Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich

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Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich Nanozyme-Linked Immunosorbent Assay Zden#k Farka, Veronika #underlová, Veronika Horá#ková, Mat#j Pastucha, Zuzana Mikušová, Antonín Hlavá#ek, and Petr Skládal Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04883 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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Analytical Chemistry

Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich Nanozyme-Linked Immunosorbent Assay Zdeněk Farka1,*, Veronika Čunderlová1,2, Veronika Horáčková1, Matěj Pastucha1,2, Zuzana Mikušová2, Antonín Hlaváček1,3, Petr Skládal1,2,* 1

CEITEC MU, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

2

Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00

Brno, Czech Republic 3

Institute of Analytical Chemistry CAS, v. v. i., 602 00 Brno, Czech Republic

*E-mail: [email protected]; *E-mail: [email protected], Telephone: +420 54949 7010.

ABSTRACT Enzyme immunoassays are widely used for detection of analytes within various samples. However, enzymes as labels suffer several disadvantages such as high production costs and limited stability. Catalytic nanoparticles (nanozymes) can be used as an alternative label in immunoassays overcoming the inherent disadvantages of enzymes. Prussian blue nanoparticles (PBNPs) are nanozymes composed of the Fe4[Fe(CN)6]3 based coordination polymer. They reveal peroxidase-like activity and are capable of catalyzing the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine in the presence of H2O2 to form intensively blue product. Here, we introduce the method for conjugation of PBNPs with antibodies and their application in Nanozyme-Linked Immunosorbent Assay (NLISA). Sandwich NLISA for detection of human serum albumin in urine was developed with limit of detection (LOD) of 1.2 ng mL−1 and working range up to 1 µg mL−1. Furthermore, the microbial contamination of Salmonella Typhimurium in powdered milk was detected with LOD of 6·103 CFU mL−1 and working range up to

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106 CFU mL−1. In both cases, a critical comparison with the same immunoassay but using native peroxidase as label was realized. The achieved results confirmed the suitability of PBNPs as universal and robust replacement of enzyme labels.

INTRODUCTION Enzyme-linked immunosorbent assay (ELISA) is considered as the golden standard for highly sensitive qualitative and quantitative detection of analytes within various samples. In ELISA, an enzyme is conjugated with either antibody or antigen to be used as a signal amplification label, which catalyzes the conversion of substrate molecules to easily detectable product. For example, the conversion of a colorless substrate to a colored or fluorescent product may be applied.1 The most commonly used enzymatic labels for immunochemical detection and imaging are peroxidases, e.g. horseradish peroxidase (HRP). However, high production costs, short shelf-lives, and decrease of activity during conjugation reactions of natural enzymes create an increasing demand for cheap and more stable nanozymes.2, 3 Nanozymes are nanomaterials mimicking catalytic activity of enzymes, e.g. peroxidases.4-6 Catalytically active nanozymes with high signal amplification and good stability generally improve the performance of immunoassays7 and have an excellent potential for the detection of single molecules in digital (immuno)assays.8 Nanozymes can be commonly synthesized from benign precursors in aqueous dispersants.9 The applicability of nanozymes for practical use is also promoted by thermal and chemical stability of inorganic nanomaterials.10 Nanozymes with high peroxidase-like activity were studied for their potential use in immunochemical assays.3-6 Initially, magnetite nanoparticles (Fe3O4) of different sizes were characterized for their activity, high turnover number and good stability.10 Later on, the peroxidase-like activity was described for CeO2,11 CuO,12 Co3O4,13 bimetallic,14 and MnO2 nanoparticles,15 graphene oxide nanoplates,16 mesoporous silica nanoparticles coated by catalytic metal complexes,7 and hemin-block copolymer micelles.17 Coordination polymers18 are solids, which have been studied recently for their exceptional catalytic properties. Prussian blue nanoparticles (PBNPs)19 are nanozymes composed of coordination polymer with a chemical formula Fe4[Fe(CN)6]3 that reveal peroxidase-like activity.20, 21 Previously, we reported the biotinylation scheme of PBNPs allowing for competitive nanozyme-linked immunosorbent assay (NLISA) of protein markers.22 However, competitive immunoassays typically possess lower sensitivity than sandwich variants.23 Here, we introduce a method for direct conjugation of PBNPs with antibodies. The nanomaterial is characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Two sandwich NLISAs were developed for the detection of human serum albumin (HSA) in urine and the detection of bacterial pathogen Salmonella Typhimurium in milk and their performance was critically compared with the corresponding classic ELISA format using similar conjugates based on the natural HRP. In


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comparison with our previous reported competitive NLISA for HSA, the limit of detection was improved by three orders of magnitude.22 This demonstrates that antibody-conjugated PBNPs can be used as an enzyme replacement and provide a universal detection system for sandwich immunoassays.

