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Review electrochemical aptasensors

Review electrochemical aptasensors. 汇报人：黄伟涛 2009 年 12 月 3 日 Electroanalysis 2009, 21, No. 11, 1237 – 1250. 1. Introduction of aptasensor. 2. Application of E-aptasensor. 3. Shortage and prospect. 4. Elicitation. Contents. 1. Introduction of aptasensor. 1.1 introduction of aptamer

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Review electrochemical aptasensors

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  1. Reviewelectrochemical aptasensors 汇报人：黄伟涛 2009年12月3日 Electroanalysis 2009, 21, No. 11, 1237 – 1250

  2. 1.Introduction of aptasensor 2. Application of E-aptasensor 3. Shortage and prospect 4.Elicitation Contents

  3. 1. Introduction of aptasensor • 1.1 introduction of aptamer • 1.1.1 origin of aptamer • 1.1.2 its advantages • 1.2 electrochemical aptasensor • 1.2.1 component of biosensor • 1.2.2 advantages of electrochemical aptasensor • 1.2.3 Annual trends on electrochemical aptasensors

  4. 1.1 introduction of aptamer • 1.1.1 origin of aptamer • Aptamer are RNA or DNA molecules (ca. 30 to 100 nucleotides) that recognize specific ligands and that are selected in vitro (selection evolution of ligands by exponential enrichment, SELEX process) from vast populations of random sequences. • In 1990, Ellington and Szostak termed the resulting motifs “aptamers,” while Tuerk and Gold dubbed the experimental process itself (SELEX). • 1.1.2 its advantages (compare to antibody) • 1)comparability —— affinity and specificity • 2)advantages • accurate, reproducible and more stable than antibodies • reversible denaturationandeasily modified • immunization and animalhosts are not necessary with aptamers

  5. selection evolution of ligands by exponential enrichment--SELEX

  6. 1.2 electrochemical aptasensor Enzyme Antibody Receptor nucleic- acid microorganisms... • 1.2.1 component of biosensor a biological recognition element (selectivity) a transducer (sensitivity) • 1.2.2 advantages of electrochemical aptasensor • Aptamers as key recognition tool elements • portable, low-cost and simple-to-operate, robust, reusable • easily miniaturized and integrated and automated, highly convenient for the developmentand applications of biosensors. • 1.2.3 Annual trends on electrochemical aptasensors(fig.1) electrochemical mass optical thermal

  7. 1.2.3 Annual trends on electrochemical aptasensors

  8. 2. Application of E-aptasensor • 2.1 Classification • 2.2Configurationandmain designs • 2.3Targets range • 2.4 Detailed introduction of electrochemical aptasensor

  9. 2.1 Classification • 2.1.1 according to labels • covalent • use of labels (Table1) • noncovalent • label-free(Table2) • 2.1.2 according to transduction principle • electrochemical (main) • optical • mass-sensitive transductions • 2.1.3 Global repartition of the different transduction modes amperometric impedimetric semiconductor field-effect principles potentiometric conductometric

  10. Amperometricaptasensors based on aptamer labels F. Other nanomaterials (CNTs, CPNTs)

  11. Aptamer label-free amperometric aptasensors

  12. 2.1.3 Global repartition of the different transduction modes Ca.1% ca. 10% ca. 32% ca. 57%

  13. 2.2Configurationandmain designs • two main configurations Targetbinding-induced aptamer conformational change Targetbinding-induced strand displacement • Main designs • Sandwich configuration(fig. a) • Signal-on & signal-off (fig. b) • G-quadruplex structure • based on covalent redox labels(fig.3) • based on noncovalent redox labels(fig.4) • label-free electrochemical aptasensors(fig.5)

  14. based on covalent redox labels Fig. 3. Main designs of electrochemical aptasensors based on covalent redox labels: sandwich amplified detection (a), signal-off aptasensors based on conformational change of labeled aptamers (b) or on labeled strand displacement (c1, c2), signal-on aptasensors based on conformational change of labeled aptamers (d1, d2) or on nonlabeled aptamer displacement (e)

  15. based on noncovalent redox labels Fig. 4. Main designs of electrochemical aptasensors based on noncovalent redox labels: labels interacting with the aptamer-target complex (a), intercalated labels (b), labels interacting with nucleic acid strands (c) or ionic labels (d1, d2).

