies of the PS, aptamer-functionalized PS (PS-apt15), and thrombin captured PS-apt15 complex (PS-apt15-th) were examined by field emission scanning electron microscopy (SEM, SU8230, USA). The PS, PS-apt15, and PS-apt15-th samples were characterized using high-resolution powder x-ray diffractometer (HR-XRD, RIGAKU, USA). Fluorescence analysis was carried out using a Tecan Infinite® M200 PRO microplate reader (TECAN, Switzerland). Fluorescence microscopy was used to acquire the images (OLYMPUS, USA). Micro syringe pumps (neMESYS Syringe Pumps, Germany) and glass syringes (Hamilton, USA) were used to inject the sample and sheath flows. The highfrequency alternating current signal generated by an RF signal generator (Agilent N5181A) and amplified by a power amplifier (Mini Circuits LZT-22+) was used to actuate the IDT.59 Conjugation of Polystyrene Microspheres with Aptamer15 (PS-apt15) Streptavidin-coated PS was separated by centrifugation for 15 min from 100 µL of a solution containing 5 mg/mL streptavidin fluoresbrite® YG microspheres, 6 µm in diameter. The collected PS microspheres were rinsed twice with binding buffer A and re-dispersed in 20 µL of the same buffer. Eighty microliters of the 10 µM biotin-modified aptamer15 (apt15) solution were added to the above solution and incubated at 25°C for 15 min for complete conjugation of apt15 to the PS. The resulting PS microspheres modified with apt15 (PS-apt15) were separated by centrifugation and rinsed twice with binding buffer A. They were then resuspended in 100 µL of the same buffer solution. The as-prepared PS-apt15 were stored at 4°C prior to use. Capturing of Thrombin with PS-apt15 In a typical experiment, 200 µL of thrombin (5 µM) in binding buffer B was added to 20 µL PS-apt15. The mixture was then incubated at 37°C for 2 h on a dry shaker (200 rpm) to ensure complete interaction and capture of the thrombin. The PS-apt15 bound to the thrombin


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were then separated by the SAW. To demonstrate the specificity of this strategy, solutions containing the non-target protein (mCardinal2) or the target protein (thrombin) in a given concentration were subjected to the assay procedure. Thrombin was captured with PS-apt15 whereas the red fluorescent protein remained free in solution and was separated by the SAW. This observation clearly confirmed the excellent specificity of this method toward the target thrombin. Results and Discussion Mechanism underlying the Affinity-Based Protein Capture and Acoustofluidic Particle Separation Technique Streptavidin-functionalized microparticles were modified with a biotinylated customized aptamer to capture the target biomolecule with an affinity for the specific aptamer to form a microparticle–aptamer–biomolecule complex. The non-target biomolecules remained free in solution (see Figure 1A). The target molecule was separated from the non-target molecules by pumping the solution mixture through an acoustofluidic device composed of a singlelayered polydimethylsiloxane (PDMS) microchannel with an IDT patterned on top of the piezoelectric lithium niobate (LiNbO3) substrate to produce a high-frequency SAW. The microparticle–aptamer–biomolecule complex carrying the target was deflected by a SAWinduced ARF and sorted through a different outlet port (see Figure 1B). The mechanism behind the deflection of a selected microparticle by a particular frequency SAW has been well described in earlier works by our group.48–50 The separated sample was collected and treated with deoxyribonuclease (DNase) to catalyze the hydrolytic cleavage of the phosphodiester linkages in the aptamer DNA backbone, thus snipping the aptamer to release the target biomolecule for analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).



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Figure 1: Schematic diagram showing the target biomolecule–microparticle complex formation and the acoustofluidic separation device used to isolate the targeted protein using surface acoustic waves (SAW). (A) A biotin-labeled aptamer conjugated to streptavidin-functionalized microparticles (green) specifically captured the target biomolecules (blue) from a complex mixture. (B) A hydrodynamically focused sample fluid, pinched by the fluid flows from sheaths 1 and 2, was intercepted by the SAW originating from the interdigitated transducer (IDT) to laterally deflect target biomolecules (blue) carrying the microparticles from their laminar flow streamlines and isolate the targets from the non-target biomolecules (red). The separated biomolecules were collected through outlet 2 as the remaining sample passed through outlet 1.

