Fe-doped carbon dots: a novel fluorescent nanoprobe for cellular hypochlorous acid imaging.

Real-time monitoring of hypochlorous acid (HClO) in biological systems is of great significance for exploring and regulating its pathological functions because abnormal production of HClO is closely related with many diseases, such as atherosclerosis, rheumatoid arthritis, and cancer. Herein, we developed a reliable fluorescent Fe-doped carbon dots (Fe-CDs) for the sensitive and selective detection of biological HClO using ferrocenecarboxylic acid and m-phenylenediamine as precursors through a one-step solvothermal procedure. The Fe-CDs exhibited excellent a wide HClO detection range from 20 nmol/L to 1000 nmol/L with corresponding limits of detection at 7.8 nmol/L. The sensing mechanism is based on the chemical oxidation of the hydroxyl groups on the surface of Fe-CDs by HClO. In addition, Fe-CDs also displayed high photoluminescence yield, excitation-independence emission, as well as good biocompatibility, enabling the successful imaging of endogenous and exogenous HClO in HeLa cells. These results revealed that Fe-CDs holds great promise as a robust fluorescent probe for investigating HClO-mediated biological events.

Simultaneous Response of Mitochondrial MicroRNAs to Identify Cell Apoptosis with Multiple Responsive Intelligent DNA Biocomputing Nanodevices.

Challenges remained in precisely real-time monitoring of apoptotic molecular events at the subcellular level. Herein, we developed a new type of intelligent DNA biocomputing nanodevices (iDBNs) that responded to mitochondrial microRNA-21 (miR-21) and microRNA-10b (miR-10b) simultaneously which were produced during cell apoptosis. By hybridizing two hairpins (H1 and H2) onto DNA nanospheres (DNSs) that had been previously modified with mitochondria-targeted triphenylphosphine (TPP) motifs, iDBNs were assembled in which two localized catalytic hairpins self-assembly (CHA) reactions occurred upon the co-stimulation of mitochondrial miR-21 and miR-10b to perform AND logic operations, outputting fluorescence resonance energy transfer (FRET) signals for sensitive intracellular imaging during cell apoptosis. Owing to the spatial confinement effects of DNSs, it was discovered that iDBNs had a high efficiency and speed of logic operations by high local concentrations of H1 and H2, making the simultaneous real-time responses of mitochondrial miR-21 and miR-10b reliable and sensitive during cell apoptosis. These results demonstrated that iDBNs were simultaneously responsive to multiple biomarkers, which greatly improved the detection accuracy to identify the cell apoptosis, demonstrating that iDBNs are highly effective and reliable for the diagnosis of major disease and screening of anticancer drugs.

Rational Design of Cascade DNA System for Signal Amplification.

System leakage critically confines the development of cascade DNA systems that need to be implemented in a strict order-by-order manner. In principle, ternary DNA reactants, composed of three single-strand DNA (ssDNA) with a strict equimolar ratio (1:1:1), have been indispensable for successfully cascading upstream entropy-driven DNA circuit (EDC) with downstream circuits, and system leakage will occur with any unbalance of the molar ratio. In this work, we proposed "splitting-reconstruction" and "protection-release" strategies on the potential downstream circuit initiator derived from upstream EDC to guide the construction of EDC-involved cascade systems independent of system leakage derived from unpurified reactants. Both the reconstructed and released downstream circuit initiators were in compliance with the principle of the cascade AND logic gate. Using these two strategies, two cascade systems─EDC2-4WJ-TMSDR and EDC3-HCR─were developed to carry out the designed order, which did not require that the ratio of 1:1:1 be maintained. Furthermore, the inherent property of the upstream EDC could transfer into the downstream circuit, endowing the developed cascade systems with a more powerful signal amplification ability for the sensitive detection of the corresponding initiator strand. These two strategies may provide new insights into the process of constructing EDC-like circuit-involved high-order DNA networks.

CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.

DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.

Electrostatic assemblies of molecularly imprinted polymers on the surface of electrospun nanofiber membranes for the point-of-care detection of thiodiglycol, a sulfur mustard poisoning metabolic marker.

