Synthesis and characterization of metabolite-nanoparticle conjugates for use in metabolite biosensor systems

Detecting specific metabolites and measuring their changing levels in various biofluids has played a key role in understanding health and diagnosing disease. While most metabolite measurements are performed via spectroscopic (colorimetric, UV, mass spectrometry-MS, nuclear magnetic resonance) methods, it is also possible to perform metabolite detection and quantification via metabolite-antibody interactions, such as those used in competitive enzyme-linked immunosorbent assays (ELISAs), and/or impedance measurements. While metabolite-specific antibodies are available, conjugated-metabolites needed for metabolite-antibody detection are rare. Here we describe a general method that allows for the efficient conjugation of different classes of metabolites (acids, amines, and aromatic compounds) to gold nanoparticles and liposomes. We also describe a method for the efficient insertion of metabolite-lipid complexes into liposomes. We extensively characterized these conjugates, confirming their identity and composition, using a wide variety of analytical (thermal gravimetric analysis), spectroscopic (dynamic light scattering, MS) and microscopic (transmission electron microscopy-TEM, cryo-EM, high resolution EM) techniques. We also demonstrated that these metabolite conjugates can successfully bind to their metabolite-specific antibodies. We used scanning electron microscopy, atomic force microscopy, and ELISA to confirm the successful binding of these NPs-conjugates (N1-acetylspermine (AcSpm), hippuric acid and creatinine) to metabolite-specific antibodies attached to silicon wafers and electrodes. We believe the methods developed here are quite general and could be used in the development of a number of different types of metabolite biosensors and portable metabolite assays.