We present the first automated synthesis of magnetic immunosorbents (MIS) using a lab-in-syringe (LIS) platform, facilitating antibody bioconjugation to magnetic beads via carbodiimide-mediated covalent binding. This approach is an efficient, reproducible alternative to traditional manual methods, minimizing pipetting steps, vortexing, and incubation with a reduced handling bias. Utilizing a 1 mL syringe pump with a 12-port multiposition valve and an internal magnetic stir bar enables precise mixing, bead dispersion, and magnetic capture for consistent bioconjugate synthesis. The LIS platform achieved a 99.6% bead recovery with 0.4 mg of MIS (1 μm in diameter), outperforming the 83% recovery of manual techniques, and maintained an 83% recovery at reduced scales of 0.2 mg, surpassing manual yields of 76%. As a proof-of-concept, MIS conjugated with anti-SARS-CoV2 antibodies (6 μg/400 mg beads) were synthesized and validated for viral RNA isolation from COVID-19-positive samples, demonstrating high immunocapture efficiency comparable to manual methods but with significantly reduced time and labor requirements. This automated synthesis of antibody-MIS enables the scalable, reproducible production of bioconjugated materials, supporting advanced applications in diagnostic assays, therapeutic delivery, and microfluidic integrations. The LIS approach thus enhances the scope of biomolecular conjugate synthesis, offering streamlined workflows that are suited for downstream analytical and bioanalytical applications. LIS is a versatile, automated system for preparing MIS that researchers can adapt for various targets, particularly when commercial products are unavailable, are prohibitively expensive, or require custom carriers.

Charles University Faculty of Pharmacy Department of Analytical Chemistry Hradec Kralove 500 03 Czech Republic

Charles University Faculty of Pharmacy Department of Biochemical Sciences Hradec Kralove 500 03 Czech Republic

Charles University Faculty of Pharmacy Department of Biological and Medical Sciences Hradec Kralove 500 09 Czech Republic

Institute of Clinical Biochemistry and Diagnostics Charles University Hospital and Faculty of Medicine in Hradec Kralove Hradec Kralove 500 03 Czech Republic

University of Pardubice Faculty of Chemical Technology Department of Biological and Biochemical Sciences Pardubice 532 10 Czech Republic

Zobrazit více v PubMed

Torres-Herrero B., Armenia I., Alleva M., Asín L., Correa S., Ortiz C., Fernández-Afonso Y., Gutiérrez L., de la Fuente J. M., Betancor L.. et al. Remote Activation of Enzyme Nanohybrids for Cancer Prodrug Therapy Controlled by Magnetic Heating. ACS Nano. 2023;17(13):12358–12373. doi: 10.1021/acsnano.3c01599. PubMed DOI PMC

Zhang L., Li Q., Liu J., Deng Z., Zhang X., Alifu N., Zhang X., Yu Z., Liu Y., Lan Z.. et al. Recent advances in functionalized ferrite nanoparticles: From fundamentals to magnetic hyperthermia cancer therapy. Colloids Surf., B. 2024;234:113754. doi: 10.1016/j.colsurfb.2024.113754. PubMed DOI

Li Z., Wan W., Bai Z., Peng B., Wang X., Cui L., Liu Z., Lin K., Yang J., Hao J.. et al. Construction of pH-responsive nanoplatform from stable magnetic nanoparticles for targeted drug delivery and intracellular imaging. Sens. Actuators, B. 2023;375:132869. doi: 10.1016/j.snb.2022.132869. DOI

Rahimkhoei V., Alzaidy A. H., Abed M. J., Rashki S., Salavati-Niasari M.. Advances in inorganic nanoparticles-based drug delivery in targeted breast cancer theranostics. Adv. Colloid Interface Sci. 2024;329:103204. doi: 10.1016/j.cis.2024.103204. PubMed DOI

Paltanea G., Manescu V., Antoniac I., Antoniac A., Nemoianu I. V., Robu A., Dura H.. A Review of Biomimetic and Biodegradable Magnetic Scaffolds for Bone Tissue Engineering and Oncology. International Journal of Molecular Sciences. 2023;24:4312. doi: 10.3390/ijms24054312. PubMed DOI PMC

