In this work, an electrochemiluminescent (ECL) biosensor was constructed on the basis of amino-modified 3,4,9,10-perylenetetracarboxylic dianhydride/luminol (PTC-NH2/Lu) nanocomposite as emitter and ...bipedal DNA walker signal amplification strategy for ultrasensitive detection of microRNA-21 (miRNA-21). The PTC-NH2/Lu nanocomposite was prepared as signal tag via π–π stacking molecular assembly, in which amino-modified 3,4,9,10-perylenetetracarboxylic dianhydride (PTC-NH2) as a novel coreaction accelerator significantly enhanced the ECL emission of luminol-H2O2 system. Moreover, target miRNA-21 triggered bipedal DNA walker was powered by toehold-mediated strand displacement reaction (TSDR) for signal amplification. Consequently, the proposed ECL biosensor achieved ultrasensitive detection of miRNA-21 with a linear range from 100 aM to 100 pM and a limit of detection of 33 aM. Simultaneously, the biosensor was also successfully applied to detect target miRNA-21 in lysates from human cancer cells. As a result, this work constructed a new signal amplification platform, exhibiting great application potential in biomedical analysis and early clinical diagnostics.
The aberrant expression of microRNAs (miRs) in cells is closely linked to the initiation and progression of various diseases. Sensitive monitoring of their level is hence vital for biomedical ...research and disease diagnosis. Herein, a highly sensitive and non-label fluorescence sensor based on multiple recycling signal amplification cascades is constructed for the detection of miR-21 in human sera. The presence of miR-21 initiates the primer-fueled target recycling process for the generation of many primer/hairpin templates for the subsequent auto-cycling primer extension (APE) amplification cycles, which result in the formation of lots of long-stem hairpins. The enzyme-based cleavage of such hairpins
polymerization/excision cycles further leads to the generation of abundant G-quadruplex strands, which associate with the thioflavin T (ThT) dye to emit remarkably magnified fluorescence for detecting miR-21 in the range of 1 pM-100 nM with a 0.32 pM detection limit without labeling the probes. Besides, the proposed assay can selectively discriminate miR-21 against other control molecules and realize the sensing of low levels of miR-21 in diluted sera. With features of high sensitivity
the triplex signal amplification cycles and simplicity in a non-label homogeneous manner, our miR sensing protocol can be a robust means for detecting various nucleic acids for the early diagnosis of diseases.
The isothermal assembly of a target-recognizable hairpin and a catalytic hairpin was driven by specific HIV-related DNA (hDNA) as a test model, outputting amplified duplexes to open a functional ...hairpin beacon (HB) tethering the templates of biemissive (green and red) silver nanoclusters (G-AgNCs and R-AgNCs). As such, their ratiometric fluorescence was remarkably lit up, achieving rapid, specific and sensitive biosensing with potential for disease diagnosis and biomolecule detection.
A novel and ultrasensitive electrochemiluminescence (ECL) immunosensor, which was based on the amplifying ECL of luminol by hemin-reduced graphene oxide (hemin-rGO) and Ag nanoparticles (AgNPs) ...decorated reduced graphene oxide (Ag-rGO), was constructed for the detection of carcinoembryonic antigen (CEA). For this proposed sandwich-type ECL immunosensor, Au nanoparticles electrodeposited (DpAu) onto hemin-rGO (DpAu/hemin-rGO) constructed the base of the immunosensor. DpAu had outstanding electrical conductivity to promote the electron transfer at the electrode interface and had good biocompatibility to load large amounts of primary antibody (Ab1), which provided an excellent platform for this immunosensor. Moreover, AgNPs and glucose oxidase (GOD) functionalized graphene labeled secondary antibody (Ag-rGO–Ab2–GOD) was designed as the signal probe for the sandwiched immunosensor. Not only did the hemin-rGO improve the electron transfer of the electrode surface, but hemin also further amplified the ECL signal of luminol in the presence of hydrogen peroxide (H2O2). With the aid of Ag-rGO–Ab2–GOD, enhanced signal was obtained by in situ generation of H2O2 and catalysis of AgNPs to ECL reaction of the luminol–H2O2 system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of CEA in the range from 0.1pgmL−1 to 160ngmL−1 with a detection limit of 0.03pgmL−1 (SN−1=3).
