Detection Of Proteins In Solution Research Articles

Gum Arabic-assisted green synthesis of gold nanoparticles as fluorescence modulator for potential analytical applications

Aim: To demonstrate a simple, eco-friendly, and cost-effective green method to synthesize gold nanoparticles (AuNPs) using the aqueous extract of gum Arabic (GA) as a reducing and stabilizing agent. Methods: Green synthesis of nanoparticles, characterization by absorption, infra-red and fluorescence spectroscopy. Results: The absorption spectrum (UV-Vis) showed an absorption peak ~522 nm corresponding to the surface plasmon resonance (SPR) absorption peak of AuNPs. Transmission electron microscopy (TEM) images revealed spherical-shaped nanoparticles with an average size of 15 nm. Fourier transform infrared (FTIR) analysis showed that the nanoparticles are coated with organic compounds that are present in GA. The fluorescence quenching properties of the AuNPs were assessed by monitoring their effects on fluorescence intensity of coumarin 153 (C153) dye. The fluorescence of the dye decreased with an increase in concentration of the nanoparticles. Upon addition of the protein bovine serum albumin (BSA) to the mixture the fluorescence increased (recovery) again. Conclusions: The fluorescence quenching and recovery (turn-on/off system) is a valuable method for protein detection in solution. By observing the effect of BSA on the quenched fluorescence, this nanoparticle system shows promise in biomedicine, drug delivery and environmental monitoring.

Atomic Force Microscopy for Protein Detection and Their Physicoсhemical Characterization.

This review is focused on the atomic force microscopy (AFM) capabilities to study the properties of protein biomolecules and to detect the proteins in solution. The possibilities of application of a wide range of measuring techniques and modes for visualization of proteins, determination of their stoichiometric characteristics and physicochemical properties, are analyzed. Particular attention is paid to the use of AFM as a molecular detector for detection of proteins in solutions at low concentrations, and also for determination of functional properties of single biomolecules, including the activity of individual molecules of enzymes. Prospects for the development of AFM in combination with other methods for studying biomacromolecules are discussed.

Single-core magnetic markers in rotating magnetic field based homogeneous bioassays and the law of mass action

In this work, we report on the effect of the magnetic nanoparticle (MNP) concentration on the quantitative detection of proteins in solution with a rotating magnetic field (RMF) based homogeneous bioassay. Here, the phase lag between 30 nm iron oxide single-core particles and the RMF is analyzed with a fluxgate-based measurement system. As a test analyte anti-human IgG is applied which binds to the protein G functionalized MNP shell and causes a change of the phase lag. The measured phase lag changes for a fixed MNP and a varying analyte concentration are modeled with logistic functions. A change of the MNP concentration results in a nonlinear shift of the logistic function with the analyte concentration. This effect results from the law of mass action. Furthermore, the bioassay results are used to determine the association constant of the binding reaction.

Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3-(3,5-bis((bis(pyridin-2-ylmethyl)amino)-methyl)-4-hydroxyphenyl)-2-(5-(dimethylamino)naphthalene-1-sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl-1-Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high-throughput electrophoresis by using a UV-based detection system. Dansyl-1-Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in-gel SDS-PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl-1-Zn(II) allowed 1-step protein staining (SDS-PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312-nm or 365-nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl-1-Zn(II) was successfully applied to protein identification by MS via in-gel tryptic digestion, Western blotting, and Native-PAGE. Accordingly, Dansyl-1-Zn(II) may facilitate highly sensitive and high-throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.

Homogeneous immunoassays based on fluorescence emission intensity variations of zinc selenide quantum dot sensors

The fluorescence emission intensity of ZnSe quantum dots (QDs) conjugated to proteins to form QD-based biomolecular sensors increases significantly upon binding of the sensors to target proteins in solution. This phenomenon enables the development of homogeneous, separation-free immunoassays for rapid quantitative detection of proteins in solution. Proof-of-principle assays were developed by dosing a solution containing a biomolecular target with a solution containing the corresponding QD-based sensor and monitoring the changes in the peak fluorescence emission intensity of the QDs. Direct immunoassays for detecting basic fibroblast growth factor (bFGF) and prostate-specific antigen (PSA) in solution were demonstrated using QD-anti-bFGF and QD-anti-PSA sensors. A competitive immunoassay for detecting human serum albumin (HSA) was also demonstrated by dosing samples containing HSA with QD-HSA sensors and free anti-HSA antibodies. The QD-HSA sensors were tested in 1000× diluted human serum and found to be unaffected by interference from other proteins. The lower limit of detection of the assays was equal to the lowest sensor concentration in the solution that can be unambiguously detected, typically less than 1nM. The dynamic range of the assays was determined by identifying the sensor concentration above which optical interference between QDs affected adversely the observed fluorescence emission intensity. The upper limit of this concentration was 2.5μM for 4nm QDs. The ZnSe QD-based sensors were stable and preserved ∼80% of their initial peak emission intensity after two months in refrigerated storage. These biosensors have potential applications in rapid sensing of target proteins for emergency and point-of-care diagnostic applications.

