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The work considers a combination of three enhancing approaches for immunochromatographic assay (ICA) and the integration of their impacts into changes of the limit of detection (LOD). Human fatty acid binding protein (FABP), an early biomarker of acute myocardial infarction, was the target analyte. Starting from the common ICA protocol with an LOD equal to 11.2 ng/mL, three approaches were realized: (1) replacement of spherical gold nanoparticles with gold nanoflowers having a branched surface (20-fold lowering the LOD); (2) enhanced labeling of immune complexes via nanoparticle aggregates (15-fold lowering); (3) in-situ growth of bound nanoparticles by reduction of gold salts (3-fold lowering). Single and combined implementations of these approaches have been studied. It has been shown that the LOD decrease for combined approaches is close to the multiplied contribution of each of them. The final LOD for FABP was 0.05 ng/mL, which is 220 times lower than the LOD for the common ICA protocol. The efficiency of the enhanced ICA with three combined approaches was confirmed by testing human serum samples for FABP presence and content. The development presents a new efficient technique for rapid sensitive detection of FABP for medical diagnostics. Moreover, the demonstrated multiple enhancements could be applied for various demanded analytes.

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Many studies have found that gold nanoparticles with branched surfaces (nanoflowers) are markers for immunosensors that provide higher sensitivity gains than the commonly used spherical gold nanoparticles. Although the analytical characteristics of nanoparticle-using systems vary significantly depending on their size and shape, the question of choosing the best gold nanoflowers remains open. This work presents a comparative study of a panel of 33 preparations of gold nanoflowers formed by varying several parameters: the size of spherical nanoparticles-nuclei, the concentrations of nuclei, and tetrachloroauric acid during growth. The sizes of the resulting particles, their sorption capacity under antibody immobilization, mobility along membranes for lateral flow assays, and the effects of these parameters on the limits of detection of lateral flow immunoassay are characterized. The optimality of preparations obtained by growing a 0.2% v/v solution of nuclei with a diameter of 10 or 20 nm with tetrachloroauric acid at a concentration of 0.12 mM was shown. With their use, lateral flow immune tests were developed to determine markers of acute myocardial infarction-fatty acids binding protein and troponins I and T. The use of gold nanoflowers obtained under the proposed protocols led to significant gains in the limits of detection-3 to 10 times under visual detection and over 100 times under instrumental detection-compared to spherical gold nanoparticles. The significant increase under instrumental detection is due to the label's low nonspecific binding.

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Highly sensitive detection of cardiac troponins I and T (cTnI and cTnT) was completed by immunochromatography with double amplification, through the binding of functionalized gold nanoparticles (GNPs). The robust nature of the approach, based on the formation of nanoparticle networks through the biotin-streptavidin interaction, was confirmed; the choice of the best assay parameters for maximal increase in ICA sensitivity was demonstrated. A bifunctional conjugate of GNPs with biotinylated specific IgG and two auxiliary conjugates, GNP-biotin and GNP-streptavidin, form three-component aggregates in the analytical zone of the test strip. The inclusion of abundant gold labels in the resulting immune complex leads to an amplified colorimetric signal. The limits of detection (LoDs) of cTnI and cTnT were 0.9 and 0.4 ng mL-1, respectively, which is 3 times lower than the LoDs of more commonly used systems. Visual LoDs were 10-fold lower in concentration. The enhancement has been realized both in single and double assay formats; analysis of cTnI and cTnT presented the same characteristics.

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Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of fluorophores, the detection of which the biosample matrix does not influence; thus, their choice simplifies and accelerates the preparation of samples. For decades, these possibilities were successfully applied in fluorescence polarization immunoassays based on differences in the polarization of fluorophore emissions excited by plane-polarized light, whether in a free state or as part of an immune complex. However, the results of recent studies demonstrate the efficacy of fluorescence polarization as a detected signal in many bioanalytical methods. This review summarizes and comparatively characterizes these developments. It considers the integration of fluorescence polarization with the use of alternative receptor molecules and various fluorophores; different schemes for the formation of detectable complexes and the amplification of the signals generated by them. New techniques for the detection of metal ions, nucleic acids, and enzymatic reactions based on fluorescence polarization are also considered.

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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.

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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.

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