MATERIAL AND METHODS Chemicals and Reagents Bovine γ-globulin (BGG), bovine serum albumin (BSA), HSA, HRP, iron(III) chloride hexahydrate, poly-L-lysine

hydrobromide

(30–70 kDa),

sodium

azide,

sodium

borohydride,

sodium

cyanoborohydride, sodium periodate, 3,3′,5,5′- tetramethylbenzidine (TMB), Tris, and Tween 20 were obtained from Sigma-Aldrich (USA). EDTA, glucose, and potassium ferrocyanide trihydrate were purchased from Lachema (Czech Republic). Poly(vinyl alcohol) (6 kDa) was purchased from Polysciences (USA). All other common chemicals were obtained in the highest quality available from Penta (Czech Republic). Phosphate buffer (PB, 50 mM NaH2PO4/Na2HPO4, pH 7.4), phosphate buffered saline (PBS, PB containing 150 mM NaCl), washing buffer (PB, 0.01% Tween 20, 0.05% NaN3), and assay buffer (0.2% BSA, 0.5% BGG, 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.2% PVA (w/w), 1% glucose, 0.05% NaN3, 0.01% Tween 20, pH 7.5) were used throughout the work. For the detection of HSA, mouse monoclonal antibody (mAb) clone AL-01 (Exbio, Czech Republic) and FITC-labeled swine polyclonal antibody (pAb-F; Sevapharma, Czech Republic) were used. The detection of Salmonella was based on mouse mAb ab8274 (Abcam, UK) and rabbit pAb 8209–4006 (AbD Serotec, UK).

Preparation of Antibody-Modified Prussian Blue Nanoparticles PBNPs and denatured BSA (dBSA) were prepared according to our previous work.22 Briefly, PBNPs were synthesized by mixing 20 mL of aqueous solution of 1 mM K4[Fe(CN)6] and 25 mM citric acid with 20 mL of 1 mM FeCl3 and 25 mM citric acid, which was preheated to 55 °C. The resulting bluecolored PBNP dispersion was stirred for 10 min and cooled down to room temperature. The molar concentration of the synthesized PBNPs was 33 nM; this was based on the amounts of Fe2+ and Fe3+ ions used in the synthesis and the average nanoparticle size determined by TEM. For the preparation of dBSA, 10 mg of BSA were reduced by 0.7 mg of NaBH4 in 3.1 mL of water and shaken for 60 min at room temperature. In order to decompose the excess of NaBH4, the mixture was heated to 80 °C for 30 min until the bubbles of H2 were no longer formed. Thus prepared dBSA was mixed in a ratio of 10:1 with phosphate buffer (0.5 M, pH 8.0) and stored at −20 °C. The dBSA-modified PBNPs (PBNP-dBSA) were prepared by mixing PBNPs (33 nM) and dBSA (0.36 mg mL−1) in a volume ratio of 1:1 with subsequent heating to 70 °C for 5 min.



To prepare the antibody-modified PBNPs, the pAb-F (alternatively pAb Serotec in case of the Salmonella assay) was diluted in 10 mM PBS to the concentration of 3 mg mL−1. The antibody was oxidized by the addition of 100 µL of 0.1 M NaIO4 per 1 mL of antibody solution and incubated at room temperature for 30 min under shaking (Thermomixer Comfort, Eppendorf, Germany). Subsequently, the oxidized pAb-F was purified with the Microcon centrifugal unit YM-10 (10 kDa MWCO; Merck Millipore, USA). The oxidized antibody was diluted in 10 mM PBS to the concentration of 0.2 mg mL−1, mixed with 16.5 nM PBNP-dBSA conjugate solution in volume ratio of 1:1 and incubated at room temperature for 2 hours followed by incubation at 4 °C overnight. The formed Schiff base was reduced by the addition of 50 µL of 1 M NaBH3CN per 1 mL of solution and incubated for 4 hours at room temperature. The free reactive groups were blocked by the addition of 50 µL of 1 M Tris-HCl per 1 mL of solution and mixed at room temperature for 30 min. The final PBNP-Ab conjugates were stored at 4 °C. The synthesis of PBNP-Ab conjugates is summarized in Figure 1.

Figure 1: Scheme of PBNP-Ab conjugate synthesis. The PBNPs are modified by denatured BSA to introduce amino-groups on their surface and the oxidized antibody is conjugated via the aldehyde groups.


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Characterization of Nanoparticles Samples of PBNPs and their conjugates were diluted tenfold with PBS and hydrodynamic diameters were estimated from dynamic light scattering utilizing Zetasizer Nano ZS (Malvern, United Kingdom). For the characterization of PBNPs by AFM, the mica (Mica Grade V-1 Muscovite, SPI Supplies, USA) was cleaned with adhesive tape and impurities from surface were removed by compressed air. A droplet of poly-L-lysine (20 µL of 10 µg mL−1 solution in 0.1 M PB, pH 6.9) was deposited on mica for 20 min at room temperature, washed by distilled water and dried. The sample of PBNPs (15 µL) was incubated on mica surface for 20 min, washed and scanned in dry state by AFM microscope Dimension Fast Scan with Fast Scan-A probe (Bruker, USA), the images were evaluated in software Gwyddion. For the TEM characterization, the dispersion of nanomaterial (~4 µL) was deposited onto 400mesh copper EM grids coated with a continuous carbon layer. Dried grids were imaged by transmission electron microscopy (Tecnai F20, FEI, USA). The dimensions of individual particles were analyzed using ImageJ software (National Institutes of Health, USA).

