An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ο-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3′ terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100 fM to 1 nM with a low detection limit of 20.3 fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

Sensitive chemiluminescence aptasensor based on exonuclease-assisted recycling amplification

We report herein an exonuclease-assisted aptamer-based target recycling amplification strategy for sensitive and selective chemiluminescence (CL) determination of adenosine. This aptasensor is based on target-induced release of aptamers from capture probes immobilized on the 96-well plate surface, and thus leading to a decreased hybridization with gold nanoparticle-functionalized reporter sequences followed by a CL signal. The introduction of exonuclease III catalyzes the stepwise removal of mononucleotides from 3′-hydroxyl termini of duplex DNAs of aptamers, liberating the adenosine. Therefore, a single copy of target adenosine can lead to the release and digestion of numerous aptamer strands from the 96-well plates and ultimately an enhanced sensitivity is achieved. Experimental results revealed that the exonuclease-assisted recycling strategy enabled the monitoring of adenosine with wide working ranges and low detection limits (LOD: 0.5 nM). This new CL strategy might create a novel technology for the detection of various targets and could find wide applications in the environmental and biomedical fields.     

A novel label-free electrochemical aptasensor for thrombin based on the {nano-Au/thionine}n multilayer films as redox probes

Herein, a novel label-free electrochemical aptasensor based on direct immobilization of the redox probes on an electrode surface was reported. Gold electrode coated Nafion was firstly modified with redox probe-thionine (Thi) through ion exchange adsorption. Then, with the help of chemisorption and electrostatic adsorption, negatively charged nano-Au and positively charged Thi were layer-by-layer (LBL) self-assembled onto the modified electrode surface, which formed {nano-Au/Thi⁺}_n multilayer films for improving the amount of redox probes and immobilizing thiolated thrombin aptamers (TBA). In the presence of target thrombin (TB), the TBA on the multilayer film could catch the TB onto the electrode surface, which resulted in a barrier for electro-transfer, leading to decrease of the current. The proposed method avoided the cubsome redox probe labeling process, increased the amount of redox probe and reduced the distance between the redox probe and electrode surface. Thus, the approach showed a high sensitivity and a wider linearity to TB in the range from 0.12 nM to 46 nM with a detection limit of 40 pM.     

Hierarchical dendritic gold microstructure-based aptasensor for ultrasensitive electrochemical detection of thrombin using functionalized mesoporous silica nanospheres as signal tags

A sensitive electrochemical approach for the detection of thrombin was designed by using densely packed hierarchical dendritic gold microstructures (HDGMs) with secondary and tertiary branches as matrices, and thionine-functionalized mesoporous silica nanospheres as signal tags. To prepare the signal tags, the positively charged thionine (as an indicator) was initially adsorbed onto the mesoporous silica nanoparticles (MSNs). Then [AuCl₄]⁻ ions were in situ reduced on the thionine-modified MSNs by ascorbic acid to construct nanogold-decorated MSNs (GMSNs). The formed GMSNs were employed as label of the aminated aptamers. The assay was carried out in PBS, pH 7.4 with a sandwich-type assay mode by using the assembled thionine in the GMSNs as indicators. Compared with the pure silica nanoparticles, mesoporous silica could provide a larger surface for the immobilization of biomolecules and improve the sensitivity of the aptasensor. Under optimal conditions, the electrochemical aptasensors exhibited a wide linear range from 0.001 to 600 ng mL⁻¹ (i.e. 0.03 pM to 0.018 μM thrombin) with a low detection limit (LOD) of 0.5 pg mL⁻¹ (≈15 fM) thrombin at 3σ. No obvious non-specific adsorption was observed during a series of analyses to detect target analyte. The precision, selectivity and stability of the aptasensors were acceptable. Importantly, the methodology was evaluated with thrombin spiked samples in blank fetal calf serum, and the recoveries were 94.2–112%, indicating an exciting potential for thrombin detection.     

A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H₂O₂. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM–50 nM with a detection limit of 33 fM (defined as S/N = 3) for TB.     

