No abstract

In this study, a novel biocompatible and inexpensive method for the rapid production of single-cell based microparticles is described. Using an HP DeskJet 550C printer, alginate microparticles containing one to several insulin-producing cells (beta-TC6) were fabricated by coprinting the cells and sodium alginate suspension into a CaCl2 solution. This method is able to generate microparticles of 30–60μm in diameter at a rate as high as 55,000particles∕s. Cell survival assays showed that more than 89% of printed cells survived the fabrication process. The printed beta-TC6 cells demonstrated continuous insulin secretion over a 6day period, which suggests that the printed cells are able to maintain normal cellular function within the microparticles. We show that the printing conditions, such as cell number, alginate concentration, and ionic strengths of CaCl2, influence cellular distribution and geometry of the printed particles. This study presents a simple and high-throughput method to encapsulate single cells, and this technique may be applied in various research investigations, including single-cell analysis, high-throughput drug screening, and stem cell differentiation at the single-cell level.

We have developed a process to incorporate an integral membrane protein, Photosystem I (PSI), into an organic thin film at an electrode surface and thereby insulate the protein complex on the surface while mimicking its natural environment. The PSI complex, which is primarily more hydrophobic on the exterior than interior, is hydrophobically confined in vivo within the thylakoid membrane. To mimic the thylakoid membrane and entrap PSI on an electrode, we have designed a series of steps using a thin self-assembled monolayer (SAM) to adsorb and orient PSI followed by exposures to longer-chained methyl-terminated alkanethiols that place exchange with components of the original SAM in the interprotein domains. In this process, PSI is first adsorbed onto a HOC(6)S/Au substrate through a short exposure to a dilute solution of the protein to achieve a protein coverage of approximately 25%. The PSI/HOC(6)S/Au substrate is then placed into a solution containing one of various longer-chained alkanethiols including C(22)SH or C(18)OC(19)SH. Changes in thickness, interfacial capacitance, infrared spectra, and surface wettability were used to assess the extent of backfilling by the long-chained thiols. The coverage of the protein layer and the solvent used for backfilling affected the rate and quality of the SAM formed in the interprotein regions. After exposure of the PSI layer to solvents containing alkanethiols, there was only minor loss of protein on the surface and no real change in protein secondary structure as evidenced by reflectance absorption infrared spectroscopy.

We report the first directed adsorption of Photosystem I (PSI) on patterned surfaces containing discrete regions of methyl- and hydroxyl-terminated self-assembled monolayers (SAMs) on gold. SAM and PSI patterns are characterized by scanning electrochemical microscopy (SECM). The insulating protein complex layer blocks the electron transfer of the SECM mediator, thereby reducing the electrochemical current significantly. Uniformly and densely packed adsorbed protein layers are observed with SECM. Pattern images correlate with our previous studies where we showed that low-energy surfaces (e.g., CH3-terminated) inhibit PSI adsorption in the presence of Triton X-100, whereas high-energy surfaces (e.g., OH-terminated) enable adsorption. Therefore, a SAM pattern with alternating methyl and hydroxyl surface regions allows PSI adsorption only on the hydroxyl surface, and this is demonstrated in the resulting SECM images.