SUPPORTING INFORMATION

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1 SUPPORTING INFORMATION Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich Nanozyme-Linked Immunosorbent Assay Zdeněk Farka, 1, * Veronika Čunderlová, 1,2 Veronika Horáčková, 1 Matěj Pastucha, 1,2 Zuzana Mikušová, 2 Antonín Hlaváček, 1,3 Petr Skládal 1,2, * 1 CEITEC MU, Masaryk University, Kamenice 5, Brno, Czech Republic 2 Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic 3 Institute of Analytical Chemistry CAS, v. v. i., Brno, Czech Republic * farka@mail.muni.cz, * skladal@chemi.muni.cz, Telephone: Table of Contents Fig. S1: Selection of capture and detection Ab for HSA detection S-2 Fig. S2: Optimization of blocking conditions and PBNP-Ab sample in NLISA S-2 Fig. S3: Time dependency of the PBNP-Ab catalyzed substrate conversion S-3 Fig. S4: Comparison of NLISA readout wavelength S-3 Fig. S5: Reproducibility of NLISA for the detection of HSA S-4 Fig. S6: Optimization of HSA detection using ELISA S-4 Table S1: Comparison of NLISA and ELISA S-5 Table S2: Comparison of immunoassays for the detection of HSA in urine S-6 Fig. S7: Optimization of Salmonella detection using NLISA S-6 Fig. S8: Detection of Salmonella using direct ELISA S-7 Table S3: Comparison of immunoassays for the detection of Salmonella S-8 References S-8 S-1

2 Figure S1: The effect of different coating and detection antibodies (indicated as coating Ab detection Ab combination) on the measured NLISA signal. The coating by 2 μg ml 1 of given antibody was followed by the binding of 10 μg ml 1 of HSA and 750 pm PBNP-Ab conjugate. The first column corresponds to HSA in concentration of 10 μg ml 1 coated directly to the microtiter plate. Figure S2: The optimization of NLISA assay for the detection of HSA. (A) Comparison of different blocking conditions (5 % powdered milk, 50 % milk or 10 mg ml 1 of BSA in PBS). (B) Comparison of uncentrifuged and centrifuged conjugates of PBNPs with antibody oxidized for either 30 or 60 min. The empty triangles represent the LOD values. S-2

3 Figure S3: Time dependency of the measured signal (absorbance at 652 nm) on the reaction time for complete sandwich based on antibody AL-01 coated in concentration 4 µg ml 1 and 750 pm PBNP-Ab conjugate. The dependencies for 100 ng ml 1 of HSA (red) and no HSA (black) are compared. Figure S4: The comparison of NLISA readout based on A652 (read after 120 min) and A450 (after 120 min and stopping the reaction using H2SO4). The empty triangles represent the LOD values. S-3

4 Figure S5: Reproducibility of NLISA assay for the detection of HSA. Each dataset corresponds to independently performed assay with PBNP-Ab conjugate from different batch. The empty triangles represent the LOD values. Figure S6: The optimization of HRP-pAb-F conjugate concentration in sandwich ELISA. The empty triangles represent the LOD values. S-4

5 Table S1: Comparison of conjugate properties and assay parameters in NLISA and ELISA. NLISA ELISA Properties and preparation of catalytic label Label PBNP HRP Turnover number towards TMB 1 Cost of 1 g of catalytic label (excluding antibody) s s 1 ~ USD a ~ USD Possibility to synthesize using common reagents Yes No Size of label 116 nm ~ 15 nm ph stability Immunoassay properties and parameters Substrate concentration 0.5 mm TMB 125 mm H 2O 2 ~ 1 mm TMB b ~ 1 mm H 2O 2 b Time of catalytic reaction 120 min 30 min HSA detection Salmonella detection LOD 1.2 ng ml 1 EC ng ml 1 LOD CFU ml 1 EC CFU ml 1 LOD 3.7 ng ml 1 EC ng ml 1 LOD CFU ml 1 EC CFU ml 1 a calculation based on cost and consumption of reagents during the PBNP synthesis b proprietary TBM substrate mixture was used, the table shows typical concentrations S-5

6 Table S2: Comparison of immunoassays for the detection of human serum albumin in urine. Method LOD Working range Reference Sandwich NLISA 1.2 ng ml ng ml 1 to 1 µg ml 1 This work Sandwich ELISA 3.7 ng ml ng ml 1 to 1 µg ml 1 This work Competitive ELISA 89 ng ml ng ml 1 to 15 µg ml 1 2 Competitive FIA 1 µg ml µg ml 1 to 10 µg ml 1 3 Competitive NLISA 0.27 µg ml 1 NA 1 Binding of specific 0.4 µg ml 1 or 1 50 µg ml 1 or µg ml 1 4 fluorescent probe 1.4 µg ml 1 Immunonephelometry 2 µg ml 1 NA 5 Immunoturbidimetry 6 µg ml 1 NA 5 RIA 16 ng ml 1 NA 5 Sandwich ELISA 6.25 ng ml ng ml 1 to 200 ng ml 1 6 FIA fluoroimmunoassay; RIA radioimmunoassay; ELISA enzyme-linked immunosorbent assay Figure S7: The optimization of NLISA assay for the detection of Salmonella Typhimurium. (A) Optimization of coating concentration. (B) The detection of heat-treated Salmonella is compared with the heat-treated and sonicated sample. The empty triangles represent the LOD values. S-6

