Graphene Oxide-enabled Tandem SPRi Signal Amplification For Sensitive Immunoassay

Surface plasmon resonance imaging (SPRi) is a powerful optical tool for high-throughput analysis of biomolecular interactions and multiplexed immunoassay with the intrinsic advantages of label-free and real-time properties. However, the insufficient sensitivity is a major disadvantage of SPRi, restricting its applications in a wide variety of practical fields. Compared to the label-based techniques such as fluorescence, the detection sensitivity of SPRi is three orders of magnitude lower for immunoassay. To enhance the sensitivity of SPRi immunoassay, some signal amplification strategies such as surface-initiated polymerization, enzyme-catalyzed deposition and bioconjugated nanomaterials have been developed. The core idea of these means is to introduce nanomaterials, reaction products, or polymers onto the sensing surface after the bio-affinity interaction to intensify the local dielectric constant change for enhanced SPRi signals and thus improving the sensitivity. These signal amplification methods boost the sensitivity of SPRi with encouraging progress, however, the specificities of these strategies are not satisfying, due to the ubiquitous nonspecific protein adsorption on sensing surface and/or poor specificities of the bio/chemical reactions used for signal intensification. Moreover, in some cases, the amplification is not quantitative as a result of variable amplification coefficient, making it impossible to accurately quantify a target over a wide concentration range. In this regard, reliable signal amplification with high specificity and stable amplification coefficient is highly demanded for SPRi-based immunoassays.Graphene oxide (GO), a novel single-atomic layer of graphite, has received considerable attention in recent years in different fields due to its unique properties. GO contains abundant oxygen-containing functional group such as hydroxyl, carboxyl and epoxy groups, which allow them to be stably dissolved/suspended in an aqueous solution and facilitate subsequent chemical modification and biomolecule coupling.In this thesis, a novel tandem SPRi signal amplification strategy was developed by exploiting GO as a unique carrier to not only conjugate antibody for direct signal amplification, but also catalyze the growth of metallic nanoparticles for secondary signal enhancement. By functionalizing GO and designing the SPRi sensing interface, as-developed signal amplification strategy ensures the specificity of immunoassay and the subsequent signal amplification and avoid the generation of false positive signals, thus showing excellent quantitative relationship to meet the requirements of accurate quantification of the target in a wide concentration range. Its feasibility was demonstrated by detecting model tumor marker in human serum, showing high sensitivity and excellent specificity. The main results are as follows:(1) Graphene oxide-enabled tandem signal amplification for sensitive SPRi immunoassay in serumIn this innovative strategy, bioconjugated GO and subsequent silver catalytic deposition are developed to realize sensitive and specific SPRi immunoassay of tumor markers in human serum. The second antibody-conjugated rGO sheet interacts with the captured target (a-Fetoprotein, AFP) on SPRi chip surface to form a sandwich immunocomplex for generating the first SPRi signal amplification. Then the rGO catalyzes the reductive deposition of silver nanoparticles in a silver enhancement solution to produce the secondary SPRi signal amplification. The nonspecific adsorption of both protein and rGO bioconjugate on the chip surface is ultimately suppressed due to the non-fouling nature of the sensing surface and the silver reductive deposition is triggered exclusively by the rGO and resultant silver particles are controllably deposited on the rGO surface. With this strategy, highly sensitive and specific immunoassay of biomarkers is accomplished in a wide concentration range with a limit of detection down to 100 pg mL-1 for a-Fetoprotein (AFP).(2) Polydopamine-functionalized graphene oxide to enable dual SPRi signal amplification for sensitive detection of biomarkerIn the part, GO is coated by a thin layer of polydopamine (PDA) to form PDA-rGO nanosheet, which is further conjugated with recognition antibody via a spontaneous reaction between the antibody and the PDA component. In the dual amplification mode, the first signal amplification comes from capture of the antibody-conjugated PDA-rGO to form sandwiched immunocomplexes on the SPRi chip, followed by a PDA-induced spontaneous gold reductive deposition on PDA-rGO in HAuCl4 solution to further enhance the SPRi signal. With this novel dual signal enhancement strategy, a detection limit as low as 500 pg mL-1 is achieved on a nonfouling SPRi chip with high specificity and a wide dynmic range for a model biomarker, carcinoembryonic antigen (CEA) in 10% human serum.