Hybrid Nanobioengineered Nanomaterial-Based Electrochemical Biosensors.

Nanoengineering biosensors have become more precise and sophisticated, raising the demand for highly sensitive architectures to monitor target analytes at extremely low concentrations often required, for example, for biomedical applications. We review recent advances in functional nanomaterials, mainly based on novel organic-inorganic hybrids with enhanced electro-physicochemical properties toward fulfilling this need. In this context, this review classifies some recently engineered organic-inorganic metallic-, silicon-, carbonaceous-, and polymeric-nanomaterials and describes their structural properties and features when incorporated into biosensing systems. It further shows the latest advances in ultrasensitive electrochemical biosensors engineered from such innovative nanomaterials highlighting their advantages concerning the concomitant constituents acting alone, fulfilling the gap from other reviews in the literature. Finally, it mentioned the limitations and opportunities of hybrid nanomaterials from the point of view of current nanotechnology and future considerations for advancing their use in enhanced electrochemical platforms.

On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors.

Human purine nucleoside phosphorylase (HsPNP) catalyzes reversible phosphorolysis of nucleosides and deoxynucleosides in the purine cascade. HsPNP has been a target on behalf of the development of new leads for the treatment of a variety of T-cell mediated disorders. Several studies on the HsPNP are focused on the identification of effective, safe, and selective inhibitors. Therefore, this study describes the development of direct, simple, reliable, and inexpensive enzymatic assays to screen HsPNP inhibitors. Initially, HsPNP was covalently immobilized on the surface of magnetic particles (MPs). Due to the versatility of the MPs as solid support for enzyme immobilization, two different methods to monitor the enzyme activity are presented. Firstly, the activity of HsPNP-MPs was assessed offline by HPLC-DAD quantifying the formed hypoxanthine. Then, HsPNP-MPs were trapped in a peek tube, furnishing a microreactor which was inserted on-flow in an HPLC-DAD system to monitor the enzyme activity by the hypoxanthine quantification. Kinetic assays provided KMapp values for the inosine substrate of 488.2 ± 49.1 and 1084 ± 111 µM for the offline and on-flow assays, respectively. For the first time, kinetic studies for Pi as substrate using the HsPNP-MPs exhibits a Michaelis-Menten kinetic, yielding KMapp values for offline and on-flow of 521.2 ± 62.9 µM and 601 ± 66.5 µM, respectively. Inhibition studies conducted with a fourth generation immucillin derivative (DI4G) were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes. Additionally, a small library containing 11 compounds was used to assess the selectivity of the developed assays. The results showed that both presented assays can be applied to selectively recognizing and characterizing HsPNP inhibitors. Particularly, the on-flow method exhibited a high throughput and performance because of its automation and represents an easy and practical approach to reuse the HsPNP-MPs. Besides, this novel enzyme activity assay model can be further applied to other biological targets.

Identification of blood plasma proteins using heparin-coated magnetic chitosan particles.

Heparin was immobilized on magnetic chitosan particles to be used as a tool for human plasma protein identification. Chitosan was magnetized by co-precipitation with Fe2+/Fe3+ (MAG-CH). Heparin was functionalized with carbodiimide and N-hydroxysuccinimide and covalently linked to MAG-CH (MAG-CH-hep). X-ray diffraction confirmed the presence of chitosan and Fe3O4 in MAG-CH. This particle exhibited superparamagnetism and size between 100-300 µm. Human plasma diluted with 10 mM phosphate buffer (pH 5.5) or 50 mM Tris-HCl buffer (pH 8.5) was incubated with MAG-CH-hep, and the proteins fixed were eluted with the same buffers containing increasing concentrations of NaCl. The proteins obtained were investigated by SDS-PAGE, LC/MS, and biological activity tests (PT, aPTT, and enzymatic chromogenic assay). Inhibitors of the serpin family, prothrombin, and human albumin were identified in this study. Therefore, MAG-CH-hep can be used to purify these proteins and presents the following advantages: low-cost synthesis, magnetic separation, ion-exchange purification, and reusability.

Bioaffinity Fishing Procedure Using Secretory Phospholipase A2 for Screening for Bioactive Components: Modulation of Pharmacological Effect Induced by sPLA2 from Crotalus durissus terrificus by Hispidulin from Moquiniastrum floribundum.

Bioaffinity capturing of molecules allows the discovery of bioactive compounds and decreases the need for various stages in the natural compound isolation process. Despite the high selectivity of this technique, the screening and identification methodology depends on the presence of a protein to capture potential ligands. However, some proteins, such as snake secretory phospholipase A2 (sPLA2), have never been investigated using this approach. The purpose of this study was to evaluate the use of a new method for screening natural compounds using a bioaffinity-guided ultrafiltration method on Crotalus durissus terrificus sPLA2 followed by HPLC-MS to identify the compounds, and this method could be used to discover new anti-inflammatory compounds from the various organisms originating from biodiversity. Different extracts were selected to evaluate their ability to inhibit sPLA2 activity. The extracts were incubated with sPLA2 and the resulting mixture was ultrafiltrated to elute unbound components. The resulting compounds were identified by HPLC-MS. We identified hispidulin as one of the components present in the Moquiniastrum floribundum leaf and evaluated the ability of this isolated compound to neutralize the inflammatory activity of sPLA2 from Crotalus durissus terrificus.

