Time-temperature Indicators Based on the Acidic Permanganate-oxalate Reaction System

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Description
The kinetic behavior of acidified aqueous reaction systems based on the oxidation of sodium oxalate by potassium permanganate was investigated by UV-Vis absorbance spectrophotometry as part of a wider project to develop time-temperature indicators based on such systems. Reaction systems…

The kinetic behavior of acidified aqueous reaction systems based on the oxidation of sodium oxalate by potassium permanganate was investigated by UV-Vis absorbance spectrophotometry as part of a wider project to develop time-temperature indicators based on such systems. Reaction systems in various eutectic salt solutions as well as salt-free aqueous solutions were prepared and measured to establish the precision and accuracy of four different reaction systems designed to run for specified periods of time at 25°C. Reaction progress was also monitored in reaction systems previously subjected to prolonged periods of storage at −80°C, as well as systems subjected to repeated freezing and thawing, to determine the effect of preliminary freeze time or multiple freeze-thaw cycles on reaction run time. Eutectic-free aqueous reaction systems showed a noticeable decrease in run time from freshly frozen to 2 week frozen, but minimal to no decrease with increasing preliminary freeze time. Perchlorate-based reaction systems showed minimal to no decrease in reaction time with increasing preliminary freeze time, and were found to be capable of tolerating up to three <5-minute excursions into room temperature before reaction time was significantly affected. It was also found that all reaction systems studied were capable of reaching their target run times within the intended ±10% limit of accuracy, as average run times for each system ranged from 0.4% to 6.9% off from the target run time. These systems were found to be reproducible with considerable precision as well, with the average coefficient of variation for each system ranging from 2.6 to 5.5.
Date Created
2024
Agent

High-Resolution 3D Printed Microfluidic Devices for Dielectrophoretic Manipulation of Proteins

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Description
Insulator-based dielectrophoresis (iDEP) has attracted considerable attention due to its ability to precisely capture and manipulate nanoparticles and biomolecules. A distinctive approach for effective manipulation of nanometer-sized proteins employing iDEP technique by generating higher electric field (E) and gradient (??2)…

Insulator-based dielectrophoresis (iDEP) has attracted considerable attention due to its ability to precisely capture and manipulate nanoparticles and biomolecules. A distinctive approach for effective manipulation of nanometer-sized proteins employing iDEP technique by generating higher electric field (E) and gradient (??2) in the iDEP microfluidic devices is delineated. Strategies to generate higher ??2 in the iDEP devices were outlined using numerical simulations. Intriguingly, the numerical simulation results demonstrated that by decreasing the post-to-post gap in the iDEP microfluidic devices, the ??2 was increased by ⁓12 fold. Furthermore, the inclusion of channel constrictions, such as rectangular constriction or curved constriction into the straight channel iDEP microfluidic device led to a significant increase in ??2. In addition, the inclusion of rectangular constrictions in the straight channel iDEP microfluidic device resulted in a greater increase in ??2 compared to the incorporation of curved constrictions in the same device. Moreover, the straight channel device with horizontal post-to-post gap of 20 μm and vertical post-to-post gap of 10 μm generated the lowest ??2 and the ??2 was uniform across the device. The rectangular constriction device with horizontal and vertical post-to-post gap of 5 μm generated the highest ??2 and the ??2 was non-uniform across the device. Subsequently, suitable candidate devices were fabricated using soft lithography as well as high resolution 3D printing and the DEP behavior of ferritin examined under various experimental conditions. Positive streaming DEP could be observed for ferritin at low frequency in the device generating the lowest ??2, whereas at higher frequency of 10 kHz no DEP trapping characteristics were apparent in the same device. Importantly, in the device geometry resulting in the highest ??2 at 10 kHz, labeled ferritin exhibited pDEPtrapping characteristics. This is an indication that the DEP force superseded diffusion and became the dominant force.
Date Created
2024
Agent

Investigating Protein Co-Crystallization for Improved Sensitivity in Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry

Description

There are limited methods and techniques to quantitatively assess protein content in single cells or small cell populations of tissues. The standard protein insulin was used to understand how potential changes in the preparation or co-crystallization process could improve sensitivity…