In this study, molecularly imprinted polymers (MIPs) were assembled on the surface of ethylene imine polymer (PEI)/poly(vinyl alcohol) (PVA) electrospun nanofiber membranes for the point-of-care testing (POCT) of thiodiglycol (TDG), a sulfur mustard poisoning metabolic marker, using concentrated gold nanoparticles (AuNPs) as the signal reporting units. The MIPs/PEI/PVA nanofiber membranes could capture TDG specifically through the recognition interaction between MIPs and TDG. Then, AuNPs were adsorbed onto the MIPs/PEI/PVA nanofiber membranes through the Au-S interaction between TDG and AuNPs to produce a visible red color. In order to improve the sensitivity, the silver-enhanced solutions were used to deepen the color of the nanofiber membranes and the software Image J was used to read the gray value as the signal response for subsequent analysis. There was a good linear relationship between the color change of the MIPs/PEI/PVA nanofiber membranes and the TDG concentration from 0.1 ng mL-1 to 1.0 µg mL-1, and the limit of detection was 38 pg mL-1. This method was applied for the selective detection of TDG in urine, showing great potential for the clinical diagnosis of mustard gas poisoning.

A General Signal Amplifier of Self-Assembled DNA Micelles for Sensitive Quantification of Biomarkers.

Owing to the excellent structural rigidity and programmable reaction sites, DNA nanostructures are more and more widely used, but they are limited by high cost, strict sequence requirements, and time-consuming preparation. Herein, a general signal amplifier based on a micelle-supported entropy-driven circuit (MEDC) was designed and prepared for sensitive quantification of biomarkers. By modifying a hydrophobic cholesterol molecule onto a hydrophilic DNA strand, the amphiphilic DNA strand was first prepared and then self-assembled into DNA micelles (DMs) driven by hydrophobic effects. The as-developed DM showed unique advantages of sequence-independence, easy preparation, and low cost. Subsequently, amplifier units DMF and DMTD were successfully fabricated by connecting fuel strands and three-strand duplexes (TDs) to DMs, respectively. Finally, the MEDC was triggered by microRNA-155 (miR-155), which herein acted as a model analyte, resulting in dynamic self-assembly of poly-DNA micelles (PDMs) and causing the recovery of cyanine 3 (Cy3) fluorescence as the DMTD dissociated. Benefiting from the "diffusion effect", the MEDC herein had a nearly 2.9-fold increase in sensitivity and a nearly 97-fold reduction in detection limit compared to conventional EDC. This amplifier exhibited excellent sensitivity of microRNAs, such as miR-155 detection in a dynamic range from 0.05 to 4 nM with a detection limit of 3.1 pM, and demonstrated outstanding selectivity with the distinguishing ability of a single-base mismatched sequence of microRNAs. Overall, the proposed strategy demonstrated that this sequence-independent DNA nanostructure improved the performance of traditional DNA probes and provided a versatile method for the development of DNA nanotechnology in biosensing.

Plasmonic Hot-Electron-Painted Au@Pt Nanoparticles as Efficient Electrocatalysts for Detection of H2O2.

Plasmon-driven catalysis of metal nanostructures has garnered wide interest. Here, a photogenerated plasmonic hot-electron painting strategy was reported to form Au@Pt composite nanoparticles (Au@Pt NPs) with high catalytic reactivity without using reducing agents. Au nanoparticles, including Au nanospheres (Au NSs), Au nanorods (Au NRs), and Au nanobipyramids (Au NBPs), generated hot electrons under localized surface plasmon resonance (LSPR) excitation, which made the platinum precursor reduced as a consequence that Pt(0) atoms were painted on the surface of Au NPs to form an asymmetric Pt shell outside the plasmonic Au core. Compared with bare Au NPs, Au@Pt NPs exhibited significantly enhanced electrocatalytic activity toward reduction of H2O2 due to the bimetallic synergistic effect and great dispersion of Au@Pt NP-modified indium tin oxide (Au@Pt NPs/ITO). It exhibited a linear detection of H2O2 in a wide concentration range from 0.5 to 1000 µM with a low detection limit of 0.11 µM (S/N = 3). Therefore, the plasmonic hot-electron-painted Au@Pt NPs represent a novel and simple method for the design of advanced noble asymmetric metal nanomaterials.

