Ultrahigh-Speed 3D DNA Walker with Dual Self-Protected DNAzymes for Ultrasensitive Fluorescence Detection and Intracellular Imaging of microRNA.

Herein, a dual self-protected DNAzyme-based 3D DNA walker (dSPD walker), composed of activated dual self-protected walking particles (ac-dSPWPs) and track particles (TPs), was constructed for ultrasensitive and ultrahigh-speed fluorescence detection and imaging of microRNAs (miRNAs) in living cells. Impressively, compared with the defect that "one" target miRNA only initiates "one" walking arm of the conventional single self-protected DNAzyme walker, the dSPD walker benefits from the secondary amplification and spatial confinement effect and could guide "one" target miRNA to generate "n" secondary targets, thereby initiating "n" nearby walking strands immediately, realizing the initial rate over one-magnitude-order faster than that of the conventional one. Moreover, in the process of relative motion between ac-dSPWPs and TPs, the ac-dSPWPs could cleave multiple substrate strands simultaneously to speed up movement and reduce the derailment rate, as well as combine with successive TPs to facilitate a large amount of continuous signal accumulation, achieving an ultrafast detection of miRNA-221 within 10 min in vitro and high sensitivity with a low detection limit of 0.84 pM. In addition, the DNA nanospheres obtained by the rolling circle amplification reaction can capture the Cy5 fluorescence dispersed in liquids, which achieves the high-contrast imaging of miRNA-221, resulting in further ultrasensitive imaging of miRNA-221 in cancer cells. The proposed strategy has made a bold innovation in the rapid and sensitive detection as well as intracellular imaging of low-abundance biomarkers, offering promising application in early diagnosis and relevant research of cancer and tumors.

High-Efficient Electrochemiluminescence of DNA-Au Ag Nanoclusters with Au NPs@Ti3C2 as a Novel Coreaction Accelerator for Ultrasensitive Biosensing.

In this work, an ultrasensitive electrochemiluminescence (ECL) biosensor was constructed based on DNA-stabilized Au Ag nanoclusters (DNA-Au Ag NCs) as the efficient luminophore and Au NPs@Ti3C2 as a new coreaction accelerator for determining microRNA-221 (miRNA-221) related to liver cancer. Impressively, DNA-Au Ag NCs were stabilized by the high affinity of the periodic 3C sequence, exhibiting an excellent ECL efficiency of 27% compared with classical BSA-Au Ag NCs (16%). Moreover, the Au NPs@Ti3C2 nanocomposites, as a new coreaction accelerator, were first introduced to accelerate the production of abundant sulfate free radicals (SO4•-) for promoting the ECL efficiency of DNA-Au Ag NCs in the DNA-Au Ag NCs/Au NPs@Ti3C2/S2O82- ternary system due to the energy band of Au NPs@Ti3C2 being well-matched with the frontier orbital of S2O82-. Furthermore, the trace target (miRNA-221) could drive the rolling circle amplification to generate an amount of output DNA with periodic 3C and 10A sequences. Through covalent bonds on the surface of poly A and Au NPs, the distance between the luminophor and the coreaction accelerator could be narrowed to further enhance the detection sensitivity. As a result, the constructed sensor has been applied for the ultrasensitive detection of miRNA-221 with a low detection limit of 50 aM and successfully monitored miRNA-221 in MHCC-97L and HeLa cell lysates. This strategy could be utilized for guiding the synthesis of light-emitting DNA-metal NCs, which has great potential in the construction of ultrasensitive biosensors for the early diagnosis of diseases.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

Electron-Accelerator-Induced Fast Electron Transfer for Enhancing Electrochemiluminescence of Gold Nanoclusters and Its Bioanalysis Application: A Novel Avenue for Developing High-Efficient Emitters.

