scholarly journals Integrating Gene Synthesis and Microfluidic Protein Analysis for Rapid Protein Engineering

2015 ◽  
Author(s):  
Matthew C Blackburn ◽  
Ekaterina Petrova ◽  
Bruno E Correia ◽  
Sebastian Josef Maerkl
Keyword(s):  
Protein Design ◽  
Solid Phase ◽  
Synthesis Method ◽  
Gene Synthesis ◽  
Sequence Specificity ◽  
Quantitative Characterization ◽  
Synthetic Genes ◽  
In Silico Modeling ◽  
On Chip ◽  
Protein Biophysics

The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology, and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification, and characterization (mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE- MITOMI we synthesized and characterized over 440 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF – DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

scholarly journals Dendrimeric Poly(Epsilon-Lysine) Delivery Systems for the Enhanced Permeability of Flurbiprofen across the Blood-Brain Barrier in Alzheimer’s Disease

2018 ◽  
Vol 19 (10) ◽  
pp. 3224 ◽  
Author(s):  
Shafq Al-azzawi ◽  
Dhafir Masheta ◽  
Anna Guildford ◽  
Gary Phillips ◽  
Matteo Santin
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Blood Brain Barrier ◽  
Solid Phase ◽  
Synthesis Method ◽  
Delivery Systems ◽  
Brain Barrier ◽  
Target Cells ◽  
Target Tissue ◽  
Blood Brain

Alzheimer’s disease (AD) is a progressive brain disorder and age-related disease characterised by abnormal accumulation of β-amyloid (Aβ). The development of drugs to combat AD is hampered by the lack of therapeutically-active molecules able to cross the blood-brain barrier (BBB). It is agreed that specifically-designed carriers, such as dendrimers, could support the drug penetration across the BBB. The aim of this study was to design biocompatible and biodegradable dendrimeric delivery systems able to carry Flurbiprofen (FP), as drug for AD treatment, across the BBB and liberate it at the target tissue. These dendrons were synthesised using solid-phase peptide synthesis method and characterised by mass spectrometry and fourier-transform infrared spectroscopy (FTIR). The results revealed successful synthesis of dendrons having FP been integrated during the synthesis at their branching ends. Cytotoxicity assays demonstrated the biocompatibility of the delivery systems, whereas HPLC analysis showed high percentages of permeability across an in vitro BBB model for FP-integrated dendrons. Results also revealed the efficiency of drug conjugates on the γ-secretase enzyme in target cells with evidence of eventual drug release by hydrolysis of the carrier. This study demonstrates that the coupling of FP to dendrimeric delivery systems can successfully be achieved during the synthesis of the poly(epsilon-lysine) macromolecules to improve the transport of the active drug across the BBB.

Electrochemical Dissolution of Goethite by Abrasive Stripping Voltammetry

1995 ◽  
Vol 60 (6) ◽  
pp. 950-959 ◽  
Author(s):  
Tomáš Grygar ◽  
Jan Šubrt ◽  
Jaroslav Boháček
Keyword(s):  
Solid Phase ◽  
Reaction Pathway ◽  
Stripping Voltammetry ◽  
Electrode Reaction ◽  
Reductive Dissolution ◽  
Quantitative Characterization ◽  
Acid Media ◽  
Qualitative And Quantitative ◽  
Spectroscopic Examination

Abrasive stripping voltammetry was applied to the investigation of the reductive dissolution of some iron(III) oxides and hydroxy-oxides, particularly goethite (α-FeOOH), in acid media. The electrode reaction directly involves the solid phase, and the reaction pathway depends on the phase composition and particle shape. This can be used for a qualitative and quantitative characterization of goethite. The results of a quantitative analysis of a mixture of goethite (α-FeOOH) and lepidocrocite (γ-FeOOH) are compared with those of IR and Moessbauer spectroscopic examination. The effects of the particle appearance (shape, crystal intergrowth) on the results of the voltammetric and chronoamperometric measurements are discussed.

