R. RUDOLF et al.: DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS 89–93 DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS RAZVOJ POSTOPKA RECIKLIRANJA HITRIH ANTIGENSKIH TESTOV Rebeka Rudolf 1,2* , Darja Feizpour 3 , @iga Jelen 1,4 , Peter Majeri~ 1,2 , Tilen [varc 1 , Matej Zadravec 1 , Timi Gomboc 1 , Aleksandra Kocijan 3 1 University of Maribor, Faculty of Mechanical Engineering, Maribor, Smetanova ul. 17, Slovenia 2 Zlatarna Celje d.o.o., Kersnikova ul. 19, Celje, Slovenia 3 Institute of Metals and Technology, Lepi pot 11, Ljubljana, Slovenia 4 Znanstveno in inovacijsko sredi{~e Pomurje, Lendavska ulica 28, 9000 Murska Sobota, Slovenia Prejem rokopisa – received: 2023-12-04; sprejem za objavo – accepted for publication: 2023-12-19 doi:10.17222/mit.2023.1063 The article presents the problem of rapid antigen tests when they become mass waste after use. Based on this, the hypothesis was made that rapid antigen tests can be recycled. Rapid antigen tests, which were used in the Covid-19 epidemic to quickly de- tect infections in the population or to confirm the presence of the Sars-Cov 2 virus in patients, were intended to limit the spread of the epidemic. To confirm the hypothesis of recycling for rapid antigen tests, the LFIA-REC ATP 150 project was prepared, which was selected for co-financing by the Norwegian Fund. Rapid antigen tests consist of a sample and conjugate pad, detectable or nitrocellulose membranes and absorbent pads and a plastic case. The function of the sample pad is to evenly absorb the sample (mucus, blood) and lead it to the conjugate pad with a steady flow. Gold nanoparticles (labels) are deposited on the conjugate pad. The key is that the gold nanoparticles are conju- gated with capture molecules capable of binding to potentially present antibodies or virus in the sample. The scope of the research problem thus required the inclusion of various characterization techniques that must be applied to the individual material in the rapid antigen test to subsequently develop an efficient recycling process for the rapid antigen tests. The result of the research presented in this paper represents a newly developed algorithm of characterization techniques, which includes a recommended description of the preparation of samples of key materials from rapid antigen tests. This algorithm suc- cessfully achieved the characterization of gold nanoparticles from rapid antigen tests. Based on the developed algorithm, the fi- nal part of the project will validate the recycling process of rapid antigen tests, so that they can be recycled, i.e. gold nanoparticles or plastic used in new products. The paper presents the algorithm of characterization techniques with a description of the preparation of material samples from rapid antigen tests. Keywords: rapid antigen tests, recycling, characterization, nanogold, plastic V prispevku je predstavljena problematika hitrih antigenskih testov, ko le-ti postanejo po uporabi masovni odpadek. Na osnovi tega je bila postavljena hipoteza, da je hitre antigenske teste mo`no reciklirati. Hitri antigenski testi, ki so se uporabljali v epidemiji Covid 19 za hitro odkrivanje oku`b v populaciji oziroma za potrditev prisotnosti virusa Sars-Cov 2 pri testirancih, so bili namenjeni za zamejitev {irjenja epidemije. Za potrditev hipoteze recikliranja hitrih antigenskih testov je bil pripravljen projekt LFIA-REC ATP 150, ki je bil izbran za sofinanciranje s strani Norve{kega fonda. Hitri antigenski testi so sestavljeni iz vzor~ne in konjugatne blazine, zaznavne oz. nitrocelulozne membrane in vpojne blazine ter iz plasti~nega ohi{ja. Funkcija vzor~ne blazine je, da enakomerno absorbira vzorec (sluz, kri) in ga z enakomernim tokom vodi do konjugatne blazine. Na konjugatni blazini so nane{eni zlati nanodelci (oznake). Klju~no je, da so zlati nanodelci konjugirani z lovilnimi molekulami, ki so se sposobne vezati na potencialno prisotna protitelesa ali virus v vzorcu. Podro~je raziskovalnega problema je tako zahtevalo vklju~itev razli~nih tehnik karakterizacije, ki jih je potrebno uporabiti za posamezen material v hitrem antigenskem testu, da bi lahko kasneje razvili u~inkovit postopek recikliranja hitrih antigenskih testov. Rezultat raziskave, ki je predstavljen v tem prispevku, predstavlja na nov razvit algoritem tehnik karakterizacije, kjer je vklju~en priporo~ljiv opis priprave vzorcev