Aptasensors

Biosensors that employ aptamers as a recognition element are called aptasensors. Aptasensors will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers. Aptamers are single-stranded RNA or DNA molecules that bind to their target molecules with high specificity and affinity. Aptamers have been developed for different appHcations. Their use as biological recognition elements 

Development of aptasensors has been carried out with various detection methods  

Aptamers are typically isolated from combinatorial libraries by a process of in vitro evolution called systematic evolution of Hgands by exponential enrichment (SELEX). 

Aptamers are high-afFmity ligands selected from DNA or RNA libraries via SELEX process. SELEX is a process used for in vitro selection of aptamers, which are highly specific in binding as well as function due to the nucleotide sequence and shape. 

A library is created, containing around 1x10 oligonucleotides. These are singlestrand nucleic acids consisting of a random sequence region flanked by a binding site. 

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It is not surprising that wide range of aptamer-based sensors (aptasensors) have been reported in the literature.118-120 Recently, electrochemical aptamer sensors gained considerable attention.121,122 Since, nucleic acids aptamers fold their structure upon binding to the target molecule, formation of the aptamer-target complex can be... 

Despite being relatively new technology, aptamers have a tremendous potential and can be envisioned to rival antibodies and other traditional recognition elements for molecular detection and recognition, due to their inherent affinity, selectivity, and ease of synthesis. In addition, the combination of aptasensors with electrochemical detection methods has the added advantage of further cost reduction and miniaturization of such systems. 

Figure 12.18 (a) Electrochemical aptasensor for thrombin based on the control of electron... 

Several electrical aptamer biosensors implemented the biocatalytic hydrolytic activities of thrombin, or the fact that proteins (e.g., thrombin) often include several binding sites for the formation of supramolecular complexes with different aptamers. The bioelectrocatalytic detection of thrombin by an electrical aptasensor was demonstrated by formation of an aptamer-thrombin complex on the electrode, followed by a thrombin-mediated hydrolysis of the nitroaniline-functionalized peptide, (22), yielding the redox-active product nitroaniline, (23), which was analyzed electrochemically76 (Fig. 12.20b). A further bioelectrocatalytic aptasensors configuration is depicted in Fig. 12.20c, where the multidentate formation of aptamer-protein supramolecular complexes was used to analyze thrombin.76 Thrombin includes two different binding sites for aptamers.77 One of the thrombin aptamers... 

Metal NPs were also used as labels to follow aptamer-substrate interactions. The supramolecular self-organization of the aptamer-substrate complexes on surfaces was implemented to develop different configurations of electrical aptasensors.88 The anticocaine aptamer was separated into two fragments (37 and 38) (Fig. 12.24a). The nucleic acid (37) was assembled on a Au electrode, whereas the nucleic acid (38) was... 

O Sullivan, C.K. (2002) Aptasensors tire future of biosensing Anal. Bioanal. Chem. 372, 44-4-8. 

Aptamer-ligand interactions can be detected by conventional assay based on, e.g., radioactive labeling, chromatography, capillary electrophoresis and mass spectrometry. These methods have been reviewed recently by Tombelli et al. [5]. Novel approach in detection of aptamer-ligand interaction is connected with aptasensors. In aptasensors,... 

Electrochemical methods of detection affinity interactions at the surfaces are rather effective due to their relative simplicity and low cost. Amperometric aptasensor based on sandwich assay was proposed by Ikebukuro et al. [42]. They used two aptamers selective to thrombin. 

The first aptasensor reported was particularly based on optical detection [66]. The 58-mer RNA aptamer selective to L-adenosine was immobilized onto the core of multimode fiber using avidin-biotin method. The detection was based on competitive binding of FITC-labeled L-adenosine with unlabeled analyte. This sensor also allowed to study the kinetics of binding and determine equilibrium constants. 

AQ/Qo = (Q - Qo)/Qo, where Q0 is charge consumption without analyte and Q is that at certain thrombin, concentration. An example of calibration curve for two independently prepared electrodes is shown in Fig. 47.3. It is seen, that results are well reproducible. Statistical analysis, performed earlier [4] revealed that standard error is approximately 11%. Interferences of this aptasensor with other compounds, human serum albumine (HSA) and human IgG are relatively low. An example is shown in Fig. 47.4, where calibration curve for thrombin is compared with those for HSA and IgG. Please note, that concentrations of HSA and IgG are much higher in comparison with that of thrombin. 

Wang Y, Wei H, Li B, Ren W, Guo S, Dong S, Wang E (2007) SERS opens a new way in aptasensor for protein recognition with high sensitivity and selectivity. Chem Comm 28 5220-5222... 

Wang YL, Lee K, Imdayaraj J (2010) SERS aptasensor from nanorod-nanoparticle jimction for protein detection. Chem Commim 46 613-615... 

Radi, A. E., Acero SSnchez, J. L., Baldrich, E., O Sullivan, C. K. (2006). Reagentless, reusable, ultrasensitive electrochanical molecular beacon aptasensor. J. Am. Chem. 

Baldrich, E., Restrepo, A., O Sullivan, C.K. (2004). Aptasensor development elucidation of critical parameters for optimal aptamer performance. Anal. Chem. 76, 7053-7063... 

Mir, M., Vreeke, M., Katakis, I. (2006). Different strategies to develop an electrochemical thrombin aptasensor. Electrochem. Commun. 8, 505-511... 

Another nucleic acid sensor, also based on the affinity between the analyte and the receptor immobilized on the surface, is reported as an example of the most recent trend in the field. This receptor, called aptamer, acts as capturing receptor for a molecule in solution, such as a protein. An aptasensor developed for a specific protein will be reported. 

New Frontiers in Nucieic Acid-Based Piezoeiectric Biosensors Aptasensors... 

With the piezoelectric transduction, an RNA aptamer has been used as biorecognition element to develop an aptasensor for the detection of Tat (trans-activator of transcription) protein [9]. 

The immunosensor for Tat protein was realized with the specific monoclonal anfibody anfi-Tat, immobilized on fhe sensor following the same procedure used for the aptamer. The same concentrations of Tat used for the aptasensor were tested. 

Comparison of the developed aptasensor with other Tat detection methods is very difficult since most of the published papers on this subject deal with Tat-derived peptides and not with the full length protein. Anyway, the results demonstrated that the use of a biosensor with a specific aptamer as biorecognition element could be an interesting approach in the detection of proteins, which has been examined here considering a model system. 

The advent of new receptors based on nucleic acids, called aptamers, is a new challenge in piezoelectric sensing, not based on the hybridization reaction. The development of aptasensors has analogies to the introduction of immunosensing almost two decades ago the direct detection of analytes in complex matrices by immobilizing an optimized receptor on the sensing surface. 

There is a limited number of studies on aptasensors (aptamer sensors), particularly for the detection of small organic molecules. An electrochemical aptasensor was developed by Kim et al. " for the detection of tetracycline using an ssDNA aptamer that selectively binds to tetracycline as the recognition element. The aptamer was highly selective for tetracycline and distinguishes minor structural... 

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