Supplementary Information

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Supplementary Information

Bioinspired Artificial “Clickase” for Catalytic Click Immunoassay of Foodborne Pathogens

Xianlong Zhang

^a

, Yongning Wu

^a,b

, Juhong Chen

^c

, Yan Yang

^a

and Guoliang Li

^a*

a

School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China

b

NHC Key Laboratory of Food Safety Risk Assessment, Food Safety Research Unit (2019RU014) of Chinese Academy of Medical Science, China National Center for Food Safety Risk Assessment, Beijing, 100021, China

c

Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA

AUTHOR INFORMATION

E-mail: [email protected] (Guoliang Li); ORCID: 0000-0001-7951-9750

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Figure

Figure S1. Optimization of synthesis time and temperature of CCN clickase through a response surface methodology.

Figure S2. The CCN clickase synthesis using various concentrations of aqueous GSH solution.

Figure S3. SEM images of CCN clickases.

Figure S4. XRD patterns of CCN clickases and GSH.

Figure S5. N₂ adsorption-desorption curves of CCN clickases. (The black is adsorption curves and the red is desorption curves).

Figure S6. Pore size distribution of the dried CCN clickases from the N₂ adsorption.

Figure S7. XPS fully scanned spectrum of the CCN clickases.

Figure S8. (A) The click reaction of Azide 1 and Alkyne 2 catalyzed by CCN clickases.

Fluorescence spectroscopy of the Azide 1, Alkyne 2, the mixture of Azide 1 and Alkyne 2, and the mixture of Azide 1 and Alkyne 2 in the presence of CCN clickases.

Figure S9. Optimization of the substrate concentrations (Azide 1 and Alkyne 2) in the CuAAC reaction catalyzed by CCN clickases.

Figure S10. Effects of different molar ratios of GSH/Cu²⁺ on catalytic activity of CCN clickases.

Figure S11. Effects of various temperature and pH on the catalytic activity of CCN clickases.

Figure S12. Effects of storage time on the catalytic activity of CCN clickases.

Figure S13. Recyclability of CCN clickases in the CuAAC reaction after recycling and reusing.

Figure S14. (a) CCN clickases catalyzed the click reaction between Azide 1 and Alkyne 2. (b) Possible catalytic mechanism of the CCN clickases-catalyzed click reaction, including (ⅰ) coordination of Alkyne 2 onto the surface of CCN clickases; (ⅱ) the deprotonation of Alkyne 2 for the formation of the CCN clickases-Cu(Ⅰ) intermediate; (ⅲ) the nucleophilic attack from the CCN clickase-Cu(Ⅰ) intermediate to Azide 1 followed by the coordination of Azide 1 onto the surface of CCN clickases; (ⅳ) cycloaddition reaction; (ⅴ) C-Cu bond protonation; (ⅵ) the produced product (Triazole 3) desorption.

Figure S15. (A) Fluorescence intensity at 395 nm in the immunoassays based on clickase@Ab2 containing various amounts of Ab2.

Figure S16. Effects of capture antibody concentration (Ab1) on the CNN clickase-based catalytic

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click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

Figure S17. Effects of blocking agent amount on the CNN clickase-based catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

Figure S18. Effects of clickase@Ab2 concentration on the CNN clickase-based catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

Figure S19. Optimization of the incubation time during the catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

Figure S20. Optimization of the catalytic click reaction time during the catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

Table

Table S1. The FT-IR spectral analysis results of CCN clickases and GSH.

Table S2. Comparison of the present study with the previously reported methods for the detections of S. Enteritidis.

Table S3. Results of the established method for the detection of S. Enteritidis in milk samples.

References

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Figure S1. Optimization of synthesis time and temperature of CCN clickase through a response surface methodology.

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Figure S2. The CCN clickase synthesis using the various concentrations of aqueous GSH solution.

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Figure S3. SEM images of CCN clickases.

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Figure S4. XRD patterns of CCN clickases and GSH.

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Figure S5. N₂ adsorption-desorption curves of CCN clickases. (The black is adsorption curves and the red is desorption curves).

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Figure S6. Pore size distribution of the dried CCN clickases from the N₂ adsorption.

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Figure S7. XPS fully scanned spectrum of the CCN clickases.

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Figure S8. (A) The click reaction of Azide 1 and Alkyne 2 catalyzed by CCN clickases.

Fluorescence spectroscopy of the Azide 1, Alkyne 2, the mixture of Azide 1 and Alkyne 2, and the mixture of Azide 1 and Alkyne 2 in the presence of CCN clickases.

