Elastomeric microparticles for acoustic mediated bioseparations
© Johnson et al.; licensee BioMed Central Ltd. 2013
Received: 14 June 2013
Accepted: 14 June 2013
Published: 28 June 2013
Acoustophoresis has been utilized successfully in applications including cell trapping, focusing, and purification. One current limitation of acoustophoresis for cell sorting is the reliance on the inherent physical properties of cells (e.g., compressibility, density) instead of selecting cells based upon biologically relevant surface-presenting antigens. Introducing an acoustophoretic cell sorting approach that allows biochemical specificity may overcome this limitation, thus advancing the value of acoustophoresis approaches for both the basic research and clinical fields.
The results presented herein demonstrate the ability for negative acoustic contrast particles (NACPs) to specifically capture and transport positive acoustic contrast particles (PACPs) to the antinode of an ultrasound standing wave. Emulsification and post curing of pre-polymers, either polydimethylsiloxane (PDMS) or polyvinylmethylsiloxane (PVMS), within aqueous surfactant solution results in the formation of stable NACPs that focus onto pressure antinodes. We used either photochemical reactions with biotin-tetrafluorophenyl azide (biotin-TFPA) or end-functionalization of Pluronic F108 surfactant to biofunctionalize NACPs. These biotinylated NACPs bind specifically to streptavidin polystyrene microparticles (as cell surrogates) and transport them to the pressure antinode within an acoustofluidic chip.
To the best of our knowledge, this is the first demonstration of using NACPs as carriers for transport of PACPs in an ultrasound standing wave. By using different silicones (i.e., PDMS, PVMS) and curing chemistries, we demonstrate versatility of silicone materials for NACPs and advance the understanding of useful approaches for preparing NACPs. This bioseparation scheme holds potential for applications requiring rapid, continuous separations such as sorting and analysis of cells and biomolecules.
KeywordsCell separation Continuous cell sorting Acoustofluidics Particle synthesis Ultrasound standing wave
The capacity to relocate PACPs to pressure nodes has been used in various approaches for focusing and separation of mammalian cells [5–11]. For example, the recently commercialized Attune® flow cytometer (Life Technologies) substitutes traditional hydrodynamic focusing with ultrasonic standing wave fields to focus cells into a single flowing stream prior to laser interrogation . To increase the high-throughput capacity of flow cytometry, Piyasena et al. recently developed multi-node acoustic focusing and demonstrated up to 37 parallel flow streams . Peterson et al. exploited the inherent contrast factor of constituents from whole blood to separate and sort positive contrast erythrocytes from negative contrast lipids within an acoustofluidic device [7, 8]. Strategies for separating two particle populations with contrast factors of the same sign can exploit differences in the magnitude of the acoustic force [9, 10]. In certain cases, the contrast factor can be adjusted by changing the density of the solution, as shown in a report separating polystyrene and PMMA microparticles by increasing the salt concentration of the media .
Herein, we report on the preparation of NACPs and demonstrate the utility of these microparticles in a new acoustophoretic separation scheme. Specifically, NACPs are prepared using two different silicone elastomers and biotinylated using two different chemical modification approaches. The newly designed NACPs are evaluated as carriers for the transport of streptavidin PACPs to pressure antinodes within acoustofluidic devices. Our results reveal the potential of this approach for cell sorting applications.
Results and discussion
Silicone microparticles as biofunctional NACPs
NACPs as carriers for acoustic-mediated separations
The separation of silicone NACPs from polystyrene microparticles demonstrated in Figure 4 encouraged further investigations aimed at evaluating potential for the use of NACPs in cell separations. We hypothesized that NACP-PACP complexes within aqueous media will transport to pressure antinodes, provided the total radiation force from NACPs in the complex is greater than the total radiation force from PACPs in the complex. To this end, we employed polystyrene microparticles as surrogates for mammalian cells and investigated separation characteristics using NACPs prepared from PDMS. The brightfield image (Figure 2B) and accompanying fluorescent image (Figure 2C) show association between streptavidin coated polystyrene and PDMS microparticles functionalized with biotin-Pluronic F108. Notably, within the acoustofluidic device, the NACP-polystyrene microparticle complexes transport in unison to the pressure antinode (Figure 5B). This supports the notion that NACPs may serve as vehicles for specific transport of positive acoustic contrast particles. Conversely, non-biotinylated PDMS microparticles did not bind streptavidin polystyrene particles. This is shown in the negative control (Figure 5A) where non-biotinylated PDMS particles (red) transport to the pressure antinode and polystyrene microparticles (green) align at the pressure node. Figure 5 suggests the feasibility of a new bioseparation technique where transport of targeted PACPs (e.g., cells) will rely on specific, well-defined interactions with the NACPs. Figure 5 shows all PACP-NACP complexes transported to the antinode at the acoustofluidic wall (e.g., ~14 NACPs and ~12 PACPs in four separate complexes). However, additional studies are required to further understand the effects of parameters, such as particle ratios, flow rates, and applied voltages on efficiency of separation.
