Developing A Multiplexed Electrochemical Aptamer-Based Biosensor For The Quantification Of Free Amino Acids
Authors:   , Dunham Hamish 
Published: October 2024

<p><strong>The demand for profiling of systemic amino acid (AA) concentrations as indicators of nutritional, metabolic and fertility status is growing. Therefore, developing an electrochemical aptamer-based (E-Ab) biosensor for the simple, and rapid quantification of multiple free-AAs in biological fluids would be of significant benefit to the medical field. Current approaches to free amino acid profiling require lab facilities, expensive equipment and a high level of expertise. Aptamers represent next generation bioreceptors and are capable of binding small-molecule targets such as AAs in complex samples, such as biological fluids. These can be integrated into electrochemical systems to produce electrochemical aptamer-based biosensors that can be used for routine quantitation of free AA’s in biological fluids.</strong></p><p>Herein, the isolation and characterisation of novel L-asparagine binding aptamers, and the development of a multiplexed E-Ab biosensor for free-AA detection is described. Conventional affinity-matrix SELEX was performed to isolate single-stranded (ss)DNA aptamers that bound the AA targets of L-arginine, L-asparagine, L-glutamic acid and L-proline. Candidate aptamers were characterised using circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). Two novel L-asparagine aptamers were identified that exhibited low-affinity for the target molecule. ssDNA aptamers reported in the literature that target AA’s were similarly characterised. As a result, L-arginine (Arg12-28), L-phenylalanine (Phe1) and L-tryptophan (Trp3a-1) aptamers were identified for testing on an E-Ab sensor.</p><p>Test aptamers were functionalised onto the gold electrode E-Ab sensor and the target and off-target molecules were applied at a range of concentrations in buffer. For the Phe1 aptamer, a concentration dependent gain in signal of 10 to 30% was observed for L-phenylalanine concentrations ranging from 10 to 100 µM. At mM range concentrations, off-target interactions are observed for structurally similar AA such as L-tryptophan and L-tyrosine. For the Trp3a-1 aptamer, a ~10% gain in signal was observed in response to L-tryptophan concentrations in the mM range. The Arg12-28 aptamer did not exhibit an appreciable sensor response to L-arginine.Although the Phe1 sensor was tested in human saliva this result was not reproduceable. However, the Phe1 aptamer was demonstrated to have decreased binding activity in lower ionic conditions such as those found in saliva the sensor response observed here when testing in saliva was greater than when tested in an idealized buffer system.</p><p>In summary, an E-Ab sensor was developed that measured L-phenylalanine at a physiologically relevant concentration range, which could be of use for individuals facing L-phenylalanine related medical challenges. There were challenges in the reproducibility of the sensor functionalisation which would need to be addressed, along with a more expansive set of AA-binding ssDNA aptamers, before such a device can be realised.</p>