Tzu Chiao Chao

Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser

Diffraction Before Destruction A bottleneck in x-ray crystallography is the growth of well-ordered crystals large enough to obtain high-resolution diffraction data within an exposure that limits radiation damage. Serial femtosecond crystallography promises to overcome these constraints by using short intense pulses that out-run radiation damage. A stream of crystals is flowed across the free-electron beam and for each pulse, diffraction data is recorded from a single crystal before it is destroyed. Redecke et al. (p. 227, published online 29 November; see the Perspective by Helliwell) used this technique to determine the structure of an enzyme from Trypanosoma brucei, the parasite that causes sleeping sickness, from micron-sized crystals grown within insect cells. The structure shows how this enzyme, which is involved in degradation of host proteins, is natively inhibited prior to activation, which could help in the development of parasite-specific inhibitors. In vivo crystallization and serial femtosecond crystallography reveal the structure of a sleeping sickness parasite protease. [Also see Perspective by Helliwell] The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the “diffraction-before-destruction” approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

Microfluidic single-cell analysis of intracellular compounds

Biological analyses traditionally probe cell ensembles in the range of 103–106 cells, thereby completely averaging over relevant individual cell responses, such as differences in cell proliferation, responses to external stimuli or disease onset. In past years, this fact has been realized and increasing interest has evolved for single-cell analytical methods, which could give exciting new insights into genomics, proteomics, transcriptomics and systems biology. Microfluidic or lab-on-a-chip devices are the method of choice for single-cell analytical tools as they allow the integration of a variety of necessary process steps involved in single-cell analysis, such as selection, navigation, positioning or lysis of single cells as well as separation and detection of cellular analytes. Along with this advantageous integration, microfluidic devices confine single cells in compartments near their intrinsic volume, thus minimizing dilution effects and increasing detection sensitivity. This review overviews the developments and achievements of microfluidic single-cell analysis of intracellular compounds in the past few years, from proof-of-principle devices to applications demonstrating a high biological relevance.

Immunoglobulin G and bovine serum albumin streaming dielectrophoresis in a microfluidic device

Dielectrophoresis (DEP) has demonstrated to be a versatile tool to manipulate micro‐ and nanoparticles with applications for positioning, separation and fractionation. Recent developments of DEP have also shown that DEP can be used for the manipulation of biomolecules, such as DNA. Here, we focus on the manipulation of proteins using insulator‐based dielectrophoresis (iDEP). We designed suitable post arrays in a microfluidic channel and use numerical simulations to calculate the electric field distribution as well as concentration of proteins according to a convection–diffusion model for both negative and positive DEP. Experimentally, we find DEP trapping of mainly protein aggregates in phosphate buffer. However, when adding a charged zwitterionic detergent, we observed DEP streamlining of immunoglobulin G (IgG) and bovine serum albumin (BSA). Our experimental observations are in excellent agreement with numerical simulations and indicate positive DEP behavior of IgG and BSA under the employed experimental conditions. Our results demonstrate DEP streaming of proteins in an iDEP device for the first time and indicate the potential of protein DEP for separation and fractionation.

Characterization and Liquid Chromatography-MS/MS Based Quantification of Hydroxylated Fullerenes

Highly water-soluble hydroxylated fullerene derivatives are being investigated for a wide range of commercial products as well as for potential cytotoxicity. However, no analytical methods are currently available for their quantification at sub-ppm concentrations in environmental matrixes. Here, we report on the development and comparison of liquid chromatography-ultraviolet/visible spectroscopy (LC-UV/vis) and liquid chromatography-mass spectrometry (LC-MS) based detection and quantification methods for commercial fullerols. We achieved good separation efficiency using an amide-type hydrophilic interaction liquid chromatography (HILIC) column (plate number >2000) under isocratic conditions with 90% acetonitrile as the mobile phase. The method detection limits (MDLs) ranged from 42.8 ng/mL (UV detection) to 0.19 pg/mL (using MS with multiple reaction monitoring, MRM). Other MS measurement modes achieved MDLs of 125 pg/mL (single quad scan, Q1) and 1.5 pg/mL (multiple ion monitoring, MI). Each detection method exhibited a good linear response over several orders of magnitude. Moreover, we tested the robustness of these methods in the presence of Suvanee River fulvic acids (SRFA) as an example of organic matter commonly found in environmental water samples. While SRFA significantly interfered with UV- and Q1-based quantifications, the interference was relatively low using MI or MRM (relative error in presence of SRFA: 8.6% and 2.5%, respectively). This first report of a robust MS-based quantification method for modified fullerenes dissolved in water suggests the feasibility of implementing MS techniques more broadly for identification and quantification of fullerols and other water-soluble fullerene derivatives in environmental samples.

Physisorbed surface coatings for poly(dimethylsiloxane) and quartz microfluidic devices.