klju~nih materialov iz hitrih antigenskih testov. Ta algoritem je uspe{no dosegel karakterizacijo nanodelcev zlata iz hitrih antigenskih testov. Na osnovi razvitega algoritma bo v sklepnem delu projekta validiran postopek recikliranja hitrih antigenskih testov, tako, da bodo lahko reciklati t.j. nanodelci zlata oziroma plastika uporabljeni v novih proizvodih. Klju~ne besede: hitri antigenski testi, recikliranje, karakterizacija, nanozlato, plastika 1 INTRODUCTION The Recycling of Rapid Antigen Lateral Flow Immunoassay (LFIA) Tests (COVID-19) (LFIA-REC) is aimed at mitigating climate change and adapting to it. The main focus of the project is not financial profitabil- ity but also aligning with leading guidelines for sustain- able development. 1 The project contributes to the Devel- opment Strategy of Slovenia by 2030 by demonstrating pathways for the efficient recycling of rapid antigen LFIA tests, enabling the use of recyclables as secondary raw materials. The use of advanced technologies for the selective refinement of nanogold (AuNP) and plastic re- cycling is anticipated, supporting the central goal of the strategy, which is the quality of life for all through bal- anced development in economic, social, and environ- mental terms. 2 Materiali in tehnologije / Materials and technology 58 (2024) 1, 89–93 87 UDK 628.39:544.77.032.18 ISSN 1580-2949 Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 58(1)89(2024) *Corresponding author's e-mail: rebeka.rudolf@um.si (Rebeka Rudolf) The project is also in line with the 2030 Agenda for Sustainable Development, particularly Goal 12, which calls for sustainable production and consumption prac- tices by reducing waste associated with rapid antigen LFIA tests. 3 Reuse of recyclables and Au nanoparticles for creating new products is also planned. Simulta- neously, the project supports the European Green Deal 4 by advocating for a sustainable and green economy and promoting recycling and the circular economy in Euro- pean efforts. Additionally, the project aligns with the goals of the Paris Agreements in Articles 4, 6, 7, 9, and 10. 5 The End of the Federal COVID-19 public-health emergency in the USA was declared on May 11 th 2023, 6 and represents the end of the COVID-19 pandemic in the western world, and with it all the precautions related to it slowed down or completely stopped. This resulted in large quantities of unused rapid antigen tests in storage facilities that are due to expire, which are worth millions and need to be recycled 7 . The recycling of LFIA tests for COVID 19 involves an innovative approach to extract valuable AuNPs from the conjugate pads, contributing to sustainability and re- source optimization. In this process, the used LFIA test strips are collected, and the conjugate pads, which con- tain embedded gold nanoparticles, are carefully sepa- rated. The LFIA test strip consists of a number of porous pads and membranes, such as the sample pad, conjugate pad, filter pads, nitrocellulose chromatographic mem- brane, and an absorbent pad. 8 These pads vary in compo- sition, depending on the specifics of each LFIA test. However, the most common choices for them are cellu- lose fibres, polyester fibres, glass fibres and nitro- cellulose. The basis of each LFIA test is a trapping mole- cule or bioreceptor and a nanomaterial label. Typically, nanomaterials such as AuNPs, magnetic, polymer, silver, carbon and fluorescent nanoparticles, as well as quantum dots are most often used for this purpose, with AuNPs being by far the most common. 9 The test strip is encased in a polymer or paper cassette, which both protects the test strip and ensures good contact between the various pads and membranes. These are typically composed of polystyrene (PS) or acrylonitrile butadiene styrene (ABS) plastic. Both the AuNPs contained in the test strip and the plastic of the cassette casing represent the high- est recycling value in the whole LFIA test. However, to effectively recycle both the AuNPs and plastics it is nec- essary to separate them. The separation device that is being developed will use mechanical grinding to break apart the membranes saturated with AuNPs and plastic casings. The resulting grindings will be directed to a vibrating table, which will sort the heavier fraction (plastic) and the lighter fraction (membranes). Both fractions will be prepared for further processing, such