Multi-protein (or enzyme) conjugates play a vital role in biosensing due to the integrated function of each component, such as biological recognition and signal amplification. In this work, a green self-assembled method for the synthesis of multi-functional protein-enzyme nanoflowers has been developed, in which no chemical modification and coupling reaction is needed to fabricate the fluorescent signal probe. The self-assembled protein-enzyme conjugates streptavidin (SA) -β-galactosidase (β-Gal)-CaHPO4 nanoflowers load sufficient enzymes without damaging their activity, which meets the requirements of signal tags for biosensing. Through integrated multi-function of biorecognition (SA) and signal amplification (β-Gal), the SA-β-Gal-CaHPO4 hybrid nanoflower-based fluorescent sensor exhibited an ultrasensitive detection of protein biomarker alpha-fetoprotein (AFP), with limits of detection at the fM level. The presented self-assembled strategy can be extensively applied to develop on-demand protein-enzyme conjugates according to the specific requirements in a variety of applications including biosensors, bioimaging, and biomedicine.
Protein-enzyme conjugate Self-assembled nanoflower Signal tag Fluorescent sensing Protein biomarker assay
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This work was supported by the National Natural Science Foundation of China (21475101, 21675119).
Compliance with ethical standards
Ethical standards and informed consent
The study was approved by the Ethical Committee of Wuhan University. Human fluid samples used in this study do not have any identifying information about all the participants that provided written informed consent.
Conflict of interest
The authors declare that they have no conflict of interest.
Somturk B, Hancer M, Ocsoy I, Ozdemir N. Synthesis of copper ion incorporated horseradish peroxidase-based hybrid nanoflowers for enhanced catalytic activity and stability. Dalton Trans. 2015;44:13845–52.CrossRefGoogle Scholar
Mittal S, Kaur H, Gautam N, Mantha AK. Biosensors for breast cancer diagnosis: a review of bioreceptors, biotransducers and signal amplification strategies. Biosens Bioelectron. 2017;88:217–31.CrossRefGoogle Scholar
Zhou Z, Hartmann M. Progress in enzyme immobilization in ordered mesoporous materials and related applications. Chem Soc Rev. 2013;42:3894–912.CrossRefGoogle Scholar
Spicer CD, Jumeaux C, Gupta B, Stevens MM. Peptide and protein nanoparticle conjugates: versatile platforms for biomedical applications. Chem Soc Rev. 2018;47:3574–620.CrossRefGoogle Scholar
Fang Y, Xu W, Wu J, Xu Z-K. Enzymatic transglycosylation of PEG brushes by β-galactosidase. Chem Commun. 2012;48:11208–10.CrossRefGoogle Scholar
Tang F, Li L, Chen D. Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery. Adv Mater. 2012;24:1504–34.CrossRefGoogle Scholar
Jia H. Enzyme-carrying electrospun nanofibers. In: Wang P, editor. Nanoscale biocatalysis: methods and protocols. Totowa: Humana Press; 2011. p. 205–12.CrossRefGoogle Scholar
Kalska-Szostko B, Rogowska M, Satuła D. Organophosphorous functionalization of magnetite nanoparticles. Colloids Surf B. 2013;111:656–62.CrossRefGoogle Scholar
Wang Q, Zhou L, Jiang Y, Gao J. Improved stability of the carbon nanotubes–enzyme bioconjugates by biomimetic silicification. Enzym Microb Technol. 2011;49:11–6.CrossRefGoogle Scholar
Ge J, Lei J, Zare RN. Protein-inorganic hybrid nanoflowers. Nat Nanotechnol. 2012;7:428–32.CrossRefGoogle Scholar
Wang X, Shi J, Li Z, Zhang S, Wu H, Jiang Z. Facile one-pot preparation of chitosan/calcium pyrophosphate hybrid microflowers. ACS Appl Mater Interfaces. 2014;6:14522–32.CrossRefGoogle Scholar
Hua X, Xing Y, Zhang X. Enhanced promiscuity of lipase-inorganic nanocrystal composites in the epoxidation of fatty acids in organic media. ACS Appl Mater Interfaces. 2016;8:16257–61.CrossRefGoogle Scholar
Thawari AG, Rao CP. Peroxidase-like catalytic activity of copper-mediated protein–inorganic hybrid nanoflowers and nanofibers of β-lactoglobulin and α-lactalbumin: synthesis, spectral characterization, microscopic features, and catalytic activity. ACS Appl Mater Interfaces. 2016;8:10392–402.CrossRefGoogle Scholar
Cui J, Zhao Y, Liu R, Zhong C, Jia S. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance. Sci Rep. 2016;6:27928.CrossRefGoogle Scholar
Li M, Luo M, Li F, Wang W, Liu K, Liu Q, et al. Biomimetic copper-based inorganic–protein nanoflower assembly constructed on the nanoscale fibrous membrane with enhanced stability and durability. J Phys Chem C. 2016;120:17348–56.CrossRefGoogle Scholar
Zhu X, Huang J, Liu J, Zhang H, Jiang J, Yu R. A dual enzyme-inorganic hybrid nanoflower incorporated microfluidic paper-based analytic device (μPAD) biosensor for sensitive visualized detection of glucose. Nanoscale. 2017;9:5658–63.CrossRefGoogle Scholar
Liu Y, Chen J, Du M, Wang X, Ji X, He Z. The preparation of dual-functional hybrid nanoflower and its application in the ultrasensitive detection of disease-related biomarker. Biosens Bioelectron. 2017;92:68–73.CrossRefGoogle Scholar
Ariza-Avidad M, Salinas-Castillo A, Capitán-Vallvey LF. A 3D μPAD based on a multi-enzyme organic–inorganic hybrid nanoflower reactor. Biosens Bioelectron. 2016;77:51–5.CrossRefGoogle Scholar
Li W, Lu S, Bao S, Shi Z, Lu Z, Li C, et al. Efficient in situ growth of enzyme-inorganic hybrids on paper strips for the visual detection of glucose. Biosens Bioelectron. 2018;99:603–11.CrossRefGoogle Scholar
Sun J, Ge J, Liu W, Lan M, Zhang H, Wang P, et al. Multi-enzyme co-embedded organic-inorganic hybrid nanoflowers: synthesis and application as a colorimetric sensor. Nanoscale. 2014;6:255–62.CrossRefGoogle Scholar
Guan Z, Zou Y, Zhang M, Lv J, Shen H, Yang P, et al. A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection. Biomicrofluidics. 2014;8(1):014110.CrossRefGoogle Scholar
Martinez-Bilbao M, Gaunt MT, Huber RE. E461H-beta-galactosidase (Escherichia coli): altered divalent metal specificity and slow but reversible metal inactivation. Biochemistry. 1995;34:13437–42.CrossRefGoogle Scholar