Fig 1.
Schematic representation of the nanoparticle and ELISA design.
Peptide 5A contains biotin and carboxyfluorescein, lower case letters denote D-amino acids. Nanoparticles were synthesized by a free-radical polymerization method in a microemulsion system. N-isopropylacrylamide, N,N-dimethylacrylamide and acrylic acid were used as monomers with methylenebisacrylamide as cross-linker. Pentafluorophenyl methacrylate (PFM) was added and the nanoparticles were adorned with peptide 5A (B). An ELISA was designed to detect nanoparticles in biological fluids using the biotin and carboxyfluorescein groups present on peptide 5A (C). Streptavidin coated plates were used to capture the peptides by binding the biotin group and bound peptides were detected with a HRP conjugated mAb anti-fluorescein.
Fig 2.
Quantification of peptide 5A in blood plasma by ELISA.
A standard curve was generated using nanoparticles decorated with peptide 5A in a concentration range of 0.3 to 20 ng/mL peptide (A). Nanoparticles containing peptide 5A were injected into the lateral tail vein of male Wistar rats (n = 3). Plasma was prepared from venous blood samples collected at different time points after injection and peptide 5A concentration was measured by ELISA (B). Data shown are the mean ± S.E.M. peptide concentration in the plasma of three individual animals. The standard curve was generated using the Hill equation.
Fig 3.
Quantification of peptide 5A in blood plasma by LC/MS.
A standard curve for the quantification of peptide by LC/MS was generated using peptide 5A in a concentration range of 4.1 to 1674 ng/mL peptide (A). Blood plasma samples of animals injected with peptide 5A decorated nanoparticles were reduced and free peptide was quantified by LC/MS (B). Data shown are the mean ± S.E.M. peptide concentration in the plasma of three individual animals.
Fig 4.
LC/MS chromatograms comparing different concentrations of peptide 5A in blood plasma matrix.
Peptide 5A peaks in plasma matrix spiked with 0, 13.6 and 45.2 ng/mL (SRM trace: 877.7 → (412.2 + 341.1 + 946.4).