as recovering AuNPs from the mem- branes, which is an important aspect of the rapid test re- cycling. Several methods were developed to recover gold nanomaterials in a laboratory setting, 10,11 while in this re- search, the recycling of AuNPs was included in an estab- lished industrial refining process for gold. This recycling initiative serves dual purposes: reduc- ing environmental impact by diverting LFIA test compo- nents from traditional waste streams and repurposing AuNPs for potential reuse in various applications. The recovered AuNPs, once purified, can be utilized in new LFIA test production or redirected to other fields, such as catalysis, sensing technologies, or nanomaterial-based applications. By incorporating recycling strategies into LFIA test disposal practices, the scientific community contributes to both environmental conservation and the sustainable utilization of precious materials, aligning with global efforts to promote circular economies and re- sponsible resource management. To ensure efficient recycling, the key is the separation of the individual materials and the complex components. Currently, suitable devices for this purpose are not avail- able, making close collaboration with industry and a swift response essential. Nanotechnology has opened a realm of possibilities in various scientific domains, and the development and characterization of nanogold nanoparticles stand as a crucial area of research. These minute entities, ranging from 1 to 100 nanometers in size, exhibit unique optical, electronic, and catalytic properties, making them highly versatile in fields such as medicine, electronics, and ma- terials science. Understanding the algorithm of charac- terizing nanogold nanoparticles is pivotal in harnessing their potential applications. The characterization of nanogold nanoparticles in- volves a comprehensive algorithm combining various techniques to elucidate their physical, chemical, and structural properties. Understanding these properties is crucial to tailoring the nanoparticles for specific applica- tions in fields like targeted drug delivery, imaging, catal- ysis, and sensor technology. 12–15 As research in nanotechnology advances, refining and expanding these characterization methods will continue to unveil the full potential of nanogold nanoparticles in various scientific and technological domains. In this paper the protocol and algorithm are estab- lished, ensuring accurate transmission electron micros- copy (TEM) and scanning TEM (STEM) characteriza- tion of recycled AuNP, while minimizing potential sample damage. 2 EXPERIMENTAL PART The mechanical separation of LFIA tests involves the opening of the cassette housing using free spinning ham- mers in a modified SM-300 cutting mill (Retsch GmbH, Germany). This breaks open the whole test, while keep- ing the cassette halves and LFIA test strips mostly intact. This was followed by vibration sifting on a cus- R. RUDOLF et al.: DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS 88 Materiali in tehnologije / Materials and technology 58 (2024) 1, 89–93 tom-made, linear-vibration sifter, which separates the cassette halves from the LFIA test strips. The chemical refining of gold requires melting the gold scrap to be melted with silver, with the final metal containing about one fourth of gold. This metal is granu- lated and put in heated nitric acid (HNO 3 )a t8 0° Ct o dissolve the silver and other metals from the gold. The silver-containing solution is filtered form the remaining gold, which is then dissolved with aqua regia (nitric and hydrochloric (HCl) acids in a volume ratio of 1:3) at 70 °C. Sodium metabisulphite is then used to precipitate the gold from the acid solution, obtaining pure gold. In an industrial setting, the used acid solution is then neu- tralized, and a flocculant is added, producing an organic sludge which still contains a small amount of gold metal and other metals, dissolved by the acids during the refin- ing process. This sludge is pumped inside a filter press, which removes the excess liquids and produces dry cakes of the sludge material, which can then be further refined to obtain the smaller amounts of gold within the cakes. A manual filter press model 500 × 500 with 30 inserted plates (EUROTecniche Srl, Mussolente, Italy) was ac- quired to facilitate this process in the project for the re- covery of gold