A novel fluorescent aptasensor based on silica nanoparticles,PicoGreen and exonuclease III as a signal amplification method for ultrasensitive detection of myoglobin

Measurement of myoglobin (Mb) in human blood serum is of great interest for quick diagnosis of acute myocardial infarction (AMI). In this study, a novel fluorescent aptasensor was designed for ultrasensitive and selective detection of Mb, based on target-induced high fluorescence intensity, complementary strand of aptamer (CS), PicoGreen (PG) dye, exonuclease III (Exo III) and silica nanoparticles coated with streptavidin (SNPs-Streptavidin). The developed aptasensor obtains characteristics of SNPs as enhancers of fluorescence intensity, Exo III as an enzyme which selectively digests the 3'-end of double-stranded DNA (dsDNA), PG as a fluorescent dye which could selectively bind to dsDNA and high selectivity and sensitivity of aptamer (Apt) toward its target. In the absence of Mb, no free CS remains in the environment of SNPs-Streptavidin, resulting in a weak fluorescence emission. In the present of Mb, dsDNA-modified SNPs-Streptavidin complex forms, leading to a very strong fluorescence emission. The developed fluorescent aptasensor exhibited high specificity toward Mb with a limit of detection (LOD) as low as 52 pM. In addition, the designed fluorescent aptasensor was efficiently used to detect Mb in human serum.     

An electrochemical aptasensor for chiral peptide detection using layer-by-layer assembly of polyelectrolyte-methylene blue/polyelectrolyte-graphene multilayer

Here we demonstrate for the first time that by physically adsorbing aptamer onto conductive film assembled via alternate adsorption of graphene/polyelectrolyte and methylene blue/polyelectrolyte, a label-free electrochemical aptasensor with high sensitivity and selectivity for peptide detection is constructed. Graphene multilayer derived from layer-by-layer assembly has played significant roles in this sensing strategy: allowing accumulation of methylene blue, facilitating electron transfer and providing much more adsorption site. As compared to previous electrochemical aptasensors, the current sensor based on graphene multilayer alternated with electroactive molecule layer offers extremely high capability for sensitive detection of target without interference of environmental surrounding. This electroactive probe-confined graphene multilayer confers great flexibility to combine with differential pulse voltammetry (DPV) together. In the presence of target d entiomer of arginine vasopressin (d-VP), the binding of peptide to aptamer block the electron transfer process of MB, leading to decreased current peak of DPV. By this way, this electrochemical aptasensor based on electroactive molecule-intercalated graphene multilayer provide highly sensitive and specific detection of d-VP with the lowest detectable concentration of 1 ng mL⁻¹ and a wide detection range from 1 to 265 ng mL⁻¹.     

A novel aptasensor strategy for protein detection based on G-quadruplex and exonuclease III-aided recycling amplification

The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.     

Label-free electrochemical aptasensor for the detection of lysozyme

This work reports the advantages of a label free electrochemical aptasensor for the detection of lysozyme. The biorecognition platform was obtained by the adsorption of the aptamer on the surface of a carbon paste electrode (CPE) previously blocked with mouse immunoglobulin under controlled-potential conditions. The recognition event was detected from the decrease in the guanine and adenine electro-oxidation signals produced as a consequence of the molecular interaction between the aptamer and lysozyme. The biosensing platform demonstrated to be highly selective even in the presence of large excess (9-fold) of bovine serum albumin, cytochrome C and myoglobin. The reproducibility for 10 repetitive determinations of 10.0 mg L⁻¹ lysozyme solution was 5.1% and 6.8% for guanine and adenine electro-oxidation signals, respectively. The detection limits of the aptasensor were 36.0 nmol L⁻¹ (if considering guanine signal) and 18.0 nmol L⁻¹ (if taking adenine oxidation current). This new sensing approach represents an interesting and promising alternative for the electrochemical quantification of lysozyme.     

An intriguing electrochemical impedance aptasensor based on a porous organic framework supported silver nanoparticles for ultrasensitively detecting theophylline

Porous organic frameworks (POFs) are excellently stable porous materials, which can be employed as host platforms to support metal nanoparticles as functional composites for various applications. Herein, a novel POF is successfully prepared via Friedel-Crafts reaction. Silver nanoparticles (Ag NPs) are embedded in the prepared POF to generate an Ag@POF composite, which not only possesses high surface area, outstanding physicochemical stability and outstretched π-conjugation skeleton, but also exhibits preferable electrochemical stability and conductivity. This composite is able to immobilize a mass of aptamer strands to fabricate an intriguing electrochemical aptasensor. Electrochemical impedance spectroscopy (EIS) is a commonly used technology to analyze the electrochemical signal variation. The Ag@POF-based biosensor shows the excellent electrochemical detection behavior through analyzing EIS. For instance theophylline as a research mode, the Ag@POF based electrochemical aptasensor reveals ultra-sensitiveness, high selectivity, remarkable stability, good repeatability and simple operability even in various real samples. Notably, this aptasensor has the sensitive detection performance with the limit of detection of 0.191 pg/mL (1.06 pmol/L) in a wide concentration range of 5.0 × 10^-4 – 5.0 ng/mL (2.78 × 10^-3 – 27.8 nmol/L).     

