Peptide Functionalization of Gold Nanoparticles for the Detection of Carcinoembryonic Antigen in Blood Plasma via SPR-Based Biosensor

Abstract

Nanoparticles functionalized with specific biological recognition molecules play a major role for sensor response enhancement in surface plasmon resonance (SPR) based biosensors. The functionalization procedure of such nanoparticles is crucial, since it influences their interactions with the environment and determines their applicability to biomolecular detection in complex matrices. In this work we show how the ζ-potential (Zpot) of bio-functionalized gold spherical NPs (Bio-NPs) is related to the SPR sensor response enhancement of an immune-sandwich-assay for the detection of the carcinoembryonic antigen (CEA), a cancer marker for colorectal carcinomas. In particular, we prepare bio-functional nanoparticles by varying the amount of peptide (either streptavidin or antibody against CEA) bound on their surface. Specific and non-specific sensor responses, reproducibility, and colloidal stability of those bio-functional nanoparticles are measured via SPR and compared to ζ-potential values. Those parameters are first measured in buffer solution, then measured again when the surface of the biosensor is exposed to blood plasma, and finally when the nanoparticles are immersed in blood plasma and flowed overnight on the biosensor. We found that ζ-potential values can guide the design of bio-functional NPs with improved binding efficiency and reduced non-specific sensor response, suitable reproducibility and colloidal stability, even in complex matrixes like blood plasma.

Keywords: SPR; biosensor; blood; functionalization; gold nanoparticles; immuno-assay; peptide; ζ-potential.

GRAPHICAL ABSTRACT

Nanoparticles with higher number of ligands on their surface (here represented as black molecule) are faster and more specific in detecting the target molecule (green one). Blue molecules represent the antibodies on the SPR biosensor surface.

Figure 1

SEM image of citrate covered NPs deposited on a silicon substrate (image taken with e_LiNE plus, Raith, Germany).

Figure 2

Schematic of the sandwich-assays using two types of functionalized NPs for the detection of CEA in the three different conditions studied in this paper. (A) Assay: Primary antibody immobilized on the sensor surface, incubated with EA in PBS_BSA and either NP functionalized with the secondary antibody specific for CEA (Ab₂-NPs) or secondary antibody and NPs functionalized with streptavidin (S-NPs) binding to the biotinylated Ab₂B. (B) Assay: Analogous to (A) except for that the sensor is exposed to plasma after CEA capture. (C) Assay: Analogous to (A) except for that the functionalized NPs are contained in blood plasma.

Figure 3

ζ-potential of Ab₂-NPs (left) and S-NPs (right) as a function of LDPN. Measurements performed at pH 8 using a ZetaPals instrument.

Figure 4

Specific SPR sensor responses for S-NPs (A) and Ab₂-NPs (B) measured for three different LDPNs. The solid lines, dashed lines and dotted lines correspond to a LDPN of 200, 50, and 20, respectively. All the measurements were performed in PBS_BSA. All the sensor response data are reference-compensated.

Figure 5

Specific SPR sensor responses to S-NPs (A) and Ab₂-NPs (C) and relative non-specific SPR sensor response (RNS) to S-NPs (B) and Ab₂-NPs (D) as a function of LDPN. The data were calculated as the mean values of SPR sensor responses at the equilibrium taken from at least 3 measurements. Measurements were performed in buffer (PBS_BSA).

Figure 6

Specific SPR sensor responses to S-NPs (A) and Ab₂-NPs (C) and relative non-specific SPR sensor response (RNS) to S-NPs (B) and Ab₂-NPs (D) as a function of LDPN. The data were calculated as the mean values of SPR sensor responses at the equilibrium taken from at least 3 measurements. Measurements were performed in buffer (PBS_BSA) after the biosensor surface was exposed to blood plasma.

Figure 7

Detection of CEA in PBS_BSA employing sandwich assay with S-NPs: calibration curve. The SPR sensor response to the S-NPs (contact time with S-NPs solution: 10min) as a function of concertation of CEA. The error bars denote the standard deviation of the sensor response for each concentration. All the sensor response data are reference-compensated.

Figure 8

Specific SPR sensor responses to S-NPs (A) and Ab₂-NPs (C) and relative non-specific SPR sensor response (RNS) to S-NPs (B) and Ab₂-NPs (D) as a function of LDPN. The data were calculated as the mean values of SPR sensor responses at the equilibrium taken from at least 3 measurements. Measurements were performed in blood plasma diluted to 30% with PBS_BSA.