Mao Y., Zhang Y., Yu Y., Zhu N., Zhou X., Li G., Yi Q., Wu Y.. Self-assembled supramolecular immunomagnetic nanoparticles through π–π stacking strategy for the enrichment of circulating tumor cells. Regenerative Biomaterials. 2023;10:rbad016. doi: 10.1093/rb/rbad016. PubMed DOI PMC

Wang Y., Li J., Liu H., Du X., Yang L., Zeng J.. Single-Probe-Based Colorimetric and Photothermal Dual-Mode Identification of Multiple Bacteria. Anal. Chem. 2023;95(5):3037–3044. doi: 10.1021/acs.analchem.2c05140. PubMed DOI

Kavruk M., Babaie Z., Kibar G., Çetin B., Yeşilkaya H., Amrani Y., Dursun A. D., Özalp V. C.. Aptamer decorated PDA@magnetic silica microparticles for bacteria purification. Microchimica Acta. 2024;191(5):285. doi: 10.1007/s00604-024-06322-3. PubMed DOI PMC

Wang X., Li S., Qu H., Hao L., Shao T., Wang K., Xia Z., Li Z., Li Q.. SERS-based immunomagnetic bead for rapid detection of H5N1 influenza virus. Influenza and Other Respiratory Viruses. 2023;17(3):e13114. doi: 10.1111/irv.13114. PubMed DOI PMC

Guo X., Hu F., Zhao S., Yong Z., Zhang Z., Peng N.. Immunomagnetic Separation Method Integrated with the Strep-Tag II System for Rapid Enrichment and Mild Release of Exosomes. Anal. Chem. 2023;95(7):3569–3576. doi: 10.1021/acs.analchem.2c03470. PubMed DOI

Dhandapani G., Nguyen V. G., Kim M. C., Noh J. Y., Jang S. S., Yoon S.-W., Jeong D. G., Huynh T. M. L., Le V. P., Song D.. et al. Magnetic-bead-based DNA-capture-assisted real-time polymerase chain reaction and recombinase polymerase amplification for the detection of African swine fever virus. Arch. Virol. 2023;168(1):21. doi: 10.1007/s00705-022-05681-7. PubMed DOI

Sadasivam M., Sakthivel A., Nagesh N., Hansda S., Veerapandian M., Alwarappan S., Manickam P.. Magnetic bead-amplified voltammetric detection for carbohydrate antigen 125 with enzyme labels using aptamer-antigen-antibody sandwiched assay. Sens. Actuators, B. 2020;312:127985. doi: 10.1016/j.snb.2020.127985. DOI

Soliman M. G., Trinh D. N., Ravagli C., Meleady P., Henry M., Movia D., Doumett S., Cappiello L., Prina-Mello A., Baldi G.. et al. Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices. J. Colloid Interface Sci. 2024;659:503–519. doi: 10.1016/j.jcis.2023.11.177. PubMed DOI

Shabatina T. I., Vernaya O. I., Shabatin V. P., Melnikov M. Y.. Magnetic Nanoparticles for Biomedical Purposes: Modern Trends and Prospects. Magnetochemistry. 2020;6(3):30. doi: 10.3390/magnetochemistry6030030. DOI

Zuo H., Wang X., Liu W., Chen Z., Liu R., Yang H., Xia C., Xie J., Sun T., Ning B.. Nanobody-based magnetic chemiluminescence immunoassay for one-pot detection of ochratoxin A. Talanta. 2023;258:124388. doi: 10.1016/j.talanta.2023.124388. PubMed DOI

Otieno, B. A. ; Krause, C. E. ; Rusling, J. F. . Bioconjugation of Antibodies and Enzyme Labels onto Magnetic Beads. In Methods in Enzymology, Kumar, C. V. , Ed.; Vol. 571; Academic Press, 2016; Chapter 7, pp 135–150. PubMed

Kamel H. H., Elleboudy N. A. F., Hasan A. N., Ali I. R., Mohammad O. S.. Nano magnetic-based ELISA and nano magnetic-based latex agglutination test for diagnosis of experimental trichinellosis. Journal of Parasitic Diseases. 2023;47(2):400–409. doi: 10.1007/s12639-023-01583-w. PubMed DOI PMC

