Ability of Antibodies Immobilized on Gold Nanoparticles to Bind Small Antigen Fluorescein.

The analytical applications of antibodies are often associated with their immobilization on different carriers, which is accompanied by a loss of antigen-binding activity for a sufficient proportion of the bound antibodies. In contrast to data on plain carriers, minimal data are available on the properties of antibodies on the surfaces of nanoparticles. Protein antigens have been predominantly investigated, for which space restrictions do not allow them to occupy all active sites of immobilized antibodies. This study considered a low-molecular-weight compound, fluorescein, as an antigen. Spherical gold nanoparticles with five different sizes, two differently charged forms of fluorescein, and three different levels of surface coverage by immobilized antibodies were tested. For gold nanoparticles with diameters from 14 to 35.5 nm with monolayers of immobilized antibodies, the percentage of molecules capable of binding carboxyfluorescein varied from 6% to 17%. The binding of aminofluorescein was more efficient; for gold nanoparticles with an average diameter of 21 nm, the percentage of active binding sites for the immobilized antibodies reached 27% compared with 13% for the carboxyfluorescein case. A fourfold reduction in the coverage of the nanoparticles' surface compared with that of the monolayer did not lead to reliable changes in the percentage of active binding sites. The obtained data demonstrate that an antigen's binding to immobilized antibodies is limited even for small antigens and depends on the size of the nanoparticles and the electrostatic repulsion near their surface.

Comparison of Three Lateral Flow Immunoassay Formats for the Detection of Antibodies against the SARS-CoV-2 Antigen.

Reliable detection of specific antibodies against pathogens by lateral flow immunoassay (LFIA) greatly depends on the composition of the detectable complex and the order of its assembly. We compared three LFIA formats for revealing anti-SARS-CoV-2 antibodies in sera with the following detected complexes in the analytical zone of the strip: antigen-antibodies-labeled immunoglobulin-binding protein (Scheme A); antigen-antibodies-labeled antigen (Scheme B); and immunoglobulin-binding protein-antibodies-labeled antigen (Scheme C). The lowest detection limit was observed for Scheme C, and was equal to 10 ng/mL of specific humanized monoclonal antibodies. When working with pooled positive sera, Scheme C had a detection limit 15 times lower than Scheme B and 255 times lower than Scheme A. Due to the high sensitivity of Scheme C, its application for the panel of human sera (n = 22) demonstrated 100% diagnostic specificity and sensitivity. These consistent results be useful for designing the format of LFIA serodiagnosis for other diseases.

Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey.

A scheme of modular competitive immunochromatography with an analyte-independent test strip and changeable specific immunoreactants has been proposed. Native (detected) and biotinylated antigens interact with specific antibodies during their preincubation in solution, that is, without the immobilization of reagents. After this, the detectable complexes on the test strip are formed by the use of streptavidin (which binds biotin with high affinity), anti-species antibodies, and immunoglobulin-binding streptococcal protein G. The technique was successfully applied for the detection of neomycin in honey. The visual and instrumental detection limits were 0.3 and 0.014 mg/kg, respectively, and the degree of neomycin revealed in honey samples varied from 85% to 113%. The efficiency of the modular technique with the use of the same test strip for different analytes was confirmed for streptomycin detection. The proposed approach excludes the necessity of finding the condition of immobilization for each new specific immunoreactant and transferring the assay to other analytes by a simple choice of concentrations for preincubated specific antibodies and the hapten-biotin conjugate.

Enhanced Lateral Flow Immunoassay with Double Competition and Two Kinds of Nanoparticles Conjugates for Control of Insecticide Imidacloprid in Honey.

Finding optimal conditions for competitive lateral flow immunoassay is a controversial task. The content of specific antibodies labeled by nanoparticles should be simultaneously high to reach intense signals and low to register an influence on the signals for minimal concentrations of the target analyte. We propose to use two kinds of complexes of gold nanoparticles in the assay, with antigen-protein conjugates and with specific antibodies. The first complex interacts both with immobilized antibodies in the test zone and with antibodies on the surface of the second complex. In this assay, the coloration is enhanced by the binding of two-colored preparations in the test zone, whereas the antigen in the sample inhibits both the binding of the first conjugate with the immobilized antibodies and with the second conjugate. This approach is realized for the detection of insecticide imidacloprid (IMD), an important toxic contaminant connected with the recent global death of bees. The proposed technique expands the working range of the assay, that is, in accordance with its theoretical analysis. The reliable change of coloration intensity is achieved for a 2.3-times-lower concentration of the analyte. The limit of IMD detection is 0.13 ng/mL for tested solutions and 1.2 µg/kg for initial honey samples. The combination of two conjugates doubles the coloration in the absence of the analyte. The developed lateral flow immunoassay is applicable for five-fold-diluted honey samples without extraction, does not require additional stages (all reagents are pre-applied to the test strip), and is implemented in 10 min.

