The preparation of self-assembled DNA nanostructure with different sizes and shapes has been one of the most promising research areas in recent years, while the application of these DNA ...nanostructures in biosensors is far from fully developed. Here, we presented a novel carrier system to construct an electrochemiluminescence (ECL) aptasensor for ultrasensitive determination of lipopolysaccharides (LPS) on the basis of self-assembled tetrahedron DNA dendrimers. Doxorubicin (Dox), a well-known intercalator of double stranded DNA (dsDNA), was conjugated with the ECL luminophore of N-(aminobutyl)-N-(ethylisoluminol) (ABEI) to form a new type of ECL indicators (Dox-ABEI), which could noncovalently attach to dsDNA through intercalation. Based on this property, self-assembled tetrahedron DNA dendrimers were employed as an efficient nanocarrier to achieve a high loading efficiency for Dox-ABEI with significantly amplified ECL signal output. Streptavidin (SA) and biotin, a typical ligand–receptor pair, has been chosen to anchor the tetrahedron DNA dendrimers on the electrode surface. Moreover, by converting LPS content into DNA output, catalyzed hairpin assembly (CHA) target recycling signal amplification strategy was also adopted to enhance the sensitivity of the ECL aptasensor. With combining the loading power of the tetrahedron DNA dendrimers for ECL indicators, the inherent high sensitivity of ECL technique and target recycling for signal amplification, the proposed strategy showed a detection limit of 0.18 fg/mL for LPS.
N-terminal pro-brain natriuretic peptide (NT-proBNP) has been demonstrated to be a sensitive and specific biomarker for heart failure (HF). Surface-enhanced Raman spectroscopy (SERS) technology can ...be used to accurately detect NT-proBNP at an early stage for its advantages of high sensitivity, less wastage and time consumption. In this work, we have demonstrated a new SERS-based immunosensor for ultrasensitive analysis of NT-proBNP by using metal–organic frameworks (MOFs)@Au tetrapods (AuTPs) immobilized toluidine blue as SERS tag. Here, MOFs@AuTPs complexes were utilized to immobilize antibody and Raman probe for their excellent characteristics of high porosity, large surface area, and good biocompatibility which can obviously enhance the fixing amount of biomolecule. To simplify the experimental operation and improve the uniformity of the substrate, Au nanoparticles functionalized CoFe2O4 magnetic nanospheres (CoFe2O4@AuNPs) were further prepared to assemble primary antibody. Through sandwiched antibody–antigen interactions, the immunosensor can produce a strong SERS signal to detect NT-proBNP fast and effectively. With such design, the proposed immunosensor can achieve a large dynamic range of 6 orders of magnitude from 1 fg mL–1 to 1 ng mL–1 with a detection limit of 0.75 fg mL–1. And this newly designed amplification strategy holds high probability for ultrasensitive immunoassay of NT-proBNP.
Loop-mediated isothermal amplification (LAMP) is an outstanding DNA amplification procedure, in which the reaction can accumulate 109 copies from less than 10 copies of input template within an hour. ...While the amplification reaction is extremely powerful, the quantitative detection of LAMP products is still analytically difficult. Besides, the type of targets that LAMP can detect is also less, which to some extent limited the application of LAMP. In this study, we are reporting for the first time an efficient and accurate detection system which employs the integration of LAMP, aptamer and the electrochemical method for the sensitive detection of Ochratoxin A (OTA). Aptamers were designed as the forward outer primer to trigger the LAMP reaction, and then the LAMP amplification products were combined with a redox active molecule methylene blue (MB) and analyzed by an electrode using differential pulse voltammograms (DPV). As the reaction progresses, the MB intercalated into double-stranded regions of LAMP amplicons reduces the free MB concentration. Hence, the peak current of reaction mixture decreased with the amplification because of the slow diffusion of MB-amplified DNA complex to the electrode surface. The peak height of the current was related to the input amount of the aptamers, providing a ready means to detection the concentration of OTA. With such design, the proposed assay showed a good linear relationship within the range of 0.001–50nM with a detection limit of 0.3pM (defined as S/N=3) for OTA.
