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 and sensitive electrogenerated chemiluminescence aptasensor for the determination of lysozyme

A novel label-free electrogenerated chemiluminescence (ECL) aptasensor for the determination of lysozyme is designed employing lysozyme binding aptamer (LBA) as molecular recognition element for lysozyme as a model analyte and Ru(bpy)(3)(2+) as an ECL signal compound. This ECL aptasensor was fabricated by self-assembling the thiolated LBA onto the surface of a gold electrode. Using this aptasensor, sensitive quantitative detection of lysozyme is realized on basis of the competition of lysozyme with Ru(bpy)(3)(2+) cation for the binding sites of LBA. In the presence of lysozyme, the aptamer sequence prefers to form the LBA-lysozyme complex, the less negative environment allows Ru(bpy)(3)(2+) cations to be less bound electrostatically to the LBAs on the electrode surface, in conjunction with the generation of a decreased ECL signal. The integrated ECL intensity versus the concentration of lysozyme was linear in the range from 6.4×10(-10) M to 6.4×10(-7) M. The detection limit was 1.2×10(-10) M. This work demonstrates that using the competition of target protein with an ECL signal compound Ru(bpy)(3)(2+) for binding sites of special aptamer confined on the electrode is promising approach for the design of label-free ECL aptasensors for the determination of proteins.     

Detection of lysozyme with aptasensor based on fluorescence resonance energy transfer from carbon dots to graphene oxide

A photoluminescent aptasensor has been developed for the detection of lysozyme based on fluorescence resonance energy transfer (FRET) between the carbon dots (CDs) and graphene oxide (GO). In the sensing system, the CDs‐labeled aptamer is adsorbed onto the GO surface and the photoluminescence (PL) signal of the CDs is effectively quenched by GO. Addition of lysozyme can cause a significant FRET inhibition and recover the PL signal of the CDs due to the specific combination of lysozyme and its aptamer and the removal of the CDs‐labeled aptamer from GO surface. Under optimal conditions, the ratio of PL intensity change at 440 nm of the sensing system before and after the addition of lysozyme shows a good linear relationship against the concentration of lysozyme in the range of 0.01–2 μg/mL, with a low detection limit of 1 × 10^?3 μg/mL. In addition, the aptasensor has good selectivity so it can distinguish lysozyme with no or little interference by many other biomolecules. It was applied to the detection of lysozyme in human sera with satisfactory recoveries. The results demonstrate the applicability of the aptasensor for monitoring lysozyme in real samples. Copyright © 2016 John Wiley & Sons, Ltd.     

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 "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.     

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.     

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.     

Design and characterization of electrochemical dopamine–aptamer as convenient and integrated sensing platform

Here, an ultrasensitive label-free electrochemical aptasensor was developed for dopamine (DA) detection. Construction of the aptasensor was carried out by electrodeposition of gold–platinum nanoparticles (Au–PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs–COOH). A designed complementary amine-capped capture probe (ssDNA1) was immobilized at the surface of PtNPs/CNTs–COOH/GC electrode through the covalent amide bonds formed by the carboxyl groups on the nanotubes and the amino groups on the oligonucleotides. DA-specific aptamer was attached onto the electrode surface through hybridization with the ssDNA1. Methylene blue (MB) was used as an electrochemical indicator that was intercalated into the aptamer through the specific interaction with its guanine bases. In the presence of DA, the interaction between aptamer and DA displaced the MB from the electrode surface, rendering a lowered electrochemical signal attributed to a decreased amount of adsorbed MB. This phenomenon can be applied for DA detection. The peak current of probe (MB) linearly decreased over a DA concentration range of 1–30 nM with a detection limit of 0.22 nM.     

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.     

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.     

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.     

A universal fluorescent aptasensor based on AccuBlue dye for the detection of pathogenic bacteria

We report a universal fluorescent aptasensor based on the AccuBlue dye, which is impermeant to cell membranes, for the detection of pathogenic bacteria. The sensor consists of AccuBlue, an aptamer strand, and its complementary strand (cDNA) that partially hybridizes to the aptamer strand. We have fabricated two models by changing the sequence of the reaction between the elements in the system. One is the “signal on” model in which the aptamer is first bound to the target, followed by the addition of cDNA and AccuBlue, at which time the cDNA hybridizes with the free unreacted aptamer and forms a double-stranded DNA (dsDNA) duplex. Such hybridization causes AccuBlue to insert into the dsDNA and exhibit significantly increased fluorescence intensity because of the specific intercalation of the AccuBlue into dsDNA rather than single-stranded DNA (ssDNA). The other model, “signal off,” involves hybridization of the aptamer with cDNA first, resulting in high fluorescence intensity on the addition of AccuBlue. When the target is added, the aptamer binds the target, causing the cDNA to detach from the dsDNA duplex and resulting in low fluorescence as a result of the liberation of AccuBlue. Because this design is based purely on DNA hybridization, and AccuBlue is impermeant to cell membranes, it could potentially be adapted to a wide variety of analytes.     

