Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb3+ Ions in Drinking and Natural Waters.

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb3+ detection was developed. The biotinylated aptamer A10 was hybridized with its complementary biotinylated oligonucleotide T10 and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3',5,5'-tetramethylbenzidine substrate, generating an optical signal. Sb3+ presenting in the sample bounded to an A10 aptamer, thus releasing T10, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb3+ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag+ at the stage of A10/T10 complex formation promoted dehybridization of the aptamer A10 and the formation of the A10/Sb3+ complex. The working range of the Ag+-enhanced microplate apta-enzyme assay for Sb3+ was determined to be 8-135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb3+ in the range of 109.0-126.2% and 99.6-106.1%, respectively.

Development of Lateral Flow Test-System for the Immunoassay of Dibutyl Phthalate in Natural Waters.

The use of a large amount of toxic synthetic materials leads to an increase in the pollution of environmental objects. Phthalates are compounds structurally related to esters of phthalic acid that are widely used in the manufacturing of synthetic packaging materials as plasticizers. Their danger is conditioned by leaching into the environment and penetrating into living organisms with negative consequences and effects on various organs and tissues. This work presents the first development of lateral flow immunoassay to detect dibutyl phthalate, one of the most common representatives of the phthalates group. To form a test zone, a hapten-protein conjugate was synthesized, and gold nanoparticles conjugated with antibodies to dibutyl phthalate were used as a detecting conjugate. The work includes the preparation of immunoreagents, selectivity investigation, and the study of the characteristics of the medium providing a reliable optical signal. Under the selected conditions for the analysis, the detection limit was 33.4 ng/mL, and the working range of the determined concentrations was from 42.4 to 1500 ng/mL. Time of the assay-15 min. The developed technique was successfully applied to detect dibutyl phthalate in natural waters with recovery rates from 75 to 115%.

Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers.

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.

Raman Scattering-Based Biosensing: New Prospects and Opportunities.

The growing interest in the development of new platforms for the application of Raman spectroscopy techniques in biosensor technologies is driven by the potential of these techniques in identifying chemical compounds, as well as structural and functional features of biomolecules. The effect of Raman scattering is a result of inelastic light scattering processes, which lead to the emission of scattered light with a different frequency associated with molecular vibrations of the identified molecule. Spontaneous Raman scattering is usually weak, resulting in complexities with the separation of weak inelastically scattered light and intense Rayleigh scattering. These limitations have led to the development of various techniques for enhancing Raman scattering, including resonance Raman spectroscopy (RRS) and nonlinear Raman spectroscopy (coherent anti-Stokes Raman spectroscopy and stimulated Raman spectroscopy). Furthermore, the discovery of the phenomenon of enhanced Raman scattering near metallic nanostructures gave impetus to the development of the surface-enhanced Raman spectroscopy (SERS) as well as its combination with resonance Raman spectroscopy and nonlinear Raman spectroscopic techniques. The combination of nonlinear and resonant optical effects with metal substrates or nanoparticles can be used to increase speed, spatial resolution, and signal amplification in Raman spectroscopy, making these techniques promising for the analysis and characterization of biological samples. This review provides the main provisions of the listed Raman techniques and the advantages and limitations present when applied to life sciences research. The recent advances in SERS and SERS-combined techniques are summarized, such as SERRS, SE-CARS, and SE-SRS for bioimaging and the biosensing of molecules, which form the basis for potential future applications of these techniques in biosensor technology. In addition, an overview is given of the main tools for success in the development of biosensors based on Raman spectroscopy techniques, which can be achieved by choosing one or a combination of the following approaches: (i) fabrication of a reproducible SERS substrate, (ii) synthesis of the SERS nanotag, and (iii) implementation of new platforms for on-site testing.

Combination of phenylboronic acid and oligocytosine for selective and specific detection of lead(ii) by lateral flow test strip.

Detection of lead (II) in water sources is of high importance for protection from this toxic contaminant. This paper presents the development and approbation of a lateral flow test strip of lead (II) with the use of phenylboronic acid as chelating agent and oligocytosine chain as receptor for the formed complexes. To locate the bound lead (II) on the test strip, phenylboronic acid was conjugated with carrier protein (bovine serum albumin) and applied as a binding line. In turn, the oligocytosine was conjugated with gold nanoparticle to provide coloration of the finally formed complexes (bovine serum albumin - phenylboronic acid - lead (II) - oligocytosine - gold nanoparticle). This combination of two binding molecules provides the «sandwich ¼ assay with direct dependence of label binding from the analyte content. The technique is characterized by high sensitivity (0.05 ng mL-1) and the absence of cross-reactions with other metal ions which are often satellite in natural waters. The developed lateral flow tests were successfully applied for lead (II) detection in water. Time of the assay was 5 min. The reached parameters confirm efficiency of the proposed technique for rapid and non-laborious testing under nonlaboratory conditions.

