Performance of Optimized Wide Pore Superficially Porous Particles for Separation of Proteins and Immunoglobulin G Antibodies.

In this study, we studied the chromatographic performance of this newly developed wide pore superficially porous particles (SPPs) with 3.5 µm particle size and 450 Å pore size, for the separation of proteins and Immunoglobulin G antibodies. We studied the selectivity of different phases (C4, SB-C18 and Diphenyl), the effect of temperature, column carryover and column chemical lifetime. We also compared our SPPs with other wide pore SPPs in similar particle sizes and sub 2 µ wide pore totally porous particles by van Deemter studies and gradient separations of proteins and immunoglobulin G antibodies. The results showed that the SPPs containing larger pore size gave better chromatographic performance.

Synthesis and optimization of wide pore superficially porous particles by a one-step coating process for separation of proteins and monoclonal antibodies.

Superficially porous particles (SPPs) with pore size ranging from 90Å to 120Å have been a great success for the fast separation of small molecules over totally porous particles in recent years. However, for the separation of large biomolecules such as proteins, particles with large pore size (e.g. ≥ 300Å) are needed to allow unrestricted diffusion inside the pores. One early example is the commercial wide pore (300Å) SPPs in 5µm size introduced in 2001. More recently, wide pore SPPs (200Å and 400Å) in smaller particle sizes (3.5-3.6µm) have been developed to meet the need of increasing interest in doing faster analysis of larger therapeutic molecules by biopharmaceutical companies. Those SSPs in the market are mostly synthesized by the laborious layer-by-layer (LBL) method. A one step coating approach would be highly advantageous, offering potential benefits on process time, easier quality control, materials cost, and process simplicity for facile scale-up. A unique one-step coating process for the synthesis of SPPs called the "coacervation method" was developed by Chen and Wei as an improved and optimized process, and has been successfully applied to synthesis of a commercial product, Poroshell 120 particles, for small molecule separation. In this report, we would like to report on the most recent development of the one step coating coacervation method for the synthesis of a series of wide pore SPPs of different particle size, pore size, and shell thickness. The one step coating coacervation method was proven to be a universal method to synthesize SPPs of any particle size and pore size. The effects of pore size (300Å vs. 450Å), shell thickness (0.25µm vs. 0.50µm), and particle size (2.7µm and 3.5µm) on the separation of large proteins, intact and fragmented monoclonal antibodies (mAbs) were studied. Van Deemter studies using proteins were also conducted to compare the mass transfer properties of these particles. It was found that the larger pore size actually had more impact on the performance of mAbs than particle size and shell thickness. The SPPs with larger 3.5µm particle size and larger 450Å pore size showed the best resolution of mAbs and the lowest back pressure. To the best of our knowledge, this is the largest pore size made on SPPs. These results led to the optimal particle design with a particle size of 3.5µm, a thin shell of 0.25µm and a larger pore size of 450Å.

Microfluidic generation of uniform water droplets using gas as the continuous phase.

Microfluidic schemes for forming uniform aqueous microdroplets usually rely on contacting the aqueous liquid (dispersed phase) with an immiscible oil (continuous phase). Here, we demonstrate that the oil can be substituted with gas (nitrogen or air) while still retaining the ability to generate discrete and uniform aqueous droplets. Our device is a capillary co-flow system, with the inner flow of water getting periodically dispersed into droplets by the external flow of gas. The droplet size and different formation modes can be tuned by varying the liquid and gas flow rates. Importantly, we identify the range of conditions that correspond to the "dripping mode", i.e., where discrete droplets are consistently generated with no satellites. We believe this is a significant development that will be beneficial for chemical and biological applications requiring clean and contaminant-free droplets, including DNA amplification, drug encapsulation, and microfluidic cell culture.

Microfluidic assembly of Janus-like dimer capsules.

We describe the microfluidic assembly of soft dimer capsules by the fusion of individual capsules with distinct properties. Microscale aqueous droplets bearing the biopolymer chitosan are generated in situ within a chip and, as they travel downsteam, pairs of droplets are made to undergo controlled cross-linking and coalescence (due to a channel expansion) to form stable dimers. These dimers are very much like Janus particles: the size, shape, and functionality of each individual lobe within the dimer can be precisely controlled. Dimers with one lobe much shorter than the other resemble a bowling pin in their overall morphology, while dimers with nearly equal-sized lobes are akin to a snowman. To illustrate the diverse functionalities possible, we have prepared dimers wherein one lobe encapsulates paramagnetic Fe2O3 nanoparticles. The resulting dimers undergo controlled rotation in an external rotating magnetic field, much like a magnetic stir bar. The overall approach described here is simple and versatile: it can be easily adapted in numerous ways to produce soft structures with designed properties.

Capturing rare cells from blood using a packed bed of custom-synthesized chitosan microparticles.

