Mario Alberto Saucedo Espinosa


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Saucedo Espinosa

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Mario Alberto

ORCID

0000-0001-8884-933X

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2019 - 201912020 - 20255
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Publications 1 - 6 of 6
  • Enrichment and Analysis of Breast Cancer Cell-Derived Extracellular Vesicles by Laser-Assisted Protein Adsorption in Thermoplastic Microchannels
    Item type: Conference Paper
    Kling, André; Nikoloff, Jonas; Saucedo Espinosa, Mario Alberto; et al. (2021)
    2021 21st International Conference on Solid-State Sensors, Actuators and Microsystems (Transducers)
    Extracellular vesicles (EVs) and their cargo proteins, DNAs and RNAs are very promising biomarkers. Accessible through liquid biopsies, EVs can reveal valuable diagnostic information on the health or disease state of a patient. Here, we propose a method for the enrichment of cancer cell-derived EVs and subsequent on-chip cargo protein analysis in thermoplastic microchannels. For this purpose, we characterized and optimized a technique for localized multiplexed surface functionalization, referred to as laser-assisted protein adsorption after photobleaching (LAPAP). Using LAPAP, we successfully immobilized antibodies in defined areas of the device to capture and analyze EVs by immunoassays. We employ the method for the identification and quantification of MCF-7 cell-derived EVs and cancer-associated proteins.
  • In-droplet separation of proteins and nucleic acids
    Item type: Other Conference Item
    Saucedo Espinosa, Mario Alberto; Hirth, Elisabeth F.; Dittrich, Petra S. (2019)
    23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2019)
    This contribution presents an electric field-driven approach for the separation of small biomolecules inside droplets. An electrophoretic separation is performed as droplets move through a microfluidic channel located between two parallel liquid-electrodes. Thin carbon-based PDMS membranes interface the electrodes with the microfluidic channel. Experiments performed with nucleic acids and proteins show that biomolecules can be focused in less than half the volume of droplets at frequencies of over 10 droplets per second. The proposed technique can be readily integrated with standard droplet generators.
  • Microfluidic Platform for Profiling of Extracellular Vesicles from Single Breast Cancer Cells
    Item type: Journal Article
    Nikoloff, Jonas M.; Saucedo Espinosa, Mario Alberto; Dittrich, Petra S. (2023)
    Analytical Chemistry
    Extracellular vesicles (EVs) are considered as valuable biomarkers to discriminate healthy from diseased cells such as cancer. Passing cytosolic and plasma membranes before their release, EVs inherit the biochemical properties of the cell. Here, we determine protein profiles of single EVs to understand how much they represent their cell of origin. We use a microfluidic platform which allows to immobilize EVs from completely isolated single cells, reducing heterogeneity of EVs as strongly seen in cell populations. After immunostaining, we employ four-color total internal reflection fluorescence microscopy to enumerate EVs and determine their biochemical fingerprint encoded in membranous or cytosolic proteins. Analyzing single cells derived from pleural effusions of two different human adenocarcinoma as well as from human embryonic kidney (SkBr3, MCF-7 and HEK293, respectively), we observed that a single cell secretes enough EVs to extract the respective tissue fingerprint. We show that overexpressed integral plasma membrane proteins are also found in EV membranes, which together with populations of colocalized proteins, provide a cell-specific, characteristic pattern. Our method highlights the potential of EVs as a diagnostic marker and can be directly employed for fundamental studies of EV biogenesis.
  • In-droplet electrokinetic separation of proteins with pH gradients
    Item type: Other Conference Item
    Saucedo Espinosa, Mario Alberto; Dittrich, Petra S. (2022)
    25th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2021)
    Controlling the motion of molecules and colloidal dispersions is a fundamental task in microfluidics, especially in droplet-based microfluidics, which has evolved into a versatile platform for biochemical assays. This paper introduces a microfluidic approach that exploits controlled pH gradients inside droplets for fast protein separations.
  • Continuous Electroformation of Gold Nanoparticles in Nanoliter Droplet Reactors
    Item type: Journal Article
    Saucedo Espinosa, Mario Alberto; Breitfeld, Maximilian; Dittrich, Petra S. (2023)
    Angewandte Chemie. International Edition
    Gold nanoparticles (AuNPs) are employed in numerous applications, including optics, biosensing and catalysis. Here, we demonstrate the stabilizer-free electrochemical synthesis of AuNPs inside nanoliter-sized reactors. Droplets encapsulating a gold precursor are formed on a microfluidic device and exposed to an electrical current by guiding them through a pair of electrodes. We exploit the naturally occurring recirculation flows inside confined droplets (moving in rectangular microchannels) to prevent the aggregation of nanoparticles after nucleation. Therefore, AuNPs with sizes in the range of 30 to 100 nm were produced without the need of additional capping agents. The average particle size is defined by the precursor concentration and droplet velocity, while the charge dose given by the electric field strength has a minor effect. This method opens the way to fine-tune the electrochemical production of gold nanoparticles, and we believe it is a versatile method for the formation of other metal nanoparticles.
  • Ultrafast Formation of Microdroplet Arrays with Chemical Gradients for Label-Free Determination of Enzymatic Reaction Kinetics
    Item type: Journal Article
    Breitfeld, Maximilian; Dietsche, Claudius L.; Saucedo Espinosa, Mario Alberto; et al. (2025)
    Small
    High throughput assays including enzymatic reactions are usually conducted in multiwell plates and analyzed in plate readers. This approach has limitations in i) upscaling and ii) the choice of reactions, as labeled compounds are required. A technique is introduced to rapidly generate dense microdroplet arrays by stream shearing (MASS). The fluid is delivered via a capillary onto a glass plate that carries a pattern of hydrophilic spots surrounded by a hydrophobic coating. Moving the glass plate shears off droplets from the fluid stream that are retained on the hydrophilic spot. Arrays of up to 24 192 homogenous droplets (coefficient of variation: 2.9 %) with defined volumes from 280 to 980 pL are generated in less than 22 min. Thereby, the droplet content is varied, and fine chemical gradients are obtained across the plate, which are analyzed with both fluorescence microscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This method is employed for label-free kinetic studies of an enzymatic reaction. For the cleavage of angiotensin II-fluorescein by the angiotensin II-converting enzyme, a maximum reaction velocity ($v_m$$_a$$_x$) of 5.7 mu m min⁻¹ is determined and the Michaelis-Menten constant ($K_M$) of 84.8 µm is found. The platform can be further upscaled for biochemical assays as required for drug discovery and protein engineering.
Publications 1 - 6 of 6