Herein, the gold nanoclusters/CaFe2O4 nanospheres (Au NCs/CaFe2O4) heterostructure as a novel electrochemiluminescence (ECL) emitter was developed. Excitingly, Au NCs/CaFe2O4 displayed highly efficient and greatly stable ECL based on the newly defined electron-accelerator p-type semiconductor CaFe2O4 NS-induced fast electron transfer; it solved one key obstacle of metal NC-based ECL emitters: sluggish through-covalent bond electron transport kinetics-caused inferior ECL performance. Specifically, on account of the energy level matching between emitter Au NCs and electron-accelerator CaFe2O4 NSs, the valence band (VB) of the electron-accelerator could provide abundant holes for rapidly transporting the electrogenerated electron from the highest occupied molecular orbital (HOMO) of Au NCs to the electrode, generating massive excited species of Au NCs for strong ECL emission. Notably, Au NCs/CaFe2O4 emerged 5.4-fold higher ECL efficiency with 3.5-fold higher electrochemical oxidation current in comparison with pure Au NCs, exhibiting great prospects in extensive lighting installations, ultrasensitive biosensing, and high-resolution ECL imagery. As applications, an ECL bioassay platform was constructed with Au NCs/CaFe2O4 as an emitter and U-like structure-fueled catalytic hairpin assembly (U-CHA) as a signal amplifier for fast and trace analysis of aflatoxin B1 (AFB1) with the detection limit (LOD) down to 2.45 fg/mL, which was 3 orders of magnitude higher than that of the previous ECL biosensors with much better stability. This study developed an entirely new avenue for enlarging the ECL performance of metal NCs, and it is a very attractive orientation for directing the reasonable design of prominent metal NC-based ECL emitters and broadening the practical application of metal NCs.

Porous Complex-Mediated Dual Emission of Porphyrins for the Electrochemiluminescence Bioassay.

Although porphyrins make up a promising class of electrochemiluminescence (ECL) luminophors, their aggregation-caused quenching (ACQ) characteristics lead to inferior ECL efficiency (ΦECL). Furthermore, current application of porphyrins is limited to cathodic emission. This work creatively exploited a cage-like porous complex (referred to as SWU-1) as the microreactor to recede the ACQ effect while modulating dual ECL emission of meso-tetra(4-carboxyphenyl)porphine (TCPP), which self-assembled with SWU-1 to form TCPP@SWU-1 nanocapsules (TCPP@SWU-1 NCs). As the microreactor, SWU-1 not only effectively constrained TCPP aggregation to improve electron-hole recombination efficiency but also improved stability of anion and cation radicals, thus significantly enhancing the dual emission of TCPP. Compared with TCPP aggregates, the resulting TCPP@SWU-1 NCs exhibited significantly enhanced anodic and cathodic emission, and their ΦECL was increased by 8.7-fold and 3.9-fold, respectively. Furthermore, black hole quencher-2 (BHQ2) can simultaneously quench anodic and cathodic signals. TCPP@SWU-1 NCs coupling BHQ2 conveniently achieved an ECL ratio detection of miRNA-126, and the limit of detection (S/N = 3) was 4.1 aM. This work pioneered the development of the cage-like porous complex SWU-1 as the microreactor to alleviate defects of the ACQ effect and mediate dual emission of TCPP. The coupling of dual-emitting TCPP@SWU-1 NCs and dual-function moderator BHQ2 created a novel single-luminophor-based ratio system for bioanalysis and provided a promising ECL analysis approach for miRNA-126.

Universal Signal Switch Based on a Mesostructured Silica Xerogel-Confined ECL Polymer for Epigenetic Quantification.

The development of a highly accurate electrochemiluminescence (ECL) signal switch to avoid nonspecific stimulus responses is currently a significant and challenging task. Here, we constructed a universal signal switch utilizing a luminophore-quencher pair of mesostructured silica xerogel-confined polymer and gold nanoparticles (Au NPs) that can accurately detect low-abundance epigenetic markers in complex sample systems. Notably, the ECL polymer encapsulated in mesostructured silica xerogel acts as a luminophore, which demonstrated a highly specific dependence on the Au NPs-mediated energy transfer quenching. To demonstrate the feasibility, we specifically labeled the 5-hydroxymethylcytosine (5hmC) site on the random sequence using a double-stranded (dsDNA) tag that was skillfully designed with the CRISPR/Cas12a activator and recombinant polymerase amplification (RPA) template. After amplification by RPA, a large amount of dsDNA tag was generated as the activator to initiate the trans-cleavage activity of CRISPR/Cas12a and subsequently activate the signal switch, allowing for precise quantification of 5hmC. The ECL signal switch improves the stability of the luminophore and prevents nonspecific stimulus responses, providing a new paradigm for constructing high-precision biosensors.

