The sensitive and quantitative analysis of mucin 1 (MUC1) is very important for the prevention and early diagnosis of cancers. In the present work, based on the mechanism of the four-way DNA branch ...migration cascades, we constructed a simple and effective signal amplification strategy for aptamer-based sensitive detection of MUC1. The specific binding of MUC1 to the aptamer sequence in the hairpin probe unfolds and switches its structure, triggering the formation of the DNA Holliday junction structure for cascaded branch migrations with the assistance of two fuel DNA duplexes. Importantly, a target analogue DNA complex can be generated in such processes for recycling the branch migration reactions for the production of substantial amounts of G-quadruplexes, which can bind the thioflavin T dye to show significantly intensified fluorescence for detecting MUC1 with a low detection limit of 2.8 nM without the involvement of any labels or enzymes. In addition, this detection strategy could be successfully applied to monitor the target MUC1 in diluted human serums with a high selectivity and acceptable accuracy to demonstrate its potential application for real samples with the advantages of simplicity and signal amplification capability.
This study demonstrates a novel approach toward development of advanced immunosensors based on chemically functionalized core−shell Fe3O4@Ag magnetic nanoparticles, and the preparation, ...characterization, and measurement of relevant properties of the immunosensor useful for the detection of carcinoembryonic antigen (CEA) in clinical immunoassay. The immunosensor based on the combination of a magnetic nanocore and an Ag metallic shell shows good adsorption properties for the attachment of the CEA antibody selective to CEA. The core−shell nanostructure presents good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of CEA, and allows detection of CEA at a concentration as low as 0.5 ng·mL-1. Importantly, the proposed methodology could be extended to the detection of other antigens or biocompounds.
The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four ...overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications.
Highly sensitive detection of molecular biomarkers plays a significant role in diagnosing various types of diseases at the early stage. We demonstrated in this paper an ultrasensitive aptamer-based ...fluorescence method for detecting mucin 1 (MUC1) in human serum via a cascaded multiple recycling signal amplification strategy. The MUC1 target molecules present in the samples cause structure switching of the hairpin aptamer probes, which initiates three cascaded recycling cycles for the cleavage of the fluorescently quenched signal probes to recover significant fluorescence for highly sensitive detection of MUC1. The developed method has a linear range from 100 fM to 1 nM for MUC1 detection. Besides, owing to the substantial signal amplification by the integrated and cascaded recycling cycles, a low detection limit of 35 fM is achieved with high selectivity. Moreover, the monitoring of trace MUC1 in human serum can also be realized with such a method, indicating its great potential for highly sensitive detection of different disease biomarkers.
A novel sandwich-type electrochemical immunosensor based on functionalized nanomaterial labels and bienzyme (horseradish peroxidase and glucose oxidase) biocatalyzed precipitation was developed for ...the detection of α-fetoprotein (AFP). The enzymes linked to functionalized nanomaterials as biocatalysts could accelerate the oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 to yield the insoluble product on the electrode surface; the mass loading of the precipitates on the device led to a significant enhanced signal. Cyclic voltammetry and electrochemical impedance spectroscopy techniques were used to monitor the enhanced precipitation of 4-CN that accumulated on the electrode surface and subsequent decrement in the electrode surface area by monitoring the reduction process of the Fe(CN)64−/3− redox couple. Under optimal conditions, the proposed immunosensor showed a high sensitivity and a wide linear range from 0.001 to 60ngmL−1 with a low detection limit of 0.33pgmL−1. Moreover, the immunosensor exhibited good selectivity, acceptable stability and reproducibility. The amplification strategy showed good promise for clinical screening of tumor biomarkers.
•Enzymatically catalyzed precipitates based on bienzyme.•Functionalized single-walled carbon nanohorns (SWCNHs) as molecular tags were first reported.•A sandwich electrochemical immunosensor for AFP detection was developed.
In this work, a novel two-dimensional (2D) ultrathin metal-organic layer (MOL) based on the aggregation-induced emission (AIE) ligand H4ETTC (H4ETTC = ...4',4''',4''',4'''-(ethene-1,1,2,2-tetrayl)tetrakis((1,1'-biphenyl-4-carboxylic acid))) was developed and used to construct a novel electrochemiluminescence (ECL) aptasensor for ultrasensitive detection of carcinoembryonic antigen (CEA). The newly synthesized AIE luminogen (AIEgen)-based MOL (Hf-ETTC-MOL) yielded a higher ECL intensity and efficiency than did H4ETTC monomers, H4ETTC aggregates and 3D bulk Hf-ETTC-MOF. This improvement occurred not only because the ETTC ligands were coordinatively immobilized in a rigid MOL matrix, which restricted the intramolecular free rotation and vibration of these ligands and then reduced the non-radiative transition, but also because the porous ultrathin 2D MOL greatly shortened the transport distances of ions, electrons, coreactant (triethylamine, TEA) and coreactant intermediates (TEA˙ and TEA˙+), which made more ETTC luminophores able to be excited and yielded a high ECL efficiency. On the basis of using the Hf-ETTC-MOL as a novel ECL emitter and rolling circle amplification (RCA) as a signal amplification strategy, the constructed ECL aptasensor exhibited a linear range from 1 fg mL-1 to 1 ng mL-1 with a detection limit of 0.63 fg mL-1. This work has opened up new prospects for developing novel ECL materials and is expected to lead to increased interest in using AIEgen-based MOLs for ECL sensing.
