Biochemistry strategies for label-free optical sensor biofunctionalization: advances towards real applicability

Abstract

Label-free biosensors, and especially those based on optical transducers like plasmonic or silicon photonic systems, have positioned themselves as potential alternatives for rapid and highly sensitive clinical diagnostics, on-site environmental monitoring, and for quality control in foods or other industrial applications, among others. However, most of the biosensor technology has not yet been transferred and implemented in commercial products. Among the several causes behind that, a major challenge is the lack of standardized protocols for sensor biofunctionalization. In this review, we summarize the most common methodologies for sensor surface chemical modification and bioreceptor immobilization, discussing their advantages and limitations in terms of analytical sensitivity and selectivity, reproducibility, and versatility. Special focus is placed on the suggestions of innovative strategies towards antifouling and biomimetic functional coatings to boost the applicability and reliability of optical biosensors in clinics and biomedicine. Finally, a brief overview of research directions in the area of device integration, automation, and multiplexing will give a glimpse of the future perspectives for label-free optical biosensors.

Keywords: Antibody immobilization; Antifouling coating; Biochemical cross-linking; Lipid membrane; Silicon photonics; Surface plasmon resonance.

Fig. 1

a Illustrative scheme of an optical biosensor system based on the evanescent field sensing mechanism: light is coupled to the transducer surface generating an electromagnetic field that penetrates evanescently into the dielectric medium where the biological interaction takes place. The biointeraction changes the refractive index of the medium, which is translated in variations of certain optical properties of the output light (intensity, wavelength, etc.). b Main optimization parameters in sensor surface biofunctionalization: bioreceptor orientation (top), grafting density (middle), and antifouling coating (bottom)

Fig. 2

a Schematics of chemical scaffolds for gold-based sensor functionalization: dextran-based polymeric layer (e.g., carboxymethyl dextran) (left) and mixed alkanethiol self-assembled monolayer (e.g., mercaptohexadecanoic acid/mercaptoundecanol, MHDA/MUOH) (right). b Schematics of chemical scaffold for silicon-based sensor functionalization: alkoxysilane monolayer (e.g., 3-aminopropyl(triethoxysilane), APTES). c Schematics of different examples of cross-linking strategies for amine-functional bioreceptors. d Schematics of different examples of cross-linking strategies for thiol-functional bioreceptors. e Schematics of cross-linking strategy based on click chemistry: copper(I)-catalyzed alkyne-azyde cycloaddition (CuAAC). Antibodies and DNA strands are only used for illustrative purposes; any receptor carrying the desired functionality could be employed indistinctly

Fig. 3

Antibody orientation strategies. a Affinity-based immobilization of intact antibodies on Protein A/G. b Affinity-based immobilization of biotinylated antibodies on avidin protein (e.g., streptavidin or neutravidin). c Affinity-based immobilization through DNA hybridization. d Immobilization of intact antibodies on a calixarene-based linker (e.g., Prolinker™). e Immobilization of recombinant antibody fragments, i.e., antigen-binding region (Fab)

Fig. 4

Antifouling strategies. a Formation of a hydration layer with hydrophilic compounds, e.g., incorporating polyethylene glycol or oligoethylene glycol moieties. b Formation of an effective charge-balanced layer with zwitterionic compounds

Fig. 5

Biomimetic lipid-based biofunctionalization. a Schematic illustration of liposome-based bioreceptor immobilization. b Schematic illustration of bioreceptor immobilization on a planar lipid bilayer. c Representative examples of liposome tethering strategies: electrostatic interactions (left), biotin/avidin system (middle), and DNA-directed (right). d Representative examples of planar lipid bilayer formation strategies: supported lipid bilayer (SLB) on hydrophilic substrate (i.e., glass) (left), SLB on hydrophilic coating on gold surface (middle), and hybrid lipid bilayer formed by hydrophobic SAM coating on gold and single lipid layer (right)