A Fluorescence Light-Up 3D DNA Walker Driven and Accelerated by Endogenous Adenosine-5'-triphosphate for Sensitive and Rapid Label-Free MicroRNA Detection and Imaging in Living Cells.

Herein, a fluorescence light-up 3D DNA walker (FLDW) was powered and accelerated by endogenous adenosine-5'-triphosphate (ATP) molecules to construct a biosensor for sensitive and rapid label-free detection and imaging of microRNA-221 (miRNA-221) in malignant tumor cells. Impressively, ATP as the driving force and accelerator for FLDW could significantly accelerate the operation rate of FLDW, reduce the likelihood of errors in signaling, and improve the sensitivity of detection and imaging. When FLDW was initiated by output DNA H1-op transformed by target miRNA-221, G-rich sequences in the S strand, anchored to AuNP, were exposed to form G-quadruplexes (G4s), and thioflavin T (ThT) embedded in the G4s emitted intense fluorescence to realize sensitive and rapid detection of target miRNA-221. Meanwhile, the specific binding of ThT to G4 with a weak background fluorescence response was utilized to enhance the signal-to-noise ratio of the label-free assay straightforwardly and cost-effectively. The proposed FLDW system could realize sensitive detection of the target miRNA-221 in the range of 1 pM to 10 nM with a detection limit of 0.19 pM by employing catalytic hairpin assembly (CHA) to improve the conversion of the target. Furthermore, by harnessing the abundant ATP present in the tumor microenvironment, FLDW achieved rapid and accurate imaging of miRNA-221 in cancer cells. This strategy provides an innovative and high-speed label-free approach for the detection and imaging of biomarkers in cancer cells and is expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

Exploring the Effect of Steric Hindrance on Trans-cleavage Activity of CRISPR-cas12a for Ultrasensitive SERS Detection of P53 DNA.

The trans-cleavage properties of Cas12a make it important for gene editing and disease diagnosis. In this work, the effect of spatial site resistance on the trans-cleavage activity of Cas12a was studied. First, we have explored the cutting effect of Cas12a when different-sized nanoparticles are linked with various spacings of DNA strands using the fluorescence method. The minimum spacing with different-sized nanoparticles that cas12a can cut was determined. We found that when the size of the nanoparticles increases, the minimum spacing that cas12a can cut gradually increases. Subsequently, we verified the conclusion using the surface-enhanced Raman scattering (SERS) method, and at the same time, we designed a SERS biosensor that can achieve ultrasensitive detection of P53 DNA with a linear range of 1 fM-10 nM and a limit of detection of 0.40 fM. Our work develops a deep study of the trans-cleavage activity of Cas12a and gives a guide for DNA design in cas12a-related studies, which can be applied in biomedical analysis and other fields.

Highly Specific Aptamer-Antibody Birecognized Sandwich Module for Ultrasensitive Detection of a Low Molecular Weight Compound.

Herein, the aptamer-antibody sandwich module was first introduced to accurately recognize a low molecular weight compound (mycotoxin). Impressively, compared with the large steric hindrance of a traditional dual-antibody module, the aptamer-antibody sandwich with low Gibbs free energy and a low dissociation constant has high recognition efficiency; thus, it could reduce false positives and false negatives caused by a dual-antibody module. As a proof of concept, a sensitive electrochemiluminescence (ECL) biosensor was constructed for detecting mycotoxin zearalenone (ZEN) based on an aptamer-antibody sandwich as a biological recognition element and porous ZnO nanosheets (Zn NSs) supported Cu nanoclusters (Cu NCs) as the signal transduction element, in which the antibody was modified on the vertex of a tetrahedral DNA nanostructure (TDN) with a rigid structure to increase the kinetics of target recognition for promoting the detection sensitivity. Moreover, the Cu NCs/Zn NSs exhibited an excellent ECL response that was attributed to the aggregation-induced ECL enhancement through electrostatic interactions. The sensing platform achieved trace detection of ZEN with a low detection limit of 0.31 fg/mL, far beyond that of the enzyme-linked immunosorbent assay (ELISA, the current rapid detection method) and high-performance liquid chromatography (HPLC, the national standard detection method). The strategy has great application potential in food analysis, environmental monitoring, and clinical diagnosis.

Electrochemiluminescence of Ultrasmall Silica Nanoparticles from Size Modulation and Multipath Surface State Adjustment for Ultrasensitive HIV-DNA Fragment Detection.

