Fe Single-Atom Carbon Dots Nanozyme Collaborated with Nucleic Acid Exonuclease III-Driven DNA Walker Cascade Amplification Strategy for Circulating Tumor DNA Detection.

Circulating tumor DNA (ctDNA), as a next-generation tumor marker, enables early screening and monitoring of cancer through noninvasive testing. Exploring the development of new methods for ctDNA detection is an intriguing study. In this work, a unique electrochemical biosensor for the ctDNA detector was constructed in the first utilizing Fe single-atom nanozymes-carbon dots (SA Fe-CDs) as a signaling carrier in collaboration with a DNA walker cascade amplification strategy triggered by nucleic acid exonuclease III (Exo III). The electrochemical active surface area of AuNPs/rGO modified onto a glassy carbon electrode (AuNPs/rGO/GCE) was about 1.43 times that of a bare electrode (bare GCE), with good electrical conductivity alongside a high heterogeneous electron transfer rate (5.81 × 10-3 cm s-1), that is, as well as the ability to load more molecules. Sequentially, the DNA walker cascade amplification strategy driven by Exo III effectively converted the target ctDNA into an amplified biosignal, ensuring the sensitivity and specificity of ctDNA. Ultimately, the electrochemical signal was further amplified by introducing SA Fe-CDs nanozymes, which could serve as catalysts for 3,3',5,5'-tetramethylbenzidine (TMB) oxidation with facile responding (Vmax = 0.854 × 10-6 M s-1) and robust annexation (Km = 0.0069 mM). The integration of the triple signal amplification approach achieved detection limits as low as 1.26 aM (S/N = 3) for a linearity spanning from 5 aM to 50 nM. In this regard, our proposal for a biosensor with exceptional assay properties in complicated serum environments had great potential for early and timely diagnosis of cancer.

3D DNAzyme walker based electrochemical biosensor for attomolar level microRNA-155 detection.

Herein, an ultrasensitive electrochemical biosensor for microRNA-155 (miR-155) detection based on the powerful catalytic and continuous walking signal amplification capability of 3D DNAzyme walker and the gold nanoparticles/graphene aerogels carbon fiber paper-based (AuNPs/GAs/CFP) flexible sensing electrode with excellent electrochemical performance was successfully constructed. In a proof-of-concept experiment, in the presence of miR-155, the DNAzyme strands anchored on the streptavidin-modified magnetic beads (MBs) silenced by locked strands can be activated, thus generating the walking arm of the 3D DNAzyme walker. Meanwhile, the substrate strands modified with Fe-MOF-NH2 nanoparticles were evenly distributed on the surface of MBs and served as tracks of the 3D DNAzyme walker. Once the DNAzyme strand was activated, the catalytic site in the substrate strand can be cleaved in the presence of Mn2+, and a large number of stumps modified with Fe-MOF-NH2 nanoparticles (output@Fe-MOF-NH2) will be generated during the continuous and efficient walking cleavage of the DNAzyme walker, driving the recognition-catalysis-release cycle process for signal amplification. Immediately afterwards, the signal was read out through the base complementary pairing of capture probe (PS) immobilized on the surface of the paper-based flexible sensing electrode AuNPs/GAs/CFP and signal probes output@Fe-MOF-NH2, thus achieving the quantitative detection of miR-155. Under optimal experimental conditions, the designed 3D DNAzyme walker-based biosensor exhibited a relatively lower limit of detection (LOD) of 56.23 aM, with a linear range of 100 aM to 100 nM. Overall, the proposed 3D DNAzyme walker biosensor exhibited good interference and reproducibility, demonstrating a promising future in the field of clinical disease diagnosis.

Duplex-specific nuclease powered 3D DNA walker and quantum dots barcodes for homogeneous electrochemical detection of microRNAs.

