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  1. Article: High Throughput Protein Nanocrystal Fractionation in a Microfluidic Sorter

    Abdallah, Bahige G / Coe Jesse / Fromme Petra / Ros Alexandra / Roy-Chowdhury Shatabdi

    Analytical chemistry. 2015 Apr. 21, v. 87, no. 8

    2015  

    Abstract: Protein crystallography is transitioning into a new generation with the introduction of the X-ray free electron laser, which can be used to solve the structures of complex proteins via serial femtosecond crystallography. Sample characteristics play a ... ...

    Abstract Protein crystallography is transitioning into a new generation with the introduction of the X-ray free electron laser, which can be used to solve the structures of complex proteins via serial femtosecond crystallography. Sample characteristics play a critical role in successful implementation of this new technology, whereby a small, narrow protein crystal size distribution is desired to provide high quality diffraction data. To provide such a sample, we developed a microfluidic device that facilitates dielectrophoretic sorting of heterogeneous particle mixtures into various size fractions. The first generation device demonstrated great potential and success toward this endeavor; thus, in this work, we present a comprehensive optimization study to improve throughput and control over sorting outcomes. First, device geometry was designed considering a variety of criteria, and applied potentials were modeled to determine the scheme achieving the largest sorting efficiency for isolating nanoparticles from microparticles. Further, to investigate sorting efficiency within the nanoparticle regime, critical geometrical dimensions and input parameters were optimized to achieve high sorting efficiencies. Experiments revealed fractionation of nanobeads from microbeads in the optimized device with high sorting efficiencies, and protein crystals were sorted into submicrometer size fractions as desired for future serial femtosecond crystallography experiments.
    Keywords crystal proteins ; crystallography ; dielectrophoresis ; fractionation ; geometry ; nanocrystals ; nanoparticles ; X-radiation
    Language English
    Dates of publication 2015-0421
    Size p. 4159-4167.
    Publishing place American Chemical Society
    Document type Article
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021%2Facs.analchem.5b00589
    Database NAL-Catalogue (AGRICOLA)

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  2. Article: Protein Crystallization in an Actuated Microfluidic Nanowell Device.

    Abdallah, Bahige G / Roy-Chowdhury, Shatabdi / Fromme, Raimund / Fromme, Petra / Ros, Alexandra

    Crystal growth & design

    2016  Volume 16, Issue 4, Page(s) 2074–2082

    Abstract: Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic ... ...

    Abstract Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies.
    Language English
    Publishing date 2016-02-25
    Publishing country United States
    Document type Journal Article
    ISSN 1528-7483
    ISSN 1528-7483
    DOI 10.1021/acs.cgd.5b01748
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Crystallization of the large membrane protein complex photosystem I in a microfluidic channel.

    Abdallah, Bahige G / Kupitz, Christopher / Fromme, Petra / Ros, Alexandra

    ACS nano

    2013  Volume 7, Issue 12, Page(s) 10534–10543

    Abstract: Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, ... ...

    Abstract Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these "difficult to crystallize" proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.
    MeSH term(s) Buffers ; Computer Simulation ; Crystallization ; Crystallography, X-Ray ; Cyanobacteria/chemistry ; Diffusion ; Hydrophobic and Hydrophilic Interactions ; Ions ; Magnesium/chemistry ; Membrane Proteins/chemistry ; Microfluidic Analytical Techniques ; Microfluidics ; Models, Theoretical ; Muramidase/chemistry ; Nanotechnology ; Photosystem I Protein Complex/chemistry ; Phycocyanin/chemistry ; Salts/chemistry ; Solvents/chemistry ; Sulfates/chemistry ; Thylakoids/chemistry
    Chemical Substances Buffers ; Ions ; Membrane Proteins ; Photosystem I Protein Complex ; Salts ; Solvents ; Sulfates ; Phycocyanin (11016-15-2) ; Muramidase (EC 3.2.1.17) ; Magnesium (I38ZP9992A)
    Language English
    Publishing date 2013-11-14
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ISSN 1936-086X
    ISSN (online) 1936-086X
    DOI 10.1021/nn402515q
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: High throughput protein nanocrystal fractionation in a microfluidic sorter.

    Abdallah, Bahige G / Roy-Chowdhury, Shatabdi / Coe, Jesse / Fromme, Petra / Ros, Alexandra

    Analytical chemistry

    2015  Volume 87, Issue 8, Page(s) 4159–4167

    Abstract: Protein crystallography is transitioning into a new generation with the introduction of the X-ray free electron laser, which can be used to solve the structures of complex proteins via serial femtosecond crystallography. Sample characteristics play a ... ...

