Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.

Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM.

A multilayer of CdSe quantum dots (QDs) was prepared on the mesoporous surface of a nanoparticulate TiO(2) film by a layer-by-layer (LBL) assembly using the electrostatic interaction of the oppositely charged QD surface for application as a sensitizer in QD-sensitized TiO(2) solar cells. To maximize the absorption of incident light and the generation of excitons by CdSe QDs within a fixed thickness of TiO(2) film, the experimental conditions of QD deposition were optimized by controlling the concentration of salt added into the QD-dissolved solutions and repeating the LBL deposition a few times. A proper concentration of salt was found to be critical in providing a deep penetration of QDs into the mesopore, thus leading to a dense and uniform distribution throughout the whole TiO(2) matrix while anchoring the oppositely charged QDs alternately in a controllable way. A series of post-treatments with (1) CdCl(2), (2) thermal annealing, and (3) ZnS-coating was found to be very critical in improving the overall photovoltaic properties, presumably through a better connection between QDs, effective passivation of QD's surface, and a high impedance of recombination, which were proved by transmission electron microscopy (TEM) and electrochemical impedance spectroscopy (EIS) experiments. With a proper post-treatment of multilayered QDs as a sensitizer, the overall power conversion efficiency in the CdSe QD-sensitized TiO(2) solar cells could reach 1.9% under standard illumination condition of simulated AM 1.5G (100 mW/cm(2)).

Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by several species of Aspergillus and Penicillium and frequently found as a natural contaminant in a wide range of food commodities. Novel and robust biorecognition agents for detecting this molecule are required. Aptamers are artificial nucleic acid ligands able to bind with high affinity and specificity to a given target molecule. In the last few years, three separate research groups have selected aptamers for ochratoxin A. While each of these three families of aptamers have been incorporated into various methods for detecting OTA, it is unclear if each aptamer candidate is better suited for a particular application. Here, we perform the first head-to-head comparison of solution-based binding parameters for these groups of aptamers. Based on our results, we provide recommendations for the appropriate choice of aptamer for incorporation into solution-based biorecognition assays and applications.

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Nano-aptamer probes were prepared and used in lateral flow colorimetric assays for the detection of Ochratoxin A (OTA). In this study, two approaches were examined using 5'-biotin-modified OTA aptamers and silver or gold nanoparticles (AgNP or AuNP). The first method used an "adsorption-desorption" approach wherein aptamers were adsorbed onto the metal nanoparticle surface. Upon the addition of OTA, the aptamer binds specifically to the target, releasing the NPs. The above solutions were applied on a lateral flow assay (LFA) and a detection limit of 6.3 nM was achieved with both metal nanoparticles. The second method used a labelled approach based on Linkage Inversion Assembled Nano-Aptasensors (LIANAs) using a DNA linker containing a 5'-5' linkage inversion (5'-5' linker) to assemble biotinylated aptamer-functionalized metal nanoparticles. In the presence of target, OTA specifically binds with its aptamer leading to release of the linker and disassembly of LIANA aggregates into dispersed nanoparticles. The same solutions were applied in LFA format and the lowest detection limit of 0.63 nM was achieved. The results indicated that the LIANA-based LFA strips were more sensitive than the "adsoprtion-desorption" LFAs. Both lateral flow assays are inexpensive, simple, and rapid to perform and produces results visible to the naked-eye.

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