•Hemin-GNs could further enhance the luminol ECL in the presence of H2O2.•Glucose oxidase was used to label the Ag-rGO–Ab2 for generating H2O2 in situ.•With the catalysis of AgNPs, amplified ECL signal could be obtained.•The immnuosensor exhibits high sensitivity, good stability and satisfying selectivity.
The monitoring and detection of molecular biomarkers play crucial roles in disease diagnosis and treatment. In this work, we proposed a target-responsive netlike hybridization chain reaction (nHCR) ...DNA nanostructure construction method, which can offer an exceptional signal enhancement, for highly sensitive fluorescence detection of cytokine, interferon-gamma (IFN-γ). The presence of the target cytokine can lead to the conformational change of the aptamer recognition hairpin probes and the liberation of the nHCR initiator strands, which further trigger the nHCR process between two dye-labeled and double hairpin-structured probes to form netlike DNA nanostructures. The formation of the DNA nanostructures brings the dyes into close proximity, resulting in significantly amplified fluorescence resonance energy transfer signals for sensitive and enzyme-free detection of IFN-γ. The present method has a detection limit of 1.2 pM and a dynamic linear range of 5 to 1000 pM for IFN-γ detection. Besides, with the high specificity of the aptamer probe and the significant signal amplification of the nHCR, such an IFN-γ detection strategy shows excellent selectivity and high sensitivity, which can be potentially applied to detect IFN-γ in human serums. With such a demonstration of the detection of IFN-γ, this proposed method can be extended for detecting different types of biomolecules.
Endotoxin, also known as lipopolysaccharide (LPS), is able to induce a strong immune response on its internalization into mammalian cells. To date, aptamer-based biosensors for LPS detection have ...been rarely reported. This work describes a new signal-on electrochemical aptasensor for the ultrasensitive detection of LPS by combining the three-way DNA hybridization process and nanotechnology-based amplification. With the help of DNA1 (associated with the concentration of target LPS), the capture probe hybridizes with DNA1 and the assistant probe to open its hairpin structure and form a ternary "Y" junction structure. The DNA1 can be released from the structure in the presence of nicking endonuclease to initiate the next hybridization process. Then a great deal of cleaved capture probe produced in the cyclic process can bind with DNA2-nanocomposite, which contains the electroactive toluidine blue (Tb) with the amplification materials graphene (Gra) and gold nanoparticles (AuNPs). Thus, an enhanced electrochemical signal can be easily read out. With the cascade signal amplification, this newly designed protocol provides an ultrasensitive electrochemical detection of LPS down to the femtogram level (8.7 fg mL(-1)) with a linear range of 6 orders of magnitude (from 10 fg mL(-1) to 50 ng mL(-1)). Moreover, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of the capture probe and the assistant probe.
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•The C-g-C3N4 possesses the better conductivity and solubility and was more favorable for chemical modification due to the existence of carboxyl group.•The novel nanocomposite ...(C-g-C3N4-PEI) possessed excellent luminous efficiency and stability in S2O82− solution.•Based on the C-g-C3N4-PEI-S2O82− ECL system and enzyme inhibition, the fabricated biosensor was developed for ultrasensitive detection of OPs.
A signal on electrochemiluminescence (ECL) biosensor was designed for sensitive organophosphate pesticides (OPs) detection based on a novel composite of carboxylated graphitic carbon nitride-poly(ethylenimine) (C-g-C3N4-PEI) and acetylcholinesterase (AChE). The C-g-C3N4-PEI nanocomposite which was prepared through covalent bonding between the COOH of C-g-C3N4 and the NH2 of PEI exhibited significantly enhanced ECL efficiency and stability. K2S2O8 as the coreactant of C-g-C3N4-PEI could be consumed by thiocholine, produced by the hydrolysis of acetylthiocholine (ATCl) in the presence of AChE. Since OPs are one of AChE inhibiter, the consumption of coreactant K2S2O8 decreased with the increasing concentration of OPs, thus enhancing ECL signal. Under the optimum conditions, the proposed biosensor for OPs detection (using ethyl paraoxon as a model OPs) exhibited a wide linear ranging from 1.0 pM to 5.0μM with a low detection limit of 0.3 pM. The novel strategy has the advantages of fine practicality, good stability and reproducibility, which might provide a new promise for OPs detection in real-life samples.