There are limited methods and techniques to quantitatively assess protein content in single cells or small cell populations of tissues. The standard protein insulin was used to understand how potential changes in the preparation or co-crystallization process could improve sensitivity and limit of detection through matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis in Bruker’s Microflex LRF using polydimethylsiloxane (PDMS) reservoirs. In addition, initial imaging tests were performed on Bruker’s RapifleX MALDI Tissuetyper to determine the instrument’s imaging capabilities on proteins of interest through the use of a single layer “Christmas tree” microfluidic device, with the aim of applying a similar approach to future tissue samples. Data on 2µM insulin determined that a 95% laser power in the Microflex corresponded to 12-15% laser power in the RapifleX. Based on the experiments with insulin, the process of mixing insulin and saturated ɑ-Cyano-4-hydroxycinnamic acid (HCCA) matrix solvent in a 1:1 ratio using 10mM sodium phosphate buffer under area analysis is most optimized with a limit of detection value of 110 nM. With this information, the future aim is to apply this method to a double layer Christmas tree device in order to hopefully quantitatively analyze and image protein content in single or small cell populations.

Date Created
2023-05
Agent

Manipulation of Nano-rods and Biomolecules with Insulator-based Dielectrophoresis

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Description
Microfluidics has enabled many biological and biochemical applications such as high-throughput drug testing or point-of-care diagnostics. Dielectrophoresis (DEP) has recently achieved prominence as a powerful microfluidic technique for nanoparticle separation. Novel electric field-assisted insulator-based dielectrophoresis (iDEP) microfluidic devices have been…

Microfluidics has enabled many biological and biochemical applications such as high-throughput drug testing or point-of-care diagnostics. Dielectrophoresis (DEP) has recently achieved prominence as a powerful microfluidic technique for nanoparticle separation. Novel electric field-assisted insulator-based dielectrophoresis (iDEP) microfluidic devices have been employed to fractionate rod-shaped nanoparticles like Single-walled carbon nanotubes (SWNTs) and manipulate biomolecules like Deoxyribonucleic acid (DNA) and proteins. This dissertation involves the development of traditional as well as 3D-printed iDEP devices for the manipulation of nm-to-µm scale analytes. First, novel iDEP microfluidic constriction-based sorting devices were developed to introduce inhomogeneous electric field gradients to fractionate SWNTs by length. SWNTs possess length-specific optical and electrical properties, expanding their potential applications for future nanoscale devices. Standard synthesis procedures yield SWNTs in large-length polydispersity and chirality. Thus, an iDEP-based fractionation tool for desired lengths of SWNTs may be beneficial. This dissertation presents the first study of DEP characterization and fractionation of SWNTs using an iDEP microfluidic device. Using this iDEP constriction sorter device, two different length distributions of SWNTs were sorted with a sorting efficiency of >90%. This study provides the fundamentals of fractionating SWNTs by length, which can help separate and purify SWNTs for future nanoscale-based applications. Manipulation of nm-scale analytes requires achieving high electric field gradients in an iDEP microfluidic device, posing one of the significant challenges for DEP applications. Introducing nm-sized constrictions in an iDEP device can help generate a higher electric field gradient. However, this requires cumbersome and expensive fabrication methods. In recent years, 3D printing has drawn tremendous attention in microfluidics, alleviating complications associated with complex fabrication methods. A high-resolution 3D-printed iDEP device was developed and fabricated for iDEP-based manipulation of analytes. A completely 3D-printed device with 2 µm post-gaps was realized, and fluorescent polystyrene (PS) beads, λ-DNA, and phycocyanin protein trapping were demonstrated. Furthermore, a nm-resolution 3D-printed iDEP device was successfully printed. In the future, these high-resolution 3D-printed devices may lead to exploring DEP characteristics of nanoscale analytes like single protein molecules and viruses. The electric field-assisted unique fractionation phenomena in microfluidic platforms will become a critical solution for nanoparticle separation and manipulating biomolecules.
Date Created
2022
Agent

Hyphenated Microfluidic and MALDI Mass Spectrometry Platform for Targeted Intracellular Protein Analysis

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Description
Understanding cellular processes can provide insight into disease pathogenesis and reveal critical information for prevention, diagnosis, and treatment. As key executors and signaling regulators, proteins carry relevant information not available from genomics and transcriptomics. Cell-to-cell differences significantly affect disease incidence…

