Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

Here, we describe the fabrication of an electrochemical immunoglobulin E (IgE) aptasensor using enzyme-linked aptamer in the sandwich assay method and thionine as redox probe. In this protocol, 5′-amine-terminated IgE aptamer and thionine were covalently attached on glassy carbon electrode modified with carbon nanotubes/ionic liquid/chitosan nanocomposite. Furthermore, another IgE aptamer was modified with biotin and enzyme horseradish peroxidase (HRP), which attached to the aptamer via biotin–streptavidin interaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were performed at each stage of the chemical modification process to confirm the resulting surface changes. The presence of IgE induces the formation of a double aptamer sandwich structure on the electrode, and the electrocatalytic reduction current of thionine in the presence of hydrogen peroxide was measured as the sensor response. Under optimized conditions and using differential pulse voltammetry as the measuring technique, the proposed aptasensor showed a low detection limit (6 pM) and high sensitivity (1.88 μA nM⁻¹). This aptasensor also exhibited good stability and high selectivity for IgE detection without an interfering effect of some other proteins such as bovine serum albumin (BSA) and lysozyme. The application of the aptasensor for IgE detection in human serum sample was also investigated. The proposed protocol is quite promising as an alternative sandwich approach for various protein assays.     

Nanocrystalline diamond impedimetric aptasensor for the label-free detection of human IgE

Like antibodies, aptamers are highly valuable as bioreceptor molecules for protein biomarkers because of their excellent selectivity, specificity and stability. The integration of aptamers with semiconducting materials offers great potential for the development of reliable aptasensors. In this paper we present an aptamer-based impedimetric biosensor using a nanocrystalline diamond (NCD) film as a working electrode for the direct and label-free detection of human immunoglobulin E (IgE). Amino (NH(2))-terminated IgE aptamers were covalently attached to carboxyl (COOH)-modified NCD surfaces using carbodiimide chemistry. Electrochemical impedance spectroscopy (EIS) was applied to measure the changes in interfacial electrical properties that arise when the aptamer-functionalized diamond surface was exposed to IgE solutions. During incubation, the formation of aptamer-IgE complexes caused a significant change in the capacitance of the double-layer, in good correspondence with the IgE concentration. The linear dynamic range of IgE detection was from 0.03 μg/mL to 42.8 μg/mL. The detection limit of the aptasensor reached physiologically relevant concentrations (0.03 μg/mL). The NCD-based aptasensor was demonstrated to be highly selective even in the presence of a large excess of IgG. In addition, the aptasensor provided reproducible signals during six regeneration cycles. The impedimetric aptasensor was successfully tested on human serum samples, which opens up the potential of using EIS for direct and label-free detection of IgE levels in blood serum.     

Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)–lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA–LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.     

Label-free electrochemical aptasensor for thrombin detection based on the nafion@graphene as platform

A sensitive label-free electrochemical aptasensor was successfully fabricated for thrombin detection with nafion@graphene as platform. With electrostatic interaction between nafion and methylene blue (MB), positive charged MB was successfully assembled on nafion@graphene modified electrode surface, which provided amounts of redox probes for electrochemical aptasensor. In the presence of thrombin, the thrombin aptamer (TBA) on the electrode surface would catch the target on the electrode interface, which made a barrier for electrons and inhibits the electro-transfer, resulting in the decreased differential pulse voltammetry signals of MB. As a result, the proposed approach showed a high sensitivity and a wider linearity to thrombin in the range 0.01–50 nM with a detection limit of 6 pM.     