•We employed the integration of LAMP, aptamer and the electrochemical method for the sensitive detection of Ochratoxin A (OTA) for the first time.•This assay was demonstrated that the LAMP reaction can be applied to ultrasensitive detection of OTA.•The prepared aptasensor exhibited low detection limit and wide linear range to OTA.
•A sandwich impedimetric electrochemical aptasensor was constructed for the determination of LPS.•The poly-T ssDNA sequences from RCA served as specific template for CuNPs formation.•CuNPs@ssDNA ...catalyzed PCV into insoluble bulky oligomer for enhanced resistance generation.•The aptasensor exhibited good performance for LPS detection with a detection limit of 4.8 fg/mL.
In this work, a novel sandwich-type impedimetric biosensor was fabricated for highly sensitive and selective detection of lipopolysaccharide (LPS) from E. coli O55:B5. LPS aptamer I was immobilized onto electrodeposited gold nanoparticles (Au) modified electrode for capturing LPS in buffer and serum. LPS aptamer II and rolling circle amplification (RCA) primer were conjugated with gold nanoparticles (AuNPs) to use as signal tags. LPS was sandwiched between AuNPs/primer/aptamer II bioconjugates and aptamer I. This was followed by extension of RCA, which resulted in long poly-thymine (T) single strand (ssDNA) sequences to adsorb copper ions (Cu2+) into the skeleton of poly-T sequences. Thus, poly-T ssDNA directly served as the specific template for copper nanoparticles (CuNPs) formation with the aid of ascorbic acid (VC). Finally, the formed CuNPs@ssDNA complex promoted the oxidation of pyrocatechol violet (PCV) to insoluble bulky oligomer (precipitation) in the presence of H2O2. The reaction significantly enhanced the resistance signal of LPS quantitative determination. The impedimetric aptasensor exhibited a wide dynamic working range of 0.01 pg/mL ˜ 100 ng/mL with a detection limit of 4.8 fg/mL. This method also showed good selectivity and reproducibility, and demonstrated the applicability of LPS detection in human serum samples, offering a promising avenue for sensitive LPS detection.
For the first time, a glucose oxidase-functionalized bioconjugate was prepared and served as a new trace label through its direct electrochemistry and electrocatalysis in a sandwich-type ...electrochemical aptasensor for ultrasensitive detection of thrombin.
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•This assay is label-free, the signal can be read out by measuring the electrochemical signal of hemin.•The hemin/G-quadruplex HRP-DNAzyme nanowires were formed via EXPAR reaction and ...HCR.•The prepared aptasensor exhibited low detection limit and wide linear range to TB.
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 0.1pM–50nM with a detection limit of 33 fM (defined as S/N=3) for TB.
In this work, a new electrochemical aptasensor based on direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) using exonuclease-catalyzed target recycling and hybridization ...chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The electrochemical signal was originated from HRP without the addition or labeling of redox probes. To construct the aptasensor, the capture probe was immobilized on gold nanoparticles (AuNPs) modified electrode for the following hybridization with the complementary thrombin binding aptamer. In the presence of thrombin, the formation of aptamer–thrombin complex would result in the dissociation of aptamer from the double-strand DNA (dsDNA). Subsequently, with the employment of exonuclease, aptamer was selectively digested and thrombin could be released for analyte recycling. The capture probe and two hairpin helper DNAs lead to the formation of extended dsDNA polymers through HCR on the electrode surface. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled HRP, resulting in significantly amplified electrochemical signal through the direct electrochemistry and electrocatalysis of HRP. The proposed strategy combined the amplification of analyte recycling and HCR, as well as the inherent electroactivity and catalytic activity of HRP, which exhibited high sensitivity for thrombin determination with an ultra-low detection limit of 1.2×10–13 M. Moreover, the detection scheme could be easily extended to the detection of other biomolecules.
•Use direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) for indicator signal and for amplification.•Combine exonuclease-catalyzed target recycling with hybridization chain reaction for signal enhancement.•Highly-sensitive electrochemical detection of thrombin.