  16. label-free electrochemical aptasensors Fig. 5. Main designs of aptamer label-free electrochemical aptasensors, based on a target-aptamer complex in a three-way junction configuration (a), on change of aptamer conformation (a, b, c1), on aptamer-complementary strand duplex dissociation (c1, c2), or on a competitive assay (d).

  17. 2.3 Targets range small ion molecules large proteins

  18. 2.4 Detailed introduction of electrochemical aptasensors • 2.4.1 Amperometric Aptasensors • 2.4.2 Aptamer Label-Free Potentiometric Aptasensor • 2.4.3 Aptamer Label-Free ISFETs • 2.4.4 Impedimetric Aptasensors Based on Labeled Aptamer and on Aptamer Label-Free Detection

  19. 2.4.1 Amperometric Aptasensors • 1 Enzyme Labels in 2004, Ikebukuro et al. reported the first electrochemical aptasensor. Thrombin was detected in a sandwich manner with the thiolated aptamer immobilized onto the gold electrode; the enzyme-labeled aptamer With GlcDH was added and the electric current generated by glucose addition was measured at 0.1 V vs. Ag/AgCl, in a 1-methoxyphenazine methosulfate (1-甲氧基吩嗪甲氨蝶呤) containing buffer.1 μMof thrombin was selectively detected [13]. With pyrroquinolinequinone glucose dehydrogenase (PQQ-GlcDH), the linear range extended from 40 nM to 100 nM, with a detection limit of 10 nM [16]. • 2 Metal Nanoparticle (NP) Labels Polsky et al [20] reported the first amplified aptasensor using electrocatalytic NPs. A sandwich configuration based on a self-assembled thiolated aptamers on gold reacting with thrombin and then with Pt NPs-labeled signaling aptamers was designed (Fig. 3a). Pt NPs were used as catalysts showing higher stability than enzymes. Pt NPs catalyzed reduction of H2O2 before linear sweep voltammetry measurements. Electrocatalytic reduction of H2O2 led to a cathodic current that could be related to thrombin concentration. Detection limit of 1 nM of thrombin was reported.

  20. 2.4.1 Amperometric Aptasensors • 3 Noncovalent Labels: Intercalated Redox Species (Fig. 4c)Target binding induced aptamer displacement from the aptamer/capture probe duplex structure that was immobilized on a gold electrode surface covered by a gold NP film. An external electroactive indicator, MB, interacted with DNA, so that target binding led to a decreased amount of adsorbed MB and a corresponding decreased redox current of the indicator that was measured by DPV(差分脉冲伏安法). Linear range extended from 5 nM to 1 mM adenosine and detection limit was of 1 nM. （Fig.4b）The beacon aptamer was covalently immobilized on a gold electrode surface. After accumulation of MB on the beacon aptamer gold film, thrombin binding led to release of the intercalated electrochemical marker, thus giving rise to a decrease in the cathodic peak current ofMBmeasured by DPV. Detection limit was given as 11 nM and the linear range extended up to 51 nM of thrombin. • 4 Aptamer Label-Free Detection A label-free and reagentless aptasensor allows direct detection of thrombin without using additional electroactive label [49]. Aptamers were immobilized by avidin-biotin interaction on a screen-printed electrode modified with gold NPs. Stripping voltammetry detection of gold NPs was performed after oxidation of gold NPs by application of an appropriate potential, mea-surement of the cathodic peak area related to gold oxide reduction. Detection limit was reported as 1 nM and the linear range extended from 10 nM to 10 mM of thrombin.

  21. 2.4.2 Aptamer Label-Free Potentiometric Aptasensor • A true potentiometric aptasensor has been recently reported. This simple and cheap device was based on poly(phenothiazine吩噻嗪) conducting polymers electropolymerized on a glassy carbon electrode. Avidin-modified polymer surfaces obtained by direct electrostatic precipitation have then been used to immobilize biotinylated anti-thrombin DNA aptamers. Measurement of the difference in the potential of the sensor in pH 3 and in pH 7.6 media gave rise to potentiometric thrombin detection in the concentration range from 109 to 106 M. • [Reference] G. Evtugyn et al. Electroanalysis 2008, 20, 1300.

