汇报人：黄伟涛2009 年 12 月 3日

Electroanalysis 2009, 21, No. 11, 1237 – 1250

Review electrochemical aptasensors

Contents

1. Introduction of aptasensor

2. Application of E-aptasensor

3. Shortage and prospect

4. Elicitation 

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

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 denaturation and easily modified immunization and animal hosts are not necessary with aptamers

selection evolution of ligands by exponential enrichment--SELEX

1.2 electrochemical aptasensor

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)

EnzymeAntibodyReceptornucleic- acid microorganisms...

electrochemical mass optical thermal

1.2.3 Annual trends on electrochemical aptasensors

2. Application of E-aptasensor

2.1 Classification

2.2 Configuration and main designs

2.3 Targets range

2.4 Detailed introduction of electrochemical aptasensor

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

amperometricimpedimetricsemiconductor field-effect principlespotentiometricconductometric

Amperometric aptasensors based on aptamer labels

F. Other nanomaterials (CNTs, CPNTs)

Aptamer label-free amperometric aptasensors

2.1.3 Global repartition of the different transduction modes

ca. 57%

ca. 32%

ca. 10%Ca.1%

2.2 Configuration and main designs

two main configurations Target binding-induced aptamer conformational change

Target binding-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)

based on covalent redox labels

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

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).

label-free electrochemical aptasensors

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

small ion molecules

large proteins

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

2.4.1 Amperometric Aptasensors

1 Enzyme Labelsin 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 μM of thrombin was selectively detected [13]. With pyrroquinoline quinone 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) LabelsPolsky 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.

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 of MB measured by DPV. Detection limit was given as 11 nM and the linear range extended up to 51 nM of thrombin.

4 Aptamer Label-Free DetectionA 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.

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.

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]).

2.4.4 Impedimetric Aptasensors Based on Labeled Aptamer and on Aptamer Label-Free Detection1. Aptamer Label-Free EISThe 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 were im-mobilized on a silicon electrode surface. Target binding led to a decrease in capacitance which could be linearly related to the logarithm of PDGF concentration. Detection limit was 40 nM.

2. EIS Detection Using Aptamer LabelsIn the impedimetric aptasensor recently described for thrombin detection [32], signal ampli-

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. A low detection limit of 30 fM of thrombin was obtained.

2.4.4 Impedimetric Aptasensors Based on Labeled Aptamer and on Aptamer Label-Free Detection3. Nanogap SensorsHigh 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.

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

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

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

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? ………………

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 )