An electrochemical aptasensor based on hybridization chain reaction with enzyme-signal amplification for interferon-gamma detection

A novel electrochemical aptasensor based on hybridization chain reaction (HCR) with enzyme-signal amplification was constructed for the detection of interferon-gamma (IFN-γ). In this aptasensor, the recognition probes which contained the sequence of IFN-γ aptamer were initially binded to IFN-γ, and the unbound recognition probes were captured on the electrode as an initiator to trigger the HCR. The two DNA hairpins bio-H1 and bio-H2 were opened by the recognition probe, and bound one by one on the electrode. The biotin was used as a tracer in the hairpins and streptavidin-alkaline phosphatase (SA-ALP) as a reporter molecule. Then, SA-ALP converted its electro-inactive substrate 1-naphthyl phosphate into an electroactive derivative 1-naphthol generating amplified electrochemical signal by differential pulse voltammetry (DPV). The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated for enzymatic dephosphorylation of 1-naphthyl phosphate. The electrochemical signal observed was inversely related to the concentration of IFN-γ. The proposed approach showed a high sensitivity for IFN-γ in a concentration range of 0.5-300 nM with a detection limit of 0.3 nM. The sensing system also provided satisfactory results for the detection of IFN-γ in the cell media.     

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices

An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3'-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H(2)O(2). The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 μM to 50 μM and the detection limit down to 20 nM (S/N=3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.     

A novel aptasensor based on silver nanoparticle enhanced fluorescence

In the present study, we report a novel aptasensor based on silver nanoparticle enhanced fluorescence for the detection of adenosine. First, the distance dependence nature of silver nanoparticle enhanced fluorescence was investigated through fluorescent dyes modified oligonucleotides to control the spacing distance between dyes and AgNP. The results showed that the fluorescence intensity reached the maximum value with the spacing distance of dyes about 8 nm from AgNP surface. The fluorescence intensity decreases when the spacing distance is either above or below this value. Based on this result, a fluorescence switch is constructed. In the "OFF" state, without the target molecules, there is a greater spacing distance between the Cy3 dyes and the AgNP giving comparatively lower fluorescence intensity. While in the "ON" state, in the presence of target molecules, the fluorescence signals increased for the conformation structure change of the aptamer which shorten the spacing distance between the Cy3 dyes and the AgNP to 8 nm. Using adenosine as target, the aptasensor produced a linear range from 200 nM to 200 μM with a correlation coefficient of 0.9949 and the detection limit was 48 nM estimated using 3σ. The aptasensor was also found to be specific in targeting adenosine. The presented method shows a new strategy of combining aptamer recognition and silver nanoparticle for fluorescence signal enhancement and increasing sensitivity.     

Colorimetric Aptasensor Using Unmodified Gold Nanoparticles for Homogeneous Multiplex Detection

Colorimetric aptasensors using unmodified gold nanoparticles (AuNPs) have attracted much attention because of their low cost, simplicity, and practicality, and they have been developed for various targets in the past several years. However, previous research has focused on developing single-target assays. Here, we report the development of a homogeneous multiplex aptasensor by using more than one class of aptamers to stabilize AuNPs. Using sulfadimethoxine (SDM), kanamycin (KAN) and adenosine (ADE) as example targets, a KAN aptamer (750 nM), an SDM aptamer (250 nM) and an ADE aptamer (500 nM) were mixed at a 1∶1∶1 volume ratio and adsorbed directly onto the surface of unmodified AuNPs by electrostatic interaction. Upon the addition of any of the three targets, the conformation of the corresponding aptamer changed from a random coil structure to a rigid folded structure, which could not adsorb and stabilize AuNPs. The AuNPs aggregated in a specific reaction buffer (20 mM Tris-HCl containing 20 mM NaCl and 5 mM KCl), which led to a color change from red to purple/blue. These results demonstrate that the multiplex colorimetric aptasensor detected three targets simultaneously while maintaining the same sensitivity as a single-target aptasensor for each individual target. The multiplex aptasensor could be extended to other aptamers for various molecular detection events. Due to its simple design, easy operation, fast response, cost effectiveness and lack of need for sophisticated instrumentation, the proposed strategy provides a powerful tool to examine large numbers of samples to screen for a small number of potentially positive samples containing more than one analyte, which can be further validated using sophisticated instruments.     

