Spherical GNPs of various sizes (10–30 nm), obtained by the reduction of chloroauric acid with ascorbic acid, are used to obtain conjugates with native and biotinylated antibodies (Ab, bAb). The process of the formation of nanoagglomerates of GNP-bAb conjugates with the participation of free streptavidin (Stvd) in a solution is studied in the range of stoichiometric ratios [Stvd] : [GNP] from 0 : 1 to 3200 : 1. Using the dynamic-light-scattering method, it is shown that at a 19–50-fold excess of free Stvd relative to GNPs in a solution, GNP-bAb conjugates form nanoagglomerates with an effective diameter 1.5–3 times larger than the size of the initial conjugate. The efficiency of the interaction of a detection reagent prepared from a GNP-bAb conjugate in the presence of various concentrations of free Stvd in a lateral flow with Stvd or bAb absorbed on a membrane is studied. It is shown that the color intensity of the reaction zones undergoes a significant change in the region of the ratio of the concentrations of GNP-bAb and Stvd, corresponding to the formation of nanoagglomerates, and depends on the composition and size of the resulting complex and the presence in its composition of Stvd molecules or biotin residues available for binding with the absorbed component. On the basis of mathematical modeling of the proposed kinetic mechanism for the formation of GNP complexes with the participation of biotin-streptavidin interaction, the predominant formation of nanoagglomerates consisting of two or three GNPs linked by Stvd molecules is shown. The use of such nanoagglomerates as a label in a lateral immunoassay can lead to a several-fold reduction in the detection limit of the assay.

The sample preparation method for liquid biosamples dried on a carrier (dried blood spot technology) has been increasingly used in various fields of bioanalysis, pharmacokinetics, therapeutic drug monitoring, toxicokinetics, metabolomics, and disease diagnostics over the past 20 years. The dried blood spot technology involves the application of a sample directly to the carrier; after drying, the sample is analyzed using modern analytical, immunochemical, and genetic methods. The advantages of this sample preparation method include minimal invasiveness; small sample volumes; ease and cost-effectiveness of sampling, transportation, and storage of samples; and high stability of analytes. The review considers the main aspects of the application of the sample preparation of biological fluids, suspensions, tissues, and organs in the form of dried spots on porous membrane carriers in analytical practice. The principles of using membrane carriers in order to obtain biological samples in the form of dried spots, approaches to their analytical application, interfering factors and limitations associated with the rheological properties of blood, types of devices for taking blood and other samples, and the current state and prospects for the development of dried spot technology are described.

We optimize the procedure for synthesizing gold nanoshells for their use as a sensitive label in an immunochromatographic assay (ICA). An immunochromatographic test system is developed to visually determine the concentration of an early cardio marker, a heart-type fatty acid-binding protein (H-FABP). Visual detection yields the detection limit of the procedure with gold nanoshells to be more 50% lower than that of gold nanoparticles; it is 0.5 ng/mL for H-FABP.

An immunochromatographic test system for the combined analysis of two cardiomarkers, such as fatty acid binding protein (FABP) and troponin I (TnI), in serum and whole blood is developed. The main stages of the design of a multiparametric system are investigated, including the influence of the antibody position and gold nanoparticle size on the results. The detection limits of the method are found to be 10 ng/mL for FABP and 1 ng/mL for TnI, and the coefficient of variation did not exceed 10%. The developed test system allows us to confirm the diagnosis of acute myocardial infraction (N = 9). The analysis time is 15 min.

The specific antibodies for viral arthritis-encephalitis and toxoplasmosis in goats is comparatively determined by the ELISA and latex agglutination reaction using strip-dried samples of serum and whole blood on a porous membrane carrier. It is shown that the use of strip-dried samples makes it possible to qualitatively and quantitatively determine specific antibodies and its results are completely consistent with those of the analysis of the liquid samples (serum). This sample preparation method can be used for the safe shipment of blood samples and following serological studies in epizootic monitoring.

A new approach based on the use of a complex conjugate is proposed for improving the sensitivity of a lateral flow immunoassay. The complex conjugate is an agglomerate of gold nanoparticles formed by binding biotinylated antibodies labeled with gold nanoparticles to streptavidin. The amplification of the analytical signal is due to the accumulation of the agglomerates of gold nanoparticles in the test zone of the strip. The proposed test system is implemented in the lateral flow immunoassay of a model protein, procalcitonin. This system makes it possible to perform the analysis in a single step without resorting to additional reagents or manipulations. The procalcitonin detection range was 0.5–100 ng/mL and the limit of detection was 0.25 ng/mL; i.e., the sensitivity of the method is four times better than that of the conventional lateral flow immunoassay, in which spherical gold nanoparticles are used as a label.

