**7. Proper output for LFIA**

The generation of a signal reflecting the formation of immune complexes during LFIA is not the final stage of the analysis. The analysis is only completed when a diagnostically meaningful conclusion is made on the basis of this signal.-

Effective use of LFIA is possible only when it is combined with modern means for documenting, storing, and processing information. In the absence of these tools, the advantages of rapid and high-performance nonlaboratory diagnostics are lost because of time-consuming processing and description of test results. Of fundamental importance is the transition from a subjective yes-no evaluation of results to automatic quantitative registration and the formation of databases that integrate the results of mass screenings or information on the dynamics of the state of patients (objects). Such systems will allow rapid collection of various indicators "at the time of request," contributing to an accurate diagnosis. Taking into account the foregoing, **Figure 11** summarizes the requirements for proper output in LFIA.-

In some cases, the developer does not need to achieve maximum sensitivity but to fix the threshold that separates the positive and negative results in accordance with the regulatory requirements for MRLs. This allows the composition of conjugates used in the analysis discussed above to be varied [63]. A qualitative "yes-no" analysis can be transformed into a semiquantitative one with a change in the number of colored bands corresponding to several threshold levels. To do this, depletion of the conjugate can be used when interacting

**Figure 11.** Main research and development tasks to obtain proper output for LFIA.-

**Figure 12.** Comparison of traditional and digital photometry as a means for registration and processing of immunochromatographic data.-

with several consecutive identical binding zones. Additional opportunities arise when using antibodies with different affinities, varying the surface density of the reagents applied in the binding zones and the distance between these zones and the beginning of the test strip. An example of an appropriate development with three thresholds of potato X virus concentrations corresponding to the degree of plant infection was described by Panferov etal. [140].-

Initially, attempts were made to create detectors for membrane tests that recorded the total intensity of the staining (brightness of the reflected light) in certain sections of the test strip using a row of light-emitting diodes and individual systems of signal transformation for each diode. However, such detectors were extremely cumbersome. Blatt etal. [141] proposed a device made from 28 photosensitive sensors located along the test strip. Nowadays, the dominant means of detecting the results of LFIA, allowing a full-color image of the test strip to be received, are digital cameras. This technology is based on the use of inexpensive portable detectors or household recording devices—such as a mobile phone camera [142]. Serially produced cameras record images with a resolution of up to 2400 dpi, which corresponds to the size of an individually characterized section of less than 1μm2 . **Figure 12** summarizes the advantages of digital photometry in LFIA.-

Trends in the transformation of LFIA from the visual to the instrumental method are summarized by Cheung etal. [5]. Reviews by Quesada-Gonzalez and Merkoci [143] and Zarei [144] present the current state of analytical technologies based on the use of mobile phones/smartphones. At the same time, a significant number of manufacturers of test systems offer portable detectors that are adapted to work with their own products [8]. Of the original solutions, mention should be made of Feng etal. [145], in which the registration tool for LFIA was Google Glasses. In recent years, a number of companies have introduced cloud technologies into practice, where external servers receive data about testing results via standard communication devices and store and process this information. Thus, since 2017, Abbott has proposed a set of tools named i-STAT Alinity for distant diagnostics. Special cartridges allow 14 parameters of blood composition by bio- and immuno-chemical techniques to be controlled.-

An extremely important means of increasing informativeness, although not related to an- increase in sensitivity, is to conduct a multiplex analysis—that is, detection of the presence- and level of several analytes using a single test strip. Data on the control of several analytes- can be discriminated in space (by the position of binding zones) or by signals (by using- different labels). Quantum dots are an effective tool for multi-analysis with different signals. The use of conjugated quantum dots with different spectral characteristics allows one- to perform highly sensitive diagnostics with simultaneous detection of, for example, three- antibiotics ("traffic light" in Taranova etal. [146]) or four mycotoxins ("rainbow" in Foubert- etal. [147]).-

Because the number of binding zones that can be sequentially located on one test strip while preserving the rapidity of the analysis and the reliability of the information obtained for each analyte is limited (usually no more than five zones), the transition to "two-dimensional immunochromatography" is promising—see **Figure 13**. This approach, combining the advantages of immunochromatographic tests and immunochips,is based on the formation of an ordered two-dimensional array of points with immunoreagents of different specificity on

**Figure 13.** Concept of 2-D immunochromatography.-

the membrane of a test strip. In such systems, interaction occurs in several dozens of binding zones. Due to this, the 2D immunochromatography increases the information content of LFIA results and reduces the consumption of reagents and materials for one analysis.-

Examples of test systems based on the principle of "two-dimensional immunochromatography" are presented in the works of Taranova etal. [104] on the detection of drugs and Safenkova etal. [148] on the detection of phytopathogens. General approaches to multizonal LFIA were discussed in Hu etal. [149], and the current state of the development of multiplex immunoassays was discussed in Li etal. [150].-