AbstractSurface modifications of microfluidic devices are of essential importance for successful bioanalytical applications. Here, we investigate three different coatings for quartz and poly(dimethylsiloxane) (PDMS) surfaces. We employed a triblock copolymer with trade name F108, poly(l-lysine)-g-poly(ethylene glycol) (PLL-PEG), as well as the hybrid coating n-dodecyl-β-d-maltoside and methyl cellulose (DDM/MC). The impact of these coatings was characterized by measuring the electroosmotic flow (EOF), contact angle, and prevention of protein adsorption. Furthermore, we investigated the influence of static coatings, i.e., the incubation with the coating agent prior to measurements, and dynamic coatings, where the coating agent was present during the measurement. We found that all coatings on PDMS as well as quartz reduced EOF, increased reproducibility of EOF, reduced protein adsorption, and improved the wettability of the surfaces. Among the coating strategies tested, the dynamic coatings with DDM/MC and F108 demonstrated maximal reduction of EOF and protein adsorption and simultaneously best long-term stability concerning EOF. For PLL-PEG, a reversal in the EOF direction was observed. Interestingly, the static surface coating strategy with F108 proved to be as effective to prevent protein adsorption as dynamic coating with this block copolymer. These findings will allow optimized parameter choices for coating strategies on PDMS and quartz microfluidic devices in which control of EOF and reduced biofouling are indispensable. FigureThe difference between static coating (left) and dynamic surface coating (right) in a microfluidic channel is shown schematically. In the static case, the surface is incubated with the coating agent prior to a specific experiment. Dynamic coating refers to the case in which the coating agent is present in the solution during a specific experiment

Dielectrophoretic sorting of membrane protein nanocrystals.

Structure elucidation of large membrane protein complexes is still a considerable challenge, yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected submicrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography.

Tuning direct current streaming dielectrophoresis of proteins

Dielectrophoresis (DEP) of biomolecules has large potential to serve as a novel selectivity parameter for bioanalytical methods such as (pre)concentration, fractionation, and separation. However, in contrast to well-characterized biological cells and (nano)particles, the mechanism of protein DEP is poorly understood, limiting bioanalytical applications for proteins. Here, we demonstrate a detailed investigation of factors influencing DEP of diagnostically relevant immunoglobulin G (IgG) molecules using insulator-based DEP (iDEP) under DC conditions. We found that the pH range in which concentration of IgG due to streaming iDEP occurs without aggregate formation matches the pH range suitable for immunoreactions. Numerical simulations of the electrokinetic factors pertaining to DEP streaming in this range further suggested that the protein charge and electroosmotic flow significantly influence iDEP streaming. These predictions are in accordance with the experimentally observed pH-dependent iDEP streaming profiles as well as the determined IgG molecular properties. Moreover, we observed a transition in the streaming behavior caused by a change from positive to negative DEP induced through micelle formation for the first time experimentally, which is in excellent qualitative agreement with numerical simulations. Our study thus relates molecular immunoglobulin properties to observed iDEP, which will be useful for the future development of protein (pre)concentration or separation methods based on DEP.

Selective trapping of single mammalian breast cancer cells by insulator-based dielectrophoresis

AbstractThe trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment. Figureᅟ

Beyond nC60: strategies for identification of transformation products of fullerene oxidation in aquatic and biological samples

Owing to their exceptional properties and versatility, fullerenes are in widespread use for numerous applications. Increased production and use of fullerenes will inevitably result in accelerated environmental release. However, study of the occurrence, fate, and transport of fullerenes in the environment is complicated because a variety of surface modifications can occur as a result of either intentional functionalization or natural processes. To gain a better understanding of the effect and risk of fullerenes on environmental health, it is necessary to acquire reliable data on the parent compounds and their congeners. Whereas currently established quantification methods generally focus on analysis of unmodified fullerenes, we discuss in this review the occurrence and analysis of oxidized fullerene congeners (i.e., their corresponding epoxides and polyhydroxylated derivatives) in the environment and in biological specimens. We present possible strategies for detection and quantification of parent nanomaterials and their various derivatives.

Towards proteome standards: the use of absolute quantitation in high-throughput biomarker discovery.

The use of proteomics to profile biological fluids and identify therein biomarkers for cancer and other diseases was initially received with considerable excitement. However, results have fallen short of the expectations. Traditionally, protein biomarkers have been identified by measurement of relative expression changes between case and control samples from which differentially expressed proteins are then considered to represent biomarker candidates. We argue that current individual proteomics-based biomarker discovery studies lack the statistical strength for the identification of high-confidence biomarkers. Instead, multi-group efforts are necessary to facilitate the generation of sufficient sample sizes. This is contingent on the ability to collate and cross-compare data from different studies, which will require the use of a common metric or standards. Though profound, the technical challenges for absolute protein quantification can be overcome. The use of matrix specific, shared standards for absolute quantitation presents an opportunity to facilitate the much needed, but currently impossible, comparisons of different studies. In addition to community-wide approaches to standardize pre-analytical biomarker research studies, it is also important to establish means to integrate experimental data from different studies in order to assess the usefulness of proposed biomarkers with sufficient statistical certainty.