An ultrasensitive fluorescent aptasensor for adenosine detection based on exonuclease III assisted signal amplification

We report here a graphene oxide (GO)-based fluorescent aptasensor for adenosine detection by employing exonuclease III (Exo III) as a signal amplifying element. In the absence of adenosine, the adenosine aptamers hybridized with the complementary DNA (cDNA), and the Exo III could not cleave the single-strand signal probes labeled with carboxylfluorescein (FAM) at its 5' ends. When the graphene oxide was finally added, it could strongly adsorb the single-strand signal probes and quenched the fluorophore effectively. In the presence of adenosine, the aptamers associated with the targets, which led to the formation of duplex DNAs between the cDNAs and the signal probes. The Exo III thereafter could digest the duplex DNAs from 3' blunt terminus of signal probes, liberating the fluorophore. Upon adding the GO, the fluorophore could not be adsorbed and quenched. By coupling cyclic enzymatic cleavage, a remarkable fluorescent increase was obtained. Due to the specific recognition ability of the aptamer for the target and the powerful quenching property of GO for signal probe, this proposed approach has a good selectivity and high sensitivity for adenosine. In the optimum conditions described, >100% signal enhancement was achieved and a limit of detection as low as 1 nM was obtained, which is lower than those of commonly used fluorescent aptamer sensors. Moreover, the biosensor exhibited an ultrahigh sensitivity and held a versatile platform for clinical diagnostics, molecular biology and drug developments.     

Amplified electrochemical aptasensor taking AuNPs based sandwich sensing platform as a model

Here, we report a sensitive amplified electrochemical impedimetric aptasensor for thrombin, a kind of serine protease that plays important role in thrombosis and haemostasis. For improving detection sensitivity, a sandwich sensing platform is fabricated, in which the thiolated aptamers are firstly immobilized on a gold substrate to capture the thrombin molecules, and then the aptamer functionalized Au nanoparticles (AuNPs) are used to amplify the impedimetric signals. Such designed aptamer/thrombin/AuNPs sensing system could not only improve the detection sensitivity compared to the reported impedimetric aptasensors but also provide a promising signal amplified model for aptamer-based protein detection. In this paper, we realize a sensitive detection limit of 0.02 nM, with a linear range of 0.05-18 nM. Meanwhile, the effect of 6-mercaptohexanol (MCH) and 2-mercaptoethanol (MCE) on the modification of the electrode is investigated.     

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.     

基于HT-SELEX筛选的节球藻毒素-R适配体的鉴定

节球藻毒素-R（nodularin-R,NOD-R）是具有强烈肝毒性的蓝藻毒素。然而,现有的NOD-R检测技术均存在一定的局限性,亟待开发一种新的检测方法。本文利用指数富集的配基系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术结合高通量测序（HT-SELEX）,筛选出NOD-R特异的高亲和力适配体。生物膜干涉（biolayer interferometry,BLI）技术对适配体的鉴定结果表明,适配体H62与NOD-R之间的亲和力最高,达到了168 nM。而且,该适配体不与其他毒素结合,具有较高的特异性。以上结果表明,适配体H62有望作为NOD-R检测新方法——适配体生物传感器的分子识别元件。     

Aptabodies - new type of artificial receptors for detection proteins

We report on a new type of artificial receptor formed by hybridization of two DNA aptamers for human thrombin (aptabody). This aptasensor based on multiwalled carbon nanotubes allowed us to detect thrombin with detection limit of 0.3 nM, which was 3 times better in comparison with conventional aptamer.     

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.     

Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer-AuNPs-HRP conjugates

Successful development of an ultrasensitive and highly specific electrochemical aptasensor for thrombin based on amplification of aptamer-gold nanoparticles-horseradish peroxidase (aptamer-AuNPs-HRP) conjugates was reported. In this electrochemical protocol, aptamer1 (Apt1) was immobilized on core/shell Fe(3)O(4)/Au magnetic nanoparticles (AuMNPs) and served as capture probe. Aptamer2 (Apt2) was dual labeled with AuNPs and HRP and used as detection probe. In the presence of thrombin, the sandwich format of AuMNPs-Apt1/thrombin/Apt2-AuNPs-HRP was fabricated. Remarkable signal amplification was realized by taking the advantage of AuNPs and catalytic reactions of HRP. Other proteins, such as human serum albumin, lysozyme, fibrinogen, and IgG did not show significant interference with the assay for thrombin. Linear response to thrombin concentration in the range of 0.1-60 pM and lower detection limit down to 30 fM (S/N=3) was obtained with the proposed method. This electrochemical aptasensor is simple, rapid (the whole detection period for a thrombin sample is less than 35 min), sensitive and highly specific, it shows promising potential in protein detection and disease diagnosis.     

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.