Synthesis, spectroscopic characterization and photophysical study of dicyanomethylene-substituted squaraine dyes

Abstract In this work, the synthesis and the photophysical study of novel symmetrical and unsymmetrical squaraine dyes is described. These dyes were prepared by base-catalyzed condensation reaction between 3H-indolium or benzothiazolium salts with dicyanomethylene squarate, derived from squaric acid. The photophysical behavior of the dyes was investigated using UV–Vis absorption and steady-state fluorescence in solution. Additionally, its association with albumin from bovine serum (bovine serum albumin [BSA]) was also investigated. The dyes present strong absorption in the red/infrared regions and fluorescence emission in the infrared region tailored by the electronic structure of the squaraine. Association experiments with bovine serum albumin indicate that the obtained squaraine dyes are suitable for protein detection in solution.

Western Blotting via Proximity Ligation for High Performance Protein Analysis

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor β by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

Description of adhering with saturation using boundary conditions of hysteresis type

The problem considered in this paper is related to the development of biosensors which serve for the quantitative detection of proteins in solutions. An important part of such sensors is a wet cell, say a tiny box, filled with water into which a solution containing a protein to be detected is injected. Special molecules called aptamers are immobilized on the bottom of the wet cell. The aptamers can selectively bind the desired protein from the solution. The change of the surface mass loading can be analyzed using acoustic waves propagating along the aptamer layer. Thus, the concentration of the protein in the solution can be estimated. In this paper, a model describing the propagation of the protein in the wet cell and its adherence to the aptamer is proposed. It is assumed for simplicity that the propagation of the injected protein is governed by a diffusion equation. The central point of the model is a boundary condition of hysteresis type posed on the bottom of the wet cell. This condition provides the monotone growth with saturation of the surface concentration of the deposited protein. The monotonicity reflects non-detachment of already adhered protein molecules, whereas the saturation corresponds to the exhaustion of free aptamer molecules. We prove the existence and the uniqueness of solutions to this problem, study their regularity, and perform numerical simulations that clarify their behavior.

Biosensors for Protein Detection: A Review

There is considerable demand for the rapid low‐cost determination of proteins, particularly in the food and beverage industry. The most widely used tests are based on colorimetric procedures in which proteins react to produce colored complexes. These methods (Lowry et al. 1951; Bradford 1976; Gornall et al. 1949; Smith et al. 1985) are dependent upon a number of factors other than absolute protein quantity, including amino acid composition, protein purity, and association state. The successful application of these methods relies on using representative calibration standards. Time‐consuming and complex methodologies such as the Kjeldahl technique and quantitative amino acid analysis procedures have been also reported. Apart from these methods, biosensors are interesting tools offering certain operational advantages over standard photometric methods, notably with respect to rapidity, ease‐of‐use, cost, simplicity, portability, and ease of mass manufacture. By appropriate recognition element selection, it is possible to detect either a particular target protein or a broad range of proteins. This review presents an overview of the different biological recognition elements and the various transduction systems that have been reported in the literature to design biosensors for protein detection. Examples are given to illustrate the different possibilities. It must be noticed that this review reports detection of proteins in solution and not proteins immobilized on membranes. Briefly, immobilized proteins can be detected by antibodies (Western blotting) or oligonucleotides such as aptamers (“Eastern blotting”) bearing reporter molecules, fluorescently labeled or radiolabeled.

Fluorescent Hydrophobic Probes Based on Intramolecular Charge Transfer State for Sensitive Protein Detection in Solution.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Fluorescent Hydrophobic Probes Based on Intramolecular Charge Transfer State for Sensitive Protein Detection in Solution

Abstract New hydrophobic intramolecular charge transfer probes, comprising an aminostyryl and a cyanopyranyl moieties, were developed for sensitive protein quantification in solution. The probes exhibited dramatic increase of fluorescence quantum yields with accompanying blue-shift of the emission when bound to protein–sodium dodecyl sulfate (SDS) complexes. Using this emission enhancement, the synthesized probes were successfully applied for determining protein concentrations in solution.

Білки та ціанінові барвники. 1. Несиметричний карбоціаніновий барвник 14К – новий флюоресцентний зонд для гомогенного визначення білків у розчині

Білки та ціанінові барвники. 1. Несиметричний карбоціаніновий барвник 14К – новий флюоресцентний зонд для гомогенного визначення білків у розчині

An ISFET-based dipstick device for protein detection using the ion-step method

A compact and easy-to-operate device for the detection of proteins in solution is described. The device consists of an ISFET pH-sensor in the middle of a Ag/AgCl electrode, on top of which a microporous composite membrane is deposited. The measurement proceeds by offering an ion step and measuring the transient change of the ISFET potential versus the Ag/AgCl electrode. The potential transient is modulated by adsorption of protein to the membrane by incubation in a protein-containing solution. Theory is developed to describe the ISFET potential transient versus the Ag/AgCl electrode starting from the ISFET and Ag/AgCl potential transients versus an external reference electrode. Conditions are formulated under which the Ag/AgCl electrode potential is sufficiently stable to be used as the reference. Both theory and preliminary measurements show that this condition is met under several characteristic measuring conditions. When the internal reference electrode is used, the ion step can be offered simply by manual transference (`dipping') of the device between vessels of different salt concentration instead of by using a complex flow-through set-up. Measurements show a remarkable absence of noise in the potential signal of the dipstick device.