  22. 2.4.3 Aptamer Label-Free ISFETs 1. Carbon Nanotubes (CNTs) • So et al. (Fig. 5b) presented the first successful demonstration of a single-walled carbon nanotube (SWCNT)-FET aptasensor. An anti-thrombinDNA aptamer was covalently linked to the side wall of carbon nanotubes that were assembled between source and drain electrodes. Target binding led to measurable decrease in conductance. Detection limit was around 10 nM , but the authors underlined that the sensitivity could be improved up to subnanomolar level by using high-quality SWCNT-FETs (with high on/off ratio). 2. Conducting Polymer Nanotubes • Thrombin detection has also been recently reported by using a label-free ion-gated apta-FET sensor based on the charge transport properties of polypyrrole nanotubes (NTs) [53]. Aptamers were covalently linked to nanotube networks onto microelectrode surfaces. Intermolecular interactions occurring during the aptamer-thrombin complex formation led to a decrease in the source-drain current. Controlled chemical functionality of conducting polymer NTs was shown to influence sensor responses. The detection limit of ca. 50 nM compared well with that of SWCNT-FETs (10 nM, [50]).

  23. 2.4.4 Impedimetric Aptasensors Based on Labeled Aptamer and on Aptamer Label-Free Detection • Aptamer Label-Free EIS The first reagentless non-faradaic impedimetric aptasensor has been designed for PDGF (血小板衍生生长因子) detection [67]. The non-faradaic impedance spectroscopy strategy does not require any reagent to be added to perform measurements.Aptamers wereim-mobilized on a silicon electrode surface. Target binding led to a decrease incapacitance which could be linearly related to the logarithm of PDGF concentration. Detection limit was 40 nM. 2. EIS Detection Using Aptamer Labels In the impedimetric aptasensor recently described for thrombin detection [32], signalampli-fication was obtained by co-immobilizing microperoxidase- 11 (MP-11) and a ferrocene-labeled aptamer on a gold electrode surface. The aptamer conformational change upon thrombin addition placed the redox aptamer molecule close to MP-11 (Fig. 3d1), thus resulting in a decrease in the film transfer resistance.Alow detection limit of 30 fM of thrombin was obtained.

  24. 2.4.4 Impedimetric Aptasensors Based on Labeled Aptamer and on Aptamer Label-Free Detection 3. Nanogap Sensors High frequency nanogap-impedance aptasensors have been reported for the label-free detection of thrombin [69, 70] or a smaller size target. For these structures, the analysis of the full frequency spectrum using appropriate equivalent circuits is not required. Nanogap sensors operate at a fixed frequency. In both cases, RNA aptamers were covalently immobilized onto the sensor surface via carbodiimide(碳二亚胺)/N-hydroxy-succinimide (N-羟基琥珀酰亚胺) coupling.

  25. 3. Shortage and prospect • 3.1 Shortage • 3.1.1 limited application for generalizable procedure • 3.1.2 Using labels is complex and expensive • 3.1.3 weak sensitivity to detection of small ligands • 3.2 Prospect • 3.2.1 availability of more aptamers • 3.2.2 aptamer microarrays • 3.2.3 search for label-free and reagentless devices

  26. 3.1 Shortage • 3.1.1 limited application for generalizable procedure • target binding-induced strand displacement appears as a more generalizable procedure than conformational change • 3.1.2 complex and expensive • Using label : complex and expensive • Label-free : simple, cost-effective and no external modification but not obviously reagentless • 3.1.3 low sensitivity to detection of small ligands • Generally, detection limits for small molecule detection are in the low μM range

  27. 3.2 Prospect • 3.2.1 availability of more aptamers with higher affinity for small molecules and appropriate miniaturization and integration • 3.2.2 aptamer microarrays are expected to play a dominant role in proteomics • 3.2.3 the search for label-free and reagentless devices remains a challenge, especially for detection of small molecules

  28. 4. Elicitation • 4.1 Search for more labels • 4.2 Other signal amplification : QDs… • 4.3 Other configuration similar to sandwich • 4.4 Design of nanogap sensor • 4.5 Why Amperometric aptasensors were reported so much? • ………………

  29. 4. Elicitation • 4.5 why Amperometric aptasensors were reported so much? • 1 由于外加电势的模式可以多种多样 • 2 有多种电极材料可供选择 • 3 根据需要可设计电解池和采用不同类型的微加工技术 • 因此安培型电化学传感器是非常有用的分析化学工具。主要用于检测电活性物质(可以进行氧化还原反应的物质)

  30. Thank You !

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