A three-way junction aptasensor for lysozyme detection

A well-designed three-way junction (TWJ) aptasensor for lysozyme detection was developed based on target-binding-induced conformational change of aptamer-complementary DNA (cDNA) as probe. A ferrocene (Fc)-tagged cDNA is partially hybridized with an anti-lysozyme aptamer to form a folded structure where there is a coaxial stacking of two helices and the third one at an acute angle. In addition, the fabrication of the sensor was achieved via the single-step method, which offered a good condition for sensing. In the absence of lysozyme, electron transfer (eT), through the coaxial two helices called "conductive path", is allowed between Fc-labeled moiety and the electrode. The binding of lysozyme to the aptamer blocks eT, leading to diminished redox signal. This aptasensor with an instinct signal attenuation factor shows a high sensitivity to lysozyme, and the response data is fitted by nonlinear least-squares to Hill equation. Detection limit is 0.2nM with a dynamic range extending to 100nM. Compared with existing electrochemical impedance spectroscopy (EIS)-based approaches, TWJ-DNA aptasensor was demonstrated to be more specific for detection and simpler for regeneration procedure.     

Electrochemical aptasensor for tetracycline detection

An electrochemical aptasensor was developed for the detection of tetracycline using ssDNA aptamer that selectively binds to tetracycline as recognition element. The aptamer was highly selective for tetracycline which distinguishes minor structural changes on other tetracycline derivatives. The biotinylated ssDNA aptamer was immobilized on a streptavidin-modified screen-printed gold electrode, and the binding of tetracycline to aptamer was analyzed by cyclic voltammetry and square wave voltammetry. Our results showed that the minimum detection limit of this sensor was 10 nM to micromolar range. The aptasensor showed high selectivity for tetracycline over the other structurally related tetracycline derivatives (oxytetracycline and doxycycline) in a mixture. The aptasensor developed in this study can potentially be used for detection of tetracycline in pharmaceutical preparations, contaminated food products, and drinking water.     

4-(dimethylamino)butyric acid@PtNPs as enhancer for solid-state electrochemiluminescence aptasensor based on target-induced strand displacement

A solid-state electrochemiluminescence (ECL) aptasensor based on target-induced aptamer displacement for highly sensitive detection of thrombin was developed successfully using 4-(dimethylamino)butyric acid (DMBA)@PtNPs labeling as enhancer. Such a special aptasensor included three main parts: ECL substrate, ECL intensity amplification and target-induced aptamer displacement. The ECL substrate was made by modifying the complex of Pt nanoparticles (PtNPs) and tris(2,2-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) (Ru-PtNPs) onto nafion@multi-walled carbon nanotubes (nafion@MWCNTs) modified electrode surface. A complementary thrombin aptamer labeled by DMBA@PtNPs (Aptamer II) acted as the ECL intensity amplification. The thrombin aptamer (TBA) was applied to hybridize with the labeled complementary thrombin aptamer, yielding a duplex complex of TBA-Aptamer II on the electrode surface. The introduction of thrombin triggered the displacement of Aptamer II from the self-assembled duplex into the solution and the association of inert protein thrombin on the electrode surface, decreasing the amount of DMBA@PtNPs and increasing the electron transfer resistance of the aptasensor and thus resulting large decrease in ECL signal. With the synergistic amplification of DMBA and PtNPs to Ru(bpy)(3)(2+) ECL, the aptasensor showed an enlarged ECL intensity change before and after the detection of thrombin. As a result, the change of ECL intensity has a direct relationship with the logarithm of thrombin concentration in the range of 0.001-30 nM. The detection limit of the proposed aptasensor is 0.4 pM. Thus, the approach is expected to open new opportunities for protein diagnostics in clinical as well as bioanalysis in general.     

A "signal-on" electrochemical aptasensor for simultaneous detection of two tumor markers

In this paper, we report a "signal-on" electrochemical aptasensor for simultaneous determination of two tumor markers MUC1 and VEGF(165), by using a ferrocene-labeled aptamer-complementary DNA (cDNA) as probe. Since the cDNA immobilized on an electrode surface can hybridize with both MUC1 aptamer and VEGF(165) aptamer to form a long double strand with ferrocene far away from the electrode surface, the probe cannot give electrochemical signal. Nevertheless, the presence of the two tumor markers will inhibit the hybridization of cDNA with the aptamers, thus the distance between ferrocene and the electrode is changed, and a "signal-on" electrochemical method to detect two tumor markers is developed. Experimental results show that the electrochemical signal increases with the addition of either tumor markers, but the biggest electrochemical signal can only be obtained when both tumor markers are present. Therefore, the proposed electrochemical aptasensor can not only detect the two markers but also distinguish their co-existence. It may also display high selectivity and sensitivity towards the detection of the tumor markers, so it might have potential clinical application in the future.     