As expected, in the absence of fluid flow, NACPs accumulate at the pressure antinodes along the acoustofluidic channel walls during activation of the PZT (Figures 4 and 5). Secondary acoustic forces contribute to the aggregation of NACPs, as previously described for lipids in milk emulsions and whole blood [7, 8, 21]. This NAPC aggregation may be reduced by introducing flow to the channel. As recently demonstrated, laminar flow within the channel enables NACPs to maintain their position at the pressure antinode while simultaneously moving along laminar streamlines to the downstream trifurcation . This capacity to couple relocation with downstream sample collection facilitates continuous sorting applications.
To the best of our knowledge, this is the first report documenting the use of NACPs as carriers for active transport of PACPs in acoustofluidic systems. Although polystyrene microparticles were used as cell surrogates in this preliminary investigation to demonstrate separation, this approach should be suitable for cell sorting based on binding of NACPs to specific cell surface antigens. Because the positive acoustic contrast factor value of cells is less than polystyrene beads , we anticipate that cell-NACP complexes should readily transport to pressure antinodes. Thus, this method holds potential as a complement to current cell sorting techniques (e.g., fluorescence-activated or magnetic-activated cell sorting). In contrast to these conventional methods, the present technique offers the possibility of enhanced selectivity and separation efficiency since ultrasound wave fields exert forces on both NACPs and PACPs in opposing directions. Given this promise, it is necessary to further examine several aspects of using NACPs in cellular separations. For example, the role of bioaffinity bond strength between particles that are being subjected to force in opposite directions may need to be studied in detail. Likewise, the features that enable the primary radiation force of NACPs to dominate that of PACPs requires further investigation. The transport of PACPs to the pressure antinodes will only occur when a complex of PACP bound to NACPs exhibits an overall negative acoustic contrast factor, which can be adjusted through the volume, density, and bulk modulus of the NACPs. In the current study, these properties have converged to favor the relocation of PACP-NACP complexes to the antinode. We anticipate that future experimental and computational investigations will reveal the optimal parameters that support efficient cell separation.
This report communicates a new approach for bioseparation that employs polysiloxane-based microparticles with a negative acoustic contrast property. Emulsifying and post-curing pre-polymers within aqueous surfactant results in stable microparticles that transport to the pressure antinode of an ultrasonic standing wave field in aqueous media. By using polysiloxanes with different chemical compositions and curing chemistries (i.e., PDMS, PVMS), we demonstrate versatility and general utility of silicone materials as negative acoustic contrast agents. Both photochemical and physical adsorption approaches are used to biofunctionalize NACPs, ultimately enabling the specific capture and transport of PACPs to an acoustic pressure antinode. These results encourage further pursuits aimed at using NACPs for cell separation, owing to potential advantages of this system such as high sensitivity, selectivity, portability and low cost.