Microparticle–Aptamer–Protein Complex Formation The target biomolecule, thrombin with a binding site I (exosite I), had an affinity for aptamer15 (apt15) attached to the polystyrene microparticles via the streptavidin–biotin interaction.51 The formation of the microparticle-apt15-thrombin complex (PS-apt15-th) was confirmed by characterizing the unmodified PS microparticles, PS-apt15 conjugate,52 and PSapt15-th complex using scanning electron microscopy (SEM) (see Figure 2A). Apt15 was observed to cover most of the PS surface via streptavidin–biotin interactions visible as a white web of DNA, whereas the thrombin captured by PS-apt15 resembled a thick web-like plant growing over apt15 to form a complex (PS-apt15-th). The ultraviolet-visible (UV-vis) spectra were analyzed to further characterize the unmodified PS, PS-apt15, and PS-apt15-th (Figure 2B). The unmodified PS showed an emission peak at 510 nm, whereas a significant decrease in the fluorescent intensities of PS-apt15 and PS-apt15-th indicated that apt15 and thrombin were successfully conjugated and captured, respectively, thereby covering most of


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the surfaces of the PS microparticles. X-ray diffraction (XRD) analysis (Figure 2C) of the samples revealed that pure PS did not produce sharp peaks but rather an amorphous halo.53,54 The significantly lower XRD intensities of the PS-apt15 and PS-apt15-th compared to PS indicated, respectively, the lower crystallinity of apt15 and the high molecular weight of natural thrombin, which had a complex amino acid composition.53

Figure 2: (A) SEM characterization of the streptavidin-functionalized polystyrene (PS) microparticles, aptamer15 (ssDNA) conjugation on PS (PS-apt15), and thrombin captured by PS-apt15 to form PS-apt15-th complex. UV-vis (B) and XRD (C) spectra of the three samples confirmed that apt15 and thrombin covered the surfaces of the PS microparticles gradually, resulting in lower intensities.

Particle Deflection by the Acoustic Waves The feasibility of the proposed concept was demonstrated by subjecting a sample containing thrombin attached to green fluorescent PS particles 6.0 µm in diameter to a SAW traveling perpendicular to the fluid flow with a frequency of ~130 MHz originating from the IDT placed parallel to the microchannel. When the IDT was turned off, the streamlines of the samples in the mixture were unaffected as the PS-apt15-th complex and buffer solution passed through outlet 1 (Figure 3A). Once the IDT was actuated, the PS-apt15-th complex 11 ACS Paragon Plus Environment


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separated from the mixture solution due to the lateral migration of the particles under the influence of the ARF and was collected at outlet 2, whereas the buffer solution passed through outlet 1 (Figure 3B). The samples collected at outlets 1 and 2 were analyzed using a hemocytometer, which showed that the PS-apt15-th particles were predominantly deflected and collected at outlet 2 when IDT was actuated; however, a negligible quantity of PS-apt15th was occasionally collected at outlet 1 due to experimental perturbations, such as the development of an odd bubble, which disturbed the laminar flow, and variations in the particle diameters that produced unexpected ARFs on the particles (see Figures 3C and 3D). The samples collected at either outlet, in the SAW off and SAW on modes, were treated with DNase, which snipped the apt15 strands and released the thrombin from the PS-apt15-th complex. The resulting solution was analyzed using SDS-PAGE. The pronounced gel band intensity clearly revealed that thrombin was efficiently deflected with the particles and collected at outlet 2 (Figure 3E).

Figure 3: Deflection of thrombin-laden PS fluorescent green microparticles by the SAW. (A) Unaffected microparticles along with the buffer solution flowed through outlet 1 when the SAW is off; however, the microparticles switched to outlet 2 as the SAW was turned on (B). The samples collected were analyzed using a hemocytometer in the SAW off (C) and SAW on (D) conditions, respectively. (E) After treating the samples with DNase, the solutions were analyzed using SDS-PAGE (13%) to confirm that thrombin was successfully switched from outlet 1 to outlet 2. A lighter band in lane 2, 3 and 6 indicated the formation of a thrombin dimer.

Separation of the Target Protein from a Complex Sample


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Target protein separation from a non-target protein was demonstrated by subjecting a solution containing the PS-apt15-th complex mixed with mCardinal2 (a red fluorescent protein) to a protocol similar to the one described above. When the IDT was turned off, the laminar flow streamlines were unaffected, and separation was not observed (Figure 4A); however, thrombin-carrying PS particles (green) were directed through outlet 2 and remained distinct from mCardinal2 (red) outlet 1 (Figure 4B) as PS particle deflection induced separation between the two proteins (Figure 4C). Hemocytometer analysis revealed that both proteins were present at outlet 1 when the SAW was off (Figure 4D); however, the PS-apt15-th complex was separated and collected at outlet 2 whereas mCardinal2 passed through outlet 1, as is evident from the red solution color in the corresponding figure (Figure 4E). Finally, SDS-PAGE analysis verified that the PS-apt15-th complex was successfully separated from the red fluorescent protein by the SAW (Figure 4F). Figure S1 shows the analysis of the samples collected at both outlets in the SAW off and SAW on modes.