Efficient and Sustainable in†situ Photo-Fenton Reaction to Remove Phenolic Pollutants by NH2 -MIL-101(Fe)/Ti3 C2 Tx Schottky-Heterojunctions.

Metal-organic frameworks (MOFs) with abundant active sites, a class of materials composed of metal nodes and organic ligands, is widely used for photocatalytic degradation of pollutants. However, the rapid recombination of photoinduced carriers of MOFs limits its photocatalytic degradation performance. Herein, Ti3 C2 Tx nanosheets-based NH2 -MIL-101(Fe) hybrids with Schottky-heterojunctions were fabricated by in situ hydrothermal assembly for improved photocatalytic activity. The photodegradation efficiencies of the NH2 -MIL-101(Fe)/Ti3 C2 Tx (N-M/T) hybrids for phenol and chlorophenol were 96.36 % and 99.83 % within 60 minutes, respectively. The N-M/T Schottky-heterojunction duly transferred electrons to the Ti3 C2 Tx nanosheets surface via built-in electric fields, effectively suppressing the recombination of photogenerated carriers, thereby improving the photocatalytic performance of NH2 -MIL-101(Fe). Moreover, the Fe-mixed-valence in the N-M/T led to improvement in the efficiency of the in situ generated photo-Fenton reactions, further enhancing the photocatalytic activity with more generated reactive oxygen species (ROS). The study proposes a highly effective removal of phenolic pollutants in wastewater.

Facile synthesis of porphyrin-MOFs with high photo-Fenton activity to efficiently degrade ciprofloxacin.

The slow conversion of Fe(Ⅱ)/Fe(III) cycle was largely limited the degradation efficiency of many photo-Fenton systems. Herein, four Fe-MOFs nanorods (namely Fe-TCPP-1, Fe-TCPP-2, Fe-TCPP-3, Fe-TCPP-4) with decreasing length-diameter ratios were synthesized in a household microwave oven, using photosensitizer porphyrin and iron ions with Fenton activity as building blocks. Among them, the Fe-TCPP-3 exhibited high photogenerated electron-hole (e--h+) separation efficiency and largest pore structure, endowing Fe-TCPP-3 with superior photo-Fenton property. In addition, Fe-TCPP-3 based photo-Fenton system was applied to efficiently degrade antibiotic ciprofloxacin (CIP) under neutral condition, due to the continuously generated reactive species (h+, e-, OH·, O2·-, 1O2) in Fe-TCPP-3 under visible-light irradiation. With irradiation for 30 min, the degradation efficiency of the system could reach about 73 %, which was about 26-fold towards the system without light irradiation. This study paved a way to modulating the photo-Fenton activity of MOF-based catalysts.

A catalyst-free co-reaction system of long-lasting and intensive chemiluminescence applied to the detection of alkaline phosphatase.

A catalyst-free co-reaction luminol-H2O2-K2S2O8 chemiluminescence (CL) system was developed, with long-life and high-intensity emission, and CL emission lasting for 6 h. A possible mechanism of persistent and intense emission in this CL system was discussed in the context of CL spectra, cyclic voltammetry, electron spin resonance (ESR), and the effects of radical scavengers on luminol-H2O2-K2S2O8 system. H2O2 and K2S2O8 co-reactants can promote each other to continuously generate corresponding radicals (OH•, 1O2, O2•-, SO4•-) that trigger the CL emission of luminol. H2O2 can also be constantly produced by the reaction of K2S2O8 and H2O to further extend the persistence of this CL system. CL emission can be quenched via ascorbic acid (AA), which can be generated through hydrolysis reaction of L-ascorbic acid 2-phosphate trisodium salt (AAP) and alkaline phosphatase (ALP). Next, a CL-based method was established for the detection of ALP with good linearity from 0.08 to 5 U·L-1 and a limit of detection of 0.049 U·L-1. The proposed method was used to detect ALP in human serum samples.

A high-integrated DNA biocomputing platform for MicroRNA sensing in living cells.