from the rapid antigen tests. For recover- ing AuNPs from the rapid-antigen-test membranes, a procedure was proposed for using aqua regia to dissolve the gold from the organic components of the membranes. Fume hoods are required to capture any fumes or gases generated during this procedure, two of which were ac- quired as a part of the project (EUROTecniche N. 2 Moplen Hood, EUROTecniche Srl, Mussolente, Italy). The gold-containing membranes, obtained from rapid antigen test separation, are kept in the fume hoods and soaked in aqua regia for 2 hours to ensure total dissolu- tion of the gold in the acid. The produced acid is filtered from the rapid test membranes and can be used further with sodium metabisulphite to precipitate and obtain the gold metal content. The sodium metabisulphite reacts with the gold chloride in the acid solution, reducing the ionic gold to its metallic form. In the framework of the project, the gold-containing acid can be used as a solution for Ultrasonic Spray Py- rolysis (USP), for the production of new AuNPs. This re- fining procedure can then be considered as reusing the initial AuNPs from the membranes for the production of new, recycled AuNPs, which can be further used in lat- eral flow tests or other products. A schematic presentation of the recycling process for rapid antigen tests is shown in Figure 1. The remaining membranes from the gold recovery can be thermally processed in a furnace to destroy the or- ganic components and additionally retrieve any poten- tially remaining gold on the membranes. As part of the project, an ammonia dissociator (model P-ASP-0750, Millivolt GmbH, Donzdorf, Germany) was acquired for producing a mixture of hydrogen and nitrogen gases from ammonia, to facilitate the thermal processing in an industrial setting. The chemical analyses of the samples obtained after the recycling procedure for the rapid antigen tests were performed using inductively coupled plasma optical emission spectroscopy (ICP-OES, Agilent 5800 VDV). Electron microscopy, particularly transmission elec- tron microscopy (TEM) and scanning transmission elec- tron microscopy (STEM), offers high-resolution imaging of nanogold particles. These techniques help visualize the morphology, size, and dispersity of the particles. Ad- ditionally, selected-area electron diffraction (SAED) and fast Fourier transform (FFT) can provide insights into the crystalline nature of the nanoparticles. The electron-dif- fraction pattern provides information about the crystal- line structure of the nanoparticles and enables identifica- tion of the crystal lattice, aiding in understanding the composition and atomic arrangement of the nanogold particles. The sample preparation for TEM involved conven- tional TEM preparation and using the drop-casting method by which a portion of the sample was placed in a small centrifuge container of a few ml containing abso- lute ethanol or deionized water. Following this, a mixture of ethanol or deionized water and the sample was applied to the carbon formvar B film on the 200 mesh Cu TEM grid using a dropper. Subsequently, the sample dried overnight in a desiccator before the analysis. The exami- nation and analysis were conducted using a JEM-2100HR and ARM 200 CF (JEOL, Tokyo, Japan) TEM with an attached energy-dispersive X-ray spec- trometers (EDS) JED-2300T and solid-state detector (SSD) (JEOL, Tokyo, Japan), operating at 80 kV, 100 kV and 200 kV, under conditions that did not damage the samples. The analyses encompassed imaging, electron diffraction, and elemental composition assessment, with EDS conducted at standard acquisition settings: high res- olution, live time mode, a 200-second acquisition time, and a probe size of either 10 nm or 25 nm at ×100,000 magnification. 