**6. Proper response for LFIA**

The response of the immunochromatographic system is the recorded signal of the label (its color or other parameters), which reflects the formation of a specific immune complex and allows for highly sensitive detection of the target analyte. Therefore, the question of proper response for LFIA is first and foremost a question of choosing a label.-

The variety of molecular or colloidal labels that can be used in LFIA is extremely large [91, 92]. According to Goryacheva etal. [92], compounds such as gold nanoparticles of various shapes and sizes, carbon nanoparticles, selenium nanoparticles, iron oxide nanoparticles, fluorescent dyes, fluorescent dye-doped nanoparticles, quantum dots, infrared emitters, up-converting emitters, nanoparticles with long-lived emission, liposomes, and enzymes may be used for this purpose. There are many articles that demonstrate the advantages of a new marker on the example of the detection of one randomly chosen analyte. However, the question of correct comparison of different labels remains open. Indeed, the differences between test systems depend not only on the label but also on the affinity of the antibodies, the regimen of intermolecular interaction, and the correctness of the choice of reactant content. Therefore, the gain achieved for one analyte does not necessarily persist after the transition to another analyte.-

In this situation, it is justified to have "passports" of analytical labels, which are determined by their own properties and can be taken into account when implementing various analytical systems. The proposed list of such parameters is summarized in **Figure 9**.-

Note that along with single-valued quantitative parameters reflecting the physical properties of a label, a number of qualitative parameters must be taken into account. Unfortunately, to date, researchers do not have universally recognized quantitative characteristics of existing labels and rules for a priori evaluation of proposed labels. Therefore, when deciding on responses (**Figure 10**), we are forced to follow the data of disparate comparisons of labels in different experimental developments.-

Even within the framework of the use of gold nanoparticles, the developer has the opportunity to choose preparations of different sizes and shapes. The well-known recommendation [93] on the preferable use of spherical gold nanoparticles with an average diameter of 30–40nm is confirmed by published experimental comparisons [49]. Serebrennikova etal. [94] showed the advantages of high-branched gold nanoparticles ("nanoflowers") as optical markers—a fivefold decrease in the detection limit of procalcitonin. These patterns were confirmed by Xu etal. [95], and the preferable use of long-tip (13–15nm) nanoflowers was stated. Ji etal. [96], using gold nanoflowers, reached the detection limit of aflatoxin B1, equal to 0.32pg./ml.-

Optical markers for immunochromatography of different chemical natures are compared in a number of works. The possibilities of using carbon nanoparticles described in Van Amerongen etal. [97, 98] and Liu etal. [99], using the example of salbutamol detection, also showed the advantages of colloidal carbon compared to colloidal gold and nanogold-polyanilinenanogold microspheres. For ractopamine detection, Hu etal. [100] showed the advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold. Effective integration of palladium nanoparticles and horseradish peroxidase with a 10-fold gain in sensitivity as compared to colloidal gold in the detection of *Listeria monocytogenes* was described by Tominaga [101]. The possibilities of high-sensitivity LFIA using graphene oxide and carboxylated graphene oxide as optical markers were shown by Yu etal. [102].-

Of great interest are fluorescent markers. In many respects, this is due to the fact that with the correct choice of the wavelengths of excitation and emission, it is possible, by increasing the intensity of the exciting light, to proportionally increase the response in the practical absence (in contrast to the colorimetry) of the nonspecific signal. The gain in sensitivity achieved in

**Figure 10.** Main research and development tasks to obtain proper responses for LFIA.-

this case is one or two orders of magnitude [103, 104]. The use of fluorescent markers in LFIA is summarized in the reviews of Pyo and Yoo [105] and Gong etal. [106]. A comparison of the analytical capabilities of quantum dot nanobeads, large-sized (50–600nm) particles with impregnated quantum dots was given in Duan etal. [107].-

Additional capabilities of high-sensitivity analysis are achieved by the registration of energy transfer with the spatial convergence of two labels—fluorescence resonance energy transfer (FRET). Systems using fluorescein isothiocyanate and gold nanoparticles were developed by Wang etal. for the detection of cancer embryonic antigens [108]. Other variants of fluorescent LFIA were also described, for example, registration of background fluorescence quenching in Chen etal. [109], silver nanoparticle-based fluorescence quenching in Jiang etal. [85], and quenching of the fluorescence of quantum dots by gold and silver nanoparticles in Anfossi etal. [84]. (See also Section 5 with their consideration as examples of competitive immunoassays with a direct dependence of the detected signal on the analyte content.)-