DNA logic computing has captured increasing interest due to its ability to assemble programmable DNA computing elements for disease diagnosis, gene regulation, and targeted therapy. In this work, we developed an aptamer-equipped high-integrated DNA biocomputing platform (HIDBP-A) with a dual-recognition function that enabled cancer cell targeting. Dual microRNAs were the input signals and can perform AND logic operations. Compared to the free DNA biocomputing platform (FDBP), the integration of all computing elements into the same DNA tetrahedron greatly improved logic computing speed and efficiency owing to the confinement effect reflected by the high local concentration of computing elements. As a proof of concept, the utilization of microRNA as the input signal was beneficial for improving the scalability and flexibility of the sequence design of the logic nano-platform. Given that the different microRNAs were over-expressed in cancer cells, this new HIDBP-A has great promise in accurate diagnosis and logic-controlled disease treatment.

Simultaneous Imaging of Dual microRNAs in Cancer Cells through Catalytic Hairpin Assembly on a DNA Tetrahedron.

Accurate detection and imaging of tumor-related microRNA (miRNA) in living cells hold great promise for early cancer diagnosis and prognosis. One of the challenges is to develop methods that enable the identification of multiple miRNAs simultaneously to further improve the detection accuracy. Herein, a simultaneous detection and imaging method of two miRNAs was established by using a programmable designed DNA tetrahedron nanostructure (DTN) probe that includes a nucleolin aptamer (AS1411), two miRNA capture strands, and two pairs of metastable catalytic hairpins at different vertexes. The DTN probe exhibited enhanced tumor cell recognition ability, excellent stability and biocompatibility, and fast miRNA recognition and reaction kinetics. It was found that the DTN probe could specifically enter tumor cells, in which the capture strand could hybridize with miRNAs and initiate the catalytic hairpin assembly (CHA) only when the overexpressed miR-21 and miR-155 existed simultaneously, resulting in a distinct fluorescence resonance energy transfer signal and demonstrating the feasibility of this method for tumor diagnosis.

A centrifugal microfluidic chip for point-of-care testing of staphylococcal enterotoxin B in complex matrices.

Staphylococcal enterotoxin B (SEB) is a typical biological toxin that causes food poisoning. Currently reported SEB detection methods have the drawbacks of sophisticated sample preparation and being time-consuming and labor-intensive. Herein, we propose a strategy based on an immune sandwich structure operating on a centrifugal microfluidic chip for point-of-care testing (POCT) of SEB. The fluorescent microparticle-labeled primary antibody (CM-EUs-Ab1), capture antibody (CAb), and goat anti-mouse IgG antibody (SAb) were modified on the bond area, T-area, and C-area, respectively. When SEB was added, it first reacted with the CM-EUs-Ab1 through the specific recognition between SEB and the Ab1. Then, under capillarity, the conjugates of SEB and the CM-EUs-Ab1 were captured by the CAb when they flowed to the T-area, and the remaining CM-EUs-Ab1 bound with the SAb in the C-area. Finally, this chip was put into a dry fluorescence detection analyzer for centrifugation and on-site detection of SEB. The fluorescence intensity ratio of the T-area to the C-area was positively correlated with the concentration of SEB. The resulting linear range was 0.1-250 ng mL-1, and the limit of detection (3σ/k) was 68 pg mL-1. This POCT platform only needs 20 µL of sample and can realize the full process of detection within 12 min. This chip also exhibits good stability for 35 days. Additionally, the proposed method has been successfully utilized for the detection of SEB in urine, milk, and juice without any pre-treatment of the samples. Thus, this platform is expected to be applied to food safety testing and clinical diagnosis.

Sensitive Logic Nanodevices with Strong Response for Weak Inputs.

Sensitive sensing is critical when developing new calculation systems with weak input signals (ISs). In this work, a "weak-inputs-strong-outputs" strategy was proposed to guide the construction of sensitive logic nanodevices by coupling an input-induced reversible DNA computing platform with a hybridization chain reaction-based signal amplifier. By rational design of the sequence of computing elements (CEs) so as to avoid cross-talking between ISs and signal amplifier, the newly formed logic nanodevices have good sensitivity to the weak ISs even at low concentrations of CEs, and are able to perform YES, OR, NAND, NOR, INHIBIT, INHIBIT-OR and number classifier operation, showing that the DNA calculation proceeds in dilute solution medium that greatly improves the calculation proficiency of logic nanodevices without the confinement of the lithography process in nanotechnology.

An ultrathin 2D Yb(III) metal-organic frameworks with strong electrochemiluminescence as a "on-off-on" platform for detection of picric acid and berberine chloride form.