Sensitive label-free electrochemical analysis of human IgE using an aptasensor with cDNA amplification

In this study, we developed an ultrasensitive label-free aptamer-based electrochemical biosensor, featuring a highly specific anti-human immunoglobulin E (IgE) aptamer as a capture probe, for human IgE detection. Construction of the aptasensor began with the electrodeposition of gold nanoparticles (AuNPs) onto a graphite-based screen-printed electrode (SPE). After immobilizing the thiol-capped anti-human IgE aptamer onto the AuNPs through self-assembly, we treated the electrode with mercaptohexanol (MCH) to ensure that the remaining unoccupied surfaces of the AuNPs would not undergo nonspecific binding. We employed a designed complementary DNA featuring a guanine-rich section in its sequence (cDNA G1) as a detection probe to bind with the unbound anti-human IgE aptamer. We measured the redox current of methylene blue (MB) to determine the concentration of human IgE in the sample. When the aptamer captured human IgE, the binding of cDNA G1 to the aptamer was inhibited. Using cDNA G1 in the assay greatly amplified the redox signal of MB bound to the detection probe. Accordingly, this approach allowed the linear range (coefficient of determination: 0.996) for the analysis of human IgE to extend from 1 to 100,000pM; the limit of detection was 0.16pM. The fabricated aptasensor exhibited good selectivity toward human IgE even when human IgG, thrombin, and human serum albumin were present at 100-fold concentrations. This method should be readily applicable to the detection of other analytes, merely by replacing the anti-human IgE aptamer/cDNA G1 pair with a suitable anti-target molecule aptamer and cDNA.     

Aptamer-based colorimetric biosensing of Ochratoxin A using unmodified gold nanoparticles indicator

This work presents an aptasensor for Ochratoxin A (OTA) using unmodified gold nanoparticles (AuNPs) indicator. The assay method is based on the conformation change of OTA's aptamer in phosphate buffered saline (PBS) containing Mg(2+) and OTA, and the phenomenon of salt-induced AuNPs aggregation. A single measurement took only five minutes. Circular dichroism spectroscopic experiments revealed for the first time that upon the addition of OTA, the conformation of OTA's aptamer in PBS buffer changed from random coil structure to compact rigid antiparallel G-quadruplex structure. This compact rigid G-quadruplex structure could not protect AuNPs against salt-induced aggregation, and thus the color change from red to blue could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of OTA concentration from 20 to 625 nM and the detection limit of 20 nM (3σ) was obtained.     

A highly sensitive aptasensor towards Plasmodium lactate dehydrogenase for the diagnosis of malaria

Finding a highly sensitive diagnostic technique for malaria has challenged scientists for the last century. In the present study, we identified versatile single-strand DNA aptamers for Plasmodium lactate dehydrogenase (pLDH), a biomarker for malaria, via the Systematic Evolution of Ligands by EXponential enrichment (SELEX). The pLDH aptamers selectively bound to the target proteins with high sensitivity (K(d)=16.8-49.6 nM). The selected aptamers were characterized using an electrophoretic mobility shift assay, a quartz crystal microbalance, a fluorescence assay, and circular dichroism spectroscopy. We also designed a simple aptasensor using electrochemical impedance spectroscopy; both Plasmodium vivax LDH and Plasmodium falciparum LDH were selectively detected with a detection limit of 1 pM. Furthermore, the pLDH aptasensor clearly distinguished between malaria-positive blood samples of two major species (P. vivax and P. falciparum) and a negative control, indicating that it may be a useful tool for the diagnosis, monitoring, and surveillance of malaria.     

Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample

A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)(6)(3-/4-). The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20ng/mL and a detection limit of 0.3ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics.     

Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin

Angiogenin (Ang), one of the most potent angiogenic factor, is related with the growth and metastasis of numerous tumors. This paper presents a very simple and label-free square-wave voltammetry (SWV) aptasensor to detect angiogenin, in which an anti-angiogenin-aptamer was used as a molecular recognition element, and the couple ferro/ferricyanide as a redox probe. At the bare gold electrode, the redox couple (K₄[Fe(CN)₆]/K₃[Fe(CN)₆]) can be very easily accessed to the electrode surface to give a very strong SWV signal. At the anti-angiogenin/Au electrode surface, when angiogenin was added to the electrochemical cell, the binding of the analyte results in less availability for a redox reaction, which led to smaller SWV current. To quantify the amount of angiogenin, current suppressions of SWV peak were monitored using the redox couple of an [Fe(CN)₆]^4−/3− probe. The plot of signal suppression against the logarithm of angiogenin concentration is linear with over the range from 0.01 nM to 30 nM with a detection limit of 1 pM. The aptasensor also showed very good selectivity for angiogenin without being affected by the presence of other proteins in serum. It is the first time to use a very simple method to detect the cancer marker. Such an aptasensor opens a rapid, selective and sensitive route for angiogenin detection and provides a promising strategy for other protein detections.     

A label-free electrochemiluminescence aptasensor for thrombin based on novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles

In the work, a label-free electrochemiluminescence (ECL) aptasensor for the sensitive and selective detection of thrombin was constructed based on target-induced direct ECL signal change by virtue of a novel assembly strategy of oligonucleotide and luminol functionalized gold nanoparticles (luminol-AuNPs). It is the first label-free ECL biosensor based on luminol and its analogs functionalized AuNPs. Streptavidin AuNPs coated with biotinylated DNA capture probe 1 (AuNPs-probe 1) were firstly assembled onto an gold electrode through 1,3-propanedithiol. Then luminol-AuNPs co-loaded with thiolated DNA capture probe 2 and thiolated thrombin binding aptamer (TBA) (luminol-AuNPs-probe 2/TBA) were assembled onto AuNPs-probe 1 modified electrode through the hybridization between capture probes 1 and 2. The luminol-AuNPs-probe 2/TBA acted as both molecule recognition probe and sensing interface. An Au/AuNPs/ds-DNA/luminol-AuNPs/TBA multilayer architecture was obtained. In the presence of target thrombin, TBA on the luminol-AuNPs could capture the thrombin onto the electrode surface, which produced a barrier for electro-transfer and influenced the electro-oxidation reaction of luminol, leading to a decrease in ECL intensity. The change of ECL intensity indirectly reflected the concentration of thrombin. Thus, the approach showed a high sensitivity and a wider linearity for the detection of thrombin in the range of 0.005-50nM with a detection limit of 1.7pM. This work reveals that luminol-AuNPs are ideal platform for label-free ECL bioassays.     

A novel fluorescent sensor based on electrosynthesized benzene sulfonic acid‐doped polypyrrole for determination of Pb(II) and Cu(II)

To develop conducting organic polymers (COPs) as luminescent sensors for determination of toxic heavy metals, a new benzene sulfonic acid‐doped polypyrrole (PPy‐BSA) thin film was electrochemically prepared by cyclic voltammetry (CV) on flexible indium tin oxide (ITO) electrode in aqueous solution. PPy‐BSA film was characterized by FTIR spectrometry, X‐ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The optical properties of PPy‐BSA were investigated by ultraviolet (UV)‐visible absorption and fluorescence spectrometry in dimethylsulfoxide (DMSO) diluted solutions. PPy‐BSA fluorescence spectra were strongly quenched upon increasing copper(II) ion (Cu²⁺) and lead(II) ion (Pb²⁺) concentrations in aqueous medium, and linear Stern–Volmer relationships were obtained, which indicated the existence of a main dynamic fluorescence quenching mechanism. BSA‐PPy sensor showed a high sensitivity for detection of both metallic ions, Cu²⁺ and Pb²⁺, with very low limit of detection values of 3.1 and 18.0 nM, respectively. The proposed quenching‐fluorimetric sensor might be applied to the determination of traces of toxic heavy metallic ions in water samples.