Figure 4: Separation of thrombin captured by PS-apt15 (PS-apt15-th complex) from mCardinal2 (a red fluorescent protein). (A) In the SAW off mode, PS-apt15-th (green) and mCardinal2 (red) flowed through outlet 1. (B) Once the IDT was actuated, PS-apt15-th was separated from mCardinal2 as the PS particles (green) were deflected from their original streamlines (C) under the influence of the ARF. Hemocytometer analysis of the collected samples revealed that both proteins passed through outlet 1 when the SAW was off (D); however, the PS particles carrying thrombin passed through outlet 2 when the SAW was on (E). The red solution shown in the figure at outlet 1 indicated the presence of mCardinal2. (F) SDS-PAGE (13%) analysis confirmed protein separation, as the bands clearly appeared in different lanes. The following flow rates were adjusted: sheath flow rate 1 (Qsheath1): 200 µL/hr, sheath flow rate 2 (Qsheath2): 1000 µL/hr, sample flow rate (Qsample): 800 µL/hr, outlet



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1 (Qoutlet1): 1200 µL/hr, and outlet 2 (Qoutlet2): 800 µL/hr. The IDT was actuated at a frequency of 129 MHz. The power input was 0.85 W.

The utility of this strategy applied to complex biological samples was tested by detecting thrombin in spiked human serum. The human serum was isolated from a blood sample, diluted in 1:5 and 1:10 ratios with distilled water, and spiked with thrombin at a concentration of 2 µM. The thrombin captured by PS-apt15 were separated from the human serum sample using experimental conditions similar to those described above, and the separated thrombin passed through outlet 2 while the remaining serum sample passed through outlet 1, unaffected by the SAW (Figure 5A). The SAW response of the PS particles laden with thrombin captured from a complex serum sample was similar to the corresponding response of thrombin captured from a simple buffer solution (Figure 5B), which confirmed the practical utility of this method for biomolecular detection in complex biological fluids. The separation of target thrombin from human serum, which contains a plethora of non-target proteins, electrolytes, hormones, antibodies, and antigens, is a significant achievement using the proposed acoustofluidic platform, which takes advantage of aptamer-based chemistry. Figure S2 shows fluorescence images of the red fluorescent protein, thrombin-captured PS-apt15, human serum, and buffer solutions.

Figure 5: Separation of thrombin from spiked human serum. (A) Actuation of the IDT separated PS-apt15-th from the human serum (light green fluorescent solution). (B) PAGE analysis of the samples collected (in the SAW on mode) at both outlets clearly revealed the separation of thrombin from complex serum samples at


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different concentrations (5% and 10%). The following flow rates were adjusted: sheath flow rate 1 (Qsheath1):100 µL/hr, sheath flow rate 2 (Qsheath2): 900 µL/hr, sample flow rate (Qsample): 500 µL/hr, outlet 1 (Qoutlet1): 1400 µL/hr, and outlet 2 (Qoutlet2): 100 µL/hr. The IDT was actuated at a frequency of 119 MHz. The power input was 0.19 W.

Conclusions In conclusion, the highly selective separation of target proteins was demonstrated using aptamer-based conjugation of a target protein to a functionalized polystyrene particle driven by a high-frequency SAW generated using an acoustofluidic platform. Each PS microsphere was modified to carry thousands of aptamer (ssDNA) molecules.25 The separation performance resulting from the interaction between the model target thrombin and its aptamer was thereby amplified. The systematic evolution of ligands by exponential enrichment (SELEX) techniques has been extensively developed to produce an aptamer to recognize specific regions within a single target molecule.55–57 Therefore, the aptamer developed by SELEX could only be used for the specific target biomolecules. Consequently, by using a customized aptamer, we achieved the selective separation of target thrombin from mCardinal2 and a human serum sample (complex biological fluid). These results demonstrated the excellent capacity of the proposed acoustofluidic separation technique to selectively capture and isolate target proteins present in complex biological fluids. Particles of different sizes can be conjugated to different target proteins to realize multiplexed isolation assays based on the variable deflection of the carrier particles by acoustic waves. Supporting Information Hemocytometer analysis of collected samples (Figure S1) and fluorescent images of the samples (Figure S2). (PDF) Acknowledgements



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This work was supported by the KUSTAR-KAIST Institute, the Creative Research Initiatives (No. 2017-013369) program of the National Research Foundation (MSIP) of Korea, the Korea Polar Research Institute (KOPRI), and the KRIBB Research Initiative program. References (1)

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