Preparation & functionalization of NACPs
Preparing PVMS particles: A mixture of 1.0 g of hydroxyl-terminated PVMS , 0.07 g vinylmethoxysiloxane homopolymer (Gelest), and between 0.02 g and 0.03g tin octoate catalyst (Gelest) was thoroughly stirred and combined with a solution of 0.5 or 0.7 wt% Pluronic® F108 (Aldrich) in ultrapure water (Mill-Q, 18MΩ resistivity). The mixture was briefly vortexed, homogenized using a PT 1200E homogenizer (Polytron) with a 3mm rotor for 5 min at 18,750 rpm, and stirred for at least 2 hr at ~50°C. The polydisperse emulsion was permitted to cure via alkoxy condensation of silanol-terminated PVMS with vinylmethoxysiloxane. Particles were left at ambient conditions for approximately one week, then filtered through a 12 μm polycarbonate membrane (Whatman, Cyclopore), and stored at ambient conditions until use. Preparing PDMS particles: A mixture comprising a 1:10 weight ratio of curing agent: base of Sylgard® 184 (Dow Chemical) was thoroughly mixed and 1 gram of the mixture was subsequently combined with 1 wt% of Pluronic F108. The mixture was homogenized as previously described. The emulsion was incubated at 45°C, stirring for at least 1.5 hrs and subsequently left at ambient conditions for at least 12 hrs to permit curing. Functionalization: For reactions with biotin-TFPA (Quanta Biodesign), ~5 × 107 PVMS microparticles were washed with 1× PBS by centrifuging and resupending the pellet in a final volume of 2 mL of 1× PBS. The microparticles were transferred to a cylindrical glass vial (2.5 cm diameter) and 3 mg biotin-TFPA in 100 μL of dimethylacetamide was added. Light irradiation occurred using an Omnicure S1000 equipped with a high pressure mercury lamp and an internal 320–500 nm filter. The associated light guide was placed ~5 mm above the stirring solution for 30 min at a light intensity of ~100 mW/cm2 at a wavelength of 365 nm, (as measured by Powermax USB sensor, Coherent). The resultant yellow solution was stored at 4°C until use. Biotinylation of Pluronic F108 surfactant followed a similarly reported protocol . Briefly, hydroxyl end groups on F108 were modified to succinimidyl carbonate using N,N’-disuccinimidyl carbonate (Aldrich) and 4-(dimethylamino)pyridine (Aldrich) and subsequently reacted with biotin-hydrazide (Aldrich). Once biotinylated, Pluronic F108 was used to prepare silicone emulsions as previously described. Subsequent addition of streptavidin (AlexaFluor® 488 or AlexaFluor® 546) to NACPs occurred by washing particles at least three times by centrifuging and resuspending the pellet in 1× PBS, and incubating with either 1 μM or 1.7 μM of streptavidin for 30 min at room temperature.
Characterization of negative acoustic contrast materials and microparticles
Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra were acquired using a Thermo Electron Nicolet 8700 spectrometer (Ge crystal, 32 scans, 4 cm2 resolution). Scanning electron microscopy (SEM) images were obtained using model FEI XL 30 SEM under ultra-high resolution mode after sputter coating the samples with approximately 6 nm of gold. Optical microscopy images were obtained using an upright Zeiss Axio Imager A2 microscope with appropriate filter set (ex 470/40, em 525/50 or ex 545/25, em 605/70 or ex 365, em 445/50).
Binding between streptavidin polystyrene microparticles (Polysciences, YG microspheres, 6 μm) and PDMS NACPs (encapsulated with rhodamine B, functionalized with biotin-F108) occurred by combining ~106 polystyrene particles and ~107 PDMS particles and incubating for 30 minutes at room temperature with end-over-end rotation. Before combining with the polystyrene microparticles, ~107 PDMS NACPs were washed three times with 1× PBS. Polystyrene particles were added directly from the manufacturer’s stock without washing. Bioseparation events within the channel were monitored through the glass lid of the acoustofluidic device using fluorescent microscopy.
Fabrication of acoustofluidic device
The acoustofluidic device (Additional file 3) was prepared using standard photolithography, deep reactive-ion etching, anodic bonding and plasma bonding. The device contained a downstream collection module and an acoustic (piezoelectric) actuation element (i.e., lead zirconate titanate, PZT, 841 WFB, d33 = 0.3 nm/V, APC International). The channel width was designed to operate at a half wavelength resonant mode (e.g., 252 μm and frequency of 2.94 MHz or 272 μm and frequency of 2.72MHz) resulting in an antinode at both channel walls and a single node in the channel center line. For the experiments, an electric signal with peak-to-peak voltage of 31 V was applied to the PZT. Prior to running experiments, the acoustofluidic channels were treated with a solution of Pluronic F108.
Positive acoustic contrast particles
Negative acoustic contrast particles
Phosphate buffered saline
This work was supported by the National Science Foundation (NSF, through the Research Triangle MRSEC: DMR-1121107 and CBET-10-50176). LMJ thanks The Hartwell Foundation (Biomedical Research Fellowship) and CWS is grateful for a NSF Graduate Research Fellowship (1106401). MS thanks the Pratt Research Fellows program at Duke University. KC thanks the National Institutes of Health (NIH RR020064, NIH RR001315). We thank Zijian Zhou at Duke University for the images of the acoustofluidic chip.
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