Methods for immobilizing receptors in microfluidic devices: A review

In this review article, we discuss state-of-the-art methods for immobilizing functional receptors in microfluidic devices. Strategies used to immobilize receptors in such devices are essential for the development of specific, sensitive (bio)chemical assays that can be used for a wide range of applications. In the first section, we review the principles and the chemistry of immobilization techniques that are the most commonly used in microfluidics. We afterward describe immobilization methods on static surfaces from microchannel surfaces to electrode surfaces with a particular attention to opportunities offered by hydrogel surfaces. Finally, we discuss immobilization methods on mobile surfaces with an emphasis on both magnetic and non-magnetic microbeads, and finally, we highlight recent developments of new types of mobile supports

Particle-modified Surface Plasmon Resonance Biosensor

Surface plasmon resonance (SPR) biosensors have attracted great attention in scientific research in the past three decades. Extensive studies on the immobilisation of biorecognition elements have been conducted in pursuit of higher sensitivity, but trialled formats have focussed on a thin layer modification next to the plasmon film, which usually requires in situ derivatization. This thesis investigates an ‘off-chip’ immobilisation strategy for SPR biosensing using silica particles and considers the implications of a particle-modified evanescent field on the signal amplitude and kinetics, for an exemplar affinity binding between immobilised IgG and its anti-IgG complement. Submicron silica particles were synthesized as carriers for the bio-recognition elements. They were then immobilised to form a sub-monolayer on the gold film of an SPR biosensor using two methods: thiolsilane coupling and physical adsorption aided by mechanical pressure. The bio-sensitivity towards an antigen/antibody interaction was lower than an SPR biosensor with an alkanethiolate SAM due to the difference in ligand capacity and position in the evanescent field. The binding kinetics of antigen/antibody pair was found to follow the Langmuir model closely in a continuous flow configuration but was heavily limited by the mass transport from the bulk to the sensor surface in a stop-flow configuration. A packed channel configuration was designed with larger gel particles as ligand carriers, packed on top of a gold film to create a column-modified SPR biosensor. This sensor has comparable bio-sensitivity to the previous sub-monolayer particle-modified systems, but the binding and dissociation of the analyte was heavily dependent on mass transport and binding equilibria across the column. A bi-directional diffusion mechanism was proposed based on a two-compartment mass transport model and the expanded model fitted well with the experimental data. The column-modified sensor was also studied by SPR imaging and analyte band formation was observed and analysed. Using the lateral resolution, a multiplexing particle column configuration was explored, and its potential in distinguishing a multicomponent analyte.Agency for Science Technology and Research, Singapor

Optimization of Surface-Protein Interactions for Next Generation Biosensors

Currently, diagnosis for serological diseases such as Ebola, HIV, and Lyme disease relies on enzyme-linked immunosorbent assays (ELISAs), which require centralized laboratories and several-day timescales to complete. However, emerging technologies such as potentiometric and electrochemical impedance biosensing can be developed into portable, label-free, point-of-care devices that require only hour timescales. Specifically, potentiometric sensing platforms can be miniaturized through cost-effective microfabrication, lend themselves to multiplexed and parallel sensing, and are easily integrated with other electronics. Despite the promise of these new label free technologies, device reliability inhibits commercialization and adoption. This work focuses on improving potentiometric sensing, primarily through understanding erroneous behavior at the sensor-solution interface. During biomolecular sensing, biomolecules in solution interact with the sensor surface. Ideally, protein recognition mechanisms are leveraged to allow only target proteins to attach to the surface, imparting signal selectivity. However, unwanted protein interactions with sensor surfaces cause signal instability and increase false-positive rates. Although commonly used to functionalize the sensing surface, carboxyl-terminated thiol self-assembled monolayers (COOH-SAMs) can have large defect densities, which in turn leads to large non-selective adsorption of proteins to hydrophobic surfaces exposed by these defects. A procedure is developed where the surface of COOH-SAMs is treated before functionalization to improve the reliability and quality of receptor attachment to the sensor surface. In this method, a preblocking protein orthogonal to the immunological system of interest is used to cover hydrophobic, non-selective sites on the sensor surface while still leaving carboxylic acid headgroups available for covalent functionalization. This methodology is advantageous when compared to standard blocking, where the receptor protein must be attached to the sensor prior to the blocking step. With traditional postblocking, non-selective adsorption and degradation of the receptor protein itself can occur, and the storage stability of the receptor must be considered since the sensor cannot be functionalized after blocking. Additionally, COOH-SAMs oxidize when exposed to ambient conditions. The impact of this degradation of sensing, as well as methods to prevent degradation, are explored. Beyond SAM-based sensors, there has been significant interest in biomedical applications of 2D materials, including potentiometric sensing. The inert basal plane of certain 2D materials, such as graphene, could lead to larger biosensing signals due to a decrease in surface pH response. However, there is conflicting literature on to what extent interaction from the substrate are transmitted through a 2D monolayer, and the subsequent effect on biomolecule interactions are unknown. Therefore, the degree to which the substrate influences graphene-protein interactions is explored. Finally, a section is dedicated to non-surface-layer sources of signal unreliability, including presentation of a model for sensor response time. The work presented in this thesis demonstrates initial steps towards reliable control over sensor-solution interfaces. Despite the current challenges facing label-free, portable biosensors, the work presented here provides a step towards reliable biosensing.Ph.D

Design and Performance of a Portable and Multichannel SPR Device

A portable multichannel surface plasmon resonance (SPR) biosensor device is presented in this study. As an optical biosensor device, the core component of its light path is a semi-cylindrical prism, which is used as the coupling unit for the excitation of the SPR phenomena. Based on this prism, a wedge-shaped incident light beam including a continuous angle range (10°) is chosen to replace the commonly-used parallel light beam in traditional SPR devices, in which the incident angle is adjusted by a sophisticated mechanical system. Thus, complicated, cumbersome, and costly mechanical structures can be avoided in this design. Furthermore, the selection of a small and high-stability semiconductor laser and matrix CCD detector as well as a microfluidic system aids in the realization of a miniaturized and multichannel device. Several different samples were used to test the performance of this new device. For ethanol with different concentrations, the sensing response was of good linear relativity with the concentration (Y = 3.17143X + 2.81518, R2 = 0.97661). Mouse IgG and goat anti-mouse IgG were used as biological samples for immunological analysis, and BSA as the control group. Good specific recognition between mouse IgG and goat anti-mouse IgG has been achieved. The detection limit of antibody to antigen coated on the sensing surface was about 25 mg/L without surface modification