SMART: A Swing-Assisted Multiplexed Analyzer for Point-of-Care Respiratory Tract Infection Testing.

Respiratory tract infections such as the ongoing coronavirus disease 2019 (COVID-19) has seriously threatened public health in the last decades. The experience of fighting against the epidemic highlights the importance of user-friendly and accessible point-of-care systems for nucleic acid (NA) detection. To realize low-cost and multiplexed point-of-care NA detection, a swing-assisted multiplexed analyzer for point-of-care respiratory tract infection testing (SMART) was proposed to detect multiple respiratory tract pathogens using visible loop-mediated isothermal amplification. By performing hand-swing movements to generate acceleration force to distribute samples into reaction chambers, the design of the SMART system was greatly simplified. By using different format of chips and integrating into a suitcase, this system can be applied to on-site multitarget and multi-sample testing. Three targets including the N and Orf genes of SARS-CoV-2 and the internal control were simultaneously analyzed (limit of detection: 2000 copies/mL for raw sample; 200 copies/mL for extracted sample). Twenty-three clinical samples with eight types of respiratory bacteria and twelve COVID-19 clinical samples were successfully detected. These results indicate that the SMART system has the potential to be further developed as a versatile tool in the diagnosis of respiratory tract infection.

Handling Detection Limits of Multiplex Lateral Flow Immunoassay by Choosing the Order of Binding Zones.

Changes in the limits of detection (LODs) for a multiplex lateral flow immunoassay (LFIA) caused by different locations of the binding zone on the test strips were studied. Due to the non-equilibrium conditions of the immune reactions in LFIAs, their analytical parameters are susceptible to the binding constants of antigen-antibody reactions and assay duration. Consequently, the integration of several tests into one multiplex assay can cause a significant worsening of the sensitivity. In this study, we propose a simple methodology for the determination of the best arrangement of binding zones, which takes into account the binding constants for immunoreagents. LFIAs of four mycotoxins, namely, aflatoxin B1, deoxynivalenol, T-2 toxin, and ochratoxin A, were integrated into a multiplex test strip. An enzyme-linked immunosorbent assay was applied to determine the equilibrium and kinetic constants of the immunoreactants for each analyte. It was found that the arrangement of binding zones with a descending order of the equilibrium association constants was optimal and provided both lower detection limits and a more uniform coloration. The selected position of the binding zones allowed decreasing the LODs down to 2 and 27 times for ochratoxin A and deoxynivalenol, respectively. The proposed approach can be applied to multiplex LFIAs for different analytes.

Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes.

The presence of pathogen-specific antibodies in the blood is widely controlled by a serodiagnostic technique based on the lateral flow immunoassay (LFIA). However, its common one-stage format with an antigen immobilized in the binding zone of a test strip and a nanodispersed label conjugated with immunoglobulin-binding proteins is associated with risks of very low analytical signals. In this study, the first stage of the immunochromatographic serodiagnosis was carried out in its traditional format using a conjugate of gold nanoparticles with staphylococcal immunoglobulin-binding protein A and an antigen immobilized on a working membrane. At the second stage, a labeled immunoglobulin-binding protein was added, which enhanced the coloration of the bound immune complexes. The use of two separated steps, binding of specific antibodies, and further coloration of the formed complexes, allowed for a significant reduction of the influence of non-specific immunoglobulins on the assay results. The proposed approach was applied for the serodiagnosis using a recombinant RBD protein of SARS-CoV-2. As a result, an increase in the intensity of test zone coloration by more than two orders of magnitude was demonstrated, which enabled the significant reduction of false-negative results. The diagnostic sensitivity of the LFIA was 62.5% for the common format and 100% for the enhanced format. Moreover, the diagnostic specificity of both variants was 100%.

Double Competitive Immunodetection of Small Analyte: Realization for Highly Sensitive Lateral Flow Immunoassay of Chloramphenicol.