3 RESULTS The Au was successfully recovered from the samples before and after the recycling procedure of rapid antigen tests, which was confirmed using the ICP-OES tech- nique. The measured concentrations of Au are stated in Table 1. Table 1: Concentrations of Au after the recycling procedure of rapid antigen tests. Sample c Au (μg/mL) Grinded LFIA soaked in aqua regia 2.4 Au solution from the membrane pad soaking in aqua regia 39.6 Au solution after evaporation of excess aqua regia 247.7 Refined AuNP suspension after USP synthesis 17.0 AuNP suspension after rotary evaporation 197.5 R. RUDOLF et al.: DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS Materiali in tehnologije / Materials and technology 58 (2024) 1, 89–93 89 Further, after using ICP-OES to confirm the presence of Au in nanoparticles, the recycled AuNPs were charac- terized using TEM and STEM. During the TEM study of different samples for the recycling procedure of rapid an- tigen tests containing AuNPs a protocol and algorithm were established as follow: Transmission Electron Microscopy (TEM) Protocol for Polymer Fiber Samples Containing Gold Nano- particles Sample Preparation: Polymer Dis- solution: Dissolve the polymer fibers containing gold nanoparticles in a suitable solvent, such as distilled or deionized water or ethanol. En- sure complete dissolution for effective nanoparticle extraction. Centrifugation : Transfer a representative portion of the poly- mer solution to a centrifuge tube and spin it at an appropriate speed and duration to sepa- rate the gold nanoparticles from the polymer matrix. Resuspension: Re-disperse the extracted gold nanoparticles in the chosen solvent to form a homogeneous solution. TEM Grid Preparation: Apply a droplet of the nanoparticle solution onto a TEM grid with a thin carbon formvar B film (200 mesh, Cu). Allow the sample to adsorb onto the grid surface. Desiccation: Place the TEM grid with the sample in a des- iccator to air-dry overnight, ensuring the for- mation of a stable and representative sample. TEM Analysis: Microscope Setup: Set up the TEM microscope (e.g., JEM-2100HR) for analysis, ensuring proper alignment and calibration of the instrument. Accelerating Voltage Selec- tion: Choose an appropriate accelerating voltage (kV) to minimize sample damage caused by the electron beam. Consider using lower kV settings for sensitive samples. Imaging: Acquire TEM images of the gold nanoparticles at various magnifications. Opti- mize imaging parameters for resolution and contrast. Electron Dif- fraction (SAED): Conduct Selected-Area Electron Diffraction (SAED) to determine the crystal structure of the gold nanoparticles. Select representative areas for diffraction analysis. Chemical Composition Analysis (EDS): Utilize the Energy-Dispersive X-ray Spec- trometer (EDS) JED-2300T to perform ele- mental composition analysis. Ensure proper calibration of the EDS detector for accurate results. Optimization for Sensitive Samples: If the sample is sensitive to the electron beam, use a lower electron dose during imag- ing and analysis. Minimize exposure time and optimize imaging conditions accordingly. Data Interpre- tation: Analyse the TEM images, electron diffrac- tion patterns, and EDS spectra to obtain in- formation about the size, shape, crystal struc- ture, and elemental composition of the gold nanoparticles. Reporting and Documenta- tion: Document the experimental details, imaging parameters, and analysis results. Include rep- resentative images and diffraction patterns in the final report. By following this protocol, polymer fiber samples containing AuNPs using TEM can be effectively pre- pared and analysed, ensuring accurate characterization while minimizing potential sample damage. Transmission Electron Microscopy (TEM) and Scanning TEM (STEM) Protocol for Rapid Antigen Test Samples Containing Gold Nanoparticles Sample Preparation: Particle Size Selection: Choose the appropriate milled particle size (250 μm or 125 μm) of polymer and conju- gate pads for analysis. This may depend on the desired resolution and distribution of gold nanoparticles. Centrifugation (Optional): If necessary, conduct a centrifugation step to isolate the gold nanoparticles from the milled particles. Use a suitable solvent for this step, and carefully separate the supernatant for fur- ther analysis. Resuspension (Optional): Re-suspend the isolated gold nanoparticles in a solvent of choice (e.g., distilled water or ethanol) for improved dispersion and unifor- mity on the TEM grid. R. RUDOLF et al.: DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS 90 Materiali in tehnologije / Materials and technology 58 (2024) 1, 89–93 Figure 1: Schematic of the recycling process for rapid antigen tests Deposition on TEM Grid: Deposit the milled polymer and conjugate pad particles onto a TEM grid with a thin carbon formvar B film (200 mesh, Cu) using a dropper, utilizing distilled or deionized wa- ter or ethanol for easier deposition. Allow the sample to adsorb onto the grid