Samira Hosseini

Principles and Mechanism of MALDI-ToF-MS Analysis

This chapter reviews the history and the background of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF-MS) analysis. The chapter covers, application of this technique in variety of different areas with the special focus on MALDI analysis of the synthetic polymers. Current chapter describes the principles behind this technique in a great detail. Important technical aspects such as soft ionization, sample preparation and mass discrimination are explained in this chapter. In that perspective, the suitable choices of matrix, ionizing agent and solvents are also discussed. Furthermore, this chapter reviews the coupled analytical techniques with MALDI analysis and their specific applications and significances.

Evaluation of the Detection Results Obtained from ELISA

This chapter presents the most commons errors that typically occur when performing ELISA. Following every error, the chapter points out the possible reasons behind such errors and possible methods to overcome the problems. The chapter also describes key important parameters in assay evaluation including sensitivity, specificity, accuracy and limit of detection (LOD). Offering methods for calculation of the evaluation parameters, the chapter also provides insights and considerations for assessing the reliability of the assay. Such analysis is essential to upgrade a newly developed assay from the analytical relevance to the clinical application. Finally, the chapter provides information in regard to the measurable units in ELISA assay.

Advantages, Disadvantages and Modifications of Conventional ELISA

Nowadays ELISA is considered to be the troy horse for the routine clinical practice. This widely applied technique offers specific detection of a wide variety of target analytes in different kinds of samples. Since the invention of the technique four decades ago, ELISA has rapidly found various applications in food quality, environmental, biotechnological, and chemical disciplines among others. In spite of its many advantages, ELISA has certain limitations such as tedious/laborious assay procedure, and insufficient level of sensitivity in bio-recognition of challenging biomolecular entities such as microRNAs. A great number of research works has shown valuable attempts in addressing such shortages of ELISA through modification strategies. This chapter is dedicated to reviewing some of the main promising alternatives to the traditional ELISA. Paper- and fiber-based ELISAs, have shown great potentials for point-of-care (POC) applications due to their cost-effectiveness. Miniaturization of ELISA within micro-devices has increased the number and type of samples that can be analyzed, while much lower sample volume is required. Multiplexing was obtained as a result of micro and nano fabrication strategies and the integration of the assay within lab-on-chip (LOC) and lab-on-compact-disk (LOCD) devices. Taking advantage from a significantly vast surface area of the spheres, ultra-sensitive diagnosis was achieved by using micro-/nano-particles with different optical proprieties, sizes, synthetic variables and compositions. ELISA on the spot made possible to measure the biomolecules in vitro. Plasmonic ELISA offered detection strategies even with the aim of the naked eyes. Finally, the digital era has opened new windows of opportunity for ELISA, as the results of immunoassays can be recorded in remote/rural areas and subsequently analyzed by digital technologies or in centralized laboratories via mass data transfer.

General Overviews on Applications of ELISA

Current chapter reviews the applications of ELISA in various different fields including food industry, vaccine development, immunology (autoimmunity and humoral immunity), diagnosis (pregnancy, cancer and infectious diseases), toxicology, drug monitoring, pharmaceutical industry, and transplantation. Different examples related to each area are explained. ELISA was found to play major roles in all the mentioned disciplines.

Fundamentals and History of ELISA: The Evolution of the Immunoassays Until Invention of ELISA

Current chapter reviews background and history of the immunoassays until invention of ELISA. In that perspective, important evolutions in the field such as side-chain theory, antigen-antibody theory, discovery of the antibody structure, invention of radioimmunoassay (RIA), and invention of enzyme linked immunosorbent assay (ELISA) are reviewed. The chapter, also describes the principles of the immune system such as antibody production in human body along with different classes of antibodies, as well as antigen-antibody coupling and specificity of such interaction. In that respect, the chapter also demonstrates sources for biomolecular interaction between the biomolecules. Dominant forces that are involved in physical interaction of the antigens and antibodies including hydrogen bonding (H-bonding), hydrophobic interaction, ionic attraction, and Van der Waals forces such as London dispersion force, dipole-dipole interaction, and ion-dipole interaction are introduced in great details.

Step by Step with ELISA: Mechanism of Operation, Crucial Elements, Different Protocols, and Insights on Immobilization and Detection of Various Biomolecular Entities

Current chapter describes the essential components of ELISA including the solid phase, the adsorbents (different types of target biomolecules), and the washing and blocking agents used in assay procedure. The chapter also reviews widely applied enzymes and substrates with their specific characteristics. To complete the assay, the chapter offers information regarding the stopping procedure and readout techniques such as colorimetric, fluorescence and luminescence, along with their reading instruments. To secure a high specificity, the chapter describes protocols for conducting different types of controls in the assay procedure. These controls are namely: positive, endogenous, negative, standard, and spike controls. The chapter subsequently describes available ELISA protocols including direct, indirect, sandwich, double sandwich, and competitive assays. Finally, this chapter is dedicated to reviewing immobilization techniques including physical, covalent, oriented strategies as well as immobilization via entrapment. In the case of covalent immobilization of the biomolecules, protein attachment via zero-length cross linkers and spacers (linear or branched) are described.