Detection of Human Serum Albumin Sandwich assay based on antibody modified PBNPs as a label was developed for the detection of human serum albumin (HSA) in urine (Figure 2). Due to the presence of HSA in urine from healthy patients,24, 25 the urine was first deproteinized using the Microcon centrifugal unit YM-10 and then spiked with known HSA concentrations. Nunc MaxiSorp 96-well microtiter plates (Thermo Fisher Scientific, USA) were coated with monoclonal anti-HSA antibody AL-01 (4 µg mL−1 in PBS, 100 µL per well) and incubated overnight at 4 °C. All following steps were carried out at room temperature; after each step, the plate was washed four times with 200 µL of the washing buffer. The plate was blocked with 200 µL of 5% powdered milk (Laktino, Czech Republic) in PBS for 60 min. Then, either standard dilutions of HSA in assay buffer or spiked and ten times diluted urine samples (1 ng mL−1 – 100 µg mL−1 of HSA) were added (100 µL per well) and incubated for 60 min. Subsequently, PBNPpAb-F conjugates in the assay buffer were added (750 pM, 100 µL per well) and incubated for 60 min. Finally, the color was developed by the addition of freshly prepared substrate solution (0.5 mM TMB and 125 mM H2O2 in 200 mM sodium acetate pH 3.5, 100 µL per well) and absorbance at 652 nm (A652) was measured after 120 min using Synergy 2 reader (BioTek, USA). Alternatively, the reaction was stopped by the addition of 100 µL of 1 M H2SO4 and the absorbance was read at 450 nm (A450).



Figure 2: Scheme of PBNP-based sandwich NLISA. The microtiter plate is coated by capture antibody (Ab1), followed by specific binding of antigen (Ag) and formation of sandwich with the detection PBNP-Ab2 label. The colorimetric readout is based on PBNP-catalyzed oxidation of TMB in the presence of H2O2.

Furthermore, the classic sandwich ELISA was developed and optimized to provide the comparison of NLISA with standard approach. The conjugation of detection pAb-F with HRP was done according to our previous work.26 The assay protocol remained the same as in the case of NLISA except the detection conjugate binding and biocatalytic step. The 100 µL of HRP-pAb-F conjugate (concentration equivalent to 2.5 µg mL–1 of the antibody) was allowed to bind for 60 min followed by the addition of commercial TMB substrate solution (Enzymo Plus, Czech Republic). The color change was read after 30 min as the A652; the end-point measurement at 450 nm was carried out as well after the addition of 100 µL of 1 M H2SO4.


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Detection of Salmonella Typhimurium Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028) was obtained from the Czech Collection of Microorganisms (CCM, Czech Republic). The previously optimized sample preparation protocol that yields Salmonella with high affinity to the used antibodies was employed.27 The stock solution of Salmonella (100 µL) was inoculated into 25 mL of low salt LB broth (Duchefa Biochemie, Netherlands) in Erlenmeyer flask and incubated aerobically overnight (37 °C, mild shaking). The bacterial suspension was centrifuged twice for 10 min at 6 800 g and resuspended in PBS. The microorganism samples were treated by heat at 80 °C for 30 min with mild shaking (900 rpm) using Thermomixer Comfort. Selected samples were also homogenized using sonicator Q700 (Qsonica, USA). The 1.6 mm Microtip probe was placed into microtube with 1 mL of Salmonella sample and amplitude of 70 % was maintained for approximately 10 min until the total energy consumed by the sample reached 1 kJ. The concentrations of thus treated bacteria are expressed as CFU mL−1 corresponding to the initial amount of viable cells. The real samples were prepared by dissolving the powdered milk using the assay buffer to the concentration of 1% and spiking it with varying Salmonella concentrations. For the Salmonella detection, the surface of the microtiter plate was coated by monoclonal antiSalmonella antibody Abcam ab8274 (2 µg mL−1) and the detection label consisted of PBNPs modified by polyclonal antibody Serotec 8209-4006. Other assay steps remained the same as in the case of HSA detection. The reference ELISA experiments were performed utilizing the same experimental conditions, the only difference was use of the conjugate of HRP with polyclonal antibody Serotec at concentration of 2.5 µg mL–1.

Data Analysis For each concentration, the mean and standard deviation were calculated from three replicate wells. A four-parameter logistic function was used for a regression analysis of the calibration curves:

y=

yMAX − yBG  c   1 +   EC50 

s

+ yBG

where c is the concentration of either HSA or Salmonella, and y is the measured absorbance at 652 nm (A652). The equation yields as parameters the maximum (yMAX) and background (yBG) signals, the concentration that reduced (yMAX – yBG) by 50 % (EC50) and the slope at the inflection point (s). The limit of detection (LOD) was calculated from the regression curve as the concentration corresponding to the yLOD value:

yLOD = yBG + 3 s0 7 ACS Paragon Plus Environment


where s0 is the standard deviation of blank measurement (no analyte present in the sample).

