Optimization Analysis of Particle Separation Parameters for a Standing Surface Acoustic Wave Acoustofluidic Chip.

Microparticle separation technology is an important technology in many biomedical and chemical engineering applications from sample detection to disease diagnosis. Although a variety of microparticle separation techniques have been developed thus far, surface acoustic wave (SAW)-based microfluidic separation technology shows great potential because of its high throughput, high precision, and integration with polydimethylsiloxane (PDMS) microchannels. In this work, we demonstrate an acoustofluidic separation chip that includes a piezoelectric device that generates tilted-angle standing SAWs and a permanently bonded PDMS microchannel. We established a mathematical model of particle motion in the microchannel, simulated the particle trajectory through finite element simulation and numerical simulation, and then verified the validity of the model through acoustophoresis experiments. To improve the performance of the separation chip, the influences of particle size, flow rate, and input power on the particle deflection distance were studied. These parameters are closely related to the separation purity and separation efficiency. By optimizing the control parameters, the separation of micron and submicron particles under different throughput conditions was achieved. Moreover, the separation samples were quantitatively analyzed by digital light scattering technology and flow cytometry, and the results showed that the maximum purity of the separated particles was ∼95%, while the maximum efficiency was ∼97%.

Experimental research on surface acoustic wave microfluidic atomization for drug delivery.

This paper demonstrates that surface acoustic wave (SAW) atomization can produce suitable aerosol concentration and size distribution for efficient inhaled lung drug delivery and is a potential atomization device for asthma treatment. Using the SAW device, we present comprehensive experimental results exploring the complexity of the acoustic atomization process and the influence of input power, device frequency, and liquid flow rate on aerosol size distribution. It is hoped that these studies will explain the mechanism of SAW atomization aerosol generation and how they can be controlled. The insights from the high-speed flow visualization studies reveal that it is possible by setting the input power above 4.17 W, thus allowing atomization to occur from a relatively thin film, forming dense, monodisperse aerosols. Moreover, we found that the aerosol droplet size can be effectively changed by adjusting the input power and liquid flow rate to change the film conditions. In this work, we proposed a method to realize drug atomization by a microfluidic channel. A SU-8 flow channel was prepared on the surface of a piezoelectric substrate by photolithography technology. Combined with the silicon dioxide coating process and PDMS process closed microfluidic channel was prepared, and continuous drug atomization was provided to improve the deposition efficiency of drug atomization by microfluidic.

Thermal Control Design and Packaging for Surface Acoustic Wave Devices in Acoustofluidics.

This article presents a thermal control design method for a surface acoustic wave (SAW) device. We designed a heat-dissipation structure and packaging scheme to solve three key issues observed in SAW devices using anisotropic crystals as piezoelectric substrates in acoustofluidics (e.g., lithium niobate): SAW chip cracking caused by thermal stress, SAW chip cracking caused by mismatched thermal expansion coefficients of the packaging materials, and enhancement of the structural strength and stability of the SAW chip. This study establishes the physical model of the designed structure and the relationship between the steady-state working temperature and the physical properties of the material. By comparing these physical properties and numerical calculations, we identified nanosilver adhesive as the most effective bonding material between the SAW chip and the heat sink. In addition to designing and fabricating, we also evaluated our SAW devices experimentally. The results not only confirmed that the abovementioned three key problems were solved but also demonstrated the significant enhancement of the stability of the SAW device.

Influence of Waterproof Films on the Atomization Behavior of Surface Acoustic Waves.

One of the reasons why commercial application of surface acoustic wave (SAW) atomization is not possible is due to the condensation of aerosol droplets generated during atomization, which drip on the interdigitated transducer (IDT), thereby causing electrodes to short-circuit. In order to solve this problem, a SU-8-2002 film coating on an IDT is proposed in this paper. The waterproof performance of the film coating was tested on a surface acoustic wave (SAW) device several times. The experimental results reveal that the film coating was robust. The experiment also investigated the effects of the SU-8-2002 film on atomization behavior and heating.

Circular gradient-diameter photonic crystal fiber with large mode area and low bending loss.

