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. 2022 Oct 5;12(44):28473-28488.
doi: 10.1039/d2ra04984h. eCollection 2022 Oct 4.

A novel immuno-device based on the specific binding of AuNP-supported CTAB with biotinylated antibody of hyaluronic acid toward an early-stage recognition of a biomarker: a bioanalytical assay in real samples using disposal biosensor technology

Ahmad Mobed  1   2   3 , Fereshteh Kohansal  3 , Sanam Dolati  2 , Mohammad Hasanzadeh  3
Affiliations

A novel immuno-device based on the specific binding of AuNP-supported CTAB with biotinylated antibody of hyaluronic acid toward an early-stage recognition of a biomarker: a bioanalytical assay in real samples using disposal biosensor technology

Ahmad Mobed et al. RSC Adv. .

Abstract

Hyaluronic Acid (HA) is a non-sulfated glycosaminoglycan, which is a potential biomarker that could be evaluated in the diagnosis of some cancers. For the first time, a novel label-free electrochemical immunosensor was developed based on modified ITO-PET (indium tin oxide-polyethylene terephthalate) electrodes for the sensitive recognition of hyaluronic acid (HA) in real samples. A disposable ITO-coated PET electrode was modified with gold nanoparticles (AuNPs) to construct a suitable substrate for the efficient immobilization of biotinylated antibodies of HA. Importantly, the encapsulation of biotinylated antibody of HA in KCC1-NH-CS2 was performed successfully, which was another innovative part of this bio-device construction. For determining the immobilization steps and optimization of the biosensor, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were used. Furthermore, the morphological characterization of each ITO electrode surface was performed by field emission scanning electron microscopy (FESEM). Specific binding of gold nanoparticles supported CTAB to ITO-PET and its bioconjugation with the biotinylated antibody of HA was studied using the electroanalysis of the sensor performance. For the better performance of the antibody to generate an immunocomplex with HA (antigen), its encapsulation was performed, which led to the excellent behavior of the immunosensor. The proposed HA immunosensor indicated excellent reproducibility, high selectivity, and long-term stability. The HA electrochemical immunosensor performed perfectly with a wide determination range (0.078 to 160 ng mL-1) and a low limit of quantification (0.078 ng mL-1) in human plasma samples. It is recommended that the designed biosensor can be used as a diagnostic tool in clinical bioassays in the near future.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1. The synthesis procedure of KCC-1-NH-CS2.
Fig. 2
Fig. 2. TEM images of KCC-1-NH-CS2 in various magnifications.
Fig. 3
Fig. 3. FESEM images of KCC-1-NH-CS2.
Fig. 4
Fig. 4. EDAX analyses of KCC-1, KCC-1-NH2 and KCC-1-NH-CS2.
Fig. 5
Fig. 5. Schematic presentation for the encapsulation of biotinylated HA antibody on KCC-1-NH-CS2.
Fig. 6
Fig. 6. The main steps in creating HA immunosensor.
Fig. 7
Fig. 7. (A and D) DPV and EIS of immunosensor fabrication steps (Bare ITO-PET electrode, ITO-PET-AuNPs (CTAB), ITO-PET-AuNPs (CTAB)-Ab (encapsulated by KCC-1-NH-CS2), ITO-PET-AuNPs (CTAB)-Ab-BSA-HA. Data information of DPV: (Tequilibration: 2 s, Ebegin: −1.0 V, Eend: 1.0 V, Estep: 0.1 V, Epulse: 0.005 V, Tpulse: 0.2 s, scan rate: 0.1 V s−1), and EIS: (Edc: 0.25 V, Eac: 0.01 V, max. frequency: 100 000 Hz, min. frequency: 0.1 Hz,tMax OCP: 50 s, stability criterion: 0.00001 mV s−1), in the presence of K4Fe(CN)6/K3Fe (CN)6/KCl 0.5 M. (B and D) DPV and EIS histograms of versus modifications of ITO-PET electrode, respectively. (RSD = 0.698%, 0.479%, respectively, n = 4).
Fig. 8
Fig. 8. (A–I) FE-SEM of ITO-PET modified AuNPs(CTAB) under different magnifications. ITO-PET-AuNPs(CTAB) were evaluated in the second step.
Fig. 9
Fig. 9. (A–I) FE-SEM of ITO-Au(CTAB)-Ab (Ab encapsulated by KCC-1-NH-CS2) under different magnifications.
Fig. 10
Fig. 10. (A–I) FE-SEM of ITO-PET-AuNPs(CTAB)-Ab-BSA (Ab encapsulated by KCC-1-NH-CS2) under different magnifications.
Fig. 11
Fig. 11. (A–I) FE-SEM of ITO-PET-AuNPs(CTAB)-Ab-BSA-Ag (Ab encapsulated by KCC-1-NH-CS2) under different magnifications.
Fig. 12
Fig. 12. (A–O) Atomic-resolution chemical mapping using energy-dispersive X-ray spectroscopy of different steps of the fabricated immunosensor: (A–C) ITO-PET bare. (D–F) ITO-PET modified AuNPs(CTAB). (G–I) ITO-PET modified AuNPs(CTAB)-Ab. (J–L) ITO-PET modified AuNPs(CTAB)-Ab-BSA. (M–O) ITO-PET modified AuNPs(CTAB)-Ab-BSA-HA.
Fig. 13
Fig. 13. (A, C and E) DPV, SWV and EIS of the fabricated immunosensor in the presence of HA at different concentrations (160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 ng mL−1), in the presence of K4Fe(CN)6/K3Fe(CN)6 0.5 M containing KCl (0.1 M). (B, D and F) Calibration curves of ITO-PET-AuNPs(CTAB)-Ab-BSA modified HA immunosensor in different concentrations. (RSD = 0.299%, 0.287%, 0.35%, n = 10, n = 10, n 0 = 7, for DPV, SWV and EIS, respectively).
Fig. 14
Fig. 14. (A and C) SWV and DPV of the fabricated immunosensor in the presence of real sample (human plasma) in the concentrations (160, 80, 40, 20, 10, 5), in the presence of K4Fe (CN)6/K3Fe (CN)6 0.5 M containing KCl (0.1 M). (B and D) calibration curves (RSD = 0.25%, 0.24%, n = 6).
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References

    1. Fallacara A. Baldini E. Manfredini S. Vertuani S. Hyaluronic acid in the third millennium. Polymers. 2018;10(7):701. - PMC - PubMed
    1. Gupta R. C. Lall R. Srivastava A. Sinha A. Hyaluronic acid: molecular mechanisms and therapeutic trajectory. Front. Vet. Sci. 2019;6:192. - PMC - PubMed
    1. Cowman M. K. Lee H.-G. Schwertfeger K. L. McCarthy J. B. Turley E. A. The content and size of hyaluronan in biological fluids and tissues. Front. Immunol. 2015;6:261. - PMC - PubMed
    1. Fraser J.; Laurent T.Turnover and metabolism of hyaluronan. In Ciba Foundation Symposium 143-The Biology of Hyaluronan: The Biology of Hyaluronan: Ciba Foundation Symposium 143, 2007; Wiley Online Library: pp 41–59 - PubMed
    2. Marcellin E. Steen J. A. Nielsen L. K. Insight into hyaluronic acid molecular weight control. Appl. Microbiol. Biotechnol. 2014;98(16):6947–6956. - PubMed
    1. Vigetti D. Karousou E. Viola M. Deleonibus S. De Luca G. Passi A. Hyaluronan: biosynthesis and signaling. Biochim. Biophys. Acta Gen. Subj. 2014;1840(8):2452–2459. - PubMed