RESULTS AND DISCUSSION Characterization of Prussian Blue Nanoparticles and their Conjugates For the first time, we describe the direct covalent conjugation of PBNPs with antibodies. The employed synthetic method provided PBNPs with cubic shape; the nanoparticles were characterized by TEM, AFM and DLS. The average size estimated using AFM was 22.1 ± 2.9 nm (Figure 3A), the size determined by from TEM was 15.3 ± 3.4 nm (Figure 3C) and DLS revealed hydrodynamic diameter of 24 nm with polydispersity index 0.213 (Figure 3D). The surface of PBNPs was modified with reductively denatured bovine serum albumin and as we reported previously, the attachment of dBSA was accompanied by slight aggregation of PBNPs.22 The size of these aggregates estimated by AFM was 107 ± 20 nm; DLS provided the hydrodynamic diameter of 92 nm with polydispersity index of 0.222. Thus prepared PBNP-dBSA were conjugated with antibodies by the reductive amination method28 resulting in the PBNP-Ab tracer. The size of the final PBNP-Ab conjugate provided by AFM was 116 ± 29 nm (Figure 3B), the hydrodynamic diameter was 128 nm with polydispersity index 0.232.

Figure 3: AFM images of (A) PBNPs (average diameter 22.1 ± 2.9 nm) and (B) PBNPdBSA-Ab (116 ± 29 nm) conjugates. (C) TEM image of PBNPs (15.3 ± 3.4 nm). (D) DLS of 8 ACS Paragon Plus Environment

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PBNPs (hydrodynamic diameter ⌀HD: 24 nm); PBNP-dBSA (⌀HD: 92 nm) and PBNP-dBSAAb (⌀HD: 128 nm).

Design of Nanozyme-Linked Immunosorbent Assay (NLISA) The synthesized conjugates of PBNPs with anti-HSA antibody were employed in the sandwich immunoassay for HSA. Due to the simple conjugation and shorter analysis time (less incubation steps), PBNPs conjugated with detection antibody were used as tracer directly instead of the universal biotin labeling protocol, which is also possible but prolongs the assay.22 The assay procedure was based on two anti-HSA antibodies – monoclonal AL-01 and polyclonal pAb-F. As expected, the most efficient binding was observed when using the mAb for the analyte capture and pAb as the label conjugated on the PBNPs (Figure S1 in the Supporting Information). This arrangement allows oriented binding of the analyte on the surface and reduces probability that the label could not bind to the analyte due to the inaccessible binding site. Furthermore, binding of HSA via the capture antibody enhanced its accessibility for the PBNP-Ab tracer, which resulted in higher signals in sandwich compared to the direct assay with HSA coated on the microtiter plate surface. The coating concentration and blocking conditions were optimized in order to achieve high sensitivity and wide working range. Unlike the competitive assays where the coating concentration needs to be fine-tuned to achieve a compromise between efficient competition and measured signal intensity,29 the sandwich format generally provides higher responses for higher coating concentrations. In agreement with theory, the measured signals were increasing with increasing coating concentration (Figure 4A). However, the background was also increasing proportionally, which lead to a constant signal-to-background ratio of 3–4:1 that did not depend significantly on the coating level. The coating from higher part of the tested scale (4 µg mL−1) was selected since the higher signals in general allow readout less prone to instrumental and pipetting errors. This leads to smaller standard deviations and hence better LOD values. The effect of different blocking conditions on the assay performance is shown in Figure S2A. Compared to the blocking with pure BSA (10 mg mL−1 in PBS), the more complex solutions of milk (50% in PBS) and powdered milk (5% in PBS) provided lower degree of non-specific binding. The powdered milk was selected due to slightly lower background and expectable higher reproducibility.



Figure 4: The optimizations of NLISA for HSA detection. (A) Optimization of coating concentration of primary anti-HSA mAb. (B) Different concentrations of PBNP-Ab conjugate are used to generate the signal in case of plate coated by 1 µg mL−1 of capture mAb. (C) Optimization of substrate concentration. The empty triangles always represent the LOD values.