In this paper, a silica-based sixfold circular gradient-diameter photonic crystal fiber (CGPCF) with five rings is proposed, and the matching conditions of air hole lattices are established considering the gradient and diameter of the air holes. This CGPCF supports endlessly single-mode propagation due to the existence of small air holes near to the fiber core based on the matching rule. Simultaneously, it exhibits an ultralarge effective mode area of 3110 µm2 in the straight case and 1105 µm2 in the bending case with a bending radius of 15 cm at the wavelength of 1550 nm. In addition, within an ultrawide communication wavelength range from 1000 to 2000 nm (from 1350 nm to 2000 nm), the effective mode area in the straight (bending) case still maintains more than 3043 µm2 (981 µm2), and the bending loss maintains an ultralow order of magnitude of 10-4 dB/m at a great degree of bending radius of 15 cm. Moreover, the rotation-symmetric structure of CGPCF can reduce the impact of bending orientation on the optical properties. The proposed CGPCF is highly meaningful for optical fiber communication, high-power laser systems, and optical amplifiers.

Improved SILAC method for double labeling of bacterial proteome.

Stable isotope labeling with amino acids in cell culture (SILAC) is a robust proteomics method with advantages such as reproducibility and easy handling. This method is popular for the analysis of mammalian cells. However, amino acid conversion in bacteria decreases the labeling efficiency and quantification accuracy, limiting the application of SILAC in bacterial proteomics to auxotrophic bacteria or to single labeling with lysine. In this study, we found that adding high concentrations of isotope-labeled (heavy) and natural (light) amino acids into SILAC minimal medium can efficiently inhibit the complicated amino acid conversions. This simple and straightforward strategy facilitated complete incorporation of amino acids into the bacterial proteome with good accuracy. High labeling efficiency can be achieved in different bacteria by slightly modifying the supplementation of amino acids in culture media, promoting the widespread application of SILAC technique in bacterial proteomics. SIGNIFICANCE: Amino acid conversion in bacteria decreases labeling efficiency, limiting the application of Stable isotope labeling with amino acids in cell culture (SILAC) in bacterial proteomics to auxotrophic bacteria or single labeling with lysine. In this study, we found that high concentrations of isotope-labeled (heavy) and natural (light) amino acids facilitate full incorporation of amino acids into the bacterial proteome with good reproducibility. This improved double labeling SILAC technique using medium supplemented with high concentrations of amino acids is suitable for quantitative proteomics research on both gram-positive and -negative bacteria, facilitating the broad application of quantitative proteomics in bacterial studies.

Proteomic analysis of the copper resistance of Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive bacterial pathogen causing a variety of diseases, including otitis media, bacteraemia and meningitis. Although copper is an essential trace metal for bacterial growth, high intracellular levels of free-copper are toxic. Copper resistance has emerged as an important virulence determinant of microbial pathogens. In this study, we determined the minimum inhibition concentration of copper for the growth inhibition of S. pneumoniae. Two-dimensional-electrophoresis coupled with mass spectrometry was applied to identify proteins involved in copper resistance of S. pneumoniae. In total, forty-four proteins with more than 1.5-fold alteration in expression (p < 0.05) were identified. Quantitative reverse transcription PCR was used to confirm the proteomic results. Bioinformatics analysis showed that the differentially expressed proteins were mainly involved in the cell wall biosynthesis, protein biosynthesis, purine biosynthesis, pyrimidine biosynthesis, primary metabolic process, and the nitrogen compound metabolic process. Many up-regulated proteins in response to the copper treatment directly or indirectly participated in the cell wall biosynthesis, indicating that the cell wall is a critical determinant in copper resistance of S. pneumoniae.

Proteomic analysis on the antibacterial activity of a Ru(II) complex against Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive pathogen that causes a variety of infection diseases in human. In this project, we determined the antibacterial activity of a Ru(II) complex X-03 against S. pneumoniae in vitro, by comparing its toxicity to host cells A549 and HBE. We performed two-dimensional gel electrophoresis (2-DE)-based proteomic analysis to characterize the protein alterations in S. pneumoniae after treatment with X-03. In total, 50 proteins exhibiting significant differential expressions were identified. RT-PCR was used to confirm the expression differences for selected proteins. Bioinformatics analysis on the proteomic alterations suggested that Ru(II) complex X-03 may obstruct bacterial fatty acid synthesis and oxidation-reduction process to suppress the growth of S. pneumoniae. Metal-uptake experiments revealed that iron-acquisition pathway in the bacterium may be interfered by X-03. These results provide useful clues for further investigations on the mechanism of the antibacterial action of metal compounds. BIOLOGICAL SIGNIFICANCE: The appearance of bacterial strains with broad antibiotic resistance is becoming an alarming global health concern. The development of novel efficient antibacterial compound is urgently needed. In the present study, we found that Ru(II) complex X-03 has a significant antibacterial activity and applied proteomic technology combined with bioinformatics analysis to investigate its antimicrobial mechanism in S. pneumoniae. Many proteins were found to be dysregulated, implicating that X-03 may affect various molecular pathways leading to the inhibition of bacterial growth. Metal-uptake experiments demonstrated that X-03 treatment reduced the iron content in the bacterium, suggesting the interference with iron acquisition systems by the complex. This disturbance in iron acquisition may directly or indirectly induce the proteomic response that involved many pathways. In addition, X-03 could selectively suppress Gram-positive bacteria but execute less cytotoxicity to Gram-negative bacteria, with almost no effect on human cells, implicating its potential to be developed as a specific antimicrobial agent. These results provide useful information for further investigations on the mechanism of the antibacterial action of metal drugs and development of efficient antibacterial drugs.

