Development of surface coating strategy for label-free immunosensing in serum

Biosensors are increasingly in high demand in medical diagnostics. To fabricate high-performance biosensors, biomolecules that acts like sensing probes should be immobilized onto solid sensor surfaces to achieve the desired sensing capabilities. For high efficiency, biosensor surfaces should maintain the high specificity and binding capacity of the immobilized biomolecules by minimizing the interference between them. To this end, several surface chemistry has been developed to realize good surface activation and further immobilization of bio-probes. Despite the progress, these strategies fail to achieve the desired surface activation control attributed to the limitation of the reactions involving the self-assembled monolayers.

Polymer coatings like functionalized polyethylene glycol (PEG) are promising candidates for activating biosensor surfaces. Unlike the self-assembled monolayers, polymers are more effective in facilitating the group densities, surface regenerative and antifouling properties of the sensor surfaces. Among them, polyelectrolytes with brush-like copolymers are promising. Application of these polymers, especially the poly-L-lysine (PLL), on the surface allows for efficient functionalization of sensors and maintains their vital properties. Moreover, recent research findings revealed that replacing the long PEG side chains with short oligo-ethylene glycol (OEG) had the advantage of enhancing the binding capacities by eliminating the back folding of the PEG sidechains.

Even though polyelectrolytes coupled with linear PEG/OEG side chains result in robust, versatile and controllable biosensor surfaces, it still has a relatively low binding capacity as the self-assembly polymer monolayer is capable of only providing a 2D interface. Additionally, the limited reaction sites per sensor unit also limits the number of the end functional groups reacting with target probe molecules. Confronted with these challenges, Wenwei Pan, Ziyu Han, Dr. Ye Chang and Professor Xuexin Duan from Tianjin University designed and synthesized a three-dimensional (3D) biosensor surface based on newly modified thorn-like polyelectrolytes (3D-PETx). Their work is currently published in the journal, Biosensors and Bioelectronics.

In brief, the 3D-PETx used in this experiment comprised PLL grafted with multitude OEG and biotin moieties. Three different 3D-PETx were synthesized with various OEG-biotin sidechains grating ratios: 10%, 20% and 30%, and tested on different substrates. Finally, their sensing performance and antifouling properties for prostate-specific antigen (PSA), streptavidin and human IgG were measured and compared to those of the conventional 2D-PET using BiLayer Interferometry, microfluidic devices, Surface Plasmon Resonance and Enzyme-Linked ImmunoSorbent Assay.

Unlike the conventional 2D polyelectrolyte copolymer, the authors found out that 3D-PETx generated a 3D surface with numerous biotin groups, thus increasing the number reaction sites per surface unit. The interactions between streptavidin and the functionalized biotin units and biotinylated proteins enabled the detection of various biomarkers. In addition, 3D-PETx provided a non-fouling surface in both buffer and undiluted human serum. As such, the authors recorded a PSA detection with a reduced LOD of 0.6 ng/mL in undiluted serum. Furthermore, it was worth noting that iterative measurements involving continuous monitoring of biomarkers could be achieved by regenerating the 3D-PETx biosensor surface using pH stimulation.

In summary, a novel 3D biosensor surface based on 3D-PETx synthesized by grafting 2D PLL-g-OEG-biotin with linear PLL was developed. The resulting 3D sensor exhibited enhanced sensing performance, protein binding capacity and antifouling property in serum and in a buffer. The biosensor surface proved versatile and could potentially support various bio-functional groups. Besides, the cross-links could be reduced by optimizing the chain length and graft ratio. The simple and reliable biosensor functionalization method demonstrated here is versatile and can be extended to various bioreceptors and substrates. Specifically, due to the successful immunosensing in undiluted human serum, the authors, in a statement to Medicine Innovates, explained that the 3D-PETx coating has the potential to be used in clinical applications.