Publications by authors named "Ana Egatz-Gomez"

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS).

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Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-β peptide (Aβ) oligomers that appear as intermediates along the Aβ aggregation into plaques are considered among the main AD neurotoxic species.

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With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets.

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Despite efforts to develop effective upconverting nanoparticle (UCNP) synthesis methods, there is still a need for approaches that are accessible and up-scalable while reproducibly providing fine control of UCNP size, crystallinity, and luminescence. This work presents a one-pot microwave-assisted strategy for synthesizing NaYF:Yb/Er UCNPs. A premixed rare earth (RE) solution in oleic acid (OA) was used to enhance repeatability while testing various synthesis conditions.

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Understanding cell-to-cell variation at the molecular level provides relevant information about biological phenomena and is critical for clinical and biological research. Proteins carry important information not available from single-cell genomics and transcriptomics studies; however, due to the minute amount of proteins in single cells and the complexity of the proteome, quantitative protein analysis at the single-cell level remains challenging. Here, we report an integrated microfluidic platform in tandem with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the detection and quantification of targeted proteins from small cell ensembles (> 10 cells).

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Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection.

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In this work, we present a luminescence platform that can be used as point of care system for determining the presence and concentration of specific oligonucleotide sequences. This sensor exhibited a limit of detection as low as 50 fM by means of: (i) the use of single-stranded DNA (ssDNA) functionalized magnetic microparticles that captured and concentrated ssDNA-upconverting nanoparticles (ssDNA-UCNPs) on a solid support, when the target sequence (miR-21-5p DNA-analogue) was in the sample, and (ii) a photoligation reaction that covalently linked the ssDNA-UCNPs and the ssDNA magnetic microparticles, allowing stringent washes. The presented sensor showed a similar limit of detection when the assays were conducted in samples containing total miRNA extracted from human serum, demonstrating its suitability for detecting small specific oligonucleotide sequences under real-like conditions.

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Article Synopsis
  • Serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) is a technique that helps in determining the structures of membrane proteins and observing changes over time, but traditional methods waste a lot of sample material.* -
  • The European XFEL produces rapid femtosecond X-ray pulses, but conventional liquid delivery methods result in over 99% sample wastage due to timing differences between pulse delivery.* -
  • A new microfluidic device that delivers protein crystal-laden droplets segmented by oil reduces sample waste by about 60%, allowing for the successful determination of a specific enzyme structure with previously unreported features.*
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The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector.

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Metallic nanostructures have the potential to modify the anti-Stokes emission of upconverting nanoparticles (UCNPs) by coupling their plasmon resonance with either the excitation or the emission wavelength of the UCNPs. In this regard gold nanoparticles (AuNPs) have often been used in sensors for UCNP luminescence quenching or enhancement, although systematic studies are still needed in order to design optimal UCNP-AuNP based biosensors. Amidst mixed experimental evidence of quenching or enhancement, two key factors arise: the nanoparticle distance and nanoparticle size.

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Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures.

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Separations of bioanalytes require robust, effective, and selective migration phenomena. However, due to the complexity of biological matrices such as body fluids or tissue, these requirements are difficult to achieve. The separations field is thus constantly evolving to develop suitable methods to separate biomarkers and fractionate biospecimens for further interrogation of biomolecular content.

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Nucleic acid biomarkers have enormous potential in non-invasive diagnostics and disease management. In medical research and in the near future in the clinics, there is a great demand for accurate miRNA, mRNA, and ctDNA identification and profiling. They may lead to screening of early stage cancer that is not detectable by tissue biopsy or imaging.

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We experimentally study the temporal dynamics of amplitude-modulated laser beams propagating through a water dispersion of graphene oxide sheets in a fiber-to-fiber U-bench. Nonlinear refraction induced in the sample by thermal effects leads to both phase reversing of the transmitted signals and dynamic hysteresis in the input-output power curves. A theoretical model including beam propagation and thermal lensing dynamics reproduces the experimental findings.

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IEF protein binary separations were performed in a 12-μL drop suspended between two palladium electrodes, using pH gradients created by electrolysis of simple buffers at low voltages (1.5-5 V). The dynamics of pH gradient formation and protein separation were investigated by computer simulation and experimentally via digital video microscope imaging in the presence and absence of pH indicator solution.

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A droplet-based electrochemical digital magnetofluidics system has been developed. The system relies on the magnetic movement, in air, of different aqueous microdroplets containing magnetic microparticles--serving as the 'sample', 'blank', 'wash' and 'reagent' solutions--into and out of a three-electrode assembly. The movement of all droplets was controlled using the magnetic fields generated by three separate external magnets positioned below the superhydrophobic surface.

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