    Abstract Protein crystallography is transitioning into a new generation with the introduction of the X-ray free electron laser, which can be used to solve the structures of complex proteins via serial femtosecond crystallography. Sample characteristics play a critical role in successful implementation of this new technology, whereby a small, narrow protein crystal size distribution is desired to provide high quality diffraction data. To provide such a sample, we developed a microfluidic device that facilitates dielectrophoretic sorting of heterogeneous particle mixtures into various size fractions. The first generation device demonstrated great potential and success toward this endeavor; thus, in this work, we present a comprehensive optimization study to improve throughput and control over sorting outcomes. First, device geometry was designed considering a variety of criteria, and applied potentials were modeled to determine the scheme achieving the largest sorting efficiency for isolating nanoparticles from microparticles. Further, to investigate sorting efficiency within the nanoparticle regime, critical geometrical dimensions and input parameters were optimized to achieve high sorting efficiencies. Experiments revealed fractionation of nanobeads from microbeads in the optimized device with high sorting efficiencies, and protein crystals were sorted into submicrometer size fractions as desired for future serial femtosecond crystallography experiments.
    MeSH term(s) Crystallography ; High-Throughput Screening Assays ; Microfluidic Analytical Techniques ; Photosystem I Protein Complex/chemistry ; Photosystem I Protein Complex/metabolism
    Chemical Substances Photosystem I Protein Complex
    Language English
    Publishing date 2015-04-21
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021/acs.analchem.5b00589
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article: Insulator-based dielectrophoresis of mitochondria.

    Luo, Jinghui / Abdallah, Bahige G / Wolken, Gregory G / Arriaga, Edgar A / Ros, Alexandra

    Biomicrofluidics

    2014  Volume 8, Issue 2, Page(s) 21801

    Abstract: Isolated mitochondria display a wide range of sizes plausibly resulting from the coexistence of subpopulations, some of which may be associated with disease or aging. Strategies to separate subpopulations are needed to study the importance of these ... ...

    Abstract Isolated mitochondria display a wide range of sizes plausibly resulting from the coexistence of subpopulations, some of which may be associated with disease or aging. Strategies to separate subpopulations are needed to study the importance of these organelles in cellular functions. Here, insulator-based dielectrophoresis (iDEP) was exploited to provide a new dimension of organelle separation. The dielectrophoretic properties of isolated Fischer 344 (F344) rat semimembranosus muscle mitochondria and C57BL/6 mouse hepatic mitochondria in low conductivity buffer (0.025-0.030 S/m) at physiological pH (7.2-7.4) were studied using polydimethylsiloxane (PDMS) microfluidic devices. First, direct current (DC) and alternating current (AC) of 0-50 kHz with potentials of 0-3000 V applied over a channel length of 1 cm were separately employed to generate inhomogeneous electric fields and establish that mitochondria exhibit negative DEP (nDEP). DEP trapping potential thresholds at 0-50 kHz were also determined to be weakly dependent on applied frequency and were generally above 200 V. Second, we demonstrated a separation scheme using DC potentials <100 V to perform the first size-based iDEP sorting of mitochondria. Samples of isolated mitochondria with heterogeneous sizes (150 nm-2 μm diameters) were successfully separated into sub-micron fractions, indicating the ability to isolate mitochondria into populations based on their size.
    Language English
    Publishing date 2014-03-03
    Publishing country United States
    Document type Journal Article
    ISSN 1932-1058
    ISSN 1932-1058
    DOI 10.1063/1.4866852
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Dielectrophoretic sorting of membrane protein nanocrystals.

    Abdallah, Bahige G / Chao, Tzu-Chiao / Kupitz, Christopher / Fromme, Petra / Ros, Alexandra

    ACS nano

    2013  Volume 7, Issue 10, Page(s) 9129–9137

    Abstract: 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 ... ...

    Abstract 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.
    MeSH term(s) Crystallography ; Electrophoresis/methods ; Membrane Proteins/chemistry ; Membrane Proteins/isolation & purification ; Microscopy, Fluorescence ; Nanoparticles ; Photosystem I Protein Complex/chemistry ; Photosystem I Protein Complex/isolation & purification
    Chemical Substances Membrane Proteins ; Photosystem I Protein Complex
    Language English
    Publishing date 2013-09-09
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ISSN 1936-086X
    ISSN (online) 1936-086X
    DOI 10.1021/nn403760q
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article: Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction.

    Abdallah, Bahige G / Zatsepin, Nadia A / Roy-Chowdhury, Shatabdi / Coe, Jesse / Conrad, Chelsie E / Dörner, Katerina / Sierra, Raymond G / Stevenson, Hilary P / Camacho-Alanis, Fernanda / Grant, Thomas D / Nelson, Garrett / James, Daniel / Calero, Guillermo / Wachter, Rebekka M / Spence, John C H / Weierstall, Uwe / Fromme, Petra / Ros, Alexandra

    Structural dynamics (Melville, N.Y.)

    2015  Volume 2, Issue 4, Page(s) 41719

    Abstract: The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still ...

    Abstract The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10-100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of the dependence of crystal quality on crystal size.
    Language English
    Publishing date 2015-08-19
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2758684-4
    ISSN 2329-7778
    ISSN 2329-7778
    DOI 10.1063/1.4928688
    Database MEDical Literature Analysis and Retrieval System OnLINE

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