Here, ultrasmall SiO2 nanoparticles (u-SiO2 NPs, <5 nm) with obvious electrochemiluminescence (ECL) phenomenon, which was absent for conventional silica nanoparticles (c-SiO2 NPs), were reported. In a finite ultrasmall volume, the u-SiO2 NPs exhibited increasing ground state energy and higher optical absorption strength due to the electron-hole confinement model and favored catalyzing the reaction through the rapid diffusion of bulk charge, resulting in apparent ECL emission. Then, Zn2+-induced u-SiO2 nanoaggregates (Zn/u-SiO2-Ov nAGG) were synthesized and exhibited improved ECL performance via multipath surface state adjustment of u-SiO2 from several aspects, including aggregation-induced ECL, the generation of oxygen vacancy (Ov), and more positive surface charge. In addition, an ECL biosensor was constructed for ultrasensitive human immunodeficiency virus-related deoxyribonucleic acid detection from 100 aM to 1 nM with a low limit of 50.48 aM, combining the ECL luminescence of Zn/u-SiO2-Ov nAGG with three-dimensional DNA nanomachine-mediated multioutput amplification for enhanced accuracy and sensitivity compared to the single-output method. Therefore, exploring the ECL of ultrasmall nanoparticles via the adjustment of size and surface state provided a valuable indication to a wider investigation and application of novel ECL materials for clinical diagnostic.

Simultaneous and Sensitive Sensing of Intracellular MicroRNA and mRNA for the Detection of the PI3K/AKT Signaling Pathway in Live Cells.

Simultaneous detection of the concentration variations of microRNA-221 (miRNA-221) and PTEN mRNA molecules in the PI3K/AKT signaling pathway is of significance to elucidate cancer cell migration and invasion, which is useful for cancer diagnosis and therapy. In this work, we show the biodegradable MnO2 nanosheet-assisted and target-triggered DNAzyme recycling signal amplification cascaded approach for the specific detection of the PI3K/AKT signaling pathway in live cells via simultaneous and sensitive monitoring of the variation of intracellular miRNA-221 and PTEN mRNA. Our nanoprobes enable highly sensitive and multiplexed sensing of miRNA-221 and PTEN mRNA with low detection limits of 23.6 and 0.59 pM in vitro, respectively, due to the signal amplification cascades. Importantly, the nanoprobes can be readily delivered into cancer cells and the MnO2 nanosheets can be degraded by intracellular glutathione to release the Mn2+ cofactors to trigger multiple DNAzyme recycling cycles to show highly enhanced fluorescence at different wavelengths to realize sensitive and multiplexed imaging of PTEN mRNA and miRNA-221 for detecting the PI3K/AKT signaling pathway. Moreover, the regulation of PTEN mRNA expression by miRNA-221 upon stimulation by various drugs can also be verified by our method, indicating its promising potentials for both disease diagnosis and drug screening.

Antibody-Protein-Aptamer Electrochemical Biosensor based on Highly Efficient Proximity-Induced DNA Hybridization on Tetrahedral DNA Nanostructure for Sensitive Detection of Insulin-like Growth Factor-1.

Herein, an antibody-protein-aptamer electrochemical biosensor was designed by highly efficient proximity-induced DNA hybridization on a tetrahedral DNA nanostructure (TDN) for ultrasensitive detection of human insulin-like growth factor-1 (IGF-1). Impressively, the IGF-1 antibody immobilized on the top vertex of the TDN could effectively capture the target protein with less steric effect, and the ferrocene-labeled signal probe (SP) bound on the bottom vertex of the TDN was close to the electrode surface for generating a strong initial signal. In the presence of target protein IGF-1 and an aptamer strand, an antibody-protein-aptamer sandwich could be formed on the top vertex of TDN, which would trigger proximity-induced DNA hybridization to release the SP on the bottom vertex of TDN; therefore, the signal response would decrease dramatically, enhancing the sensitivity of the biosensor. As a result, the linear range of the proposed biosensor for target IGF-1 was 1 fM to 1 nM with the limit of detection down to 0.47 fM, which was much lower than that of the traditional TDN designs on electrochemical biosensors. Surprisingly, the use of this approach offered an innovative approach for the sensitive detection of biomarkers and illness diagnosis.