In the present study, toluidine blue–graphene (Tb–Gra) nanocomposites were prepared to design a Lable-free electrochemical aptasensor for highly sensitive detection of thrombin based on ...layer-by-layer (LBL) technology. The nanocomposites with excellent redox electrochemical activities were first immobilized on the gold nanoparticles (nano-Au) modified glassy carbon electrodes (GCE). Then, the LBL structure was performed by electrostatic adsorption between the positively charged Tb–Gra and negatively charged nano-Au, which formed {Tb–Gra/nano-Au}n multilayer films for electroactive species enrichment and biomolecule immobilization. Subsequently, the thiolated thrombin binding aptamer (TBA) was assembled on the nano-Au surface through Au–S bond. In the presence of target thrombin (TB), the TBA on the multilayer could catch the thrombin onto the electrode surface, which resulted in a barrier for electro-transfer, leading to the decrease of the electrochemical signal of Tb–Gra nanocomposites. Under the optimal conditions, a wide detection range from 0.001nM to 80nM and a low detection limit of 0.33 pM (defined as S/N=3) for thrombin were obtained. In addition, the sensor exhibited excellent selectivity against other proteins.
► The aptasensors exhibited low detection limit and wide linear range to thrombin. ► The Tb–Gra nanocomposites combined the advantages of graphene and toluidine blue. ► The aptasensors showed good fabrication reproducibility and sufficient stability.
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► The hemin/G-quadruplex simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme. ► The PdNPs-RGs were adopted to further enhance the electrochemical signal. ► The avidin–biotin ...system was employed to improve the sensitivity of thrombin analysis. ► The prepared aptasensor exhibited low detection limit and wide linear range to TB.
In this work, an advanced sandwich-type electrochemical aptasensor for thrombin was proposed by integrating hemin/G-quadruplex with functionalized graphene-Pd nanoparticles composites (PdNPs-RGs). The hemin/G-quadruplex formed by intercalating hemin into thrombin binding aptamer (TBA), firstly acted as a NADH oxidase, assisting the oxidation of NADH to NAD+ accompanying with the generation of H2O2 in the presence of dissolved O2. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme that rapidly bioelectrocatalyze the reduction of the produced H2O2. At the same time, the Pd nanoparticles supported on p-iodoaniline functionalized graphene were also adopted to catalyze the reduction of H2O2. Thus, with the dual catalysis, a dramatically amplified electrochemical signal could be obtained. Besides, the avidin–biotin system for binding aptamer sequences on electrodes not only improved the sensitivity of thrombin analysis but also obtained an acceptable repeatability of the aptasensor. With several factors mentioned above, a wide linear ranged from 0.1pM to 50nM was acquired with a relatively low detection limit of 0.03pM (defined as S/N=3). These excellent performances provided our approach a promising way for ultrasensitive assay in electrochemical aptasensors.
A new type of multifunctional metal-organic framework (MOF) has been synthesized by encapsulating hemin into the nano-sized Fe-MIL-88 MOFs (hemin@MOFs) and first applied in an electrochemical ...aptasensor to detect thrombin (TB) with the aid of an enzyme for signal amplification. The gold nanoparticle functionalized hemin@MOFs (Au/hemin@MOFs) have not only simultaneously served as redox mediators and solid electrocatalysts, but have also been utilized as an ideal loading platform to immobilize a large number of biomolecules. In this aptasensor, Au/hemin@MOFs conjugated with glucose oxidase (GOD) and thrombin binding aptamer (TBA II) were used as the secondary aptamer bioconjugates (Au/hemin@MOF-TBA II-GOD bioconjugates), and TB was sandwiched between Au/hemin@MOF-TBA II-GOD bioconjugates and the amino-terminated TBA I which was self-assembled on the gold nanoparticle (AuNP) modified electrode. The GOD could oxidize glucose into gluconic acid accompanied by the generation of H2O2. The generated H2O2 on the electrode surface was further electrocatalyzed by hemin@MOFs to amplify the electrochemical signal of hemin contained in hemin@MOFs. Therefore, the synthesized hemin@MOFs represented a new paradigm for multifunctional materials since it combined three different functions including serving as catalysts, redox mediators and loading platforms within a single material. With such an ingenious design, a wide linear range of 0.0001 nM to 30 nM was acquired with a relatively low detection limit of 0.068 pM for TB detection.