During preparation, the centrifugation was used to conveniently separate PBNP-Ab conjugates from the unbound antibody that might potentially compete with the conjugate for the binding on HSA. The comparison of centrifuged and uncentrifuged conjugates of two PBNP-Ab samples (based on two different antibody oxidation times) is shown in Figure S2B. For both conjugates, significant signal decrease was observed after the centrifugation. Even though high centrifugation speed (14 000 g) and long centrifugation time (30 min) were used, substantial amount of PBNPs remained in the supernatant, while the sedimented particles were rather difficult to resuspend. On the other hand, no significant interference from the unseparated free antibody was observed in the case of uncentrifuged particles, therefore this sample was used for the further immunoassays. The concentration of detection PBNP-Ab conjugate substantially influenced the overall measured signals (Figure 4B), however it did not affect the signal-to-background ratio, as there were more particles that could bind non-specifically to the surface. Unlike enzymes, the nanoparticle labels catalyze conversions of high concentrations of substrate for longer times; such conditions usually cause denaturation of natural biocatalysts. The optimization of substrate concentration is shown in Figure 4C, the time dependency of the product formation is shown in Figure S3. The readout after 120 min was selected as a compromise between the overall measured signal and assay time requirements. Stopping the reaction by H2SO4 provided higher absorbance values, however, it did not affect the LOD values as the errors were increased proportionally (Figure S4). To evaluate the reproducibility, the assay was performed multiple times with different batches of PBNP-Ab conjugate. Only small variations in the LOD (1.3 – 4.0 ng mL−1) and EC50 (28.3 – 77.3 ng mL−1) values were observed (Figure S5), which confirms the high reliability of both conjugate preparation and assay itself; the accuracy of results is further ensured by measuring the calibration curve as a part of each microtiter plate with unknown samples. The ability to analyze real samples was demonstrated on the detection of HSA in human urine. Because of the high sensitivity of the developed NLISA and trace amounts of HSA in urine from


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healthy patients, the HSA was first removed from the urine sample by filtration through a 10 kDa centrifugal microfilter, followed by spiking of known HSA concentrations. The calibration curve comparing HSA detection in buffer and urine is shown in Figure 5A. Possible interferences from various ions present in urine, which might potentially catalyze the substrate conversion, are suppressed by the washing step; therefore no large difference in the response was observed when analyzing HSA in buffer and in the urine samples. To compare the capabilities of PBNP- and enzyme-based labels, the ELISA based on same combination of antibodies was developed, optimized (Figure S6) and employed for the analysis of HSA in spiked urine (Figure 5B). The LOD of NLISA (1.2 ng mL−1) was slightly better than in the case of ELISA (3.7 ng mL−1). The similar results suggest that the assay is limited mainly by the affinity of the selected antibodies towards the target analyte and not by the detection step. However, the catalytic nanoparticles provide several practical advantages compared to the enzymatic label, such as simple and cheap preparation, higher stability and possibility to function at much higher substrate concentrations. The PBNPs alone provide practically unlimited shelf-life (several years at 4 °C), the stability-limiting element is the antibody as the biorecognition molecule. Further enhancements of the label stability could be achieved by functionalizing the PBNPs by artificial aptamers or molecularly imprinted polymers. The detailed comparison of NLISA and ELISA with respect to properties and preparation of catalytic label and assay performance is shown in Table S1. The overall analysis time was 4 h starting from the coated and blocked plate. The concentration range typical for microalbuminuria, which is a diagnostic hallmark for diabetic nephropathy, is from 20 to 200 µg mL−1 in the 24 h specimens. The optimized assay provided LOD of 1.2 ng mL−1 of HSA in urine with working range up to 1 µg mL−1. Therefore, the parameters of developed NLISA are sufficient to discriminate the positive microalbuminuria samples.30 The achieved detection limit is better than values reported for competitive ELISA (LOD 89 ng mL−1),31 competitive fluoroimmunoassays (LOD 1 µg mL−1),32 competitive NLISA (LOD 0.27 µg mL−1),22 method based on specific fluorescence probe (LOD 0.4 µg mL−1),33 immunonephelometry (LOD 2 µg mL−1),34 immunoturbidimetry (LOD 6 µg mL−1),34 and the semiquantitative dipstick method (LOD 10 µg

mL−1).35

The

developed

sandwich

NLISA

−1 34

provided

LOD −1 36

comparable

to

radioimmunoassay (LOD 16 ng mL ) and sandwich ELISA (LOD 6.25 ng mL ) as summarized in Table S2.



Figure 5: (A) Calibration curve of sandwich NLISA for the detection of HSA in buffer (LOD 1.3 ng mL−1; EC50 57 ng mL−1) and spiked urine (LOD 1.2 ng mL−1; EC50 43 ng mL−1) under the optimized conditions (4 µg mL−1 coating, 750 pM PBNP-Ab). (B) Calibration curve of standard ELISA for the HSA detection in buffer (LOD 5.3 ng mL−1; EC50 142 ng mL−1) and spiked urine (LOD 3.7 ng mL−1; EC50 152 ng mL−1). The empty triangles represent the LOD values.

