Simple analytical devices suitable for the analysis of various biochemical and immunechemical markers are highly desirable and can provide laboratory diagnoses outside standard hospitals. This study focuses on constructing an easily reproducible do-it-yourself ELISA plate reader biosensor device, assembled from generally available and inexpensive parts. The colorimetric biosensor was based on standard 96-well microplates, 3D-printed parts, and a smartphone camera as a detector was utilized here as a tool to replace the ELISA method, and its function was illustrated in the assay of TNFα as a model immunochemical marker. The assay provided a limit of detection of 19 pg/mL when the B channel of the RGB color model was used for calibration. The assay was well correlated with the ELISA method, and no significant matrix effect was observed for standard biological samples or interference of proteins expected in a sample. The results of this study will inform the development of simple analytical devices easily reproducible by 3D printing and found on generally available electronics.

Military Faculty of Medicine University of Defence Trebesska 1575 CZ 50001 Hradec Kralove Czech Republic

Zobrazit více v PubMed

Ahirwar R., Bhattacharya A., Kumar S. Unveiling the underpinnings of various non-conventional ELISA variants: A review article. Expert Rev. Mol. Diagn. 2022;22:761–774. doi: 10.1080/14737159.2022.2117615. PubMed DOI

Daurat G. Yes, we should keep ABO agglutination test within bedside transfusion checks. Transfus. Clin. Biol. 2008;15:322–326. doi: 10.1016/j.tracli.2008.09.029. PubMed DOI

Björkman C., Uggla A. Serological diagnosis of Neospora caninum infection. Int. J. Parasit. 1999;29:1497–1507. doi: 10.1016/S0020-7519(99)00115-0. PubMed DOI

Dey M.K., Iftesum M., Devireddy R., Gartia M.R. New technologies and reagents in lateral flow assay (LFA) designs for enhancing accuracy and sensitivity. Anal. Methods. 2023;15:4351–4376. doi: 10.1039/D3AY00844D. PubMed DOI

Ince B., Uludag I., Demirbakan B., Özyurt C., Özcan B., Sezgintürk M.K. Lateral flow assays for food analyses: Food contaminants, allergens, toxins, and beyond. TrAC-Trends Anal. Chem. 2023;169:24. doi: 10.1016/j.trac.2023.117418. DOI

Silva G.B.L., Campos F.V., Guimaraes M.C.C., Oliveira J.P. Recent Developments in Lateral Flow Assays for Salmonella Detection in Food Products: A Review. Pathogens. 2023;12:1441. doi: 10.3390/pathogens12121441. PubMed DOI PMC

Cavalier E. Determination of parathyroid hormone: From radioimmunoassay to LCMS/MS. Clin. Chem. Lab. Med. 2023;61:946–953. doi: 10.1515/cclm-2022-0942. PubMed DOI

Liu R., Zhang S.X., Wei C., Xing Z., Zhang S.C., Zhang X.R. Metal Stable Isotope Tagging: Renaissance of Radioimmunoassay for Multiplex and Absolute Quantification of Biomolecules. Acc. Chem. Res. 2016;49:775–783. doi: 10.1021/acs.accounts.5b00509. PubMed DOI

Guo M., Chen Y., Mo X.H., Wei H., Li Y.Y., Jia Y.J., Hu F.D., Du Y.L. Review-Electrochemical Immunosensors for Depression Markers Detection: Development in Recent Years. J. Electrochem. Soc. 2024;171:11. doi: 10.1149/1945-7111/ad1c79. DOI

Mehta D., Gupta D., Kafle A., Kaur S., Nagaiah T.C. Advances and Challenges in Nanomaterial-Based Electrochemical Immunosensors for Small Cell Lung Cancer Biomarker Neuron-Specific Enolase. ACS Omega. 2023;9:33–51. doi: 10.1021/acsomega.3c06388. PubMed DOI PMC

Evtugyn G., Hianik T. Electrochemical Immuno- and Aptasensors for Mycotoxin Determination. Chemosensors. 2019;7:10. doi: 10.3390/chemosensors7010010. DOI

Bergua J.F., Alvarez-Diduk R., Idili A., Parolo C., Maymo M., Hu L., Merkoci A. Low-Cost, User-Friendly, All-Integrated Smartphone-Based Microplate Reader for Optical-Based Biological and Chemical Analyses. Anal. Chem. 2022;94:1271–1285. doi: 10.1021/acs.analchem.1c04491. PubMed DOI