Host-Guest Recognition-Mediated Supramolecular Aggregation-Induced Electrochemiluminescence of Iridium(III) Complexes for Nucleic Acid Bioassay.

Currently reported aggregation-induced electroluminescence (AIECL) is usually based on the electrostatic integration of luminous monomers, and its application is still limited by the low ECL efficiency and poor structural stability of electrostatic integration-based AIECL emitters. Herein, host-guest recognition-mediated supramolecular AIECL was creatively developed to overcome the defects of electrostatic-integration-based AIECL. Cucurbit[8]uril (CB[8]) as the host recognized tris (2-phenylpyridine) iridium(III) [Ir(ppy)3] as the guest to form a novel supramolecular complex Ir-CB[8]. CB[8] can not only provide a large hydrophobic cavity to efficiently load Ir(ppy)3 and enrich coreactant tripropylamine but also utilize its carbonyl-laced portals to form intramolecular hydrogen bonds to stabilize the supramolecular structure, so Ir-CB[8] revealed excellent AIECL performance. The AIECL emitter Ir-CB[8] coupled the efficient DNA walker to construct a sensing system for miRNA-16 detection. Au nanoparticles@norepinephrine (AuNPs@NE) trapped by single-strand S1 was developed to significantly quench the ECL emission of Ir-CB[8]. When the target microRNA-16 (miRNA-16) existed, H1 was opened and the sequential assembly from H2 to H7 was triggered, forming "windmill"-like DNA walker with six Pb2+-dependent leg DNA. The assembled DNA walker, which was centered on DNA structure, had high efficiency and biocompatibility and can cut S1 to keep the DNA fragment-carrying quencher AuNPs@NE away from the electrode surface, thus restoring the ECL emission of Ir-CB[8] and realizing ultrasensitive detection of miRNA-16. Supramolecular AIECL mediated by host-guest recognition provides a new way for constructing AIECL emitters with excellent structural stability and AIECL efficiency, and an Ir-CB[8] coupling "windmill"-like DNA walker builds a promising ECL-sensing system for bioassay.

CsPbBr3 Perovskite Quantum Dots Encapsulated by a Polymer Matrix for Ultrasensitive Dynamic Imaging of Intracellular MicroRNA.

Herein, CsPbBr3 perovskite quantum dots (CPB PQDs)@poly(methyl methacrylate) (PMMA) (CPB@PMMA) nanospheres were used as energy donors with high Förster resonance energy transfer (FRET) efficiency and exceptional biocompatibility for ultrasensitive dynamic imaging of tiny amounts of microRNAs in living cells. Impressively, compared with traditional homogeneous single QDs as energy donors, CPB@PMMA obtained by encapsulating numerous CPB PQDs into PMMA as energy donors could not only significantly increase the efficiency of FRET via improving the local concentration of CPB PQDs but also distinctly avoid the problem of cytotoxicity caused by divulged heavy metal ions entering living cells. Most importantly, in the presence of target miRNA-21, DNA dendrimer-like nanostructures labeled with 6-carboxy-tetramethylrhodamine (TAMRA) were generated by the exposed tether interhybridization of the Y-shape structure, which could wrap around the surface of CPB@PMMA nanospheres to remarkably bridge the distance of FRET and increase the opportunity for effective energy transfer, resulting in excellent precision and accuracy for ultrasensitive and dynamic imaging of miRNAs. As proof of concept, the proposed strategy exhibited ultrahigh sensitivity with a detection limit of 45.3 aM and distinctly distinguished drug-irritative miRNA concentration abnormalities with living cells. Hence, the proposed enzyme-free CPB@PMMA biosensor provides convincing evidence for supplying accurate information, which could be expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

AND Logic Gate-Regulated DNAzyme Nanoflower for Monitoring the Activity of Multiple DNA Repair Enzymes.

