A label-free electrochemical aptasensor for the detection of kanamycin in milk
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Kanamycin is detected based on the conformational change of the aptamer attached to the electrode surface and the corresponding SWV current change in [Fe(CN)6]3−/4−solution.Keywords:
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To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer–target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.
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SELEX Aptamer Technique
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Given that a split aptamer provides a chance for the development of a sandwich assay for targets with only one aptamer, it has received extensive attention in biosensing. However, due to the lack of binding mechanisms and reliable methods, there were still a few split aptamers that bind to proteins. In this work, cardiac biomarker myoglobin (Myo) was selected as a model, a new strategy of engineering split aptamers was explored with atomic force spectroscopy (AFM), and split aptamers against target protein could be achieved by choosing the optimal binding probability between split aptamers and target. Then, the obtained split aptamers were designed for Myo detection based on dynamic light scattering (DLS). The results demonstrated that the obtained split aptamers could be used to detect targets in human serum. The strategy of engineering split aptamers has the advantages of being intuitive and reliable and could be a general strategy for obtaining split aptamers.
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This chapter discusses the progress made in the aptamer-based colorimetric assay. Aptamer generation involves three steps, which include binding, separation, and amplification. Since aptamers show a high binding affinity with their target molecules, aptamer-based sensors have been used to detect and screen for various diseases. Several aptamers are synthesized against various metal ions such as lead, mercury, and arsenic. Aptamers and gold nanoparticles (AuNPs) have proved to be suitable candidates in the biosensor, and various sensors have used these two in order to improve the detection method. Using the AuNP-based colorimetric aptasensor, researchers detected various targets and disease biomarkers with different levels of sensitivities. Among aptamer-based colorimetric assays, DNA aptamers have been involved predominantly in the development of this assay and proved with several targets. However, the limitation with this assay is that when preferring the protein as the target, it may tend to bind nonspecifically with the AuNP.
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Aptamers are nucleic acids that can bind to various molecules. Because they have some features that are lacking in antibodies, aptamers could serve as alternatives to antibodies. For the purpose of biosensing, we focused on aptamers that undergo structural changes on binding to their target molecules. We constructed an aptamer-based bound/free (B/F) separation system that uses a designed aptamer named the "capturable aptamer". The capturable aptamer changes its structure upon recognizing its target molecule thereby exposing a specific single-strand region. The oligonucleotide that is complementary to this exposed region, named the "capture DNA" is immobilized on a support. This design permits the exclusive capture by the capture DNA of the aptamer bound to its target, and subsequent removal of any unbound aptamer and contaminants by B/F separation. The removal of unbound contaminants or aptamers results in highly sensitive detection at similar levels to those achievable by sandwich-based immunoassay. We describe the construction of a thrombin-detection system by using a capturable aptamer, and we discuss the potential of capturable aptamers in clinical diagnostics.
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Linear range
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Aptamers have emerged as research hotspots of the next generation due to excellent performance benefits and application potentials in pharmacology, medicine, and analytical chemistry. Despite the numerous aptamer investigations, the lack of comprehensive data integration has hindered the development of computational methods for aptamers and the reuse of aptamers. A public access database named AptaDB, derived from experimentally validated data manually collected from the literature, was hence developed, integrating comprehensive aptamer-related data, which include six key components: (i) experimentally validated aptamer-target interaction information, (ii) aptamer property information, (iii) structure information of aptamer, (iv) target information, (v) experimental activity information, and (vi) algorithmically calculated similar aptamers. AptaDB currently contains 1350 experimentally validated aptamer-target interactions, 1230 binding affinity constants, 1293 aptamer sequences, and more. Compared to other aptamer databases, it contains twice the number of entries found in available databases. The collection and integration of the above information categories is unique among available aptamer databases and provides a user-friendly interface. AptaDB will also be continuously updated as aptamer research evolves. We expect that AptaDB will become a powerful source for aptamer rational design and a valuable tool for aptamer screening in the future. For access to AptaDB, please visit http://lmmd.ecust.edu.cn/aptadb/.
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When aptamers first emerged almost two decades ago, most were RNA species that bound and tagged or inhibited simple target ligands. Very soon after, the 'selectionologists' developing aptamer technology quickly realized more potential for the aptamer. In recent years, advances in aptamer techniques have enabled the use of aptamers as small molecule inhibitors, diagnostic tools and even therapeutics. Aptamers are now being employed in novel applications. We review, herein, some of the recent and exciting applications of aptamers in cell-specific recognition and delivery.
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Aptamers are small structured RNA or DNA oligonucleotides that bind target molecules with high avidity and affinity. Aptamer selection begins with a hugely complex library of aptamer sequences that are enriched for a specific target using the SELEX process, Systematic Evolution of Ligands by EXponentional enrichment. An important consideration in the SELEX process is the protocol used to fold aptamers into their active conformations. The folding conditions include multiple variables, such as temperature, buffer components, incubation time and aptamer concentration. Aptamer folding protocols vary widely across the aptamer field, and most published folding conditions primarily describe only temperature and folding time. To understand how variations in folding conditions impact aptamer function, we developed a novel high-throughput assay to interrogate the optimal folding conditions of several published aptamers. The Aptamer Fluorescence Binding and Internalization (AFBI) assay is a cell-based platform that uses a 96-well plate format to rapidly and efficiently screen multiple fluorescent-labelled aptamers against hundreds of conditions. The AFBI assay can be applied to rapidly determine aptamer binding constants (Kd) on cells, time course of aptamer internalization and cross reactivity of aptamers against different cell types. Using the AFBI assay, we screened several different folding parameters against published aptamers. We found that the buffer components contributed significantly more to an aptamer's function than any other examined factor. The concentration of an aptamer during folding was important for some aptamers but not others, including aptamers that originated from the same selection. Most surprising was that most temperature protocols had little impact on aptamer function after folding, the exception being that high temperature (95°C) often attenuated aptamer function. In summary, our data using the AFBI assay revealed that aptamer folding is more dependent on buffer components than the temperature protocol. Furthermore, aptamers derived from the same selection may have different optimal folding conditions. These data demonstrate that optimal aptamer folding protocols need to be more carefully interrogated on a per aptamer basis and reported in detail to allow for efficacious and reproducible results.
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Folding (DSP implementation)
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SELEX Aptamer Technique
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