Ultrasensitive one-step rapid detection of ochratoxin A by the folding-based electrochemical aptasensor

A one-step electrochemical aptasensor using the thiol- and methylene blue- (MB-) dual-labeled aptamer modified gold electrode for determination of ochratoxin A (OTA) was presented in this research. The aptamer against OTA was covalently immobilized on the surface of the electrode by the self-assembly effect and used as recognition probes for OTA detection by the binding induced folding of the aptamer. Under the optimal conditions, the developed electrochemical aptasensor demonstrated a wide linear range from 0.1 pg mL⁻¹ to 1000 pg mL⁻¹ with the limit of detection (LOD) of 0.095 pg mL⁻¹, which was an extraordinary sensitivity compared with other common methods for OTA detection. Moreover, as a practical application, this proposed electrochemical aptasensor was used to monitor the OTA level in red wine samples without any special pretreatment and with satisfactory results obtained. Study results showed that this electrochemical aptasensor could be a potential useful platform for on-site OTA measurement in real complex samples.     

A novel signal-on fluorescent aptasensor for ochratoxin A detection based on RecJf exonuclease-induced signal amplification

This article describes a simple and homogeneous fluorescent aptasensor for the detection of ochratoxin A (OTA). With its high specificity and simplicity; RecJ_f exonuclease is used to cleave DNA strand of the FAM-aptamer/OTA complex and realize target recycling signal amplification. In order to avoid the loss of reaction system, magnetic beads (MBs) are added only once at the last experimental step. This proposed fluorescent aptasensor showed the higher sensitivity in the range of 0.1–100 ng/mL with LOD of 0.056 ng/mL, and the good selectivity against other interfering toxins. The feasibility of the prepared aptasensor was studied by detecting OTA in spiked liquor and cereal samples. The obtained average recoveries ranged from 92% to 115%. This study provides a promising application with convenience and rapidness in the aptasensor fabrication for food safety analysis.     

Label-free electrochemical aptasensor constructed by layer-by-layer technology for sensitive and selective detection of cancer cells

Here, a cytosensor was constructed with ferrocene-appended poly(allylamine hydrochloride) (Fc-PAH) functionalized graphene (Fc-PAH-G), poly(sodium-p-styrenesulfonate) (PSS) and aptamer (AS1411) by layer-by-layer assembly technology. The hybrid nanocomposite Fc-PAH-G not only brings probes on the electrode and also promotes electron transfer between the probes and the substrate electrode. Meanwhile, LBL technology provides more effective probes to enhance amplified signal for improving the sensitivity of the detection. While AS1411 forming G-quardruplex structure and binding cancer cells, the current response of the sensing electrode decreased due to the insulating properties of cellular membrane. Differential pulse voltammetry (DPV) was performed to investigate the electrochemical detection of HeLa cells attributing to its sensitivity of the current signal change. The as-prepared aptasensor showed a high sensitivity and good stability, a widely detection range from 10 to 10⁶ cells/mL with a detection limit as low as 10 cells/mL for the detection of cancer cells.     

Dual amplification strategy of highly sensitive thrombin amperometric aptasensor based on chitosan-Au nanocomposites

A highly sensitive and selective electrochemical aptasensor for thrombin was developed. By introducing chitosan-gold nanoparticles and horseradish peroxidase (CS-AuNPs-HRP) conjugates to the sensitive union, the thrombin detection signal was dual amplified. The capture probe was prepared by immobilizing an anti-thrombin aptamer on core-shell Fe(3)O(4)-Au magnetic nanoparticles (AuMNPs) and which was served as magnetic separation material as well. The detection probe was prepared from another anti-thrombin aptamer, horseradish peroxidase (HRP), thiolated CS nanoparticle and gold nanoparticle (CS-AuNPs-HRP-Apt2). In the presence of thrombin, the sandwich structure of AuMNPs-Apt1/thrombin/Apt2-CS-AuNPs-HRP was formed and abundant HRP was captured in it. The resultant conjugates are of magnetic characters and were captured onto the surface of a screen printed carbon electrode (SPCE) to prepare the modified electrode by a magnet located on the outer flank of the SPCE. It was demonstrated that the oxidation of hydroquinone (HQ) with H(2)O(2) was dramatically accelerated by the captured HRP. The electrochemical signal, which correlated to the reduction of BQ (the oxidation product of HQ), was amplified by the catalysis of HRP toward the reaction and the enrichment of HRP on the electrode surface. Under optimized conditions, ultrasensitive and high specific detection for thrombin was realized with the proposed assay strategy. The signal current was linearly correlated to the thrombin concentration in the range of 0.01-10 pM with a detection limit of 5.5 fM (S/N = 3). These results promise extensive applications of this newly proposed signal amplification strategy in protein detection and disease diagnosis.     

Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer–target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H₂O₂ by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1 × 10⁻¹⁰ M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol.     

A novel resonance Rayleigh scattering aptasensor for dopamine detection based on an Exonuclease III assisted signal amplification by G - quadruplex nanowires formation

A new method was constructed for detecting dopamine based on aptamer-specific recognition and resonance Rayleigh scattering (RRS) of G - quadruplex nanowires (G - wires). The dopamine aptamer was used to recognize the target of dopamine, and the Exonuclease III was applied to cleave the hairpin DNA, and the G - wire formation was induced in the presence of K⁺ and Mg²⁺. This phenomenon was confirmed by polyacrylamide gel electrophoresis. Thus, a quantitative relationship between the RRS intensity of the G - wires and the dopamine concentration was established. The experimental conditions were optimized, such as the concentration of Mg²⁺, reaction temperature, reaction time and the concentration of Exonuclease III in the reaction system, and the interference substances were investigated, such as uric acid, ascorbic acid and serotonin. Under the optimal conditions, there was a good linear relationship between the RRS and the logarithm of dopamine concentration in the range from 5.0 × 10^-11 M to 1.0 × 10^-8 M (r = 0.995), with a detection limit of 1.2 × 10^-11 M. The novel method for dopamine detection showed excellent selectivity and high sensitivity, and could be used to detect dopamine in mice brain tissues.     

Label‐Free Electrochemical Thrombin Aptasensor Based on Ag Nanoparticles Modified Electrode

A novel label‐free electrochemical method for protein detection based on redox properties of silver was developed. As recognition elements, thrombin‐binding aptamers were used. Screen printed electrodes modified with silver nanoparticles (AgNP) were employed as a sensing platform for aptasensor devices. The oxidation of silver upon polarization served as a basis for analytical response. Three different thrombin binding aptamers with various surface concentrations were studied. Linear range of aptasensor response corresponded to the 10⁻⁹ M to 10⁻⁷ M thrombin concentration range and the detection limit was 10⁻⁹ M.     

Ultrasensitive electrochemical immunosensor for zeranol detection based on signal amplification strategy of nanoporous gold films and nano-montmorillonite as labels

Nano-montmorillonites belong to aluminosilicate clay minerals with innocuity, high specific surface area, ion exchange, and favorable adsorption property. Due to the excellent properties, montmorillonites can be used as labels for the electrochemical immunosensors. In this study, nano-montmorillonites were converted to sodium montmorillonites (Na-Mont) and further utilized for the immobilization of thionine (TH), horseradish peroxidase (HRP) and the secondary anti-zeranol antibody (Ab₂). The modified particles, Na-Mont-TH-HRP-Ab₂ were used as labels for immunosensors to detect zeranol. This protocol was used to prepare the immunosensor with the primary antibody (Ab₁) immobilized onto the nanoporous gold films (NPG) modified glassy carbon electrode (GCE) surface. Within zeranol concentration range (0.01–12 ng mL⁻¹), a linear calibration plot (Y = 0.4326 + 8.713 X, r = 0.9996) was obtained with a detection limit of 3 pg mL⁻¹ under optimal conditions. The proposed immunosensor showed good reproducibility, selectivity, and stability. This new type of immunosensors with montmorillonites and NPG as labels may provide potential applications for the detection of zeranol.     

Ultrasensitive electrochemical immunosensor of carcinoembryonic antigen based on gold-label silver-stain signal amplification

A novel gold-label silver-stain electrochemical immunosensor based on polythionine-gold nanoparticles (PTh-Au NPs) modified glassy carbon electrode (GCE) as a platform and secondary antibody labeled Au NPs (Ab₂-Au NPs) as immumoprobe for carcinoembryonic antigen (CEA) detection. The sandwich-type biosensor adopted anodic stripping voltammetry to detect silver stripping signal when the Ab₂-Au NPs of the formed immunocomplexes were stained with silver.