Limitations for colorimetric aggregation assay of metal ions and ways of their overcoming.

The development of analytical methods for the determination of metal ions in water is one of the priority tasks for efficient environmental monitoring. The use of modified gold nanoparticles and the colorimetric detection of their aggregation initiated by ions binding with specific receptors on the nanoparticle surface has high potential for simple testing. However, the limits of this approach and the parameters determining the assay sensitivity are not clear, and the possibilities of different assay formats are estimated only empirically. We have proposed a mathematical description of the aggregation processes in the assay and have estimated the detection limits of an aptamer-based assay of Pb2+ ions theoretically and experimentally. In the studied assay, gold nanoparticles modified with G,T-enriched aptamer were used, and their aggregation caused by the interaction with Pb2+ ions was controlled via a color change. The experimentally determined limit of Pb2+ detection was 700 ppb, which was in good agreement with theoretical calculations. An examination of the model showed that the limiting parameter of the assay is the binding constant of the aptamer-Pb2+ ion interaction. To overcome this limitation without searching for alternate receptors, two methods have been proposed, namely additional aggregation-causing components or centrifugation. These approaches lowered the detection limit to 150 ppb and even to 0.4 ppb. The second value accords with regulatory demands for the permissible levels of water source contamination, and the corresponding approach has significant competitive potential due to its rapidity, simple implementation, and the visual assessment of the assay results.

A highly sensitive label-free amperometric biosensor for norfloxacin detection based on chitosan-yttria nanocomposite.

Here, non-invasive and label-free detection of trace-level of norfloxacin (NF) in human urine samples has been reported using the electrochemical technique. Nanostructured yttrium oxide (nY2O3) was synthesized at low-temperature using a one-step hydrothermal process. These nY2O3 were characterized by various methods including XRD, FT-IR, Raman spectroscopy, and TEM. A biosensing platform based on nY2O3 modified with chitosan (CH) was fabricated for the detection of NF. The nanocomposite film (CH-Y2O3/ITO) was characterized by FE-SEM, contact angle measurements, and electrochemical techniques. Further, fluoroquinolones antibodies (anti-FQ) were used to modify the CH-Y2O3/ITO electrode via covalent interaction. Non-specific sites were blocked by bovine serum albumin (BSA), those present on the anti-FQ/CH-Y2O3/ITO electrode surface. The response study of BSA/anti-FQ/CH-Y2O3/ITO bioelectrode towards NF detection revealed a wide range (1 pM-10 µM) with a lower detection limit of 3.87 pM using differential pulse voltammetry (DPV). The sensitivity obtained is as high as 10.14 µA µM-1 cm2 with a fast response time of ~10 min. Moreover, the diagnostic performance of the fabricated sensor was evaluated to detect NF in urine spiked sample. The recovery of NF from the spiked sample was observed from 90.5 to 101.1%, with a maximum relative standard deviation of 7.04. The obtained results of the fabricated bioelectrode (BSA/anti-FQ/CH-Y2O3/ITO) was validated with ELISA. The results were found better when compared with earlier described biosensors and commercially existing ELISA in terms of sensitivity and lower detection limit.

Progress in rapid optical assays for heavy metal ions based on the use of nanoparticles and receptor molecules.

This review (with 230 refs.) covers recent progress in rapid optical assays for heavy metals (primarily lead and mercury as the most relevant) based on the use of nanoparticles and receptor molecules. An introduction surveys the importance, regulatory demands (such as maximum permissible concentrations) and potential and limitations of various existing methods. This is followed by a general discussion on the use of nanoparticles in optical assays of heavy metals (including properties, basic mechanisms of signal generation). The next sections cover methods for the functionalization of nanoparticles with (a) sulfur-containing compounds (used for modification of nanoparticles or added to the reaction medium), (b) nitrogen-containing compounds (such as amino acids, polypeptides, and heterocyclic molecules), and (c) oxygen-containing species (such as hydroxy and carbonyl compounds). This is continued by a specific description of specific assays based on the use of aptamers as receptors, on the use of deoxyribozymes as synthetic reaction catalysts, of G-quadruplex aptamers, of aptamers in logic gate-type of assays of linear (unstructured) aptamers ("hairpins"), and on the use of aptamers in lateral flow assays. A next section covers assays based on the employment of antibodies as receptors (used in the immunoassay development). The properties of various nanoparticles and their applicability in optical assays are also discussed in some detail. Final sections discuss the selectivity of assays, potential interferences by other cations, methods for their elimination, and also matrix effects and approaches for sample pretreatment. A concluding section discusses current challenges and future trends. Analysis based on enzyme inhibition assay is not treated here but enzyme-like action of some receptor molecules such as DNAzymes is discussed. Graphical abstract Schematic presentation of main principles of application of various nanoparticles with receptor molecules (S-, N-, O-containing, heterocyclic compounds, proteins, antibody, aptamers) for heavy metals ions detection. The included methods cover optical assays with description of mechanisms of interactions and signal generation.