Multiplex microRNAs (miRNAs) detection is beneficial for early diagnosis and prognosis of cancer. Herein, duplex-specific nuclease (DSN) powered 3D DNA walker and quantum dots (QDs) barcodes were designed for the simultaneous detection of miRNAs in a homogeneous electrochemical sensor. In the proof-of-concept experiment, the effective active area of the as-prepared graphene aerogel-modified carbon paper (CP-GAs) electrode was ∼14.30 times larger than that of the traditional glassy carbon electrode (GCE), endowing the enhanced capability of loading more metal ions for ultrasensitive detection of miRNAs. In addition, DSN-powered target recycling and DNA walking strategy assured the sensitive detection of miRNAs. After the introduction of magnetic beads (MNs) and electrochemical double enrichment strategies, the integration of triple signal amplification methods yielded good detection results. Under optimal conditions, towards simultaneous detection of microRNA-21 (miR-21) and miRNA-155 (miR-155), a linear range of 10-16-10-7 M and a sensitivity of 10 aM (miR-21) and 2.18 aM (miR-155) were achieved, respectively. It was worth mentioning that the prepared sensor can detect miR-155 down to 0.17 aM, which was also extremely advantageous among the sensors reported so far. What's more, through verification, the prepared sensor had good selectivity and reproducibility, and exhibited good detection ability in complex serum environments, showing great potential in early clinical diagnosis and screening.

An ultrasensitive electrochemical biosensor for microRNA-21 detection via AuNPs/GAs and Y-shaped DNA dual-signal amplification strategy.

Herein, a gold nanoparticles/graphene aerogels (AuNPs/GAs) modified electrochemical biosensor with catalytic hairpin assembly (CHA) and Y-shaped DNA nanostructure dual-signal amplification approaches for ultrasensitive microRNA-21 (miR-21) detection was successfully constructed, which displayed an ultra-wide detection linear range from 5 fM to 50 nM, as well as a relatively low detection limit (LOD) of 14.70 aM (S/N = 3). Furthermore, the sensing strategy had excellent specificity among highly homologous miRNA family members and exhibited satisfactory analytical performance for miRNA detection.

A disposable and sensitive non-enzymatic glucose sensor based on a 3D-Mn-doped NiO nanoflower-modified flexible electrode.

Herein, nanoflower-shaped Mn-doped NiO nano-enzyme composites with high catalytic performance and excellent conductivity were grown on 3D flexible carbon fiber cloth (CFC) via hydrothermal and calcination methods to construct an efficient flexible glucose-sensitive detection electrode. For electrochemical-based sensors, high conductivity is a prerequisite for reliable data acquisition. To avoid the problems associated with using insulating Nafion or paraffin binders, we adopted a strategy of directly growing Mn-doped NiO onto the electrode surface, thereby avoiding interference due to the oxidization of species present in real samples at higher redox potentials, since Ni2+/Ni3+ has low redox potential. Therefore, the electrode has a linear range of 3-5166 µM for glucose detection, with a detection limit as low as 0.28 µM, showing excellent selectivity and reproducibility. The composite-modified electrode provides accurate detection results with real human serum samples, which are in full agreement with those of commercial blood glucose meters. In addition, we tested the glucose content in tea and sorghum fermentation broth at different stages, further expanding the application range of the Mn-NiO sensors. The nano-enzyme sensor fabricated herein offers a new idea for further integration into wearable flexible electronic devices for accurate glucose detection.

Catalytic Hairpin Assembly-Driven Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive Detection of MicroRNA Based on the Ratios of Fe-MOFs and MB-GA-UiO-66-NH2.