7 Figure S8: Calibration curve of direct ELISA for the detection of Salmonella Typhimurium (LOD CFU ml 1 ; EC CFU ml 1 ). The plate was coated over night with varying concentrations of Salmonella in PBS, the other assay steps were the same as in sandwich assay. The empty triangle represents the LOD value. S-7

8 Table S3: Comparison of immunoassays for the detection of Salmonella. Method Sample matrix LOD Reference (CFU ml 1 ) PBNP-based sandwich NLISA Powdered milk This work Sandwich ELISA Powdered milk This work Sandwich ELISA Milk Sandwich ELISA Milk CL-based sandwich ELISA Meat CL microarray-based ELISA Water Polycarbonate membrane-enhanced Water ELISA Immunochromatographic strip Milk Ab-SWCNT-HRP-based ELISA Milk 10 3 and Au NP-based colorimetric Milk immunoassay Immunoassay based on QDs and IMS Apple juice Aptamer-based fluorescence immunoassay Buffer CL chemiluminescence; SWCNT single-walled carbon nanotube; QD quantum dot; IMS immunomagnetic separation References 1. Čunderlová, V.; Hlaváček, A.; Horňáková, V.; Peterek, M.; Němeček, D.; Hampl, A.; Eyer, L.; Skládal, P., Catalytic nanocrystalline coordination polymers as an efficient peroxidase mimic for labeling and optical immunoassays. Microchim. Acta 2016, 183 (2), Zhao, L. X.; Lin, J. M.; Li, Z. J., Comparison and development of two different solid phase chemiluminescence ELISA for the determination of albumin in urine. Anal. Chim. Acta 2005, 541 (1-2), Hlaváček, A.; Bouchal, P.; Skládal, P., Biotinylation of quantum dots for application in fluoroimmunoassays with biotin-avidin amplification. Microchimica Acta 2012, 176 (3-4), Kessler, M. A.; Meinitzer, A.; Petek, W.; Wolfbeis, O. S., Microalbuminuria and borderline-increase albumin excretion determined with a centrifugal analyzer and the Albumin Blue 580 fluorescence assay. Clin. Chem. 1997, 43 (6), Busby, D. E.; Bakris, G. L., Comparison of Commonly Used Assays for the Detection of Microalbuminuria. J. Clin. Hypertens. 2004, 6, Watts, G. F.; Bennett, J. E.; Rowe, D. J.; Morris, R. W.; Gatling, W.; Shaw, K. M.; Polak, A., Assessment of Immunochemical Methods for Determining Low Concentrations of Albumin In Urine. Clin. Chem. 1986, 32 (8), S-8

9 7. Wang, W. B.; Liu, L. Q.; Song, S. S.; Tang, L. J.; Kuang, H.; Xu, C. L., A Highly Sensitive ELISA and Immunochromatographic Strip for the Detection of Salmonella typhimurium in Milk Samples. Sensors 2015, 15 (3), Wu, X. L.; Wang, W. B.; Liu, L. Q.; Kuang, H.; Xu, C. L., Monoclonal antibody-based cross-reactive sandwich ELISA for the detection of Salmonella spp. in milk samples. Anal. Methods 2015, 7 (21), Magliulo, M.; Simoni, P.; Guardigli, M.; Michelini, E.; Luciani, M.; Lelli, R.; Roda, A., A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157 : H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes pathogen bacteria. J. Agric. Food Chem. 2007, 55 (13), Wolter, A.; Niessner, R.; Seidel, M., Detection of Escherichia coli O157 : H7, Salmonella typhimurium, and Legionella pneumophila in water using a flow-through chemiluminescence microarray readout system. Anal. Chem. 2008, 80 (15), Jain, S.; Chattopadhyay, S.; Jackeray, R.; Abid, C.; Kohli, G. S.; Singh, H., Highly sensitive detection of Salmonella typhi using surface aminated polycarbonate membrane enhanced-elisa. Biosens. Bioelectron. 2012, 31 (1), Chunglok, W.; Wuragil, D. K.; Oaew, S.; Somasundrum, M.; Surareungchai, W., Immunoassay based on carbon nanotubes-enhanced ELISA for Salmonella enterica serovar Typhimurium. Biosens. Bioelectron. 2011, 26 (8), Wu, W. H.; Li, J.; Pan, D.; Song, S. P.; Rong, M. G.; Li, Z. X.; Gao, J. M.; Lu, J. X., Gold Nanoparticle-Based Enzyme-Linked Antibody-Aptamer Sandwich Assay for Detection of Salmonella Typhimurium. ACS Appl. Mater. Interfaces 2014, 6 (19), Zhao, Y.; Ye, M. Q.; Chao, Q. G.; Jia, N. Q.; Ge, Y.; Shen, H. B., Simultaneous Detection of Multifood-Borne Pathogenic Bacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation in Food Samples. J. Agric. Food Chem. 2009, 57 (2), Duan, Y. F.; Ning, Y.; Song, Y.; Deng, L., Fluorescent aptasensor for the determination of Salmonella typhimurium based on a graphene oxide platform. Microchim. Acta 2014, 181 (5-6), S-9