**3. Proper sample for LFIA**

Some types of liquid samples, characterized by the LFIA method, do not require sample preparation: urine, blood serum, natural and drinking water, milk and juices. Their analysis can be initiated by contacting the test strip with the sample as is. To accelerate the movement of the fluid (blood serum and milk), the sample can be diluted immediately before analysis [9]. However, in most cases, the analysis should be preceded by sample preparation.-

The main difficulty of sample preparation is the need for a short period to destroy the matrix structures that interfere with the analyte molecules contained in it to interact with antibodies. Actions that separate matrix components that interfere with analysis, or to destroy these components, are also reasonable. Such complex types of matrices may be tested as tissues of organisms, food and agricultural products, soil, and so on. Sample preparation is extremely important to easily detect the target compounds in these matrices.-

The requirements for sample preparation were studied in detail with respect to other analytical methods—liquid and gas chromatography, enzyme immunoassay, and so on. However, the accumulated research results cannot be transferred to LFIA without further development. The main advantage of LFIA—rapidity—cannot be lost because of the long (lasting several hours) extractions recommendedin many chromatographic techniques. Work with samples cannot begin from complex procedures that require expensive equipment.-

An additional feature of sample preparation for LFIA is that many analytes are extracted efficiently only with organic solvents and water-organic mixtures, but not with aqueous-salt solutions. (Such situations are usually associated with the hydrophobicity of the compounds and their surroundings in the samples.) However, these solvents inactivate antibodies; it means that the extract cannot be directly used as is as a sample for LFIA.-As a result, the extracts are either significantly diluted (which is accompanied by a loss in sensitivity), or by means of additional steps, the analyte is transferred to another medium.-

The complexity described above determines the tasks that should be solved for effective sample preparation—see their summation in **Figure 3**. In **Figure 3** and the following ones, we depict


 With respect to proper samples, the success of the developments offered directly by test- system manufacturers should be noted. Alexeter Technologies (United States) uses special- adhesives placed at the beginning of the test strip, which allow one to collect target molecules of the analyte from a large surface area by simple contact. In many cases, portable- homogenizers and low-speed centrifuges are proposed for completing the analytical laboratory. In the case of the 4MycoSensor test systems (Unisensor, Belgium), mycotoxins are- extracted from the ground grain in a special Mycobuffer on a shaker for 3min (5min for- corn). Similar solutions are offered by other manufacturers. A special aqueous two-phase- system for the concentration of protein analytes, containing polyethylene glycol, potassium- phosphate, and phosphate-buffered saline, was used by Chiu etal. [10]. With its help, a 100 fold reduction in the detection limit was achieved. Concentration of samples combined with- dialysis was used by Tang etal. [11] on the examples of myoglobin detection (fourfold signal- growth) and nucleic acid of HIV (10-fold growth). Mosley etal. [12], using the examples of- *Chlamydia trachomatis* and human immunoglobulin M analyses, showed that the formation- of an aqueous two-phase system on the test strip by applying a PEG-potassium phosphate- and UCON-50-HB-5100-potassium phosphate obtained a 10-fold reduction in detection limits. In Jue etal. [13], micellar two-phase systems were used for this purpose, which reduced- the detection limit of bacteriophage M13 by a factor of 10. An original solution based onconcentrating the analytes in an electric field was proposed by Kim etal. [14]. Using a conventional 9-V battery and commercial tests for choriogonadotropin, they acquired a 25-fold- concentration of the target compound.-

**Figure 3.** Main research and development tasks to obtain proper samples for LFIA.-

**Figure 4.** Advantages of magnetic immunosorbents application in LFIA.-

Efficient approaches for sample preparation are pseudo-homogeneous analytical techniques, where a dispersed carrier with immobilized receptor moleculesis added to a large volume of tested samples. This carrier quickly and efficiently, without diffusion restrictions, captures the analyte from the entire volume of the sample, and then the carrier is separated from the solution rapidly. Note that when the separated carrier is then redissolved in a small volume, the analyte is not only concentrated but also cleared from the organic solvent, thus excluding the influence of this solvent on LFIA.-Antibodies, immobilized on a carrier, are often more stable to the denaturing influence of organic substances than free antibodies. According to the data of Urusov etal. [15], when working with magnetic immunosorbents, the content of methanol in the test sample can be increased from 10 to 30%.-

The use of particles of iron oxide and other carriers with magnetic properties is extremely promising for immunochromatography because of the simple and rapid separation of the carrier by- contact with a permanent magnet. The principle of such an analysis is shown in **Figure 4**, and- approaches to the production of magnetic immunosorbents are systematized in the review [16].-

Liu etal. [17] showed that the combination of magnetic concentration and immunochromatography yields a 25–50-fold gain in the detection limit of aflatoxin M1in milk compared to the- variants in which magnetic or gold nanoparticles are used as conventional labels. A 40-fold- gain in the detection limit was demonstrated by Lu etal. [18] upon the detection of *Listeria monocytogenes*. In Petrakova etal. [19], using the examples of zearalenone and T-2 toxin, the- authors showed that magnetic nanoparticles can be used as directly detectable optical markers.- Razo etal. [20] combined the use of magnetic immunosorbents to bind analytes, potato virus X,- and functionalized gold nanoparticles, which, thanks to the biotin-streptavidin reaction, provide- the formation of aggregates of two kinds of nanoparticles. This analysis was 32 times more sensitive than the nonenhanced one. As a whole, the described gains in sensitivity with the use of magnetic immunosorbents did not exceed two orders of magnitude. A greater concentration requires- a significant increase in the consumption of immunoreagents and/or time for binding the analyte.-

Concentration can also be achieved if LFIA is preceded by a stage with a transverse flow of large volumes of samples through a small volume of a membrane with antibodies or other binding reagents applied to it (immunofiltration). Such analyses usually complete the detection of binding results directly in the filtration zone [21, 22]. Note that the use of LFIA for control of toxicants in solid foods is associated with a certain restriction. To correctly determine the content of the unevenly distributed analyte, several samples of large volumes are selected from different parts of the tested object and combined for subsequent extraction [23, 24]. However, the small volume of liquid absorbed by the test strip allows only a small part of the analyte molecules present in the extract to be taken into account (even with magnetic concentration). Immunofiltration concentration will overcome this limitation and come close to obtaining the proper samples for highly sensitive analyses.-