 Extremely promising is the use of surface-enhanced Raman spectroscopy (SERS) for detection of- optical labels. SERS signals are based on the increase of optical absorption for reporter molecules- by orders of magnitude after their immobilization on the surface of nanoparticles. The possibility of such highly sensitive analyses is demonstrated in the works of Sanchez-Purra etal. [110],- Fu etal. [111], and Marks etal. [112]. Clarke etal. [113] described the combination of SERS registration with rapid vertical flow technology as an additional means of increasing sensitivity. In- Maneeprakorn etal. [114], SERS detection with 4-aminothiophenol as a signal reporter lowered- the detection limit by 300 times compared to traditional LFIA.-In Cho etal. [115], the transition to- SERS based on silver-intensifying gold nanoparticles led to a 1000-fold decrease in the detection- limit. Blanco-Covian etal. [116] proposed the use of a combination of Au @ Ag core-shell nanoparticles and rhodamine B isothiocyanate in LFIA, which allowed them to perform highly sensitivedetection of pneumolysin with a detection limit of 1pg/ml, recording the surface-enhanced resonance Raman scattering (SERRS).-

Note that optical recording methods allow us to evaluate only labels that are in the upper- layers of the test strip and are not shielded by membrane fibers. The loss of the optical- signal depends on the properties of the material but is usually estimated [93] as about one- order of magnitude. In this regard, the work of Jacinto etal. [117] is extremely interesting. They offer an electromagnetic relocation of reporter particles for amplifying an optical signal and describing the fourfold reduction in the detection limit of human chorionic- gonadotropin.-

This restriction is excluded for analytical methods in which registration of a label is based on other physical principles. Thus, Wang etal. [118] developed the Thermal Contrast Amplification Reader for the registration of gold nanoparticles, which, for systems of influenza and malaria diagnostics and detection of *Clostridium difficile*, showed eight times lower detection limits as compared to an optical reader. Zao etal. [119] improved the detection limit by two orders of magnitude for photoacoustic analysis compared to colorimetric measurements. The magnetic properties of the nanodispersed label in LFIA were recorded by Barnett- etal. [120], Chen etal. [121], Lago-Cachon etal. [122], and other authors. Several variants of LFIA with electrochemical detection are presented in the literature, the most recent of which (the work of Zhao etal. [123]) is based on the use of a serial glucometer as a registrar. Just recently, Lin etal. proposed LFIA of myoglobin based on pressure measurement for oxygen generated by platinum nanolabels from hydrogen peroxide [124].-

The capabilities of high-sensitivity detection in LFIA are not limited to the choice of a label. Additional reserves provide **amplification of the recorded signal**, which can be provided by-


The existing variety of developments in this area is summarized in a review of Shan etal. [125]. The systems that implement the aggregation of several types of functionalized nanoparticles- cause particular interest. Such approaches are described, for example, by Choi etal. [126] with a 100-fold gain in sensitivity for the detection of troponin I using two kinds of gold- nanoparticles; by Razo etal. [20] with the generation of an optical signal by complexes of iron- oxide nanoparticles (also used as a concentrating agent) and gold nanoparticles with a 32-fold- decrease in the detection limit of potato virus X; by Taranova etal. [127] with a 30-fold gain- in the analysis of procalcitonin due to biotin-streptavidin aggregation of gold nanoparticles;- by Shi etal. [128] with complexation of gold nanoparticles of two sizes in the analysis of imidaclothiz; by Zhong etal. [129] with the formation of two layers of antibody conjugates with- gold nanoparticles in the detection of melamine; and by Shen etal. [130] with aggregation ofgold nanoparticles using polyamidoamine dendrimer, which lowered the detection limit of- rabbit immunoglobulin G 20 times.-

The growth of the size of gold nanoparticles with the help of the catalyzed reaction of their surface between HAuCl4and NH2 OH was examined by Bu etal. [131] as a means of amplification for LFIA.-The layered build-up of gold nanoparticles was described by Li etal. [132]. Anfossi etal. [133] and Panferov etal. [134, 135] considered the possibilities of silver enhancement (restoration of the silver salt on the surface of a gold nanoparticle with an increase in its size) in LFIA.-In a study by Rodriguez etal. [136], the optimal regimes of silver and gold enhancements were determined to enhance the signal from the gold nanoparticles. Enzymatic amplification using alkaline phosphatase was studied by Panferov etal. [137] for LFIA of potato virus X and by Kim etal. [138] for LFIA of C-reactive protein. A feature of the latest development was the use of a water-swellable polymer for the accumulation of a colored product. An original polymerization-based amplification approach for enhancing staining was described by Lathwal and Sikes [139].-

The basic requirement for amplification approaches is the maintenance of low laboriousness of analysis. Variants using additional reagents, although considered in development, should be finally transformed into devices of dry chemistry, in which all components of the test strip are applied to its membranes.-
