Study On Amperometric And Multiplexed Aptasensor

Aptamers are oligonucleotides selected from random sequence nucleic acid libraries using systemic evolution of ligands by exponential enrichment (SELEX). Because of their high affinities and specificities in bingding to amino acids, protein, inorganic ions or medicine,aptamers have been widely used for medical diagnosis, drug testing, Biological sensors and so on. Electrochemical aptasensor, which are based on the specificity of aptamer-target recognition with electrochemical transduction for analytical purposes, have received particular attention in recent years. This research focuses on the details as follow:Part 1 Aptamer/Nanoparticle-Based Sensitive, Multiplexed Electronic Coding of Proteins and Small Biomolecules through a Backfilling Strategywe report a novel aptamer/nanoparticle-based backfilling strategy for one-spot simultaneous detection of proteins and small biomolecules, employing lysozyme and adenosine triphosphate (ATP) as the model target analytes. Our approach relies on target-in-duced release of aptamers from the DNA duplexes on a sensing surface, followed by backfilling hybridization of the resulting single-stranded DNA molecules with aptamers conjugated to the electrochemically encoded nanoparticles. Subsequent unique electrochemical (EC) signatures of the acid-dissolved nanoparticles at distinct potential positions with well-resolved peaks thus reflect the identities and concentrations of lysozyme and ATP. Compared with previously reported multiplexed detection schemes, our new analysis route for proteins and small biomolecules offers three clear advantages. First, simultaneous determination of biomolecules with distinct molecular weights (lysozyme,14.3 kDa versus ATP,507.2 Da) is achieved for the first time with electrochemically encoded nanoparticle tags. Second, in contrast to other common "signal off" assay formats for lysozyme and ATP based on target-induced displacement or conformational changes of the corresponding aptamers, our multiplexed method is a "signal on" configuration, which exhibits improved sensitivity. Third, the analytical signal amplification by the nanoparticle labels composed of a large number of metal ions and the high sensitivity of the voltammetric stripping detection lead to low-concentration determination of the target analytes. The combination of these advantages facilitates the detection of biomolecules with distinct sizes in a multiplexed and sensitive manner.Part 2 A reagentless, disposable and multiplexed electronic biosensing platform:Application to molecular logic gateswe demonstrate a reagentless, multiplexed, disposable and yet sensitive electronic sensing platform for simultaneous determination of biomolecules with significant difference in size (proteins and small molecules). Our novel multiplexed sensing platformrelies on a single-step, target-induced displacement of two types of redox-tags (methylene blue,MB and ferrocene, Fc) conjugated signaling aptamers confined on a disposable screen printed carbon electrode (SPCE), followed by electrochemical (EC)monitoring of the surface-remained redox-tags. The redoxtags, MB and Fc, exhibit two well-resolved peaks, whose positions and sizes reflect the identities and concentrations of the target analytes, thrombin and adenosine triphosphate (ATP), respectively. On the other hand, there has been an increasing research focus on the development of molecular logic gates because of their important roles inmolecular computation and "autonomously regulated" chemical systems. Based on our new multiplexed sensing protocol, an "AND" molecular logic gate operation is further realized by employing the target analytes, thrombin and ATP, as the inputs and the current intensity suppressions from the redox-tags as the outputs, respectively. Moreover, intense research efforts have been put in employing aptamers as recognition elements in biodetections due to their distinct advantages such as high selectivity, stability, versatile tar get binding capability (proteins, drugs, amino acids, etc.) and long shelf life over antibodies. Besides, SPCEs are promising materials for the fabrication of an EC biosensor device due to their disposable, easy mass productive and cost-effective nature, and EC methods can transduce molecular recognition events in a fast, sensitive and miniaturized fashion as well. Coupling the advantages of EC transduction, SPCEs, and the distinct EC labels with the superiority of aptamers would potentially facilitate the fabrication ofmultiplexed point-of-care devices and the design of robust logic gate operations.Part 3 A reagentless and disposable electronic genosensor:from multiplexed analysis to molecular logic gateswe report herein a reagentless, disposable, sensitive and multiplexed genosensor for one-spot simultaneous monitoring of two gene biomarkers from the salmonella typhimurium pathogen (gyrB gene) and the colorectal tumor (K-ras gene), respectively. This multiplexed genosensor is based on changes in current intensities from two distinct redox-tags (methylene blue, MB and ferrocene, Fc) conjugated to stem-loop probes on a disposable screen printed carbon electrode (SPCE) upon hybridization with the corresponding target DNAs. The redox-tags exhibit two well-resolved peaks, whose positions (MB:-0.28 V and Fc:+0.25 V) and sizes reflect the identities and concentrations of the target sequences. Moreover, molecular logic gates have received increasing attention owing to its important roles in developing molecular computations and "'autonomously regulated" chemical systems. Most of the molecular logic gates reported to date, however, are mainly based on optical outputs, which suffer the limitations of cumbersomely interfacing the optical outputs with non-molecular-based technologies. In response, based on our multiplexed DNA sensing strategy, we show that a robust"AND" molecular logic gate operation can be designed by employing the gyrB and K-ras genes as inputs and the distinct current suppressions from the redox-tags as the electronic outputs, respectively.