In this work, a novel ratio electrochemical biosensing platform based on catalytic hairpin assembly target recovery to trigger dual-signal output was developed for ultrasensitive detection of microRNA (miRNA). To achieve the ratiometric dual-signal strategy, methylene blue (MB), an electrochemical indicator, was ingeniously loaded into the pores of graphene aerogel (GA) and metal-organic framework (MOF) composites with high porosity and large specific surface area, and another electrochemical indicator Fe-MOFs with distinct separation of redox potential was selected as a signal probe. Concretely, with the presence of the target miRNA, the CHA process was initiated and the signal probe was introduced to the electrode surface, producing abundant double-stranded H1-H2@Fe-MOFs-NH2. Then, the measurement and analysis of the prepared ratiometric electrochemical biosensor by differential pulse voltammetry (DPV) showed that the introduction of the target miRNA led to an increase in the oxidation peak signal of Fe-MOFs (+0.8 V) and a decrease in the oxidation peak signal of MB (-0.23 V). Therefore, the peak current ratio of IFe-MOFs/IMB could be employed to accurately reflect the actual concentration of miRNA. Under optimal conditions, the detection limit of the proposed biosensor was down to 50 aM. It was worth noting that the proposed biosensor exhibited excellent detection performance in a complex serum environment and tumor cell lysates, showing great potential in biosensing and clinical diagnosis.

Sandwich-type microRNA biosensor based on graphene oxide incorporated 3D-flower-like MoS2 and AuNPs coupling with HRP enzyme signal amplification.

A sandwich electrochemical biosensing strategy for ultrasensitive detection of miRNA-21 was developed by using graphene oxide incorporated 3D-flower-like MoS2 (3D MoS2-rGO) nanocomposites as the substrate and horseradish peroxidase (HRP)-functionalized DNA strand 1 (S1)-gold nanoparticles (S1-AuNPs-HRP) as signal amplification probes. Herein, 3D MoS2-rGO nanocomposites not only had a large specific surface area and excellent conductivity, but also provided more attachment sites for electrodepositing AuNPs. In the presence of target miRNA, a sandwich structure was formed, and the determination of the miRNA-21 was carried out by measuring the DPV response of H2O2 mediated by hydroquinone (HQ) at a potential of + 0.052 V (vs AgCl reference electrode). Under the optimal experimental conditions, the as-prepared biosensor enabled the ultrasensitive detection of miRNA-21 from 5 fM to 0.5 µM with the low detection limit of 0.54 fM (S/N = 3), comparable or lower than previous reported methods for miRNA-21 detection, which benefited from the synergistic amplification of 3D MoS2-rGO and AuNPs-HRP. The prepared biosensor showed satisfactory selectivity, reproducibility, and stability towards miRNA-21 detection. The biosensor was feasible for accurate and quantitative detection of miRNA-21 in normal human serum samples with RSD below 5.86%, which showed a great potential in clinical analysis and disease diagnosis.

MXene-MoS2 heterostructure collaborated with catalyzed hairpin assembly for label-free electrochemical detection of microRNA-21.

Abnormal expression of microRNAs is greatly associated with the occurrence of various cancer types, revealing great potential of microRNA as biomarkers for cancer diagnosis and prognosis. Herein, a MXene-MoS2 heterostructure enhancing electrochemical biosensor coupled with catalytic hairpin assembly (CHA) amplification approach for label-free determination of microRNA-21 (miR-21) was successfully assembled. In particular, the unique micro-nano heterostructure with large specific area and favorable electroconductivity exhibited the ability of excellent confinement effect. Thus, rendered the MXene-MoS2 heterostructure the ability to trigger more target recycling reaction, giving new vitality to the traditional CHA amplification method. Meanwhile, thionine (Thi) and gold nanoparticles (AuNPs) were anchoring at the surface of MXene-MoS2 heterostructure, respectively, empowered the sensor the capability of capture probes fixation and miR-21 label-free determination. When numerous electronegative double-stranded DNA generated, the electron transfer was greatly hindered, resulting in signal decrease. Accordingly, the design denoted a broad dynamic range from 100 fM to 100 nM and a detection limit of about 26 fM, comparable or lower than previous reported methods for miR-21 detection. Furthermore, the sensing platform supplied satisfactory selectivity, reproducibility and stability towards the miR-21 detection. The real sample determination also showed a promising performance under clinical circumstance. Finally, from the clinical standpoint, the proposed biosensor is a considerable platform toward early disease detection and monitoring.