A new scheme of reagents interaction for lateral flow immunoassay (LFIA) is proposed, which combines the features of competitive and sandwich assay and provides highly sensitive detection of low-molecular-weight analytes. Namely, the antigen in the sample interferes with the formation of the antibody (on the membrane)-hapten-protein-antibody (on the nanoparticle-marker) complex, competing with hapten-protein conjugate in both reactions. The proposed scheme was modelled using COPASI software, with a prediction of limit of detection (LOD) decrease by one order of magnitude compared to the standard competitive LFIA. This feature was experimentally confirmed for the detection of chloramphenicol (CAP) in honey. When tested in spiked honey, the visual LOD was 50 ng/mL for the common scheme and 5 ng/mL for the proposed scheme. Instrumental LOD was 300 pg/mL (1.2 µg/kg in conversion per sample weight of honey) in the standard scheme and 20 pg/mL (80 ng/kg in conversion per sample weight of honey) in the proposed scheme.

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.

Retention of Activity by Antibodies Immobilized on Gold Nanoparticles of Different Sizes: Fluorometric Method of Determination and Comparative Evaluation.

Antibody-nanoparticle conjugates are widely used analytical reagents. An informative parameter reflecting the conjugates' properties is the number of antibodies per nanoparticle that retain their antigen-binding ability. Estimation of this parameter is characterized by a lack of simple, reproducible methods. The proposed method is based on the registration of fluorescence of tryptophan residues contained in proteins and combines sequential measurements of first the immobilized antibody number and then the bound protein antigen number. Requirements for the measurement procedure have been determined to ensure reliable and accurate results. Using the developed technique, preparations of spherical gold nanoparticles obtained by the most common method of citrate reduction of gold salts (the Turkevich-Frens method) and varying in average diameter from 15 to 55 nm have been characterized. It was shown that the number of antibodies (immunoglobulins G) bound by one nanoparticle ranged from 30 to 194 during adsorptive unoriented monolayer immobilization. C-reactive protein was considered as the model antigen. The percentage of antibody valences that retained their antigen-binding properties in the conjugate increased from 17 to 34% with an increase in the diameter of gold nanoparticles. The proposed method and the results of the study provide tools to assess the capabilities of the preparations of gold nanoparticles and their conjugates as well as the expediency of seeking the best techniques for various practical purposes.

Lateral Flow Serodiagnosis in the Double-Antigen Sandwich Format: Theoretical Consideration and Confirmation of Advantages.

Determination of the presence in the blood of antibodies specific to the causative agent of a particular disease (serodiagnosis) is an effective approach in medical analytical chemistry. Serodiagnostics performed in the lateral flow immunoassay format (immunochromatography) meet the modern requirements for point-of-care testing and are supported by existing technologies of large-scale diagnostic tests production, thus increasing the amount of attention in a tense epidemiological situation. For traditional lateral flow serodiagnostics formats, a large number of nonspecific immunoglobulins in the sample significantly reduces the degree of detectable binding. To overcome these limitations, an assay based on the formation of immobilized antigen-specific antibody-labeled antigen complexes detection was proposed. However, the requirements for its implementation, providing maximum sensitivity, have not been established. This article describes the mathematical model for the above assay. The influence of the ratio of reagent concentrations on the analysis results is considered. It is noted that the formation of specific antibody complexes with several labeled antigens is the main limiting factor in reducing the detection limit, and methods are proposed to minimize this factor. Recommendations for the choice of the assay conditions, following from the analysis of the model, are confirmed experimentally.

Molecular basis for the recognition of steroidogenic acute regulatory protein by the 14-3-3 protein family.

Steroidogenesis in adrenals and gonads starts from cholesterol transport to mitochondria. This is mediated by the steroidogenic acute regulatory protein (STARD1), containing a mitochondrial import sequence followed by a cholesterol-binding START domain. Although mutations in this protein have been linked to lipoid congenital adrenal hyperplasia (LCAH), the mechanism of steroidogenesis regulation by STARD1 remains debatable. It has been hypothesized to involve a molten-globule structural transition and interaction with 14-3-3 proteins. In this study, we aimed to address the structural basis for the 14-3-3-STARD1 interaction. We show that, while the isolated START domain does not interact with 14-3-3, this interaction is enabled by STARD1 phosphorylation at Ser57, close to the mitochondrial peptide cleavage site. Biochemical analysis of the STARD1 affinity toward 14-3-3 and crystal structures of 14-3-3 complexes with Ser57 and Ser195 phosphopeptides suggest distinct roles of site-specific phosphorylations in recruiting 14-3-3, to modulate STARD1 activity, processing and import to the mitochondria. Phosphorylation at Ser195 creates a unique conditional site that could only bind to 14-3-3 upon partial unfolding of the START domain. Overall, our findings on the interaction between 14-3-3 and STARD1 may have potential clinical implications for patients with LCAH.

Design of Multiplex Lateral Flow Tests: A Case Study for Simultaneous Detection of Three Antibiotics.