Despite the progress that has been made in diverse DNA-based nanodevices to in situ monitor the activity of the DNA repair enzymes in living cells, the significance of improving both the sensitivity and specificity has remained largely neglected and understudied. Herein, we propose a regulatable DNA nanodevice to specifically monitor the activity of DNA repair enzymes for early evaluation of cancer mediated by genomic instability. Concretely, an AND logic gate-regulated DNAzyme nanoflower was rationally designed by the self-assembly of the DNA duplex modified with both apurinic/apyrimidinic (AP) site and methyl lesion site. The DNAzyme nanoflower could be reconfigured under the repair of AP sites and O6-methylguanine sites by apurinic/apyrimidinic endonuclease 1 (APE1) and O6-methylguanine methyltransferase (MGMT) to produce a fluorescent signal, realizing the sensitive monitoring of the activity of APE1 and MGMT. Compared to the free DNAzyme duplex, the fluorescent response of the DNAzyme nanoflower increased by 60%, due to the effective enrichment of the DNA probes by the nanoflower structure. More importantly, we have demonstrated that the dual-enzyme activated strategy allows imaging of specific cancer cells in the AND logic gate manner using MCF-7 as a cancer cell model, improving the specificity of cancer cell imaging. This AND logic gate-regulated multifunctional DNAzyme nanoflower provides a simple tool for simultaneously visualizing multiple DNA repair enzymes, holding great potential in early clinical diagnosis and drug discovery.

Solvent Regulation Induced Cathode Aggregation-Induced Electrochemiluminescence of Tetraphenylethylene Nanoaggregates for Ultrasensitive Zearalenone Analysis.

Zearalenone (ZEN) is an extremely hazardous chemical widely existing in cereals, and its high-sensitivity detection possesses significant significance to human health. Here, the cathodic aggregation-induced electrochemiluminescence (AIECL) performance of tetraphenylethylene nanoaggregates (TPE NAs) was modulated by solvent regulation, based on which an electrochemiluminescence (ECL) aptasensor was constructed for sensitive detection of ZEN. The aggregation state and AIECL of TPE NAs were directly and simply controlled by adjusting the type of organic solvent and the fraction of water, which solved the current shortcomings of low strength and weak stability of the cathode ECL signal for TPE. Impressively, in a tetrahydrofuran-water mixed solution (volume ratio, 6:4), the relative ECL efficiency of TPE NAs reached 16.03%, which was 9.2 times that in pure water conditions, and the maximum ECL spectral wavelength was obviously red-shifted to 617 nm. In addition, "H"-shape DNA structure-mediated dual-catalyzed hairpin self-assembly (H-D-CHA) with higher efficiency by the synergistic effect between the two CHA reactions was utilized to construct a sensitive ECL aptasensor for ZEN analysis with a low detection limit of 0.362 fg/mL. In conclusion, solvent regulation was a simple and efficient method for improving the performance of AIECL materials, and the proposed ECL aptasensor had great potential for ZEN monitoring in food safety.

Single-Atom Iron Doped Carbon Dots with Highly Efficient Electrochemiluminescence for Ultrasensitive Detection of MicroRNAs.

Herein, single-atom iron doped carbon dots (SA Fe-CDs) were successfully prepared as novel electrochemiluminescence (ECL) emitters with high ECL efficiency, and a biosensor was constructed to ultrasensitively detect microRNA-222 (miRNA-222). Importantly, compared with the conventional without single-atom doped CDs with low ECL efficiency, SA Fe-CDs exhibited strong ECL efficiency, in which single-atom iron as an advanced coreactant accelerator could significantly enhance the generation of reactive oxygen species (ROS) from the coreactant S2O82- for improving the ECL efficiency. Moreover, a neoteric amplification strategy combining the improved strand displacement amplification with Nt.BbvCI enzyme-induced target amplification (ISDA-EITA) could produce 4 output DNAs in every cycle, which greatly improved the amplification efficiency. Thus, a useful ECL biosensor was built with a detection limit of 16.60 aM in the range of 100 aM to 1 nM for detecting traces of miRNA-222. In addition, miRNA-222 in cancer cell lysate (MHCC-97L) was successfully detected by using the ECL biosensor. Therefore, this strategy provides highly efficient single-atom doped ECL emitters for the construction of sensitive ECL biosensing platforms in the biological field and clinical diagnosis.

Dual-Ligand Europium-Organic Gels as a Highly Efficient Anodic Annihilation Electrochemiluminescence Emitter for Ultrasensitive Detection of MicroRNA.

