**5. Proper interaction for LFIA**

Because LFIA is a fast analysis, all the processes that should be performed during the time of reagents' movement along the test strip and proper conditions for the interaction of these reagents are necessary (**Figure 7**).-


These general requirements remain little studied. Studies of the localization of reagents and immune complexes in a 3D membrane structure are limited [52, 53]. A significant variation in reaction media causes problems with mobility and nonspecific sorption of reagents on commercial membranes, the structure and coating of which are established by manufacturers. The developer can only compare several membranes and select reagents that affect the release of dried components and the speed of the flow. An example of such recommendations is provided by Lee etal. [54]. The contribution of fast nonspecific processes of formation of the so-called "protein corona" on the surface of gold nanoparticles to the effectiveness of immune interactions in LFIA is described in a recent paper by de Plug etal. [55]. Choi etal. [56] characterized the effects of temperature and humidity on the analytical characteristics of test systems and somewhat unexpectedly found that the transition to room temperature, conditioned by the requirements of point-of-care diagnostics, may be accompanied by a deterioration in sensitivity. In their work, the analysis at 37–40°C and relative humidity beyond 60% was three times more sensitive. Posthuma-Trumpie etal. [57] focused on the effects of the composition of solutions used in the manufacture of test systems on the analysis parameters. Interesting opportunities for further development are provided by the use of so-called nanomotors for enhanced reagent mixing, which has so far been described only for other types of immunoassays [58, 59].-

**Figure 7.** Main research and development tasks to obtain proper interaction for LFIA.-

More accessible tools are the choice of concentrations of reagents applied to the test strip and their locations. By varying these parameters, it is possible to provide extremely sensitive detection or to select the threshold of discrimination between positive and negative samples (cut-off level) that meets the regulatory requirements for the maximum permissible level of contamination. A number of works have been published with analyses of the individual effects of these parameters on the analytical characteristics [60, 61] and with the application of multiparametric optimization procedures [62]. Hsieh etal. [52] described a general scheme for the consideration of various factors in the course of LFIA optimization.-

In Zvereva etal. [63], the possibility to change the cut-off level by varying the composition of the hapten-protein and the antibody-(gold nanoparticles) conjugates is considered. Using an example of competitive LFIA of chloramphenicol, it was shown that by reducing the load of immunoreagents on carriers, it was possible to shift the detection limit by two orders of magnitude. For sandwich analysis, Liu etal. [64] showed theoretically and experimentally the optimality of the antibody: the nanoparticle ratio was equal to 30:1, but the universality of these recommendations requires further study.-

Fu etal. proposed the use of a two-dimensional paper network to control the sequence of interactions in LFIA and, using the example of choriogonadotropin, showed the gain achieved in sensitivity [65]. Similar problems were solved in Rivas etal. [66] using wax-printed pillars as delay barriers (three-fold gain for human IgG detection) and Choi etal. [67] by incorporating agarose into the test strip to achieve flow control (10-fold gain for detecting dengue viral RNA). A sponge shunt was applied by Tang etal. [68] to reduce the fluid flow rate during LFIA (10-fold signal enhancement in nucleic acid testing of Hepatitis B virus). Liu etal. [69] considered the use of a pencil made from polyethylene glycols for the application of reagents to control the rate of their subsequent release. Shin etal. [70] developed a rotary device for this purpose, the rotation of which makes it possible to initiate a reaction and then sequentially introduce into the system the necessary reagents. The volume of reagents introduced into the system during analysis can be controlled by the vertical flow immunoassay method proposed by Oh etal. [71] and successfully implemented by them for the detection of C-reactive protein. For the same antigen, Rey etal. [72] described an approach to managing the kinetics of interactions that allowed exclusion of the so-called hook effect (falsely low results for very high concentrations of the analyte). The existing variety of approaches to controlling the order of interaction of reagents in test systems is summarized in Jeong etal. [73].-

The position of the binding zone influences the degree of equilibrium reached for the reactions occurring during the flow of reactants along the test strip. Moving these zones along the test strip, we can adjust the assay sensitivity. Theoretical aspects of this approach were considered by Ragavendar etal. [74]. However, despite successful overlapping of monotests in multitests with a sequential arrangement of binding zones [75, 76], general practical recommendations for ensuring a highly sensitive detection of all analytes have not yet been formulated.-

