A novel portable immuno-device for the recognition of lymphatic vessel endothelial hyaluronan receptor-1 biomarker using GQD-AgNPrs conductive ink stabilized on the surface of cellulose

Abstract

Lymphatic vessel endothelium expresses various lymphatic marker molecules. LYVE-1, the lymphatic vessel endothelial hyaluronan (HA) receptor, a 322-residue protein belonging to the integral membrane glycoproteins which is found on lymph vessel wall and is completely absent from blood vessels. LYVE-1 is very effective in the passage of lymphocytes and tumor cells into the lymphatics. As regards cancer metastasis, in vitro studies indicate LYVE-1 to be involved in tumor cell adhesion. Researches show that, in neoplastic tissue, LYVE-1 is limited to the lymphovascular and could well be proper for studies of tumor lymphangiogenesis. So, the monitoring of LYVE-1 level in human biofluids has provided a valuable approach for research into tumor lymphangiogenesis. For the first time, an innovative paper-based electrochemical immune-platform was developed for recognition of LYVE-1. For this purpose, graphene quantum dots decorated silver nanoparticles nano-ink was synthesized and designed directly by writing pen-on paper technology on the surface of photographic paper. This nano-ink has a great surface area for biomarker immobilization. The prepared paper-based biosensor was so small and cheap and also has high stability and sensitivity. For the first time, biotinylated antibody of biomarker (LYVE-1) was immobilized on the surface of working electrode and utilized for the monitoring of specific antigen by simple immune-assay strategy. The designed biosensor showed two separated linear ranges in the range of 20-320 pg ml^-1 and 0.625-10 pg ml^-1, with the acceptable limit of detection (LOD) of 0.312 pg ml^-1. Additionally, engineered immunosensor revealed excellent selectivity that promises its use in complex biological samples and assistance for biomarker-related disease screening in clinical studies.

Scheme 1. Synthesis process of conductive AgNPrs–GQDs nano-ink.

Scheme 2. Fabrication process of the immunosensor for the monitoring of LYVE-1.

Fig. 1. FESEM images related to working electrode modified by (A) AgNPrs–GQDs nano-ink (B) AgNPrs–GQDs nano-ink–Ab, and (C) AgNPrs–GQDs nano-ink–Ab–BSA, (D) AgNPrs–GQDs nano-ink–Ab–BSA–Ag, with different magnifications to describe various levels of enlargement.

Fig. 2. (A and B) CVs and EIS of paper-based electrodes modified with AgNPrs/GQD nano-ink, AgNPrs/GQD nano-ink/biotin-Ab, AgNPrs/GQD nano-ink/biotin-Ab/BSA10%, AgNPrs/GQD nano-ink/biotin-Ab/BSA10%/LYVE-1 Ag. (C and D) Histograms of peak current versus types of modified paper-based electrodes. Supporting electrolyte was K₄Fe(CN)₆/K₃Fe(CN)₆/KCl (0.1 M).

Fig. 3. (A and B) ChA of immunosensor in different concentrations of LYVE-1 Ag (1.25, 2.5, 5, 10, 20, 80 and 320 pg ml⁻¹). (C–F) Calibration curves of immunosensor in different forms (I vs. C and I vs. log C), respectively. Supporting electrolyte was 0.1 M K₄Fe(CN)₆/K₃Fe(CN)₆ containing 0.1 M KCl (t = 100 s, E = 0.25 V).

Fig. 4. (A and B) ChAs of immunosensor in plasma samples (0.625, 1.25, 5, 10, 20, 40, 160 and 320 pg ml⁻¹). (C–F) Calibration curves of immunosensor in different forms (I vs. C and I vs. log C_LYVE-1), respectively. Supporting electrolyte is 0.1 M K₄Fe(CN)₆/K₃FE(CN)₆ containing 0.1 M KCl.