In this study, a novel europium dual-ligand metal-organic gel (Eu-D-MOGs) with high-efficient anodic annihilation electrochemiluminescence (ECL) was synthesized as an ECL emitter to construct a biosensor for ultrasensitive detection of microRNA-221 (miR-221). Impressively, compared to the ECL signal of europium single-ligand metal-organic gels (Eu-S-MOGs), the ECL signal of Eu-D-MOGs was significantly improved since the two organic ligands could jointly replace the H2O and coordinate with Eu3+, which could remarkably reduce the nonradiative vibrational energy transfer caused by the coordination between H2O and Eu3+ with a high coordination demand. In addition, Eu-D-MOGs could be electrochemically oxidized to Eu-D-MOGs•+ at 1.45 V and reduced to Eu-D-MOGs•- at 0.65 V to achieve effective annihilation of ECL, which overcame the side reaction brought by the remaining emitters at negative potential. This benefited from the annihilation ECL performance of the central ion Eu3+ caused by its redox in the electrochemical process. Furthermore, the annihilation ECL signal of Eu3+ could be improved by sensitizing Eu3+ via the antenna effect. In addition, combined with the improved rolling circle amplification-assisted strand displacement amplification strategy (RCA-SDA), a sensitive biosensor was constructed for the sensitive detection of miR-221 with a low detection limit of 5.12 aM and could be successfully applied for the detection of miR-221 in the lysate of cancer cells. This strategy offered a unique approach to synthesizing metal-organic gels as ECL emitters without a coreactant for the construction of ECL biosensing platforms in biomarker detection and disease diagnosis.

Nanoconfined Silver Nanoclusters Combined with X-Shaped DNA Recognizer-Triggered Cascade Amplification for Electrochemiluminescence Detection of APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1), as a vital base excision repair enzyme, is essential for maintaining genomic integrity and stability, and its abnormal expression is closely associated with malignant tumors. Herein, we constructed an electrochemiluminescence (ECL) biosensor for detecting APE1 activity by combining nanoconfined ECL silver nanoclusters (Ag NCs) with X-shaped DNA recognizer-triggered cascade amplification. Specifically, the Ag NCs were prepared and confined in the glutaraldehyde-cross-linked chitosan hydrogel network using the one-pot method, resulting in a strong ECL response and exceptional stability in comparison with discrete Ag NCs. Furthermore, the self-assembled X-shaped DNA recognizers were designed for APE1 detection, which not only improved reaction kinetics due to the ordered arrangement of recognition sites but also achieved high sensitivity by utilizing the recognizer-triggered cascade amplification of strand displacement amplification (SDA) and DNAzyme catalysis. As expected, this biosensor achieved sensitive ECL detection of APE1 in the range of 1.0 × 10-3 U·µL-1 to 1.0 × 10-10 U·µL-1 with the detection limit of 2.21 × 10-11 U·µL-1, rendering it a desirable approach for biomarker detection.

Fluorescence Biosensing Based on Bifurcated DNA Scaffold-Aggregated Ag Nanocluster via Responsive Conformation Switch of Quasi-Molecular Beacon.

To address the limitations of typical hairpin-structural molecular beacons, exploring the ability of a quasi-molecular beacon (qMB) to create label-free fluorescence biosensors is intriguing and remains a challenge. Herein, we propose the first example of modular qMB with the feature of a stimulation-responsive conformation switch to develop an aggregated Ag nanocluster (aAgNC) in a bifurcated DNA scaffold for fluorescently sensing a specific initiator (I*). This qMB was well designed to program four functional modules: I*-recognizable element adopting metastable stem-loop bihairpin structure and two DNA splits (exposed C3GT4 and locked C4AC4T) of aAgNC template that is separated by a tunable hairpin spacer for the customized combination of selective recognition and signaling readout. When presenting I* in an assay route, the specific hybridization induces the directional disassembly of the bihairpin unit, on which the qMB is configurationally switched to liberate the locked split. Thus, the bifurcated parent template pair of C3GT4/C4AC4T is proximal, affording in situ nucleation and clustering of emissive aAgNC. By collecting the fluorescence signal, the quantitative detection of I* is achieved. Benefiting from the ingenious programming of qMB, the recognizing and signaling integration actuates the construction of a facile and convenient fluorescent biosensor featuring rapid reaction kinetics, a wide linear range, high sensitivity, and specificity. This would provide a new paradigm to exploit versatile qMB-based biosensing platforms via stimulation-responsive conformation switches for developing various DNA-scaffolded Ag clusters.