Herein, a rigid 3D DNA nanopillar was used to investigate the influence of spatial organization on the cascade activity in multienzyme systems, realizing controllable regulation of the mimic enzyme ...ratio and spacing for acquiring a high-efficiency enzyme cascade catalytic platform. Initially, the ratio of mimic enzyme AuNPs (glucose oxidase-like activity) and hemin/G-quadruplex DNAzyme (peroxidase-like activity) fixed at the designed position was adjusted by changing the number of edges in a DNA polyhedron, resulting in an optimal mimic enzyme ratio of 1 : 4 with a quadrangular prism as the scaffold. Notably, the DNA nanopillar formed by quadrangular prism layer-by-layer assembly acted as a track for directional and controllable movement of a bipedal DNA walker based on the toehold mediated strand displacement reaction (TSDR), which endowed the assay system with continuous enzyme spacing regulation compared with previous enzyme cascade systems that induced inflexible operation. Furthermore, enzyme mimetics in this work circumvented the drawbacks of natural enzymes, such as time-consuming purification processes and poor thermal stability. As a proof of concept, the proposed dual regulation strategy of cascade enzymes was applied in the ultrasensitive electrochemical detection of Pb
2+
, which provided a new route to fabrication of high-performance artificial enzyme cascade platforms for ultimate application in bioanalysis and biodiagnostics.
A rigid 3D DNA nanopillar was used to investigate the influence of spatial organization on the cascade activity in multienzyme systems, realizing controllable regulation of the mimic enzyme ratio and spacing for efficient cascade catalytic platform.
A facile and universal aptamer-based label-free approach for selective and sensitive fluorescence detection of proteins and small biomolecules by using the SYBR Green I (SGI) dye is developed. This ...robust versatile biosensing strategy relies on fluorescence turn-off changes of SGI, resulting from target-induced structure switching of aptamers. Upon binding with the targets, the aptamers dissociate from the respective cDNA/aptamer duplexes, leading to the release of the dsDNA-intercalated SGI into solution and the quenching of the corresponding fluorescence intensities. Such target-induced conformational changes and release of aptamers from the DNA duplexes essentially lead to the change in the fluorescence signal of the SGI and thus constitute the mechanism of our aptamer-based label-free fluorescence biosensor for specific target analyses. Under optimized conditions, our method exhibits high sensitivity and selectivity for the quantification of ATP and thrombin with low detection limits (23.4 nM and 1.1 nM, respectively). Compared with previous reported methods for aptamer-based detection of ATP and thrombin, this label-free approach is selective, simple, convenient and cost-efficient without any chemical labeling of the probe or the target. Therefore, the present strategy could be easily applicable to biosensors that target a wide range of biomolecules.
In this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease III (Exo III)-catalyzed target recycling (ECTR) ...amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo III to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specific affinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a “mix-and-detect” fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules.
•Un-modified hairpin aptamer probes and SYBR Green I dye are employed to achieve label-free fluorescent detection of ATP.•The presence of ATP leads to re-configuration of the hairpin aptamer probes.•Exonuclease III cleaves the re-configured hairpin aptamer probes to release the target ATP and initiate target recycling amplification.•The proposed method offers selective and sensitive detection of ATP down to the low nanomolar level.
Herein, by directly introducing mismatched reactant DNA, high-reactivity and high-threshold enzyme-free target recycling amplification (EFTRA) is explored. The developed high-efficiency EFTRA ...(HEEFTRA) was applied as a programmable DNA signal converter, possessing higher conversion efficiency than the traditional one with perfect complement owing to the more negative reaction standard free energy (Δ
G
). Once traces of input target miRNA interact with the mismatched reactant DNA, amounts of ferrocene (Fc)-labeled output DNA could be converted
via
the EFTRA. Impressively, the Fc-labeled output DNA could be easily captured by the DNA tetrahedron nanoprobes (DTNPs) on the electrode surface to form triplex-forming oligonucleotide (TFO) at pH = 7.0 for sensitive electrochemical signal generation and the DTNPs could be regenerated at pH = 10.0, from which the conversion efficiency (
N
) will be accurately obtained, benefiting the selection of suitable mismatched bases to obtain high-efficiency EFTRA (HEEFTRA). As a proof of concept, the HEEFTRA as an evolved DNA signal converter is successfully applied for the ultrasensitive detection of miRNA-21, which gives impetus to the design of other signal converters with excellent efficiency for ultimate applications in sensing analysis, clinical diagnosis, and other areas.
Herein, by directly introducing mismatched reactant DNA, high-reactivity and high-threshold enzyme-free target recycling amplification (EFTRA) is explored.
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