Density Control and Patterning of Biosensor Surfaces Using Modified Poly-L-Lysine Polymers
Biosensors and materials for biomedical applications generally require precise chemical functionalization to bestow their surfaces with desired properties, such as specific molecular recognition and antifouling properties. Consequently, tailoring the chemistry at the biosensing interface has a crucial role in obtaining the best selectivity and sensitivity.1 Especially for DNA biosensor, either biological or artificial probes, as well as antifouling moieties, need a defined type of chemistry to be anchored with respect to their chemical modification and the type of substrate, affecting the biodistribution.2 In addition, control of the surface probe density is required for achieving high sensitivity.3 Traditional methods have the applicability limited to specific substrates and aim to control the density at the surface modification step, with the drawback of having to assess the hybridization efficiency each time.
Recently, polyelectrolytes were used in biosensing to tailor the probe type and distribution. Furthermore, the ensemble of substrates for biosensing purpose has been increased thanks to the multiple nature of polymers and their electrostatic interactions. The chemi/physisorption of modified polyelectrolytes provides advantages for the immobilization of biomolecules and for biosensing applications. At physiological pH, poly(L- lysine) (PLL) polymers readily and strongly adsorb onto a variety of metal oxide surfaces through multivalent electrostatic interactions between the positively charged lysine side-chains and a negatively charged surface.4 As a result, PLL polymers, which are easy to functionalize thanks to the amino groups in the side chain, allow the accommodation of the grafted functional moieties over the substrate, maintaining their adsorption properties.
Based on this approach, biorecognition surfaces were prepared by deposition of modified PLL polymers grafted with various fractions of oligo(ethylene glycol) (OEG, antifouling) and maleimide (Mal) moieties (PLL-OEG-Mal), so that both the type of functionalization and the control over the density is achieved at the same time, during the synthetic step, verified by 1H-NMR. PLL-OEG-Mal polymers were self-assembled at the substrate and coupled to thiol-peptide nucleic acid (PNA) probes, forming a real-time DNA biosensor. A linear relationship between the probe density and the PLL grafting density was found monitoring the frequency shift of the hybridization step for the complementary DNA versus the density of Mal group coupled to the PLL backbone. In order to establish the absolute probe density values at the biosensor surfaces, cyclic voltammetry experiments using Methylene Blue-functionalized DNA were performed, providing a density of 1.24 × 1012 probes per cm2 per % of grafted Mal, thus confirming the validity of the density control in the synthetic PLL modification step without the need of further surface characterization.5,6 Thanks to their advantages, modified PLL polymers can be used to tune the surface properties, accommodating several clickable side groups as linkers and antifouling moieties, and forming different architectures as layer-by-layer and µ-arrays, as well as their application in soft lithography and pillar structures.6
1 D. Bizzotto, I. J. Burgess, T. Doneux, T. Sagara, H. Z. Yu, ACS Sensors 2018, 3, 5–12.
2 S. H. North, E. H. Lock, C. R. Taitt, S. G. Walton, Anal. Bioanal. Chem. 2010, 397, 925–933.
3 V. Biagiotti, A. Porchetta, S. Desiderati, K. W. Plaxco, G. Palleschi, F. Ricci, Anal. Bioanal. Chem. 2012, 402, 413–421.
4 S. Pasche, S. M. De Paul, J. Vörös, N. D. Spencer, M. Textor, Langmuir 2003, 19, 9216–9225.
5 J. Movilli, A. Rozzi, R. Ricciardi, R. Corradini, J. Huskens, Bioconjug. Chem. 2018, 29, 4110-4118.
6 J. Huskens, R. Ricciardi, J. Movilli, D. D. Iorio, A. Marti Morant, World Patent WO 2018/222034, 2018.