Understanding cellular processes can provide insight into disease pathogenesis and reveal critical information for prevention, diagnosis, and treatment. As key executors and signaling regulators, proteins carry relevant information not available from genomics and transcriptomics. Cell-to-cell differences significantly affect disease incidence and drug responses, generating a need for protein analysis at the single-cell level. However, quantitative protein analysis at the single-cell level remains challenging due to the low protein amount in a single cell and the proteome complexity. It requires sensitive detection techniques and appropriate sample preparation and delivery to the detection area. Here, a microfluidic platform in tandem with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been developed for targeted intracellular protein analysis. The elastomeric multi-layer microfluidic platform, termed MIMAS, was designed as a series of 8.75 nL wells separated by pneumatic valves. The MIMAS platform allows cell loading, sample processing on-chip, and further in situ mass spectrometry analysis. The sample processing includes cell lysis, immunocapture, tryptic digestion and MALDI matrix solution loading for co-crystallization. This work demonstrates that the MIMAS approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 from MCF-7 breast cancer cells using an isotope-labeled peptide. The limit of detection was determined as 11.22 nM, equivalent to 5.91 x 10^7 protein molecules per well. Moreover, the MIMAS platform design was improved, allowing the successful quantification of Bcl-2 protein in small cell ensembles down to ~10 cells in 4 nL wells. Furthermore, the MIMAS platform was integrated with laser capture microdissection (LCM) for protein analysis from post-mortem human tissues. Intracellular amyloid-β peptide (Aβ), a hallmark of Alzheimer’s Disease, was assessed from human brain tissue using the LCM-MIMAS. The successful detection of Aβ from small cell ensembles (20 sliced pyramidal cells) demonstrated the LCM-MIMAS capability of assessing intracellular proteins from specific tissue cell subpopulations. The MIMAS approach is a promising tool for intracellular protein analysis from small cell ensembles, with the potential for single-cell analysis. It allows for protein analysis towards the understanding of biological phenomena for clinical and biological research.
Date Created
2022
Agent

Microfluidic Platforms for the Separation of Particles

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Description
Microfluidic platforms have been exploited extensively as a tool for the separation of particles by electric field manipulation. Microfluidic devices can facilitate the manipulation of particles by dielectrophoresis. Separation of particles by size and type has been demonstrated by insulator-based…

Microfluidic platforms have been exploited extensively as a tool for the separation of particles by electric field manipulation. Microfluidic devices can facilitate the manipulation of particles by dielectrophoresis. Separation of particles by size and type has been demonstrated by insulator-based dielectrophoresis in a microfluidic device. Thus, manipulating particles by size has been widely studied throughout the years. It has been shown that size-heterogeneity in organelles has been linked to multiple diseases from abnormal organelle size. Here, a mixture of two sizes of polystyrene beads (0.28 and 0.87 μm) was separated by a ratchet migration mechanism under a continuous flow (20 nL/min). Furthermore, to achieve high-throughput separation, different ratchet devices were designed to achieve high-volume separation. Recently, enormous efforts have been made to manipulate small size DNA and proteins. Here, a microfluidic device comprising of multiple valves acting as insulating constrictions when a potential is applied is presented. The tunability of the electric field gradient is evaluated by a COMSOL model, indicating that high electric field gradients can be reached by deflecting the valve at a certain distance. Experimentally, the tunability of the dynamic constriction was demonstrated by conducting a pressure study to estimate the gap distance between the valve and the substrate at different applied pressures. Finally, as a proof of principle, 0.87 μm polystyrene beads were manipulated by dielectrophoresis. These microfluidic platforms will aid in the understanding of size-heterogeneity of organelles for biomolecular assessment and achieve separation of nanometer-size DNA and proteins by dielectrophoresis.
Date Created
2021
Agent

Numerical Modeling of Hydrodynamic Flow Focusing in a Microfluidic Device for Time-Resolved Serial Crystallography

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Description

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL) has enabled the determination of protein structures and protein reaction intermediates in millisecond to microsecond time resolutions. Mix-and-Inject crystallography (MISC) at XFELs enables fast mixing in the magnitude of milliseconds…