Detection of Salmonella Typhimurium To demonstrate the universal applicability of PBNPs as labels in immunoassays, Salmonella Typhimurium was detected as a representative example of large microbial antigen. The assay was optimized based on the findings obtained during the detection of HSA, the optimization of coating concentration is shown in Figure S7A. Also in this case, the coating concentration influenced the total measured signals but did not have effect on the signal-to-background ratio. It was previously shown that the selected antibodies exhibit higher affinity towards heat-treated samples compared to the native Salmonella,27 therefore heat-treatment and sonication were tested for the sample preparation (Figure S7B). The sonicated Salmonella provided wider working range compared to the heat-treated sample, however similar LOD values were achieved. The heat-treatment is the preferable way of sample preparation due to lower requirements on the equipment and time. Detection in powdered milk (diluted by assay buffer) spiked with the microbe was carried out to demonstrate the ability of NLISA to detect Salmonella in real samples (Figure 6A). The presence of milk matrix during the Salmonella binding step served for further blocking of the surface and allowed to decrease the level of non-specific binding with the minimum effect on the LOD. The optimized assay provided LOD of 6·103 CFU mL−1 in powdered milk with working range up to 106 CFU mL−1. The obtained results are better than values for traditional ELISAs that provide typical


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LODs in the range between 104 and 106 CFU mL−1.37-39 The sandwich ELISA developed here utilizing the same combination of capture and detection antibodies provided LOD of 3·103 CFU mL−1 in powdered milk (Figure 6B). This is in agreement with the results for the detection of HSA, which suggest that the affinity of antibodies has more significant impact on the LOD than the used label. Another assay scheme based on direct ELISA was also tested and compared (Figure S8), however, the LOD did not improve compared to the sandwich arrangement.

Figure 6: (A) Calibration curve for the NLISA-based detection of Salmonella Typhimurium in buffer (LOD 2·103 CFU mL−1; EC50 4·104 CFU mL−1) and spiked powdered milk (LOD 6·103 CFU mL−1; EC50 5·104 CFU mL−1). (B) Calibration curve for the ELISA-based detection of Salmonella Typhimurium in buffer (LOD 3·103 CFU mL−1; EC50 9·104 CFU mL−1) and spiked powdered milk (LOD 3·103 CFU mL−1; EC50 1·105 CFU mL−1). The empty triangles represent the LOD values. The LOD can be further improved by a shortened cultivation-based pre-enrichment; however, such assays have higher demands on the analysis time (~ day instead of several hours).40 A faster way to concentrate the analyte and separate it from a complex matrix is immunomagnetic separation. This approach is often used in biosensor-based detection.41 It is not regularly used in immunoassays, though microfluidics-based ELISA utilizing magnetic beads was recently reported for the detection of Creactive protein.42 Various nanoparticle-based approaches were recently reported to improve the properties of traditional ELISA for the Salmonella detection.43 One of the most popular ways is to improve the catalytic properties of the label. For example, conjugation of detection antibody and HRP on singlewalled carbon nanotubes44 allowed to reach LOD of 103 and 104 CFU mL−1 for direct and sandwich assay, respectively. This method provided higher catalytic activities than simple conjugates of HRP with antibody, however it was still limited by the instability of the enzyme label. The PBNPs, on the



other hand, provide not only comparable LOD, but can be used to catalyze higher substrate concentrations (allowing to reach higher measured signals) and are significantly more stable (which provides longer shelf-life of the label). The comparison of immunoassays for Salmonella detection without pre-enrichment steps is shown in Table S3.

CONCLUSIONS The straightforward method for conjugation of Prussian blue nanoparticles with antibodies was developed and applied in the Nanozyme-Linked Immunosorbent Assay. The assay protocol is based on the oxidation of TMB forming blue product, therefore common colorimetric reader can be used without special requirements on the instrumentation. The universal applicability of PBNPs as labels is demonstrated by the development of two sandwich immunoassays. Human serum albumin was detected in urine for the diagnosis of albuminuria. The assay provided LOD of 1.2 ng mL−1 and working range of three orders of magnitude up to 1 µg mL−1. NLISA was also employed for the detection of bacterial pathogen Salmonella Typhimurirum in powdered milk with LOD of 6·103 CFU mL−1 and working range up to 106 CFU mL−1. For a comparison, the same antibodies were also used in standard sandwich ELISA formats using natural HRP enzyme. Comparable assay parameters were achieved, which suggests that the assay is limited mainly by the affinity of the antibodies towards the target analyte and not by the detection step. However, PBNPs provide several practical advantages compared to the enzymatic label, such as easy synthesis using cheap precursors and higher stability. The results achieved in the NLISA are also highly comparable with other recent nanoparticle-based immunoassays, including those based on gold nanoparticles, quantum dots and carbon nanotubes (Table S3).

ASSOCIATED CONTENT Supporting Information Optimization of NLISA assay, reference ELISA measurements, comparison of assay parameters with literature.

AUTHOR INFORMATION ORCID Zdeněk Farka: 0000-0002-6842-7081


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Antonín Hlaváček: 0000-0003-3358-3858 Petr Skládal: 0000-0002-3868-5725

Author Contributions Z.F. and V.Č. contributed equally to the work.

ACKNOWLEDGEMENTS We thank Miroslav Peterek for taking the TEM images. CIISB research infrastructure project LM2015043 funded by MEYS CR is gratefully acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography and CF Nanobiotechnology. This research has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601) and by Grant Agency of the Czech Republic (P20612G014 GACR).

REFERENCES 1. 2.