surface. Desiccation: Place the TEM grid with the sample in a des- iccator to air-dry overnight, ensuring the for- mation of a stable and representative sample. TEM/STEM Analysis: Microscope Setup: Prepare the TEM microscope (e.g., JEM-2100HR or ARM 200 CF) for analysis, ensuring proper alignment and calibration of the instrument. Accelerating Voltage Selec- tion: Choose an appropriate accelerating voltage (kV) to minimize sample damage by the electron beam. Consider using lower kV (e.g., 80 kV or 100 kV) settings for sensitive samples. R. RUDOLF et al.: DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS Materiali in tehnologije / Materials and technology 58 (2024) 1, 89–93 91 Figure 2: TEM bright-field (BF) image (top left at lower magnification and top right at higher magnifications of individual Au particles in the milled material of rapid antigen test sample) and STEM dark-field (DF) with EDS point analysis in atomic/weight % (bottom left) and with EDS mapping analysis (bottom right) in the milled material of rapid antigen test sample with milled particles’ size 250 μm. Imaging: Acquire TEM/STEM images of the gold nanoparticles on the milled polymer and con- jugate pad particles at various magnifica- tions. Optimize imaging parameters for reso- lution and contrast. Electron Dif- fraction (SAED): Perform SAED to determine the crystal structure of the gold nanoparticles. Select representative areas for diffraction analysis. Chemical Composition Analysis (EDS): Employ the EDS JED-2300T or SDD to con- duct elemental composition analysis. Cali- brate the EDS detector for accurate results. Optimization for Sensitive Samples: If the sample is sensitive to the electron beam, use a lower electron dose during imag- ing and analysis. Minimize exposure time and optimize imaging conditions accordingly. Data Interpre- tation: Analyze the TEM images, electron-diffrac- tion patterns, and EDS spectra to obtain in- formation about the size, shape, crystal struc- ture, and elemental composition of the gold nanoparticles. Reporting and Documenta- tion: Document the experimental details, imaging parameters, and analysis results. Include rep- resentative images and diffraction patterns in the final report. By following this protocol, effective preparation and analysis of rapid antigen test samples containing AuNPs using TEM can be achieved, ensuring accurate character- ization, while minimizing potential sample damage. The result of considering a protocol and algorithm written above produced TEM and STEM results pre- sented in the Figure 2. 4 DISCUSSION Using the gold-containing acid solution from the re- fining of rapid test membranes in USP has produced ir- regular and spherical AuNPs, as well as triangular, pen- tagonal and hexagonal particle shapes 16 . ICP-OES (Table 1) and TEM/STEM/EDS (Figure 2) confirmed the presence of Au in recycling nanoparticles from the rapid antigen tests. Further TEM and STEM analyses (Figure 2) revealed that the size of the AuNPs predomi- nantly ranged below 100 nm, with some reaching dimen- sions in the few-hundred-nanometer range. The average size of particles falling below 100 nm ranged from ap- proximately 10 nm to 50 nm. Among the AuNPs with a size below 100 nm, the majority exhibited a spherical morphology, while larger particles exceeding 100 nm displayed irregular shapes. The feasibility of the proposed refining process and re-using the gold-containing acid solutions in USP for new AuNP production was confirmed. However, there are several areas where gold loss can occur in the com- plete process, from the test milling, membrane separa- tion and gold recovery from the membranes. In the rapid test milling and membrane-separation process, the gold loss is not known due to the different types of rapid tests available, which have differing AuNP contents. As the AuNP markers adhere quite strongly on the test mem- branes, we expect the gold loss in this part of the overall rapid test recycling to be marginal. The larger portion of gold loss is expected in treating the membranes with ac- ids. As the membranes are highly absorbent for liquids, they need to be drained off, for obtaining the maximum amount of gold. Even when drained off, some gold con- tent may remain in the membranes. Additionally, a study for evaluating the AuNP recovery from test membranes has shown about 17 % gold loss during the acid