To investigate the strong electrochemiluminescence (ECL) of ultrathin two dimensional metal-organic frameworks (2D MOFs) is crucial. In this work, we reported the strong ECL behavior of 2D Yb-MOFs, which exhibited thickness-dependent ECL. The thinner the 2D Yb-MOFs, the stronger the ECL signals. The corresponding ECL emission mechanism was investigated in detail, which was ascribed to the thinner 2D Yb-MOF with larger specific surface area, provided more luminophores, better electronic conductivity and superior fluorescence quantum yield, which yielded a higher ECL intensity. Considering the excellent ECL performances above, the ultrathin 2D Yb-MOF-1 was selected as new ECL emitter so that a sensor could be fabricated to realize the "on-off-on" detection of picric acid (PA) and berberine chloride form (BCF). The proposed sensor strategy exhibited a good analytical performance, where the linear range for PA detection was from 0.1 µM to 1 µM with a limit of 81.3 nM, and that for BCF detection from 0.05 µM to 1 µM with a limit of 36.5 nM. This study carves out a novel avenue for exploiting excellent ECL materials.

Preparation of a molecularly imprinted test strip for point-of-care detection of thiodiglycol, a sulfur mustard poisoning metabolic marker.

Conventional methods for the detection of the sulfur mustard poisoning metabolic marker, thiodiglycol (TDG), require expensive instruments and reagents as well as professional operators. To address these problems, a novel test strip based on a molecularly imprinted sensitive membrane (MIM) was developed in this work for point-of-care (POC) detection of TDG. The TDG test strip was prepared conveniently by coating molecular imprinted polymers (MIPs) on a nitrocellulose membrane. When the sample contained TDG, the MIPs could specifically bind with TDG. A great number of AuNPs (AuNPs) could then be adsorbed on the test strip via the formation of an Au-S bond between TDG and AuNPs, giving the test strip the obvious red color of AuNPs. In the absence of TDG, the test strip exhibited much lighter color because it could not adsorb AuNPs. By monitoring the color change of the test strip, TDG could be detected from 1.0 ng/mL to 100.0 µg/mL with a detection limit of 0.23 ng/mL (3σ) under the optimal conditions (the molar ratio of TDG to MAA was 1:2; the eluent was chloroform; the elution time was 50 min; the reaction time between MIPs and TDG was 15 min; the adsorption time of AuNPs was 40 min; the temperature of the reaction system was 35 °C). This method has excellent selectivity and has been used to detect TDG in urine, showing great potential for POC detection of TDG in clinical samples.

Ultrasensitive ratiometric electrochemiluminescence for detecting atxA mRNA using luminol-encapsulated liposome as effectively amplified signal labels.

It is an advantageous way to quickly identify the toxicity of Bacillus anthracis (B. anthracis) by detecting the transcription product of the atxA gene. Herein, a novel ultrasensitive ratiometric electrochemiluminescence (ECL) biosensor with competitive mechanism and double amplified signal ways was proposed for detecting the atxA mRNA. The K2S2O8 was used as cathodic emitter and silver metal-organic gels (AgMOG) was used as ECL enhancer. The AgMOG could accelerate the electro-catalytic reduction of S2O82- to SO4˙-, which reacted with dissolved oxygen, resulting in strong cathodic ECL. Meanwhile, luminol was encapsulated in liposome as anodic amplified signal labels and the luminol anion radical also reacted with dissolved oxygen to create the anodic ECL emission. We immobilized luminol-encapsulated liposomes on the surface of AgMOG through the hybridization of DNA and mRNA. This would provide a competitive mechanism involving dissolved oxygen between K2S2O8 and luminol. Benefiting from the competitive mechanism and amplified signal ways, this ratiometric biosensor achieved a wide linear relationship range from 10 to 300 fM with a low limit of detection (8.13 fM). Considering the accessible operation, favorable performance, and high universality of this strategy, this work may be used to analyze other mRNAs of bacteria.

A crosslinked submicro-hydrogel formed by DNA circuit-driven protein aggregation amplified fluorescence anisotropy for biomolecules detection.