ELISA and Lateral Flow Immunoassay for the Detection of Food Colorants: State of the Art.

Food colorants are inalienable part of human life. Since ancient times, they have become firmly established and have undergone a number of changes. During this time, the attitude towards them has changed. The desire to color the food now raises questions about the various side effects of their use. With the development of methods of toxicology and obtaining data on the toxicity of food coloring, restrictions and standards for their content in food have emerged. The development of methods for the analysis of dyes in various food products is becoming increasingly in demand. Development of screening immunoanalytical methods such as enzyme-linked immunoassay and immunochromatographic one suitable for simultaneous analysis of a large number of samples at the same time allows to avoid serious costs when operating the chromatographic equipment. Reduction of the analysis time and simple sample preparation is what unites immunoassay techniques. This review shows the possibilities of modern immunoanalytical methods for analyzing samples and determining food dyes belonging to different classes.

Adsorption of proteins on gold nanoparticles: One or more layers?

Adsorption of proteins on nanoparticles is a complex and poorly studied process. The mechanisms of protein layer formation can fundamentally differ depending on the composition of the medium, the nanoparticles' structure, the protein's nature, and other factors. In particular, monolayer or multilayer immobilization may occur. In the present work, the composition of conjugates of bovine serum albumin and immunoglobulin G with gold nanoparticles obtained by the Turkevich-Frens method are analyzed. The composition was studied by protein fluorescence measurement for particles ranging in size from 20 to 48 nm, depending on the pH of the immobilization medium (from 4 to 5 to 8-10). It was found that a pH shift of the immobilization medium from acidic to alkaline values is accompanied by a change in the mechanism of protein adsorption on the gold surface. In acidic pH conditions (4-5), effective binding of bovine serum albumin and gold nanoparticles occurs throughout the entire range of studied protein concentrations. In alkaline pH conditions (8-10), however, effective binding occurs only at concentrations of >10 µg/mL. This effect is not observed for immunoglobulin G, which is efficiently adsorbed onto nanoparticles throughout the entire range of studied concentrations and pH values. For acidic pH values, the surface of the particles is saturated with the amount of bound proteins, which approximately corresponds to the amount the monolayer is filled. For neutral and alkaline pH values, saturation is not observed and the amount of adsorbed protein certainly exceeds the monolayer filling, leading to multilayer immobilization.

Development of Immunochromatographic Assay for Determination of Tetracycline in Human Serum.

Determining antibiotic concentration in human blood provides useful pharmacokinetic information. Commonly used methods such as ELISA require a long time to obtain results and thus cannot be applied when information is needed immediately. In this study, a novel antibody-based lateral flow technique was developed for tetracycline detection in human serum. Contrary to tests developed to analyze food samples, the features of work with serum as analyzed probe were studied for the first time here. The application of labeled and unlabeled specific antibodies was compared. For this purpose, specific and anti-species antibodies were labeled with gold nanoparticles and used for antigen⁻antibody interaction on the membrane surface with observed staining in the test zone. For both schemes, optimal conditions were established to provide the best sensitivity. The developed assay has a limit of visual detection as low as 35 and 11 ng/mL for the direct and indirect labeled antibodies, respectively. The limit of instrumental detection is from 0.4 to 3.5 ng/mL for diluted and undiluted sera. The use of indirect antibody labeling showed a small increase in sensitivity compared to traditional direct antibody labeling. The developed method showed no cross-reactivity with antibiotics of other classes. The method was used to test samples of serum. The results showed high correlation with the data obtained by ELISA (R² = 0.98968). The assay provides a quick assessment of the amount of antibiotics in the blood and keeps them under control throughout the duration of therapy.

Quantum-dot-based immunochromatographic assay for total IgE in human serum.

To rapidly quantify total immunoglobulin E levels in human serum, we developed a novel quantum-dot-based immunochromatographic assay that employs digital recording of fluorescence. It can detect IgE levels of 5-1000 kU/L, with a coefficient of variation ranging from 2.0 to 9.5%. The assay can be processed in 10 min. The developed assay was tested on 95 serum samples. The correlation coefficient between the IgE values obtained by the proposed assay and those obtained by a commercial ELISA kit was 0.9884. Our assay thus shows promise as a new diagnostic tool for IgE detection.

Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk.

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

Advantages of soybean peroxidase over horseradish peroxidase as the enzyme label in chemiluminescent enzyme-linked immunosorbent assay of sulfamethoxypyridazine.

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.