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions.

Ligand-target interactions play a central role in drug discovery processes because these interactions are crucial in biological systems. Small molecules-proteins interactions can regulate and modulate protein function and activity through conformational changes. Therefore, bioanalytical tools to screen new ligands have focused mainly on probing ligand-target interactions. These interactions have been evaluated by using solid-supported proteins, which provide advantages like increased protein stability and easier protein extraction from the reaction medium, which enables protein reuse. In some specific approaches, precisely in the ligand fishing assay, the bioanalytical method allows the ligands to be directly isolated from complex mixtures, including combinatorial libraries and natural products extracts without prior purification or fractionation steps. Most of these screening assays are based on liquid chromatography separation, and the binding events can be monitored through on-line or off-line methods. In the on-line approaches, solid supports containing the immobilized biological target are used as chromatographic columns most of the time. Several terms have been used to refer to such approaches, such as weak affinity chromatography, high-performance affinity chromatography, on-flow activity assays, and high-performance liquid affinity chromatography. On the other hand, in the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others. After the incubation step, the supernatant or the eluate from the binding assay is analyzed by liquid chromatography coupled to various detectors. Regardless of the selected bioanalytical approach, the use of solid supported proteins has significantly contributed to the development of automated and reliable screening methods that enable ligands to be isolated and characterized in complex matrixes without purification, thereby reducing costs and avoiding time-laborious steps. This review provides a critical overview of recently developed assays.

Synthesis and Inhibition Evaluation of New Benzyltetrahydroprotoberberine Alkaloids Designed as Acetylcholinesterase Inhibitors.

Secondary metabolites from natural products are a potential source of acetylcholinesterase inhibitors (AChEIs), which is a key enzyme in the treatment of many neurodegenerative diseases. Inspired by the reported activities of isoquinoline-derivative alkaloids herein we report the design, one step synthesis and evaluation by capillary enzyme reactor (ICER) of benzyl analogs (1a-1e) of the tetrahydroprotoberberine alkaloid stepholidine, which is abundant in Onychopetalum amazonicum. Docking analysis based on the crystal structure of Torpedo californica AChE (TcAChE) indicated that π-π interactions were dominant in all planned derivatives and that the residues from esteratic, anionic and peripheral subsites of the enzyme played key interaction roles. Due to the similarities observed when compared with galantamine in the AChE complex, the results suggest that ligand-target interactions would increase, especially for the N-benzyl derivatives. From a series of synthesized compounds, the alkaloids (7R,13aS)-7-benzylstepholidine (1a), (7S,13aS)-7-benzylstepholidine (1b), and (S)-10-O-benzylstepholidine (1d) are reported here for the first time. The on flow bioaffinity chromatography inhibition assay, based on the quantification of choline, revealed the N-benzylated compound 1a and its epimer 1b to be the most active, with IC50 of 40.6 ± 1 and 51.9 ± 1 µM, respectively, and a non-competitive mechanism. The proposed approach, which is based on molecular docking and bioaffinity chromatography, demonstrated the usefulness of stepholidine as a template for the design of rational AChEIs and showed how the target-alkaloid derivatives interact with AChE.

Cathepsin D immobilized capillary reactors for on-flow screening assays.

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ±â€¯7.49 µmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.

New trends in LC protein ligand screening.

The diversity of small molecules available to produce truly innovative drugs associated with the wealth of known biological targets calls for key strategies in protein ligand screening. This review encompasses the recently developed bioaffinity-based strategies. A critical view of the use of zonal, frontal, and nonlinear chromatography with immobilized proteins is given. The association of these elution modes with the ligand fishing method, which uses nanomagnetics particles, is also addressed. A series of applications and how these new screening strategies can be used to determine the function, affinity, and activity parameters of proteins is discussed.

Driving forces for the adsorption of a His-tag Chagas antigen. A rational approach to design bio-functional surfaces.

In order to rationally design a bio-functional surface based on the adsorption of a His-tag antigen, three requirements have to be considered: the bio-recognition element, the driving forces for the adsorption process and the detection mode of the bio-recognition event. This work is focused on the study of the adsorption mechanism of the His-tag H49 Chagas antigen on Ni(II) modified substrates. In order to construct the bio-functional surface, the gen of the H49 Chagas antigen was modified to incorporate His6 moiety at the N-terminal (His6-H49). Then, its physical adsorption and bio-affinity interaction with the solid substrate was studied by reflectometry. Besides His-Ni(II) bio-affinity interactions, His6-H49 was also physically adsorbed on Ni(II) modified substrates, leading to randomly oriented antigens. These loosely attached bio-molecules were partially removed using conditions of electrostatic repulsion. On the other hand, bio-affinity interactions, resulting in site-oriented molecules on the substrate, were only removable by specific competitors for Ni(II) surface sites. Finally, the surface bio-activity was determined from the peak separations of voltammetry waves due to the change of the electron transfer kinetics of a redox probe through the bio-functional surface (working electrode).