Protein is an excellent molecular mass amplifier without fluorescence quenching effect for fluorescence anisotropy (FA) assay. However, in traditional protein amplified FA methods, the binding ratio between amplifier and dye-modified probe is 1:1 or one target can only induce FA change of one fluorophore on probe, resulting in low sensitivity. Herein, we developed a simple FA strategy with high accuracy and sensitivity by using a crosslinked submicro-hydrogel that was formed through a catalyzed hairpin assembly (CHA) assisted protein aggregation as a novel FA amplifier. In the presence of catalyst, the CHA process was initiated through the toehold-mediated strand exchange reaction, which led to the formation of a dye and biotin-labeled Y-shaped H1-H2 duplex (YHD) and recycling of catalyst. With the introduction of streptavidin, a crosslinked submicro-hydrogel was formed by strong binding affinity between biotin on YHD and streptavidin, resulting in an increased FA of fluorescent dye. After rational design of the catalyst sequence, this method has been utilized for the detection of miRNA-145, staphylococcal enterotoxin B (SEB) and ATP with an LOD of 2.5 nM, 92 pg mL-1 and 3.6 µM, respectively. Moreover, this FA assay has been successfully applied for direct detection of target in biological samples, demonstrating its practicality in complex biological systems.

Europium coordination polymer particles based electrospun nanofibrous film for point-of-care testing of copper (II) ions.

Excess free copper in serum has been identified to induce neurodegenerative diseases such as Alzheimer's disease, thus it is very important to determine copper (II) ions (Cu2+) content for human health test. Herein we developed a point-of-care testing (POCT) platform through a luminescence "on-off" recognition mechanism of serum copper. Microsized europium coordination polymer particles (Eu-CPs), which was prepared with citric acid (CA) and europium nitrate hexahydrate through a hydrothermal route, were then successfully loaded with the mixture of 2,6-pyridinedicarboxylic acid (DPA) and poly(vinyl alcohol) (PVA) to form electrospun nanofibrous films (ENFFs). The as-prepared Eu-CPs/DPA/PVA ENFFs exhibited red emission at 618 nm when exciting at 280 nm, with the quantum yields of 22.2% owing to the antenna effect from DPA to Eu3+. Furthermore, the strong luminescence could be selectively quenched by Cu2+ through coordination with DPA to interrupt the antenna effect. With that, Cu2+ was successfully detected in the range of 2-45 µM with a detection of limit of 1.3 µM, well matching with the requirement of clinic test of excess free copper induced neurodegenerative diseases. As a proof of concept at last, this POCT platform was used to detect free copper in spiked serum samples with a recovery of 101.1%-105.2%, demonstrating that this platform provides significant potential for use in clinical test.

Hierarchical Hybridization Chain Reaction for Amplified Signal Output and Cascade DNA Logic Circuits.

In this work, we propose a three-layer hierarchical hybridization chain reaction (3L hHCR) composed of 1stHCR, 2ndHCR, and 3rdHCR to achieve robust signal amplification efficiency and broaden the applied range of HCR-based systems. In principle, the execution of superior HCR generates the formation of the initiator (named as 2ndI or 3rdI) of the subordinate HCR that relies on the introduction of the target sequence (1stI). To avoid the high background signal of the 3L hHCR system, a strategy of "splitting reconstruction" was adopted. The initiator of the subordinate HCR was designed as two separate fragments (splitting) that are obtained together (reconstruction) for the motivation of the subordinate HCR after the completion of the superior HCR. The implementation of the entire 3L hHCR system generates significant fluorescence recovery that derives from the impediment of Förster resonance energy transfer between fluorophore and quencher; thus, ultrasensitive detection of 1stI in the range of 50 pM to 10 nM can be achieved. Surprisingly, when the concentration of 1stI is lower than 1 nM, the 3L hHCR shows excellent ability to discriminate against various concentrations of 1stI, which is better than that of the 2L hHCR I system. Due to the hierarchical self-assembly mechanism, the 3L hHCR can also be reliably operated as a cascade AND logic gate with a high specificity and molecular keypad lock with a prompt error-reporting function. Furthermore, the 3L hHCR-based molecular keypad lock also shows potential application in the accurate diagnosis of cancer. The 3 L hHCR shows visionary prospects in biosensing and the fabrication of advanced biocomputing networks.