Anal Chim Acta. 2017 Apr 29;964:170-177.

Biofunctional Polyelectrolytes Assembling on Biosensors – A Versatile Surface Coating Method for Protein Detections

Ziyu Han, Yanyan Wang, and Xuexin Duan*

State Key Laboratory of Precision Measuring Technology & Instruments, College of Precision Instrument and Opto-electronics Engineering, Tianjin University, Tianjin 300072, China.



This paper reports a surface functionalization strategy for protein detections based on biotin-derivatized poly(L-lysine)-grafted oligo-ethylene glycol (PLL-g-OEGx-Biotin) copolymers. Such strategy can be used to attach the biomolecule receptors in a reproducible way simply by incubation of the transducer element in a solution containing such copolymers which largely facilitated the sensor functionalization at an industrial scale. As the synthesized copolymers are cationic in physiology pH, surface biotinylation can be easily achieved via electrostatic adsorption on negatively charged sensor surface. Biotinylated receptors can be subsequently attached through well-defined biotin-streptavidin interaction. In this work, the bioactive sensor surfaces were applied for mouse IgG and prostate specific antigen (PSA) detections using quartz crystal microbalance (QCM), optical sensor (BioLayer Interferometry) and conventional ELISA test (colorimetry). A limit of detection (LOD) of 0.5 nM was achieved for PSA detections both in HEPES buffer and serum dilutions in ELISA tests. The synthesized PLL-g-OEGx-Biotin copolymers with different OEG chain length were also compared for their biosensing performance. Moreover, the surface regeneration was achieved by pH stimulation to remove the copolymers and the bonded analytes, while maintaining the sensor reusability as well. Thus, the developed PLL-g-OEGx-Biotin surface assembling strategy is believed to be a versatile surface coating method for protein detections with multi-sensor compatibility.

PMID: 28351633



Among various types of transducers, immobilizations of the bioreceptors on sensor surface, while preventing the non-specific adsorption of unwanted species, is one of the most considerable issues. Different functionalization strategies for immobilizing biomolecules will result in different orientation, uniformity and surface density of biomolecule probes, which, therefore, lead to different sensor performance. Recently, we developed a new supramolecular surface functionalization approach for biosensors based on self-assembling of bioengineered polyelectrolytes (PLL-g-OEGx-Biotin) directly on sensor surface [1].

In this work, we synthesized and tested PLL-g-OEGx-Biotin with different OEG length as supramolecular linkers for protein detections on different sensor platform and compared their sensing results to deeply understand the performance of such coating for biosensing applications. After PLL-g-OEGx-Biotin and Streptavidin (SAv) were assembled on the surface, the end SAv units can be used to attach any biotinylated bio-receptors for various protein detections through well-defined biotin-streptavidin interactions the biotin group coverage can be precisely controlled during the synthesis of the copolymers, the surface density of the consequently immobilized receptors (e.g. antibodies, peptides, and nucleotides, etc.) can be optimized under control, which is important for various types of biosensors. The functionalized sensors were used to detect IgG and PSA in their diluted buffers or serum. Different transducer principles acoustic sensor (Quartz crystal microbalance (QCM)), Optical sensor (BioLayer Interferometry) and a conventional ELISA test (colorimetric) were used to demonstrate the compatibility of such coatings. We further compared the length of the linker molecule (the number of OEG units); a special focus is on understanding their influences on the densities of the probes and protein bioactivities. Moreover, pH stimulation is applied to achieve the surface regeneration and sensor reusability of PLL-g-OEGx-Biotin functionalized biosensors [2].



Fig. 1. The chemical structure, AFM characterization and sensing strategy demonstration. PLL-g-OEGx-Biotin copolymers consist of three compositions. The first component is the PLL backbone, on which the amine side chains are cationic in physiology pH. The second component is the grafted OEG short chains. Since protein non-specific binding is more likely to occur on hydrophobic surfaces. The grafted OEG short chains create a uniform, hydrophilic, electroneutral barrier against protein non-specific adsorption. The third component is the biotin units on the end of OEG side chains which are able to specifically capture SAv for further biomarker immobilizations. AFM was applied to study the surface profile of PLL-g-OEGx-Biotin assembling followed by the protein immobilizations. Fig. 1(b) shows the AFM height images of the PLL-g-OEGx-Biotin assembling. The PET films are rather uniform with a mean roughness of 0.145 nm and 0.195 nm for PLL-g-OEG4-Biotin and PLL-g-OEG12-Biotin, respectively, which is crucial to biosensor performance. To verify the feasibility of our sensing scheme, PLL-g-OEGx-Biotins based coating method was first tested for mouse IgG detections on a commercial QCM platform. Fig. 1(c) shows the responses of the QCM frequency shifting to a serial dilutions of mouse IgG. It is also interesting to note that, PLL-g-OEG12-Biotin initialized QCM chip performed higher IgG binding capability (nearly 5 times enhanced compared to that of PLL-g-OEG4-Biotin), due to the longer OEG linkers and the higher flexibility of the immobilized antibodies of OEG12.



Fig. 2. Multiple sensor compatibility of PLL-g-OEGx-Biotin for PSA detection. To further develop the multiple sensor compatibility of the proposed coating method, human PSA detection was employed on both mass loading based QCM (Fig. 2(a)) and light interference based BioLayer Interferometry (Fig. 2(b)). Since human PSA has a higher binding affinity, a lower LOD is required for reliable detection. The frequency and wavelength shift results fit well with the Langmuir equation on both QCM and BioLayer Interferometry, with a calculated KD of 12.73±1.25 nM and 8.86± 0.95 nM, revealing that the sensing performance of this coating method and sensing strategy are ensured for low concentration analyte detection on both mass loading based QCM and light interference based optical sensor. To promote the demonstrated strategy further at an industrial and commercial scale, the proposed coating method was applied for antibodies detection in universal ELISA tests. Fig. 2(c) shows the ELISA testing results of a serial concentrations of human PSA in different serum dilutions, indicating that the antibodies immobilization via our coating method can be potentially developed to other biomarker detection by universal ELISA test.



Fig. 3. Real-time measurement on BioLayer Interferometry for reverse detection of mouse IgG and sensor regeneration. As the PLL backbone is no longer positively charged under solution pH>12 (also the silicon and SiO2 surface is no longer negatively charged under solution pH<2), the PLL-g-OEGx-Biotin along with the bonded analytes can be gradually peeled off from the surface by simple pH stimulation. As a direct proof for sensor regeneration and reusability, the multi-times surface functionalization, IgG detection and surface regeneration on the BioLayer Interferometry were conducted. Fig. 3 shows the real-time sensor response of the IgG detections and regenerations. The repeatable result indicates that single sensor tip can be reused many times, while still maintains similar performance. Compared with other sensor regeneration strategy, pH stimulation is a rather mild method without compromising the sensor performance, which is of vital importance for multiple usages of the sensor chips.



[1] Duan, X., Mu, L., Sawtelle, S. D., Rajan, N. K., Han, Z., Wang, Y., … & Reed, M. A. (2015). Functionalized Polyelectrolytes Assembling on Nano-BioFETs for Biosensing Applications. Advanced Functional Materials, 25(15), 2279-2286.

[2] Han, Z., Wang, Y., & Duan, X. (2017). Biofunctional polyelectrolytes assembling on biosensors-A versatile surface coating method for protein detections. Analytica Chimica Acta, 964, 170-177.