Wild, D., The Immunoassay Handbook, Fourth Edition. Elsevier: Oxford, 2013. Lavery, C. B.; MacInnis, M. C.; MacDonald, M. J.; Williams, J. B.; Spencer, C. A.; Burke, A. A.; Irwin, D. J. G.; D'Cunha, G. B., Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots. J. Agric. Food Chem. 2010, 58 (15), 8471-8476. 3. Wei, H.; Wang, E. K., Nanomaterials with enzyme-like characteristics (nanozymes): nextgeneration artificial enzymes. Chem. Soc. Rev. 2013, 42 (14), 6060-6093. 4. Kuah, E.; Toh, S.; Yee, J.; Ma, Q.; Gao, Z. Q., Enzyme Mimics: Advances and Applications. Chem. Eur. J. 2016, 22 (25), 8404-8430. 5. Ragg, R.; Tahir, M. N.; Tremel, W., Solids Go Bio: Inorganic Nanoparticles as Enzyme Mimics. Eur. J. Inorg. Chem. 2016, 2016 (13-14), 1906-1915. 6. Nasir, M.; Nawaz, M. H.; Latif, U.; Yaqub, M.; Hayat, A.; Rahim, A., An overview on enzymemimicking nanomaterials for use in electrochemical and optical assays. Microchim. Acta 2017, 184 (2), 323-342. 7. Kumari, S.; Dhar, B. B.; Panda, C.; Meena, A.; Sen Gupta, S., Fe-TAML Encapsulated Inside Mesoporous Silica Nanoparticles as Peroxidase Mimic: Femtomolar Protein Detection. ACS Appl. Mater. Interfaces 2014, 6 (16), 13866-13873. 8. Walt, D. R., Optical Methods for Single Molecule Detection and Analysis. Anal. Chem. 2013, 85 (3), 1258-1263. 9. Shokouhimehr, M.; Soehnlen, E. S.; Khitrin, A.; Basu, S.; Huang, S. P. D., Biocompatible Prussian blue nanoparticles: Preparation, stability, cytotoxicity, and potential use as an MRI contrast agent. Inorg. Chem. Commun. 2010, 13 (1), 58-61. 10. Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.; Feng, J.; Yang, D. L.; Perrett, S.; Yan, X., Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2 (9), 577-583.



11. Xu, C.; Qu, X. G., Cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications. NPG Asia Mater. 2014, 6, 16. 12. Chen, W.; Chen, J.; Liu, A. L.; Wang, L. M.; Li, G. W.; Lin, X. H., Peroxidase-Like Activity of Cupric Oxide Nanoparticle. ChemCatChem 2011, 3 (7), 1151-1154. 13. Dong, J. L.; Song, L. N.; Yin, J. J.; He, W. W.; Wu, Y. H.; Gu, N.; Zhang, Y., Co3O4 Nanoparticles with Multi-Enzyme Activities and Their Application in Immunohistochemical Assay. ACS Appl. Mater. Interfaces 2014, 6 (3), 1959-1970. 14. He, W. W.; Liu, Y.; Yuan, J. S.; Yin, J. J.; Wu, X. C.; Hu, X. N.; Zhang, K.; Liu, J. B.; Chen, C. Y.; Ji, Y. L.; Guo, Y. T., Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays. Biomaterials 2011, 32 (4), 1139-1147. 15. Liu, X.; Wang, Q.; Zhao, H. H.; Zhang, L. C.; Su, Y. Y.; Lv, Y., BSA-templated MnO2 nanoparticles as both peroxidase and oxidase mimics. Analyst 2012, 137 (19), 4552-4558. 16. Song, Y. J.; Qu, K. G.; Zhao, C.; Ren, J. S.; Qu, X. G., Graphene Oxide: Intrinsic Peroxidase Catalytic Activity and Its Application to Glucose Detection. Adv. Mater. 2010, 22 (19), 22062210. 17. Qu, R.; Shen, L. L.; Chai, Z. H.; Jing, C.; Zhang, Y. F.; An, Y. L.; Shi, L. Q., Hemin-Block Copolymer Micelle as an Artificial Peroxidase and Its Applications in Chromogenic Detection and Biocatalysis. ACS Appl. Mater. Interfaces 2014, 6 (21), 19207-19216. 18. Kitagawa, S.; Kitaura, R.; Noro, S., Functional porous coordination polymers. Angew. Chem. Int. Ed. 2004, 43 (18), 2334-2375. 19. Herren, F.; Fischer, P.; Ludi, A.; Halg, W., Neutron-Diffraction Study of Prussian Blue, Fe4[Fe(CN)6]3.xH2O - Location Of Water-Molecules And Long-Range Magnetic Order. Inorg. Chemistry 1980, 19 (4), 956-959. 20. Zhang, X. Q.; Gong, S. W.; Zhang, Y.; Yang, T.; Wang, C. Y.; Gu, N., Prussian blue modified iron oxide magnetic nanoparticles and their high peroxidase-like activity. J. Mater. Chem. 2010, 20 (24), 5110-5116. 21. Zhang, W. M.; Ma, D.; Du, J. X., Prussian blue nanoparticles as peroxidase mimetics for sensitive colorimetric detection of hydrogen peroxide and glucose. Talanta 2014, 120, 362-367. 22. 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), 651-658. 23. Jackson, T. M.; Ekins, R. P., Theoretical Limitations on Immunoassay Sensitivity - Current Practice and Potential Advantages of Fluorescent Eu3+ Chelates as Nonradioisotopic TracersTheoretical Limitations on Immunoassay Sensitivity - Current Practice and Potential Advantages of Fluorescent Eu3+ Chelates as Nonradioisotopic Tracers. J. Immunol. Methods 1986, 87 (1), 13-20. 24. Datta, P.; Dasgupta, A., An Improved Microalbumin Method (mu ALB_2) with Extended Analytical Measurement Range Evaluated on the ADVIA (R) Chemistry Systems. J. Clin. Lab. Anal. 2009, 23 (5), 314-318. 25. Tsai, J. Z.; Chen, C. J.; Settu, K.; Lin, Y. F.; Chen, C. L.; Liu, J. T., Screen-printed carbon electrode-based electrochemical immunosensor for rapid detection of microalbuminuria. Biosens. Bioelectron. 2016, 77, 1175-1182. 26. Farka, Z.; Juřík, T.; Pastucha, M.; Skládal, P., Enzymatic Precipitation Enhanced Surface Plasmon Resonance Immunosensor for the Detection of Salmonella in Powdered Milk. Anal. Chem. 2016, 88 (23), 11830-11836. 27. Farka, Z.; Juřík, T.; Pastucha, M.; Kovář, D.; Lacina, K.; Skládal, P., Rapid Immunosensing of Salmonella Typhimurium Using Electrochemical Impedance Spectroscopy: the Effect of Sample Treatment. Electroanalysis 2016, 28 (8), 1803-1809. 28. Algar, W. R.; Prasuhn, D. E.; Stewart, M. H.; Jennings, T. L.; Blanco-Canosa, J. B.; Dawson, P. E.; Medintz, I. L., The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry. Bioconjugate Chem. 2011, 22 (5), 825-858. 29. Hlaváček, A.; Farka, Z.; Hübner, M.; Horňáková, V.; Němeček, D.; Niessner, R.; Skládal, P.; Knopp, D.; Gorris, H. H., Competitive Upconversion-Linked Immunosorbent Assay for the Sensitive Detection of Diclofenac. Anal. Chem. 2016, 88 (11), 6011-6017.