A new format for the express method of visual semiquantitative analysis based on the principle of gradient lateral flow immunoassay with gold nanoparticles as a label is proposed. Several test bands with an increasing concentration of specific antibodies immobilized on the membrane are formed in the analytic zone of the test strip. The antigen content is semiquantitatively evaluated visually by counting the number of visible colored bands in the analytic zone. By the example of the human chorionic gonadotropin hormone, the possibility of noninstrumental semiquantitative determination of the antigen in the range from 50 to 10000 mU/mL for 5–10 min was shown in five concentration ranges: under 100, 100–300, 300–500, 500–800, and above 800 mU/mL. The developed semiquantitative express method allows visually determining the concentration range of human chorionic gonadotropin in the urine by the number of colored bands for diagnosing pregnancy and its course.

In this study, new types of nanogold labels for lateral flow immunoassay of model antigen procalcitonin based on photometric and surface-enhanced Raman scattering methods of detection were obtained. The linear range of procalcitonin determination applying gold nanoflowers as a label was between 0.5 and 10 ng/mL. The limit of photometric detection was achieved at 0.1 ng/mL that was five times lower than the sensitivity of traditional lateral flow immunoassay with spherical gold nanoparticles as a label. In addition, the conjugate of polyclonal antibodies against procalcitonin with spherical gold nanoparticles labeled with 4-mercaptobenzoic acid as Raman reporter molecule was prepared and used as an immunoprobe in lateral flow immunoassay based on surface-enhanced Raman spectroscopy to improve assay sensitivity.

New sampling format of whole cows’ milk in strip-dried form was proposed. Few methodological issues of whole milk progesterone ELISA using samples dried on a membrane carrier in a form of strip were investigated and optimized: width of a strip, shape of punched/cut-off part of membrane, sample application method. It was shown that distribution of the hormone along narrow strip was even except the initial part of a strip (the first 0.5 × 0.5cm piece) where recovered concentration of progesterone was higher. Storage stability of progesterone in strip-dried whole cows’ milk samples at 4°C, ambient temperature, 37°C and 60°C was investigated. Rising of the detected progesterone concentration over storage period at elevated temperatures was observed predominantly in milk samples with low hormone concentration (from non-pregnant cows). Strip-dried whole milk samples can be used for collection, transportation, storage and ELISA analysis of progesterone level which is correlated with reproductive status of cows.

The modification of hydrophilic fiberglass and cellulose membrane materials with zinc oxide has been investigated. The surfaces of zinc oxide nanoparticles was hydrophilized by modification with 3-amino-propyltriethoxysilane. It was shown using methods of Fourier transform IR-spectroscopy and scanning electron microscopy that coating with hydrophilized zinc oxide nanoparticles did not change the structure of glass fibers and membrane material, while the treatment of cellulose-based membrane materials caused irreversible changes in the structure of fibers and the material as a whole.

A simple method for the rapid determination of prostate-specific antigen (PSA) in serum is reported using a lateral flow immunoassay with gold nanoparticles as the label. The method uses the intensity of colored test lines to determine PSA from 0.3 to 30ng/mL. The limit of detection was 0.3ng/mL and the coefficient of variation was less than 10%. The analysis time was approximately 20min. The novel method showed good correlation with enzyme linked immunosorbent assay (ELISA) measurements of prostate-specific antigen concentration in human serum with a linear regression coefficient of 0.985. The developed system was stable for at least 12 months when stored from +4 to 30 degrees C and has potential application for clinical practice.

The detection of antibodies to bovine infectious diseases (enzootic bovine leucosis, viral diarrhea, and infectious rhinotracheitis) is performed by ELISA in dry whole blood samples obtained on porous membranes using a new sampling format. Several commercial test systems are used to diagnose the infections. The obtained data are compared to the results of the analysis of liquid samples of blood serum. The results of the analysis of dried and liquid samples are in full concordance. The new sampling format makes it possible to avoid using a cold chain for the transportation of samples to diagnostic laboratories and to simplify the storage of these samples.

PCR and ELISA methods for the detection of bovine leucosis in dried blood spots on porous membranes were compared. Dry samples were analyzed through real-time PCR, using several diagnostic test systems. Nineteen and 20 samples were identified as positive by PCR and ELISA, respectively. Fourteen of these samples were identified as positive by both methods. When using PCR and ELISA, 26 samples were identified as positive for leucosis, which amounted to 47% of the total number of tested samples. The results of the analysis of dried and native samples were in good agreement. The obtained results showed that whole blood sampling in the form of dried spots applied on membrane can be used as a convenient and reliable way to obtain dry samples of biological fluids with the purpose of screening herds for infectious diseases, in particular for bovine leucosis.