Design Techniques for Microfluidic Devices Implementation Applicable to Chemical Analysis Systems

2019 ◽  
pp. 195-222 ◽  
Author(s):  
Reinaldo Lucas dos Santos Rosa ◽  
Antonio Carlos Seabra
Keyword(s):  
Chemical Analysis ◽  
Solid Phase ◽  
Microfluidic Devices ◽  
Water Quality Monitoring ◽  
Analysis Process ◽  
Micro Total Analysis Systems ◽  
Total Analysis ◽  
On Chip ◽  
Separation Cell ◽  
High Level

This chapter provides a guide for microfluidic devices development and optimization focused on chemical analysis applications, which includes medicine, biology, chemistry, and environmental monitoring, showing high-level performance associated with a specific functionality. Examples are chemical analysis, solid phase extraction, chromatography, immunoassay analysis, protein and DNA separation, cell sorting and manipulation, cellular biology, and mass spectrometry. In this chapter, most information is related to microfluidic devices design and fabrication used to perform several steps concerning chemical analysis, process preparation of reagents, samples reaction and detection, regarding water quality monitoring. These steps are especially relevant to lab-on-chip (LOC) and micro-total-analysis-systems (μTAS). μTAS devices are developed in order to simplify analytical chemist work, incorporating several analytical procedures into flow systems. In the case of miniaturized devices, the analysis time is reduced, and small volumes (nL) can be used.

scholarly journals Protein-assisted RNA fragment docking (RnaX) for modeling RNA–protein interactions using ModelX

2019 ◽  
Vol 116 (49) ◽  
pp. 24568-24573 ◽  
Author(s):  
Javier Delgado Blanco ◽  
Leandro G. Radusky ◽  
Damiano Cianferoni ◽  
Luis Serrano
Keyword(s):  
Conformational Changes ◽  
Protein Design ◽  
Protein Interactions ◽  
Rna Binding ◽  
Structural Information ◽  
Conformational Dynamics ◽  
Computational Cost ◽  
Regulation Of Transcription ◽  
Sequence Specificity ◽  
Protein Interfaces

RNA–protein interactions are crucial for such key biological processes as regulation of transcription, splicing, translation, and gene silencing, among many others. Knowing where an RNA molecule interacts with a target protein and/or engineering an RNA molecule to specifically bind to a protein could allow for rational interference with these cellular processes and the design of novel therapies. Here we present a robust RNA–protein fragment pair-based method, termed RnaX, to predict RNA-binding sites. This methodology, which is integrated into the ModelX tool suite (http://modelx.crg.es), takes advantage of the structural information present in all released RNA–protein complexes. This information is used to create an exhaustive database for docking and a statistical forcefield for fast discrimination of true backbone-compatible interactions. RnaX, together with the protein design forcefield FoldX, enables us to predict RNA–protein interfaces and, when sufficient crystallographic information is available, to reengineer the interface at the sequence-specificity level by mimicking those conformational changes that occur on protein and RNA mutagenesis. These results, obtained at just a fraction of the computational cost of methods that simulate conformational dynamics, open up perspectives for the engineering of RNA–protein interfaces.

scholarly journals Microfluidic Flow-through SPME Chip for Online Separation and MS Detection of Multiple Analyses in Complex Matrix

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 120
Author(s):  
Yujun Chen ◽  
Tao Gong ◽  
Cilong Yu ◽  
Xiang Qian ◽  
Xiaohao Wang
Keyword(s):  
Microfluidic Chip ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
Sample Pretreatment ◽  
Annular Channel ◽  
Calibration Method ◽  
Extraction Process ◽  
Single Chip ◽  
Calibration Methods ◽  
On Chip

Simplifying tedious sample preparation procedures to improve analysis efficiency is a major challenge in contemporary analytical chemistry. Solid phase microextraction (SPME), a technology developed for rapid sample pretreatment, has flexibility in design, geometry, and calibration strategies, which makes it a useful tool in a variety of fields, especially environmental and life sciences. Therefore, it is important to study the coupling between the microfluidic electrospray ionization (ESI) chip integrated with the solid phase microextraction (SPME) module and the electrospray mass spectrometer (MS). In our previous work, we designed a solid phase microextraction (SPME) module on a microfluidic chip through geometric design. However, automation and calibration methods for the extraction process remain unresolved in the SPME on-chip domain, which will lead to faster and more accurate results. This paper discusses the necessity to design a micromixer structure that can produce different elution conditions on the microfluidic chip. By calculating the channel resistances, the microfluidic chip’s integrated module with the micromixer, SPME, and ESI emitters optimize the geometry structure. We propose the annular channel for SPME to perform the resistances balance of the entire chip. Finally, for SPME on a single chip, this work provides a quantitation calibration method to describe the distribution of the analytes between the sample and the extraction phase before reaching the adsorption equilibrium.

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