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL) has enabled the determination of protein structures and protein reaction intermediates in millisecond to microsecond time resolutions. Mix-and-Inject crystallography (MISC) at XFELs enables fast mixing in the magnitude of milliseconds in order to achieve desired reaction time points. For these experiments, numerical simulations of a hydrodynamic flow mixer capable of fast mixing by diffusion has been developed using both COMSOL Multiphysics 5.6 and QuickerSims Computational Fluid Dynamics (CFD) Toolbox for MATLAB. These simulation programs were compared by calculations of mixing times and concentration flow profiles. Mixing times in the range of 1-10 ms were calculated in COMSOL under certain flow rate conditions whereas mixing times in the range of 6-15 ms were calculated with QuickerSims. From these mixing times, reaction intermediates can be varied from sub-millisecond to several hundred millisecond time points for a MISC experiment. Explanations for the discrepancies between the two models were attributed to variations in parameter definitions and meshing. Further analysis on the mixing characteristics were investigated by calculating an analytical solution to the convection-diffusion equation for fluid flow in a two-dimensional rectangular channel. The concentration profile along the width of the channel for the analytical solution was compared with the numerical solution obtained with COMSOL and QuickerSims. Upon comparison, it was determined that the diffusion coefficient may not be a significant factor for the disagreement between the two hydrodynamic flow models.

Date Created
2021-05
Agent

Capacitive Sensor for the Detection of Microdroplets in Serial Femtosecond Crystallography

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Description

Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which are then fired out as a jet in the beam…

Microfluidic devices represent a growing technology in the world of analytical chemistry. Serial femtosecond crystallography (SFX) utilizes microfluidic devices to generate droplets of an aqueous buffer containing protein crystals, which are then fired out as a jet in the beam of an X-ray free electron laser (XFEL). A crucial part of the device is its method of droplet detection. This project presents a design for a capacitive sensor that uses a unique electrode configuration to detect the difference in capacitance between the aqueous and oil phases. This design was developed using MATLAB and COMSOL Multiphysics simulations and printed using high-resolution 3D printing. Results show that this design can successfully distinguish between the two immiscible liquids, confirming it as a possible detection method in future SFX experiments.

Date Created
2021-05
Agent

Electric Field Driven Migration and Separation in the Microenvironment

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Description
Novel electric field-assisted microfluidic platforms were developed to exploit unique migration phenomena, particle manipulation, and enhanced droplet control. The platforms can facilitate various analytical challenges such as size-based separations, and delivery of protein crystals for structural discovery with both high…

Novel electric field-assisted microfluidic platforms were developed to exploit unique migration phenomena, particle manipulation, and enhanced droplet control. The platforms can facilitate various analytical challenges such as size-based separations, and delivery of protein crystals for structural discovery with both high selectivity and sensitivity. The vast complexity of biological analytes requires efficient transport and fractionation approaches to understand variations of biomolecular processes and signatures. Size heterogeneity is one characteristic that is especially important to understand for sub-micron organelles such as mitochondria and lipid droplets. It is crucial to resolve populations of sub-cellular or diagnostically relevant bioparticles when these often cannot be resolved with traditional methods. Herein, novel microfluidic tools were developed for the unique migration mechanism capable of separating sub-micron sized bioparticles by size. This based on a deterministic ratchet effect in a symmetrical post array with dielectrophoresis (DEP) for the fast migration allowing separation of polystyrene beads, mitochondria, and liposomes in tens of seconds. This mechanism was further demonstrated using high throughput DEP-based ratchet devices for versatile, continuous sub-micron size particle separation with large sample volumes. Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) revolutionized protein structure determination. In SFX experiments, a majority of the continuously injected liquid crystal suspension is wasted due to the unique X-ray pulse structure of XFELs, requiring a large amount (up to grams) of crystal sample to determine a protein structure. To reduce the sample consumption in such experiments, 3D printed droplet-based microfluidic platforms were developed for the generation of aqueous droplets in an oil phase. The implemented droplet-based sample delivery method showed 60% less sample volume consumption compared to the continuous injection at the European XFEL. For the enhanced control of aqueous droplet generation, the device allowed dynamic triggering of droplets for further improvement in synchronization between droplets and the X-ray pulses. This innovative technique of triggering droplets can play a crucial role in saving protein crystals in future SFX experiments. The electric field-assisted unique migration and separation phenomena in microfluidic platforms will be the key solution for revolutionizing the field of organelle separation and structural analysis of proteins.
Date Created
2020
Agent

Sample delivery enabled by 3D printing for reduced sample consumption and mix-and-inject serial crystallography at x-ray free electron lasers

Description
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL…

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements.

To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.

For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.
Date Created
2019
Agent
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