Latest Updates in Dengue Fever Therapeutics: Natural, Marine and Synthetic Drugs

In this paper, we review the history of Dengue, the mechanism of infection, the molecular characteristics and components of Dengue, the mechanism of entry to the target cells, cyclization of the genome and replication process, as well as translation of the proteins for virus assembly. The major emphasis of this work is on natural products and plant extracts, which were used for as palliative or adjuvant treatment of Dengue. This review article also summarizes latest findings in regards to the marine products as effective drugs to target different symptoms of Dengue. Furthermore, an update on synthetic drugs for treating Dengue is provided in this review. As a novel alternative, we describe monoclonal antibody therapy for Dengue management and treatment.

4 – Medical applications

Interfacial properties of synthetic, biodegradable composite materials are of crucial importance for clinical outcome of fabricated medical implants, tissue engineering scaffolds, and drug delivery carriers. In this chapter we outline selected types of synthetic composite biomaterials with specific fillers dispersed into polymer matrices. By definition, the choice of both polymer and filler components, together with unavoidable release of biodegradation products in tissue proximity, would have significant implications on physiological environment and tissue regeneration at tissue–biomaterial interface. In addition, careful choice of composite components will determine immunological response on surgical implantation. This chapter explains some fundamental aspects of composite biomaterials in regard to their clinical performance from the recent studies. A new aspects and strategies for biological activation of fillers by protein surface attachment are highlighted in separate sections in this chapter. At the end of the chapter we explain a novel type of polymer composite hydrogels, called “double networks” or “interpenetrated networks” with their unique properties and their potential for fabrication of new generation biomedical devices with high degree of control over material properties.

Fundamentals of Biosensors and Application of MALDI-ToF-MS in Bio-diagnostic Domain

In this chapter, fundamentals of biosensor devices are explained with a special emphasis on optical biosensors such as enzyme-linked immunosorbent assay (ELISA). Current chapter introduces a novel polymeric material that can be used as a substitution of poly methyl methacrylate (PMMA), which is one of the mostly applied materials in fabrication of ELISA well plates. Proposed polymeric material is a copolymer synthesized in free-radical polymerization reaction with methyl methacrylate (MMA) and methacrylic acid (MAA) as monomers. Potential analytical platforms made from this copolymer can overcome some of the major drawbacks of the conventional platforms made from PMMA. In order to have a close control over the chemical and physical properties of the developed material, this copolymer was synthesized in different compositions by variation of the monomers concentrations in polymerization reaction. All different composition of this copolymer has been thoroughly analyzed by MALDI-ToF-MS and results are discussed in a great detail. MADLI has been used as a powerful technique to provide essential information regarding structural formation of the polymer chains in each composition as well as end-groups analysis of the respective chains. Present chapter connects analytical data obtained from MALDI analysis with the application of these platforms as bioreceptor surfaces for antibody immobilization and subsequent virus detection. Developed platforms have been investigated by MALDI in respect to their chemical and physical properties and their suitability for their application in virus detection. Current chapter also explains that a carful design of the macromolecule is possible by controlling the reaction parameters. In that path, MALDI analysis plays a vital role in confirmation of the reaction’s outcome. This chapter is dedicated to the MALDI analysis of a linear polymer chains with 2 monomers involved in synthesis reaction.

Fundamentals of Tissue Engineering and Application of MALDI-ToF-MS in Analysis of the Scaffold Materials

In this chapter, fundamental aspects of regenerative medicine are explained with special emphasis on application of polymeric materials for tissue engineering. In that regard, current chapter describes synthesis and processing of a biodegradable polymeric system in different compositions that can be applicable in tissue engineering. Copolymers of polyoctanediol citrate acid-sebacate acid P(OCS) were polymerized via polyesterification reaction in different molar ratios of the monomers in order to have a range of properties in developed materials. Different compositions have further been processed into the scaffolds for tissue engineering application. Developed scaffolds, were carefully analyzed by MALDI-ToF-MS and resultant spectra are interpreted in a great detail. Data obtained from MALDI analysis confirm that copolymer compositions match the expected structures, which were pre-determined in polymerization reaction. Furthermore, MALDI spectra provides a clear picture of the molecular structure that, in turn, plays a crucial role in understanding of the material properties. In a separate section, a detailed analysis is extracted from MALDI results regarding the suitability of the proposed materials for their application in tissue engineering. This chapter is dedicated to the MALDI analysis of a linear polymer system with 3 monomers involved in synthesis of P(OCS) materials.