Integration of binding peptide selection and multifunctional particles as tool-box for capture of soluble proteins in serum

Abstract

In this paper, we report on a general approach for the detection of a specific tumoural biomarker directly in serum. Such detection is made possible using a protein-binding peptide selected through an improved phage display technique and then conjugated to engineered microparticles (MPs). Protein biomarkers represent an unlimited source of information for non-invasive diagnostic and prognostic tests; MP-based assays are becoming largely used in manipulation of soluble biomarkers, but their direct use in serum is hampered by the complex biomolecular environment. Our technique overcomes the current limitations as it produces a selective MP--engineered with an antifouling layer--that 'captures' the relevant protein staying impervious to the background. Our system succeeds in fishing-out the human tumour necrosis factor alpha directly in serum with a high selectivity degree. Our method could have great impact in soluble protein manipulation and detection for a wide variety of diagnostic applications.

Keywords: microparticles for diagnosis; phage display selection; soluble cancer biomarker.

Figure 1.

Graphical scheme for the design and development of MP-based bioassay for fishing-out of soluble biomarkers. (a) Experimental scheme for selection of phage-displayed peptides with high affinity and specificity for rhTNFα performed on magnetic nickel-coated beads. (b) Graphical representation of integrated system for detection or fishing-out of any soluble biomarker in complex biological medium. This MP-based bioassay consists of selective capturing of target protein due to the presence of a specific binding peptide (previously selected by modified phage display procedure). The binding event is then detected by immunofluorescence measurements.

Figure 2.

Results from de novo selection from a random PhD12 peptide library using NiMB complexed to hexahistidine-tagged recombinant human TNFα (6His-rhTNFα). (a) Binding specificity versus 6His-rhTNFα of the six best phage selected after three rounds of selection panning using the NiMB::6His-rhTNFα. (b) List of the resulting peptide sequences with the strongest specificity against rhTNFα target shows a consensus sequence in at least four clones. Conserved amino acids are reported in red.

Figure 3.

Design, construction and performances of microfluidic device used for PhD selection. (a) Layout and picture of the microfluidic chip (a glass/PDMS device) used for phage selection, showing the design of the channels, external magnets and flow path. The continuous flow separation of phages is based on the transfer of target protein-conjugated magnetic beads in a continuous washing buffer by pumping through a 100 μm deep PDMS channel with a strong magnetic field perpendicularly applied. (b) Evaluation of microfluidic device washing efficiency by measuring the specific (ΦG6) and non-specific (ΦWT) residual bound phage on NiMB after one round of washing. The percentage of bound phages before and after washing step in the microfluidic device is reported as fluorescence recovery evaluated by CLSM on single particles and pfu by standard titration. (Online version in colour.)

Figure 4.

Characterization of TNFα G6 peptide binding. TNFα–hG6 peptide complex with greatest interaction energy among those individuated by MD and AMBER optimization. Most of the protein (3L9J as PDB ID) is represented as cartoon. Protein residues belonging to region I and interacting with the peptide, and the peptide itself are represented by the accessible surface in blue and orange, respectively.

Figure 5.

ITC studies. Calorimetric curve for rhTNFα protein (25 μM) titration with G6 peptide (1 mM) on the top. Raw and integrated data are shown in the upper and lower panels, respectively. In the lower section, data fitting was achieved with a single-binding-site model. (Online version in colour.)

Figure 6.

Characterization of PEG-MP and evaluation of their capability in rhTNFα fish out. (a) TEM image of PEG-MP. (b) Chromatographic traces of rhTNFα produced by the UV detection of collected supernatant from PEG-MP and MP after incubation of the protein. (c) Evaluation of G6-PEG-MP capture efficiency. The binding event is detected by measuring the fluorescence intensity of FITC conjugated anti-human TNFα (monoclonal 1 : 100) directly on particles (image panel). Particles conjugated with UNR peptide were used as a control of unspecific signal. The experiment was performed in PBS buffer and in human serum.