The presented study is focused on the impact of binding zone location on immunochromatographic test strips on the analytical parameters of multiplex lateral flow assays. Due to non-equilibrium conditions for such assays the duration of immune reactions influences significantly the analytical parameters, and the integration of several analytes into one multiplex strip may cause an essential decrease of sensitivity. To choose the best location for binding zones, we have tested reactants for immunochromatographic assays of lincomycin, chloramphenicol, and tetracycline. The influence of the distance to the binding zones on the intensity of coloration and limit of detection (LOD) was rather different. Basing on the data obtained, the best order of binding zones was chosen. In comparison with non-optimal location the LODs were 5-10 fold improved. The final assay provides LODs 0.4, 0.4 and 1.0 ng/mL for lincomycin, chloramphenicol, and tetracycline, respectively. The proposed approach can be applied for multiplexed assays of other analytes.

Triple Immunochromatographic System for Simultaneous Serodiagnosis of Bovine Brucellosis, Tuberculosis, and Leukemia.

An immunochromatographic test system has been developed for the simultaneous rapid multiplex serodiagnostics of bovine brucellosis, tuberculosis, and leukemia. The test system is based on the use of a conjugate of gold nanoparticles with the chimeric protein Cysteine-A/G and three analytical zones with immobilized pathogen antigens: Brucella abortus lipolysaccharide, recombinant proteins MPB64 and MPB83-MPB63 of Mycobacterium bovis, and recombinant protein p24 of the bovine leukemia virus. Prototypes of the test system were tested on 98 samples of sera from healthy and infected animals. The diagnostic sensitivity of the developed test system was 92% for brucellosis, 92% for tuberculosis, and 96% for leukemia. False positive test results were not observed.

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.

Mathematical Model of Serodiagnostic Immunochromatographic Assay.

This article describes the mathematical model for an immunochromatographic assay for the detection of specific immunoglobulins against a target antigen (antibodies) in blood/serum (serodiagnosis). The model utilizes an analytical (non-numerical) approach and allows the calculation of the kinetics of immune complexes' formation in a continuous-flow system using commonly available software, such as Microsoft Excel. The developed model could identify the nature of the influence of immunochemical interaction constants and reagent concentrations on the kinetics of the formation of the detected target complex. On the basis of the model, recommendations are developed to decrease the detection limit for an immunochromatographic assay of specific immunoglobulins.

Theoretical and Experimental Comparison of Different Formats of Immunochromatographic Serodiagnostics.

In this study, a comparative theoretical and experimental analysis of two immuno-chromatographic serodiagnostics schemes, which differ in the immobilization of immunoreagents and the order of the formation of immune complexes, is performed. Based on the theoretical models, the assays are characterized to determine which scheme has a higher quantity of the detected complex and thus ensures the sensitivity of the analysis. The results show that for the effective detection of low-affinity antibodies, the scheme involving the immobilization of the antigen on gold nanoparticles and the antibody-binding protein on the test strip was more sensitive than the predominantly used scheme, which inverts the immunoreagents' locations. The theoretical predictions were confirmed by the experimental testing of sera collected from tuberculosis patients.

Express immunochromatographic detection of antibodies against Brucella abortus in cattle sera based on quantitative photometric registration and modulated cut-off level.

An immunochromatographic test system was developed for rapid detection of the levels of specific IgG antibodies to Brucella abortus lipopolysaccharide, as a tool for diagnosis of brucellosis in cattle. The pilot test strips were examined using blood sera from sick (78 samples) and healthy (35 samples) cows. The results obtained by immunochromatographic assay, using a portable optical densitometer for digital video detection, correlate well with the results obtained by immunoenzyme assay and are in agreement with the results of the disease diagnosis. The new test system allows detection of antibodies within 10 min and can be proposed as an alternative to the methods available for serodiagnosis of brucellosis.

Development and application of a label-free fluorescence method for determining the composition of gold nanoparticle-protein conjugates.

A method was developed for determining the composition of the conjugates between gold nanoparticles and proteins based on the intrinsic fluorescence of unbound protein molecules. The fluorescence was evaluated after separation of the conjugates from the reaction mixture by centrifugation. Gold nanoparticles obtained using the citrate technique (average diameter 24 nm) were conjugated at pH 5.4 with the following four proteins: human immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant streptococcal protein G (protein G), and Kunitz-type soybean trypsin inhibitor (STI). The compositions of these conjugates were determined using the developed method. The conjugate compositions were dependent on the concentration of the added protein, and in all cases reached saturation. The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively. Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively. The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.