Page 16 of 18

Page 17 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30. Doumas, B. T.; Peters, T., Serum and urine albumin: A progress report on their measurement and clinical significance. Clin. Chim. Acta 1997, 258 (1), 3-20. 31. 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), 199-207. 32. Hlaváček, A.; Bouchal, P.; Skládal, P., Biotinylation of quantum dots for application in fluoroimmunoassays with biotin-avidin amplification. Microchimica Acta 2012, 176 (3-4), 287293. 33. Kessler, M. A.; Meinitzer, A.; Petek, W.; Wolfbeis, O. S., Microalbuminuria and borderlineincrease albumin excretion determined with a centrifugal analyzer and the Albumin Blue 580 fluorescence assay. Clin. Chem. 1997, 43 (6), 996-1002. 34. Busby, D. E.; Bakris, G. L., Comparison of Commonly Used Assays for the Detection of Microalbuminuria. J. Clin. Hypertens. 2004, 6, 8-12. 35. Marshall, S. M.; Shearing, P. A.; Alberti, K., Micral-Test Strips Evaluated for Screening for Albuminuria. Clin. Chem. 1992, 38 (4), 588-591. 36. 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), 1544-1548. 37. 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), 4933-4939. 38. Roda, A.; Mirasoli, M.; Roda, B.; Bonvicini, F.; Colliva, C.; Reschiglian, P., Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection. Microchim. Acta 2012, 178 (1-2), 7-28. 39. 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), 5281-5292. 40. Wu, X. L.; Wang, W. B.; Liu, L. Q.; Kuang, H.; Xu, C. L., Monoclonal antibody-based crossreactive sandwich ELISA for the detection of Salmonella spp. in milk samples. Anal. Methods 2015, 7 (21), 9047-9053. 41. Liebana, S.; Brandao, D.; Alegret, S.; Pividori, M. I., Electrochemical immunosensors, genosensors and phagosensors for Salmonella detection. Anal. Methods 2014, 6 (22), 8858-8873. 42. Liu, D.; Li, X. R.; Zhou, J. K.; Liu, S. B.; Tian, T.; Song, Y. L.; Zhu, Z.; Zhou, L. J.; Ji, T. H.; Yang, C. Y., A fully integrated distance readout ELISA-Chip for point-of-care testing with sample-in-answer-out capability. Biosens. Bioelectron. 2017, 96, 332-338. 43. Farka, Z.; Juřík, T.; Kovář, D.; Trnková, L.; Skládal, P., Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem. Rev. 2017, 117 (15), 997310042. 44. 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), 3584-3589.



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Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich

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