Chen C., Liang H., Peng F., Zhong S., Lu Y., Guo G., Li L.. Determination of echinococcosis IgG antibodies using magnetic bead-based chemiluminescence immunoassay. Journal of Immunological Methods. 2023;520:113513. doi: 10.1016/j.jim.2023.113513. PubMed DOI

Lin C.-T., Kuo S.-H., Lin P.-H., Chiang P.-H., Lin W.-H., Chang C.-H., Tsou P.-H., Li B.-R.. Hand-powered centrifugal microfluidic disc with magnetic chitosan bead-based ELISA for antibody quantitation. Sens. Actuators, B. 2020;316:128003. doi: 10.1016/j.snb.2020.128003. DOI

Momeni M., Shamloo A., Hasani-Gangaraj M., Dezhkam R.. An experimental study of centrifugal microfluidic platforms for magnetic-inertial separation of circulating tumor cells using contraction-expansion and zigzag arrays. Journal of Chromatography A. 2023;1706:464249. doi: 10.1016/j.chroma.2023.464249. PubMed DOI

Yin D., Shi A., Zhou B., Wang M., Xu G., Shen M., Zhu X., Shi X.. Efficient Capture and Separation of Cancer Cells Using Hyaluronic Acid-Modified Magnetic Beads in a Microfluidic Chip. Langmuir. 2022;38(36):11080–11086. doi: 10.1021/acs.langmuir.2c01740. PubMed DOI

Horak D., Svobodova Z., Autebert J., Coudert B., Plichta Z., Kralovec K., Bilkova Z., Viovy J. L.. Albumin-coated monodisperse magnetic poly­(glycidyl methacrylate) microspheres with immobilized antibodies: Application to the capture of epithelial cancer cells. J. Biomed. Mater. Res., Part A. 2013;101A(1):23–32. doi: 10.1002/jbm.a.34297. PubMed DOI

Jin K., Hu C., Hu S., Hu C., Li J., Ma H.. “One-to-three” droplet generation in digital microfluidics for parallel chemiluminescence immunoassays. Lab Chip. 2021;21(15):2892–2900. doi: 10.1039/D1LC00421B. PubMed DOI

Hsu W., Shih Y.-T., Lee M.-S., Huang H.-Y., Wu W.-N.. Bead Number Effect in a Magnetic-Beads-Based Digital Microfluidic Immunoassay. Biosensors. 2022;12:340. doi: 10.3390/bios12050340. PubMed DOI PMC

Jiang D., Qi R., Wu S., Li Y., Liu J.. Polyoxometalate functionalized magnetic metal–organic framework with multi-affinity sites for efficient enrichment of phosphopeptides. Anal. Bioanal. Chem. 2024;416(19):4289–4299. doi: 10.1007/s00216-024-05365-y. PubMed DOI

Zhang X., Wang B., Luo Y., Ding C.-F., Yan Y.. An amino-rich polymer-coated magnetic nanomaterial for ultra-rapid separation of phosphorylated peptides in the serum of Parkinson’s disease patients. Anal. Bioanal. Chem. 2024;416(14):3361–3371. doi: 10.1007/s00216-024-05287-9. PubMed DOI

de Souza Schwarz P., dos Santos B. P., Birk L., Eller S., de Oliveira T. F.. Development of an innovative analytical method for forensic detection of cocaine, antidepressants, and metabolites in postmortem blood using magnetic nanoparticles. Anal. Bioanal. Chem. 2024;416(13):3239–3250. doi: 10.1007/s00216-024-05273-1. PubMed DOI

Llandro J., Palfreyman J. J., Ionescu A., Barnes C. H. W.. Magnetic biosensor technologies for medical applications: a review. Medical & Biological Engineering & Computing. 2010;48(10):977–998. doi: 10.1007/s11517-010-0649-3. PubMed DOI

Grabarek Z., Gergely J.. Zero-length crosslinking procedure with the use of active esters. Anal. Biochem. 1990;185(1):131–135. doi: 10.1016/0003-2697(90)90267-D. PubMed DOI

