Advanced Lab-on-Fiber Optrodes Assisted by Oriented Antibody Immobilization Strategy

Abstract

Lab-on-fiber (LoF) optrodes offer several advantages over conventional techniques for point-of-care platforms aimed at real-time and label-free detection of clinically relevant biomarkers. Moreover, the easy integration of LoF platforms in medical needles, catheters, and nano endoscopes offer unique potentials for in vivo biopsies and tumor microenvironment assessment. The main barrier to translating the vision close to reality is the need to further lower the final limit of detection of developed optrodes. For immune-biosensing purposes, the assay sensitivity significantly relies on the capability to correctly immobilize the capture antibody in terms of uniform coverage and correct orientation of the bioreceptor, especially when very low detection limits are requested as in the case of cancer diagnostics. Here, we investigated the possibility to improve the immobilization strategies through the use of hinge carbohydrates by involving homemade antibodies that demonstrated a significantly improved recognition of the antigen with ultra-low detection limits. In order to create an effective pipeline for the improvement of biofunctionalization protocols to be used in connection with LoF platforms, we first optimized the protocol using a microfluidic surface plasmon resonance (mSPR) device and then transferred the optimized strategy onto LoF platforms selected for the final validation. Here, we selected two different LoF platforms: a biolayer interferometry (BLI)-based device (commercially available) and a homemade advanced LoF biosensor based on optical fiber meta-tips (OFMTs). As a clinically relevant scenario, here we focused our attention on a promising serological biomarker, Cripto-1, for its ability to promote tumorigenesis in breast and liver cancer. Currently, Cripto-1 detection relies on laborious and time-consuming immunoassays. The reported results demonstrated that the proposed approach based on oriented antibody immobilization was able to significantly improve Cripto-1 detection with a 10-fold enhancement versus the random approach. More interestingly, by using the oriented antibody immobilization strategy, the OFMTs-based platform was able to reveal Cripto-1 at a concentration of 0.05 nM, exhibiting detection capabilities much higher (by a factor of 250) than those provided by the commercial LoF platform based on BLI and similar to the ones shown by the commercial and well-established bench-top mSPR Biacore 8K system. Therefore, our work opened new avenues into the development of high-sensitivity LoF biosensors for the detection of clinically relevant biomarkers in the sub-ng/mL range.

Keywords: biosensing; cancer biomarker; lab-on-fiber technology; optical fiber biosensor; oriented antibody; surface plasmon resonance.

Figure 1

Schematic representation of the exploited approach: biofunctionalization protocol optimization using a gold chip (GC)-based mSPR device (a) and its successive transfer to a BLI-based platform that exploited a plastic optical fiber (ARG2 biosensor) (b) and a LoF platform based on a highly sensitive OFMT (c) for Cripto-1 detection evaluation.

Figure 2

Basic schematic of an OFMT (a); SEM image of an OFMT (b); zoomed image of the nanopattern of a phase-gradient OFMT (c); reflection spectrum of a realized phase-gradient MT probe (d).

Figure 3

Optimization parameters: (i) antibody-to-dye ratio for the labeling, (ii) temperature, and (iii) NaIO₄ for the oxidation reaction (a). Graphical representation of the optimized protocol (b).

Figure 4

Sensorgrams obtained following immobilization of 1B4 by aldehyde coupling (oriented) on active FC2 (a) and following the random immobilization of 1B4 active FC2 (b) on the GC biosensor.

Figure 5

Sensorgrams related to the binding of Cripto-1 at different concentrations (0.05–0.5–2.5–5–12.5 nM) to oriented 1B4 (a) and the same antibody randomly immobilized (b).

Figure 6

Sensorgrams related to the binding of Cripto-1 spiked at different concentrations (0.05–0.5–2.5–5–12.5 nM) in DMEM to oriented 1B4 (a) and to the same antibody randomly immobilized (b).

Figure 7

Normalized interferograms obtained for Cripto-1 detection at different concentrations (12.5–50–500 nM) by 1B4 immobilized in oriented (a) and random (b) configurations.

Figure 8

Real-time sensorgram showing the variations in the barycentric wavelength of the resonance of an OFMT during the biofunctionalization phase (a). Sensorgram related to the detection of Cripto-1 at increasing concentrations in PBS in the range 0.05–5 nM (b).

Figure 9

Real-time sensorgram showing the variations in the barycentric wavelength of the resonance of an OFMT during the biofunctionalization phase without the oriented 1B4 antibody (a). Probe immersion at increasing concentrations of Cripto-1 in PBS in the range of 0.05 and 0.5 nM (b).