heating 16 , due to evaporation of the acids and the gold chloride inside the fume hood. A modification of the process is proposed, where less acid volume is used and the gold-containing membranes are filtered several times through the acid solution. In this way, the heating of the acids and gold loss are avoided, as there would be less acid and gold chloride evaporation from the obtained so- lution. A negative factor of such a process is gold adher- ence to the membranes and not being able to completely drain the membranes, resulting in less gold in the pro- duced solution. The proposed use of the aqua regia with dissolved gold from membranes in the USP production of new AuNPs is a new development in the conventional gold-refining process, resulting from the rapid antigen test recycling project. In using the prepared solution di- rectly in the USP process, the gold precipitation with so- dium metabisulphite used in the conventional refining is not necessary, resulting in less potential gold loss. How- ever, some additional steps are required for preparing this gold solution for the USP process. It was shown that high acid volumes detrimentally affect the AuNP forma- tion in the USP process, obtaining less-uniform AuNP sizes and shapes than with lower acid volumes. 16 The proposed modification of filtering the test membranes several times through the acid solution is also expected to reduce the acid volumes in the final solution to be used in USP, thus optimizing the AuNP formation to pro- duce more uniform particle sizes and shapes. 5 CONCLUSIONS The process of grinding and breaking apart rapid an- tigen LFIA tests makes it possible to separate with AuNP saturated membranes from the plastic housing and use them for USP precursor preparation. The refining pro- cess successfully utilizes USP to prepare recycled AuNPs from LFIAs. The algorithm for characterizing the AuNPs inte- grates diverse techniques to reveal their properties, cru- cial for customizing their applications in medicine, imag- ing, and sensor technology as nanotechnology advances. The algorithm for characterization of AuNPs is pre- sented and proposed, and with following this algorithm, characterization of recycled AuNPs from LFIAs was successfully achieved. This study offers guidance for establishing recycling processes, applicable not only to AuNPs but also for other metal nanoparticle residues that have minimal or no acid content. R. RUDOLF et al.: DEVELOPMENT OF THE RECYCLING PROCEDURE FOR RAPID ANTIGEN TESTS 92 Materiali in tehnologije / Materials and technology 58 (2024) 1, 89–93 Acknowledgment This research was funded by Norway Grants and a corresponding Slovenian contribution. The Recycling of Rapid Antigen LFIA Tests (COVID-19) (LFIA-REC) has benefitted from a 675,000.00 grant from Norway and Slovenia. The aim of the project is to establish the reuse of individual test components in the role of recyclates, which are useful directly to produce new products. Abbreviations Acrylonitrile butadiene styrene (ABS), energy dispersive X-ray spectrometers (EDS), inductively cou- pled plasma optical emission spectroscopy (ICP-OES), lateral flow immunoassay (LFIA), nanogold (AuNPs), polystyrene (PS), scanning electron microscopy (SEM), Selected Area Electron Diffraction (SAED), solid-state detector (SSD), STEM dark-field (DF), TEM bright-field (BF), transmission electron microscopy (TEM), Ultra- sonic Spray Pyrolysis (USP). 6 REFERENCES 1 Recycling of Rapid Antigen LFIA Tests (COVID-19) (LFIA-REC) Available online: https://www.norwaygrants.si/en/projects/pro- jects-of-the-programme-climate-change-mitigation-and-adapta- tion/lfia-rec/ 2 Strategija Razvoja Slovenije 2030 Available online: https://www.gov.si/assets/ministrstva/MKRR/Strategija-razvoja- Slovenije-2030/Strategija_razvoja_Slovenije_2030.pdf 3 Agenda Za Trajnostni Razvoj Do Leta 2030 Available online: https://www.gov.si/assets/ministrstva/MZZ/Dokumenti/multi- laterala/razvojno-sodelovanje/publikacije/Agenda_za_trajnostni_razv oj_2030.pdf 4 The European Green Deal Available online: https://commis- sion.europa.eu/strategy-and-policy/priorities-2019-2024/euro- pean-green-deal_en 5 Paris Agreement Available online: https://treaties.un.org/pages/ ViewDetails.aspx?src=TREATY&mtdsg_no=XXVII-7-d&chap- ter=27&clang=_en 6 U.S. COVID Public Health Emergency Is Ending. 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