Because synchronous movement in the flow of antigen, antibody, and immune complex molecules is difficult to provide, an alternative is to start the analysis with a quick (several minutes)preincubation of the analyte molecules in the sample with the free or labeled antibodies that- are specific to analyte. A number of commercial systems operate on this principle, such as testsfor antibiotic control in food produced by Bioo Scientific, United States, and Nankai Biotech,-China. Developing this idea, it is possible to implement universal test strips without compounds specific for a concrete analyte. The combination of such test strips with specific reagentsadded during the incubation stage with the sample allows adaptation of the consumption of- test strips to the tasks being solved. Such strips are manufactured by D-r Fuke, Germany, for- the detection of immunoglobulin E against various allergens: a complex of immobilized streptavidin, a biotinylated allergen from a preincubation mixture, specific immunoglobulins E,and colloidal gold-labeled anti-species antibodies is detected in the analytic zone of these tests.-

The problem of the polyvalence of antibody-nanoparticle conjugates in competitive LFIA noted in the previous section can be solved by replacing the conjugate of analyte-specific antibodies with gold nanoparticles by a combination of native specific antibodies and labels conjugated with anti-species antibodies. It gives possibility to vary the content of antigen-binding sites and the marker independently and therefore combine the high-sensitivity of competitive immunodetection (requiring a low content of specific antibodies) and the intensity of the detected signal (achieved with a high label content). This principle was implemented in our developments in the immunodetection of mycotoxins and demonstrated gains in sensitivity from one to three orders of magnitude [50, 77, 78].-

Note that the implementation of competitive analysis in LFIA involves another problem. Visual out-of-laboratory diagnostics makes it possible to distinguish only assay results consisting of the presence or absence of a colored line in the analytical zone. For a visible disappearance of color, the sample must contain a sufficient number of analyte molecules to block all binding sites for labeled specific antibodies (**Figure 8**). In this respect, analysis formats with a direct dependence of the detected signal on the analyte content are preferred. For these

**Figure 8.** Limitations of competitive immunoassay and one of the ways to overcome them.-

formats already small concentrations of the analyte ensure the coloration of the analytical zone in contrast to the absence of color in the absence of the analyte (see **Figure 8**).-

However, the implementation of such an analysis for low molecular monovalent antigens- is not an easy task. Its solutions for various types of immunoassay are summarized in the- reviews of Fan and He [79] and Liu etal. [80]. Unfortunately, many of these approaches, such- as idiometric assay [81] and immunoassay using anti-metatype antibodies [82] require the- production of antibodies not simply against the target analyte but against more complex antigenic structures, which limits their widespread use. A more universal idea is to use quenching- of fluorescence caused approaching between donor and acceptor in the binding zone of the- test strip. Such pairs can be two kinds of nanoparticles attached to different immunoreagents.- Thus, Shi etal. [83] successfully used for this purpose quantum dots and gold nanoparticles- in the analysis of ractopamine, Anfossi etal. [84]—quantum dots and gold or silver nanoparticles in the analysis of fumonisin, and Jiang etal. [85]—ruthenium-doped silicon nanoparticles- and silver nanoparticles in the analysis of ochratoxin A.-Another perspective approach is open- sandwich immunoassay (OSI). The given assay is based on the association of the separated- VH and VL chains of the antibody and reinforcement of this association after addition of the- target antigen [86]. This approach with the use of so named Quenchbodies is implemented in- different versions, mainly with fluorescent detection [87, 88], and it seems promising for LFIA.-

In our works, two types of immunoassay for low molecular compounds with direct analytesignal dependence are described. They do not require special reagents. In Urusov etal. [89],

**Figure 9.** Characteristics of labels that determine their applicability and competitive potential in LFIA.-

an assay was described in which labeled antibodies in the absence of the antigen in the sample completely bind in the first zone to the immobilized analyte. The appearance of the analyte in the sample blocks some of the antigen-binding sites of the antibodies and allows them- to reach the second binding zone on the test strip, ensuring the appearance of staining (see- **Figure 9**). For the case of deoxynivalenol detection, the proposed approach is 60 times more- sensitive than the traditional LFIA.-In Berlina etal. [90], an analysis of the food colorant Sudan- was described based on the use of two conjugates of gold nanoparticles with (i) antibodies- specific to Sudan and (ii) Sudan-ovalbumin conjugates. In the absence of Sudan, the conjugated Sudan-ovalbumin was coated with antibodies on the surface of the gold nanoparticle.- So the interaction with the anti-mouse IgG in the test area is prevented. The added Sudan- displaced the Sudan-ovalbumin causing the binding of labeled anti-Sudan antibodies in the- test area and the appearance of coloration.-