Based on the autonomous replicated and catabolic Mg2+-ligation DNAzyme units as robust biocatalysts for autocatalytic amplification, an enzyme- and label-free electrochemical aptasensor was developed ...for the sensitive detection of protein using mucin 1 as tested model.
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•A sensitive, enzyme- and label-free impedimetric aptasensor was developed for mucin 1.•Mucin 1 aptamer sequence was tethered in a hairpin DNA for affinity recognition and combination.•Autocatalytic replicated Mg2+-ligation DNAzyme was used as robust biocatalyst for amplification.•Hairpin DNA was unfolded and switched into pH-responsive i-motifs at slightly acidic conditions.•The conformation change brought significantly varied Faraday impedance for mucin 1 quantization.
Herein, an enzyme- and label-free electrochemical aptasensor was developed for the amplified detection of protein (mucin 1, MUC1 as tested model) by utilizing autocatalytic and catabolic Mg2+-ligation DNAzyme as robust biocatalyst. The MUC1-affinity aptamer sequence is encoded in DNA hairpin 1 (HP1). The combined product of HP1 with MUC1 triggers the formation of active Mg2+-dependent DNAzyme in the presence of sequence-specific ssDNA (S1) and hairpin 2 (HP2) that especially consists of S1 and the other ssDNA (S2). When introducing Mg2+, HP2 as the substrate strand of this DNAzyme is cleaved at Mg2+-recognizable site, releasing S1 to repeatedly activate the autonomous replication and catabolism of new DNAzyme units. As a result, the multiple liberated S2 was further incubated in the modified electrode surface to unfold C-rich hairpin 3 (HP3) at neutral pH. While the formed duplex with negative charges is dehybridized due to the configuration switch of unfolded HP3 into positively charged i-motifs at decreased pH, leading to enhanced electrostatic attraction to redox pairs Fe(CN)63−/4− and accelerated electron transfer in the electrode surface. Thus, the Faraday impedance (Ret) significantly decreased with MUC1-responsive variations for the electrochemical quantification of MUC1 in the range of 10 fg·mL−1 to 10 ng·mL−1, achieving high sensitivity down to 3.33 fg·mL−1, excellent specificity, reproducibility and stability. This strategy has proven to be promising application in actual biological samples.
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.
Here, we discovered that rigidifying the tetraphenylethylene (TPE)-based ligand H4TCBPE (H4TCBPE = 1,1,2,2-tetra(4-carboxylbiphenyl)ethylene) into Hf-based metal–organic framework (Hf-TCBPE) could ...lead to a stronger electrochemiluminescence (ECL) emission in comparison to H4TCBPE aggregates and H4TCBPE monomers. Due to the lack of close-packed TCBPE chromophores in Hf-TCBPE, which was required for aggregation-induced ECL (AI-ECL) enhancement, we defined this unprecedented phenomenon as matrix coordination-induced ECL (MCI-ECL) enhancement. The strong ECL intensity of Hf-TCBPE not only originated from the fixation of the TCBPE ligand between Hf6 clusters that restricted the intramolecular free motions of TCBPE and suppressed the nonradiative relaxation but also stemmed from the high porosity of Hf-TCBPE that rendered both internal and external TCBPE chromophores able to be excited. Considering the unique ECL characteristic of Hf-TCBPE, we combined the new ECL indicator of Hf-TCBPE as well as the phosphate-terminal ferrocene (Fc)-labeled hairpin DNA (Fc-HP3) aptamer together as a signal probe (Hf-TCBPE/Fc-HP3), which was employed to construct a novel “off–on” ECL sensor for ultrasensitive mucin 1 (MUC1) detection with the assistance of the exonuclease III (Exo III)-assisted recycling amplification strategy. As expected, the ECL sensor displayed a desirable linear response range from 1 fg/mL to 1 ng/mL and the detection limit down to 0.49 fg/mL. The MCI-ECL enhancement demonstrated by the Hf-TCBPE developed a new and promising strategy to design and synthesize high-performance metal–organic framework (MOF)-based ECL materials for constructing ultrasensitive ECL sensors.
