Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. To gain further insight into the effect of these highly fluorinated amino acids on helix formation exclusively, we measured the helix propensity of three highly fluorinated amino acids: (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), and (S)-pentafluorophenylalanine (Pff). We have developed a short chemoenzymatic synthesis of Hfl with extremely high enantioselectivity (>99%). To measure the helix propensity (w) of the amino acids, alanine-based peptides were synthesized, purified, and investigated by circular dichroism spectroscopy (CD). On the basis of the CD data, the helix propensity of hydrocarbon amino acids can decrease up to 24-fold (1.72 kcal.mol-1.residue-1) upon fluorination. This difference in helix propensity has previously been overlooked in estimating the magnitude of the fluoro-stabilization effect (which has been estimated to be 0.32-0.83 kcal.mol-1.residue-1 for Hfl), resulting in a gross underestimation. Therefore, the full potential of the fluoro-stabilization effect should provide even more stable proteins than the fluoro-stabilized proteins to date.

Chan-Lam coupling is one of the most popular and easy methods to perform arylation of amines (N-arylations). This cross-coupling is generally performed by reacting aryl boronate derivatives with a variety of substrates involving nitrogen containing functional groups such as amines, amides, ureas, hydrazine, carbamates. This article summarizes the synthetic applications of this reaction and the efforts of scientists to develop novel and efficient methodologies for this reaction.

Ion-pairing interactions are important for protein stabilization. Despite the apparent electrostatic nature of these interactions, natural positively charged amino acids Lys and Arg have multiple methylenes linking the charged functionality to the backbone. Interestingly, the amino acids Lys and Orn have positively charged side chains that differ by only one methylene. However, only Lys is encoded and incorporated into proteins. To investigate the effect of side chain length of Lys on ion-pairing interactions, a series of 12 monomeric alpha-helical peptides containing potential Glu-Xaa (i, i+3), (i, i+4) and (i, i+5) (Xaa = Lys, Orn, Dab, Dap) interactions were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. At pH 7, no Glu-Xaa (i, i+5) interaction was observed, regardless of the Xaa side chain length. Furthermore, only Lys was capable of supporting Glu-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Glu-Xaa (i, i+4) interactions. Side chain conformational analysis by molecular mechanics calculations showed that the side chain length of Lys enables the Glu-Xaa (i, i+3) interaction with lower energy conformations compared to residues with side chain lengths shorter than that of Lys. Furthermore, these calculated low energy conformers were consistent with conformations of intra-helical Glu-Lys salt bridges in a non-redundant protein structure database. Importantly, the CD spectra for peptides with Glu-Lys interactions did not alter significantly upon changing the pH because of a greater contribution to these interactions by forces other than electrostatics. Incorporating side chains just one methylene shorter (Orn) resulted in significant pH dependence or lack of interaction, suggesting that nature has chosen Lys to form durable interactions with negatively charged functional groups.

On-chip droplet splitting is one of the fundamental droplet-based microfluidic unit operations to control droplet volume after production and increase operational capability, flexibility, and throughput. Various droplet splitting methods have been proposed, and among them the acoustic droplet splitting method is promising because of its label-free operation without any physical or thermal damage to droplets. Previous acoustic droplet splitting methods faced several limitations: first, they employed a cross-type acoustofluidic device that precluded multichannel droplet splitting; second, they required irreversible bonding between a piezoelectric substrate and a microfluidic chip, such that the fluidic chip was not replaceable. Here, we present a parallel-type acoustofluidic device with a disposable microfluidic chip to address the limitations of previous acoustic droplet splitting devices. In the proposed device, an acoustic field is applied in the direction opposite to the flow direction to achieve multichannel droplet splitting and steering. A disposable polydimethylsiloxane microfluidic chip is employed in the developed device, thereby removing the need for permanent bonding and improving the flexibility of the droplet microfluidic device. We experimentally demonstrated on-demand acoustic droplet bi-splitting and steering with precise control over the droplet splitting ratio, and we investigated the underlying physical mechanisms of droplet splitting and steering based on Laplace pressure and ray acoustics analyses, respectively. We also demonstrated droplet tri-splitting to prove the feasibility of multichannel droplet splitting. The proposed on-demand acoustic droplet splitting device enables on-chip droplet volume control in various droplet-based microfluidic applications.

We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid 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-apt15-th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS-apt15-th complex was formed, 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-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.

We have designed a pumpless acoustofluidic device for the concentration and separation of different sized particles inside a single-layered straight polydimethylsiloxane (PDMS) microfluidic channel. The proposed device comprises two parallel interdigitated transducers (IDTs) positioned underneath the PDMS microchannel. The IDTs produce high-frequency surface acoustic waves that generate semipermeable virtual acoustic radiation force field walls that selectively trap and concentrate larger particles at different locations inside the microchannel and allow the smaller particles to pass through the acoustic filter. The performance of the acoustofluidic device was first characterized by injecting into the microchannel a uniform flow of suspended 9.9 μm diameter particles with various initial concentrations (as low as 10 particles/mL) using a syringe pump. The particles were trapped with ∼100% efficiency by a single IDT actuated at 73 MHz. The acoustofluidic platform was used to demonstrate the pumpless separation of 12.0, 4.8, and 2.1 μm microparticles by trapping the 12 and 4.8 μm particles using the two IDTs actuated at 73 and 140 MHz, respectively. However, most of the 2.1 μm particles flowed over the IDTs unaffected. The acoustofluidic device was capable of rapidly processing a large volume of sample fluid pumped through the microchannel using an external syringe pump. A small volume of the sample fluid was processed through the device using a capillary flow and a hydrodynamic pressure difference that did not require an external pumping device.

We present a method for size-independent manipulation of elastic polystyrene (PS), poly(methyl methacrylate) (PMMA), and fused silica (FS) microspheres that uses travelling surface acoustic waves (TSAWs). Normally incident TSAWs originating from an interdigitated transducer (IDT) were used to separate similar-sized pairs of PS and PMMA or PS and FS elastic particles by producing distinct lateral deflections across laminar streamlines in a continuous flow microfluidic channel. Elastic particles with similar diameters but different acoustic impedances exhibit significantly different deflection characteristics when exposed to TSAW-based acoustic radiation forces (ARFs). For instance, exposing a mixture of PS and PMMA particles with similar diameters ($5 mm) to TSAWs with frequencies of 140MHz and 185 MHz produces larger deflections of the PS and PMMA particles, respectively. This difference arises because of the resonance of the elastic particles with the incoming acoustic waves at certain frequencies. Similar particle deflection characteristics were observed for mixtures of PS and FS particles with comparable diameters (3/3, 4.8/5, 10/10 mm). This difference in deflection distances was used to experimentally characterize the non-linear behavior of the ARFs acting on particles (3-10 mm) exposed to a range of TSAW frequencies (120-205 MHz). Our experimental results can be explained by plotting the acoustic radiation force factor (F F ) against the TSAW frequency (f TSAW ) and the dimensionless Helmholtz number (1 < k < 4), which is calculated by using a theoretical model of an elastic microsphere suspended in a fluid.

Bio-hybrid micro-swimmers, composed of biological entities integrated with synthetic constructs, actively transport cargo by converting chemical energy into mechanical work. Here, using isolated and demembranated flagella from green algae \textit{Chlamydomonas...