Chemical interference with iron transport systems to suppress bacterial growth of Streptococcus pneumoniae.

Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II) complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II) level in the bacterium. When the piuA gene (SPD_0915) was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II). Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II) complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.

Proteomic analysis of putative heme-binding proteins in Streptococcus pyogenes.

Streptococcus pyogenes is an important human bacterium with high pathogenicity. Heme is a major source of iron that plays a critical role in bacterial survival and virulence. In this study, heme-affinity chromatography, two-dimensional-electrophoresis and mass spectrometry were combined to identify putative heme-binding proteins and heme-regulatory proteins. In total, 68 heme-regulatory proteins and 284 putative heme-binding proteins were identified, among which 37 proteins showed expression alterations in response to heme deficiency. Bioinformatics analysis revealed that several key metabolic pathways had changed in the absence of heme, among which glycolysis was a major pathway impaired under heme-deficient conditions. New potential heme-binding proteins were successfully identified in this study providing novel clues for the study of the heme transport mechanism. Heme-binding proteins may play fundamental roles in many important biological pathways and thus contribute to bacterial pathogenicity.

Varied metal-binding properties of lipoprotein PsaA in Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, otitis media, and meningitis. Manganese and zinc ions are essential for this bacterium, playing regulatory, structural, or catalytic roles as the critical cofactors in the bacterial proteins and metabolic enzymes. Lipoprotein PsaA has been found to mediate Mn(2+) and Zn(2+) transportation in Streptococcus pneumoniae. In the present work, we conducted a systemic study on the contributions from key amino acids in the metal-binding site of PsaA using various spectroscopic and biochemical methods. Our experimental data indicate that four metal-binding residues contribute unequally to the Mn(2+) and Zn(2+) binding, and His139 is most important for both the structural stability and metal binding of the protein. PsaA-Mn(2+) has a lower thermal stability than PsaA-Zn(2+), possibly due to the different coordination preferences of the metals. Kinetics analysis revealed that PsaA-Mn(2+) binding is a fast first-order reaction, whereas PsaA-Zn(2+) binding is a slow second-order reaction, implying that PsaA kinetically prefers binding Mn(2+) to Zn(2+). The present results provide complementary information for understanding the mechanisms of metal transport and bacterial virulence via lipoproteins in Streptococcus pneumoniae.

Putative cobalt- and nickel-binding proteins and motifs in Streptococcus pneumoniae.

Cobalt and nickel play important roles in various biological processes. The present work focuses on the enrichment and identification of Co- and Ni-binding motifs and proteins in Gram-positive bacteria. Immobilized metal affinity column (IMAC) was used to partially enrich putative metal-binding proteins and peptides from Streptococcus pneumoniae, and then LTQ-Orbitrap mass spectrometry (MS) was applied to identify and characterize the metal-binding motifs and proteins. In total, 208 and 223 proteins were isolated by Co- and Ni-IMAC columns respectively, in which 129 proteins were present in both preparations. Based on the gene ontology (GO) analysis, the putative metal-binding proteins were found to be mainly involved in protein metabolism, gene expression regulation and carbohydrate metabolism. These putative metal-binding proteins form a highly connected network, indicating that they may synergistically work together to achieve specific biological functions. Putative Co- and Ni-binding motifs were identified with H(X)nH, M(X)nH and H(X)nM derived from the identified 51 Co-binding peptides and 66 Ni-binding peptides. Statistics of frequency of amino acids in the metal-binding motifs showed that cobalt and nickel prefer to bind histidine and methionine, but not cysteine. These results obtained by a systematic metalloproteomic approach provide important clues for the further investigation of metal homeostasis and metal-related virulence of bacteria.