Volpe C., Vadstein O., Andersen G., Andersen T. Nanocosm: A well plate photobioreactor for environmental and biotechnological studies. Lab Chip. 2021;21:2027–2039. doi: 10.1039/D0LC01250E. PubMed DOI

Bagheri N., Cinti S., Caratelli V., Massoud R., Saraji M., Moscone D., Arduini F. A 96-well wax printed Prussian Blue paper for the visual determination of cholinesterase activity in human serum. Biosens. Bioelectron. 2019;134:97–102. doi: 10.1016/j.bios.2019.03.037. PubMed DOI

Seddaoui N., Amine A. Smartphone-based competitive immunoassay for quantitative on-site detection of meat adulteration. Talanta. 2021;230:10. doi: 10.1016/j.talanta.2021.122346. PubMed DOI

Wang X., Wang H.Y., Wan X.Y., Li M.J., Tang D.P. Smartphone-based photoelectrochemical immunoassay for carcinoembryonic antigen based on BiOCl/CuBi2O4 heterojunction. Anal. Chim. Acta. 2023;1279:8. doi: 10.1016/j.aca.2023.341826. PubMed DOI

Li H., Ying Y., Cao Z., Liu G.Y., Wang J. Research Progress on Rapid Detection Technology Based on Smartphone and Lateral Flow Immunoassay. Anal. Chim. Acta. 2022;50:1–11. doi: 10.19756/j.issn.0253-3820.201488. DOI

Ben-Baruch A. Tumor Necrosis Factor α: Taking a Personalized Road in Cancer Therapy. Front. Immunol. 2022;13:6. doi: 10.3389/fimmu.2022.903679. PubMed DOI PMC

Mikail M., Wilson A. Low Serum Tumor Necrosis Factor-α Antagonist Concentrations in Patients With Inflammatory Bowel Disease Who Achieve Healing From Pyoderma Gangrenosum. Inflamm. Bowel Dis. 2021;27:E141–E143. doi: 10.1093/ibd/izab148. PubMed DOI PMC

Cahn R.T., Zinn Z., Kolodney M.S. Tumor necrosis factor inhibitors and methotrexate are associated with decreased COVID-19-related hospitalization: Follow up of “Clinical outcomes of COVID-19 in patients taking tumor necrosis factor inhibitors and methotrexate”. J. Am. Acad. Dermatol. 2023;88:1385–1386. doi: 10.1016/j.jaad.2023.02.002. PubMed DOI PMC

Vercellini P., Debenedetti F., Rossi E., Colombo A., Trespidi L., Crosignani P.G. Tumor necrosis factor in plasma and peritoneal fluid of women with and without endometriosis. Gynecol. Obstet. Investig. 1993;36:39–41. doi: 10.1159/000292591. PubMed DOI

Cascio A., Gervasi F., Giordano S., Palazzolo B., Salsa L. Plasma levels of tumor necrosis factor-alpha and interferon-gamma in Sicilian children with Mediterranean spotted fever. Int. J. Clin. Lab. Res. 1997;27:135–138. doi: 10.1007/BF02912448. PubMed DOI

Keane H.M., Sheron N., Goka J., Hughes R.D., Williams R. Plasma inhibitory activity against tumour necrosis factor in fulminant hepatic failure. Clin. Sci. 1996;90:77–80. doi: 10.1042/cs0900077. PubMed DOI

Ebrahimi M., Norouzi P., Davami F., Bonakdar A., Marzabad M.A., Tabaei O. Direct detection of TNF-α by copper benzene tricarboxylate MOFs/gold nanoparticles modified electrochemical label-free immunosensor using FFT admittance voltammetry. J. Electroanal. Chem. 2022;925:12. doi: 10.1016/j.jelechem.2022.116897. DOI

Bari S.M.I., Reis L.G., Nestorova G.G. Calorimetric sandwich-type immunosensor for quantification of TNF-α. Biosens. Bioelectron. 2019;126:82–87. doi: 10.1016/j.bios.2018.10.028. PubMed DOI

Yola M.L., Atar N. Novel voltammetric tumor necrosis factor-alpha (TNF-alpha) immunosensor based on gold nanoparticles involved in thiol-functionalized multi-walled carbon nanotubes and bimetallic Ni/Cu-MOFs. Anal. Bioanal. Chem. 2021;413:2481–2492. doi: 10.1007/s00216-021-03203-z. PubMed DOI

Parandakh A., Ymbern O., Jogia W., Renault J., Ng A., Juncker D. 3D-printed capillaric ELISA-on-a-chip with aliquoting. Lab Chip. 2023;23:1547–1560. doi: 10.1039/D2LC00878E. PubMed DOI

