Nanoplasmonic sensing platform for lectin detection

Abstract

Mannose-binding lectin (MBL) is a collagen-like serum protein and a major component of the innate immune system. The main function of MBL is activation of the complement system lectin pathway and the subsequent inflammatory mechanisms. The incidence and outcome of many human diseases are associated with and influenced by the activity and concentrations of MBL in the body fluids. Increased MBL serum concentration and activity have been associated with inflammatory autoimmune diseases, cerebral ischemia, transplant rejection and diabetic nephropathy, while low MBL plasmatic levels are associated with a higher risk, severity, and frequency of infections, and are correlated with unexplained recurrent spontaneous abortions. MBL serum levels are typically measured using ELISA, but this technique requires a number of incubation and washing steps, and is typically carried out in a centralised lab, leading to lengthy turnaround times. A rapid, simple MBL assay capable of being adopted to a point-of-care setting would be beneficial for a number of diagnostic and therapeutic applications. In this study we aimed to develop an alternative MBL detection assay for direct measurement in complex biological fluids (e.g. human serum), consisting of a fast, one-step, wash-free process. To achieve this goal, we used the nanoplasmonic properties of gold nanorods (GNRs): the longitudinal plasmon band can lie in the near-infrared region, where biological fluids exhibit the highest optical transparency, and is highly sensitive to local refractive index changes of the surrounding medium. Binding of biomolecules to the gold nanorods’ surface leads to changes in the spectral properties of the localized surface plasmon resonance (LSPR) peak. The first step was to demonstrate the feasibility of LSPR-based detection assay using a cheap lectin not naturally present in human serum in order to avoid complex interactions with the molecules in serum. We selected soybean agglutinin (SBA) as a model analyte, which has a high affinity for a sugar (N-acetylgalactosamine) that is easily conjugated to the polymers used. We studied the effect of GNRs coated with different polymeric ligand chemistry and lengths on lectin binding response in buffer compared to human serum (Chapter 2). The results showed that plasmonic optical responses in complex biological media can be significantly affected by biocorona formation, and that macromolecular engineering of the polymer tethers can be used to optimise device performance. In Chapter 3, the same polymeric ligand with mannose attached were employed for MBL detection resulting in absence of signal. We achieved binding using a PEG polymer as linker, and initially tested and optimized a twostep functionalization and used complex sugar structures. Besides, we tested a one-step functionalization approach which resulted in higher MBL binding sensitivity, and therefore we used these mannose-coated GNRs (Man-GNRs) for further assay development. After characterization of the stability and mannose grafting density, the binding was first tested using recombinant human MBL (rhMBL) in buffer and in MBL-depleted serum leading to an aggregation response and red LSPR shift. Subsequently, we investigated the binding of native MBL (nMBL) naturally present in human serum (Chapter 4) and we studied the impact of mannose-coated GNR dose, and the sensitivity and specificity of nMBL detection. Man-GNRs showed to be able to detect MBL concentrations as low as 160 ng.mL-1 in just 15 minutes by using a microplate reader, enabling the its measurement within the physiological and pathological range directly from complex biofluids. We tested the Man-GNRs’ performance in human donors serum samples and found a complex MBL binding behaviour. The blue shift observed for nMBL detection was in stark contrast to the red shift observed for the recombinant version of the same lectin. Therefore, in Chapter 5 we analysed and compared the composition of the two lectins. The preliminary results showed that Man-GNRs is effectively able to detect nMBL in serum, but further studies are needed to understand the MBL oligomeric structures bound and explain the LSPR peak behaviour. Finally, in future perspective we identified the next steps needed for the further development of this assay. In conclusion, we provided proof-of-concept for a fast (15 minutes), one-pot, single-step assay where Man-GNRs are directly added to human serum and the LSPR shift is measured by using a microplate reader. This assay requires low sample volume (40 µL) and shows promise for adaptation into a rapid point-ofcare diagnostic. Due to the importance of MBL in infective, inflammatory and autoimmune diseases, this MBL assay faster and simpler than ELISA can be a valuable screening tool to detect immune system deficiencies and for the investigation of recurrent infections during child- and adulthood. Moreover, in current clinical practice, this nanoplasmonic assay may be employed as a routine test to be performed together with other blood tests to monitor patients at increased risk of developing diabetes complications and cardiovascular disease; and the course of therapy in immunosuppressed patients.