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Figure S9. Optimization of the substrate concentrations (Azide 1 and Alkyne 2) in the CuAAC reaction catalyzed by CCN clickases.

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Figure S10. Effects of different molar ratios of GSH/Cu²⁺ on catalytic activity of CCN clickases.

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Figure S11. Effects of various temperature and pH on the catalytic activity of CCN clickases.

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Figure S12. Effects of storage time on the catalytic activity of CCN clickases.

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Figure S13. Recyclability of CCN clickases in the CuAAC reaction after recycling and reusing.

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Figure S14. (a) CCN clickases catalyzed the click reaction between Azide 1 and Alkyne 2. (b) Possible catalytic mechanism of the CCN clickases-catalyzed click reaction, including (ⅰ) coordination of Alkyne 2 onto the surface of CCN clickases; (ⅱ) the deprotonation of Alkyne 2 for the formation of the CCN clickases-Cu(Ⅰ) intermediate; (ⅲ) the nucleophilic attack from the CCN clickase-Cu(Ⅰ) intermediate to Azide 1 followed by the coordination of Azide 1 onto the surface of CCN clickases; (ⅳ) cycloaddition reaction; (ⅴ) C-Cu bond protonation; (ⅵ) the produced product (Triazole 3) desorption.

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Figure S15. (A) Fluorescence intensity at 395 nm in the immunoassays based on clickase@Ab2 containing various amounts of Ab2.

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Figure S16. Effects of capture antibody concentration (Ab1) on the CNN clickase-based catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

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Figure S17. Effects of blocking agent amount on the CNN clickase-based catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

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Figure S18. Effects of clickase@Ab2 concentration on the CNN clickase-based catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

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Figure S19. Optimization of the incubation time during the catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

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Figure S20. Optimization of the catalytic click reaction time during the catalytic click immunoassay with (the red is experimental group) and without (the black is control group) antigens.

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Table S1. The FT-IR spectral analysis results of CCN clickases and GSH.

Chemical bonds CCN clickases (cm^-1) GSH (cm^-1)

ν(-NH-) 3246.8 3149.4

ν(C-N) 1206.2 1201.2

ν(-SH) 2598.2 2608.1

ν(C=O) 1685.2 1674.1

ν(-NH₂) 3406.1 3364.2

ν(-OH) 2927.2 2885.3

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Table S2. Comparison of the present study with the previously reported methods for the detection of S. Enteritidis.

Analytical methods Target LOD Ref.

Magnetic nanoparticles-based immunochromatographic assay

S. Enteritidis 1.95×10⁵ ¹

Two-dimensional nanosheet-based immunochromatographic assay

S. Enteritidis 1×10³ ²

Surface-enhanced Raman scattering -based lateral flow strip biosensor

S. Enteritidis 2.7× 10¹ ³

Ampicillin-magnetite nanoparticles- based lateral flow immunoassay

S. Enteritidis 1×10² ⁴

Nanozyme enhanced colorimetric immunoassay

S. Enteritidis 3.4 ×10¹ ⁵

Gold nanoparticles-enabled colorimetric ELISA

S. Enteritidis 1.21×10¹ ⁶

CCN clickases-based fluorescent immunoassay

S. Enteritidis 1.18×10¹ This study

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Table S3. Results of the established method for the detection of S. Enteritidis in milk samples Food samples Spiked concentration

(CFU/mL)

Detected concentration (CFU/mL)

Recovery (%) RSD (%)

1 1×10² 121 121.0 0.35

2 1×10³ 1035 103.5 1.22

3 1×10⁴ 9653 96.5 3.17

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References

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(3) Liu, H.-b.; Du, X.-j.; Zang, Y.-X.; Li, P.; Wang, S. SERS-Based Lateral Flow Strip Biosensor for Simultaneous Detection of Listeria monocytogenes and Salmonella enterica Serotype Enteritidis. J. Agric. Food Chem. 2017, 65, 10290-10299.

(4) Bu, T.; Yao, X.; Huang, L.; Dou, L.; Zhao, B.; Yang, B.; Li, T.; Wang, J.; Zhang, D. Dual recognition strategy and magnetic enrichment based lateral flow assay toward Salmonella enteritidis detection. Talanta 2020, 206, 120204. DOI: 10.1016/j.talanta.2019.120204.

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(6) Gao, B.; Chen, X.; Huang, X.; Pei, K.; Xiong, Y.; Wu, Y.; Duan, H.; Lai, W.; Xiong, Y.

Urease-induced metallization of gold nanorods for the sensitive detection of Salmonella enterica Choleraesuis through colorimetric ELISA. J. Dairy Sci. 2019, 102, 1997-2007.