Singh H., Shimojima M., Fukushi S., Van A.L., Sugamata M., Yang M. Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology. Bio-Med. Mater. Eng. 2015;26:S45–S53. doi: 10.3233/BME-151288. PubMed DOI

Singh H., Shimojima M., Shiratori T., Van An L., Sugamata M., Yang M. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases. Sensors. 2015;15:16503–16515. doi: 10.3390/s150716503. PubMed DOI PMC

Bauer M., Kulinsky L. Fabrication of a Lab-on-Chip Device Using Material Extrusion (3D Printing) and Demonstration via Malaria-Ab ELISA. Micromachines. 2018;9:27. doi: 10.3390/mi9010027. PubMed DOI PMC

Damas P., Reuter A., Gysen P., Demonty J., Lamy M., Franchimont P. Tumor necrosis factor and interleukin-1 serum levels during severe sepsis in humans. Crit. Care Med. 1989;17:975–978. doi: 10.1097/00003246-198910000-00001. PubMed DOI

Ferrajoli A., Keating M.J., Manshouri T., Giles F.J., Dey A., Estrov Z., Koller C.A., Kurzrock R., Thomas D.A., Faderl S., et al. The clinical significance of tumor necrosis factor-alpha plasma level in patients having chronic lymphocytic leukemia. Blood. 2002;100:1215–1219. doi: 10.1182/blood.V100.4.1215.h81602001215_1215_1219. PubMed DOI

Xu D.D., Huang X.W., Guo J.H., Ma X. Automatic smartphone-based microfluidic biosensor system at the point of care. Biosens. Bioelectron. 2018;110:78–88. doi: 10.1016/j.bios.2018.03.018. PubMed DOI

Omidfar K., Ahmadi A., Syedmoradi L., Khoshfetrat S.M., Larijani B. Point-of-care biosensors in medicine: A brief overview of our achievements in this field based on the conducted research in EMRI (endocrinology and metabolism research Institute of Tehran University of medical sciences) over the past fourteen years. J. Diabetes Metab. Disord. 2020:1–5. doi: 10.1007/s40200-020-00668-0. PubMed DOI PMC

Poschenrieder A., Thaler M., Junker R., Luppa P.B. Recent advances in immunodiagnostics based on biosensor technologies-from central laboratory to the point of care. Anal. Bioanal. Chem. 2019;411:7607–7621. doi: 10.1007/s00216-019-01915-x. PubMed DOI

Kulkarni M.B., Ayachit N.H., Aminabhavi T.M. Biosensors and Microfluidic Biosensors: From Fabrication to Application. Biosensors. 2022;12:543. doi: 10.3390/bios12070543. PubMed DOI PMC

Hwang C., Lee W.J., Kim S.D., Park S., Kim J.H. Recent Advances in Biosensor Technologies for Point-of-Care Urinalysis. Biosensors. 2022;12:1020. doi: 10.3390/bios12111020. PubMed DOI PMC

Pohanka M. Current trends in digital camera-based bioassays for point-of-care tests. Clin. Chim. Acta. 2024;552:9. doi: 10.1016/j.cca.2023.117677. PubMed DOI

Syed S., Rahaman A., Mondal A., Shaligram S., Pawar S.P. Diagnosis of infectious diseases: Complexity to convenience. Sens. Diagn. 2024;3:354–380. doi: 10.1039/D3SD00236E. DOI

Taron W., Phooplub K., Sanchimplee S., Piyanamvanich K., Jamnongkan W., Techasen A., Phetcharaburanin J., Klanrit P., Namwat N., Khuntikeo N., et al. Smartphone-based fluorescent ELISA with simple fluorescent enhancement strategy for Opisthorchis viverrini (Ov) antigen detection in urine samples. Sens. Actuator B Chem. 2021;348:14. doi: 10.1016/j.snb.2021.130705. DOI

Wang C., Wu Z., Liu B.C., Zhang P.L., Lu J.H., Li J.F., Zou P., Li T.T., Fu Y.S., Chen R.A., et al. Track-etched membrane microplate and smartphone immunosensing for SARS-CoV-2 neutralizing antibody. Biosens. Bioelectron. 2021;192:10. doi: 10.1016/j.bios.2021.113550. PubMed DOI PMC

Berg B., Cortazar B., Tseng D., Ozkan H., Feng S., Wei Q.S., Chan R.Y.L., Burbano J., Farooqui Q., Lewinski M., et al. Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays. ACS Nano. 2015;9:7857–7866. doi: 10.1021/acsnano.5b03203. PubMed DOI