Horstkotte B., Suárez R., Solich P., Cerdà V.. In-syringe-stirring: a novel approach for magnetic stirring-assisted dispersive liquid-liquid microextraction. Anal. Chim. Acta. 2013;788:52–60. doi: 10.1016/j.aca.2013.05.049. PubMed DOI

Maya F., Horstkotte B., Estela J. M., Cerdà V.. Lab in a syringe: fully automated dispersive liquid-liquid microextraction with integrated spectrophotometric detection. Anal. Bioanal. Chem. 2012;404(3):909–917. doi: 10.1007/s00216-012-6159-4. PubMed DOI

Horstkotte B., Solich P.. The Automation Technique Lab-In-Syringe: A Practical Guide. Molecules. 2020;25(7):1612. doi: 10.3390/molecules25071612. PubMed DOI PMC

Gemuh C. V., Horstkotte B., Solich P.. Lab-In-Syringe with Bead Injection Coupled Online to High-Performance Liquid Chromatography as Versatile Tool for Determination of Nonsteroidal Anti-Inflammatory Drugs in Surface Waters. Molecules. 2021;26(17):5358. doi: 10.3390/molecules26175358. PubMed DOI PMC

Suárez R., Horstkotte B., Cerdà V.. In-syringe magnetic stirring-assisted dispersive liquid-liquid microextraction for automation and downscaling of methylene blue active substances assay. Talanta. 2014;130:555–560. doi: 10.1016/j.talanta.2014.06.063. PubMed DOI

Gemuh C. V., Macháček M., Solich P., Horstkotte B.. Renewable sorbent dispersive solid phase extraction automated by Lab-In-Syringe using magnetite-functionalized hydrophilic-lipophilic balanced sorbent coupled online to HPLC for determination of surface water contaminants. Anal. Chim. Acta. 2022;1210:339874. doi: 10.1016/j.aca.2022.339874. PubMed DOI

Kasetsirikul S., Umer M., Soda N., Sreejith K. R., Shiddiky M. J., Nguyen N.-T.. Detection of the SARS-CoV-2 humanized antibody with paper-based ELISA. Analyst. 2020;145(23):7680–7686. doi: 10.1039/D0AN01609H. PubMed DOI

El-Daly M. M.. Advances and challenges in SARS-CoV-2 detection: a review of molecular and serological technologies. Diagnostics. 2024;14(5):519. doi: 10.3390/diagnostics14050519. PubMed DOI PMC

Sreejith K. R., Umer M., Dirr L., Bailly B., Guillon P., von Itzstein M., Soda N., Kasetsirikul S., Shiddiky M. J. A., Nguyen N.-T.. A Portable Device for LAMP Based Detection of SARS-CoV-2. Micromachines. 2021;12(10):1151. doi: 10.3390/mi12101151. PubMed DOI PMC

Matejkova N., Smela D., Beranek M., Capek J., Michalcova L., Michalkova L., Svoboda J., Skeren M., Svobodova Z., Sopha H.. et al. Open-channel microfluidic device for TiO2NTs@Fe3O4NPs-assisted viral RNA extraction and amplification-free RNA fluorescence status evaluation. Microchemical Journal. 2024;206:111554. doi: 10.1016/j.microc.2024.111554. DOI

Ramos I. I., Marques S. S., Magalhães L. M., Barreiros L., Reis S., Lima J. L. F. C., Segundo M. A.. Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection. Talanta. 2019;204:542–547. doi: 10.1016/j.talanta.2019.06.023. PubMed DOI

Staros J. V., Wright R. W., Swingle D. M.. Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 1986;156(1):220–222. doi: 10.1016/0003-2697(86)90176-4. PubMed DOI

Hacker S. M., Backus K. M., Lazear M. R., Forli S., Correia B. E., Cravatt B. F.. Global profiling of lysine reactivity and ligandability in the human proteome. Nat. Chem. 2017;9(12):1181–1190. doi: 10.1038/nchem.2826. PubMed DOI PMC

Agarwal P., Bertozzi C. R.. Site-specific antibody–drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 2015;26(2):176–192. doi: 10.1021/bc5004982. PubMed DOI PMC