We describe batch generation of uniform multifunctional chitosan microparticles for isolation of rare cells, such as circulating tumor cells (CTCs), from a sample of whole blood. The chitosan microparticles were produced in large numbers using a simple and inexpensive microtubing arrangement. The particles were functionalized through encapsulation of carbon black, to control autofluorescence, and surface attachment of streptavidin, to enable interactions with biotinylated antibodies. These large custom modified microparticles (≈164 µm diameter) were then packed into a microfluidic channel to demonstrate their utility in rare cell capture. Blood spiked with breast cancer (MCF-7) cells was first treated with a biotinylated antibody (anti-EpCAM, which is selective for cancer cells like MCF-7) and then pumped through the device. In the process, the cancer cells were selectively bound to the microparticles through non-covalent streptavidin-biotin interactions. The number density of captured cells was determined by fluorescence microscopy at physiologically relevant levels. Selective capture of the MCF-7 cells was characterized, and compared favorably with previous approaches. The overall approach using custom synthesized microparticles is versatile, and can allow researchers more flexibility for rare cell capture through simpler and cheaper methods than are currently employed.

A new approach to in-situ "micromanufacturing": microfluidic fabrication of magnetic and fluorescent chains using chitosan microparticles as building blocks.

An in situ microfluidic assembly approach is described that can both produce microsized building blocks and assemble them into complex multiparticle configurations in the same microfluidic device. The building blocks are microparticles of the biopolymer chitosan, which is intentionally selected because its chemistry allows for simultaneous intraparticle and interparticle linking. Monodisperse chitosan-bearing droplets are created by shearing off a chitosan solution at a microfluidic T-junction with a stream of hexadecane containing a nonionic detergent. These droplets are then interfacially crosslinked into stable microparticles by a downstream flow of glutaraldehyde (GA). The functional properties of these robust microparticles can be easily varied by introducing various payloads, such as magnetic nanoparticles and/or fluorescent dyes, into the chitosan solution. The on-chip connection of such individual particles into well-defined microchains is demonstrated using GA again as the chemical "glue" and microchannel confinement as the spatial template. Chain flexibility can be tuned by adjusting the crosslinking conditions: both rigid chains and semiflexible chains are created. Additionally, the arrangement of particles within a chain can also be controlled, for example, to generate chains with alternating fluorescent and nonfluorescent microparticles. Such microassembled chains could find applications as microfluidic mixers, delivery vehicles, microscale sensors, or miniature biomimetic robots.

Thermo-induced formation of unimolecular and multimolecular micelles from novel double hydrophilic multiblock copolymers of N,N-dimethylacrylamide and N-isopropylacrylamide.

Two novel double hydrophilic multiblock copolymers of N,N-dimethylacrylamide and N-isopropylacrylamide, m-PDMAp-PNIPAMq, with varying degrees of polymerization (DPs) for PDMA and PNIPAM sequences (p and q) were synthesized via consecutive reversible addition-fragmentation chain transfer (RAFT) polymerizations using polytrithiocarbonate (1) as the chain transfer agent (Scheme 1), where PDMA is poly(N,N-dimethylacrylamide) and PNIPAM is poly(N-isopropylacrylamide). The DPs of PDMA and PNIPAM sequences were determined by 1H NMR, and the block numbers, i.e., number of PDMAp-PNIPAMq sequences (n), were obtained by comparing the molecular weights of multiblock copolymers to that of cleaved products as determined by gel permeation chromatography (GPC). m-PDMA42-PNIPAM37 and m-PDMA105-PNIPAM106 multiblock copolymers possess number-average molecular weights (Mn) of 4.62x10(4) and 9.53x10(4), respectively, and the polydispersities (Mw/Mn) are typically around 1.5. Block numbers of the obtained multiblock copolymers are ca. 4, which are considerably lower than the numbers of trithiocarbonate moieties per chain of 1 (approximately 20) and m-PDMAp precursors (approximately 6-7). PDMA homopolymer is water soluble to 100 degrees C, while PNIPAM has been well known to exhibit a lower critical solution temperature (LCST) at ca. 32 degrees C. In aqueous solution, m-PDMA42-PNIPAM37 and m-PDMA105-PNIPAM106 multiblock copolymers molecularly dissolve at room temperature, and their thermo-induced collapse and aggregation properties were characterized in detail by a combination of optical transmittance, fluorescence probe measurements, laser light scattering (LLS), and micro-differential scanning calorimetry (micro-DSC). It was found that chain lengths of PDMA and PNIPAM sequences exert dramatic effects on their aggregation behavior. m-PDMA105-PNIPAM106 multiblock copolymer behaves as protein-like polymers and exhibits intramolecular collapse upon heating, forming unimolecular flower-like micelles above the thermal phase transition temperature. On the other hand, m-PDMA42-PNIPAM37 multiblock copolymer exhibits collapse and intermolecular aggregation, forming associated multimolecular micelles at elevated temperatures. The intriguing aggregation behavior of this novel type of double hydrophilic multiblock copolymers argues well for their potential applications in many fields such as biomaterials and biomedicines.